The Staphylococcus aureus secretome - TI Pharma

The Staphylococcus aureus secretome

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978-90-902-5004-5 (printed version)
978-90-367-4182-8 (digital version)
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Cover
Cover designed by Feiko Beckers (www.feikobeckers.com). The cover illustrates the typical
cluster of Staphylococcus aureus cells represented by ‘LEGO-tires’. On the back: a signpost
with the different paths that a newly synthesized protein can take depending on the nature of
its signal peptide.
2

RIJKSUNIVERSITEIT GRONINGEN
The Staphylococcus aureus secretome
Proefschrift
ter verkrijging van het doctoraat in de
Medische Wetenschappen
aan de Rijksuniversiteit Groningen
op gezag van de
Rector Magnificus, dr. F. Zwarts,
in het openbaar te verdedigen op
woensdag 27 januari 2010
om 13:15 uur
door
Mark Jan Jacobus Bernhard Sibbald
geboren op 18 mei 1975
te Bolsward
3

Promotor : Prof. dr. J.M. van Dijl
Co-promotor : Dr. J-.Y.F. Dubois
Beoordelingscommissie : Prof. dr. Tarek Msadek
: Prof. dr. Wim Quax
: Prof. dr. Arnold Driessen
4

Paranimfen: Thijs R.H.M. Kouwen
Monika A. Chlebowicz
Voor Regina
Voor Pap
“I’ll always remember the chill of November”
“Carpe diem - seize the day”
“Look around, hear the sounds
Cherish your life while you’re still around”
“Seize the day and don't you cry, now it's time to say goodbye
Even though I'll be gone, I will live on”
-Dream Theater – “A Change Of Seasons”-
“Thank you for the inspiration, thank you for the smiles
All the unconditional love that carried me for miles
It carried me for miles
But most of all: thank you for my life”
-Dream Theater – “Best Of Times”-
5

The work described in this thesis was performed in the laboratory of Molecular Bacteriology,
Department of Medical Microbiology of the University Medical Center Groningen and University of
Groningen, Groningen, the Netherlands.
Printing of this thesis was financially supported by the Graduate School for Drug Exploration
(GUIDE), the Juriaanse Stichting, Nederlandse Vereniging voor Medische Microbiologie
(NVvM/NVMM), DSM Nutritional Products Ltd, and Biomade. Their support is highly appreciated.
6

Table of contents
Chapter 1. General Introduction
Chapter 2. Mapping the pathways to staphylococcal pathogenesis by comparative secretomics
(Sibbald et al., MMBR, 2006)
Chapter 3. Proteogenomics uncovers extreme heterogeneity in the Staphylococcus aureus
exoproteome due to genomic plasticity and variant gene regulation (Ziebandt et al., in
revision)
Chapter 4. Characterization of Staphylococcus aureus secretion mutants (Sibbald et al., to be
submitted)
Chapter 5. Synthetic effects of secG and secY2 mutations on exoproteome biogenesis in
Staphylococcus aureus (Sibbald et al., in revision)
Chapter 6. Partially overlapping substrate specificities of sortases A and C of staphylococci
(Sibbald et al., submitted)
Chapter 7. The large mechanosensitive channel MscL determines bacterial susceptibility to the
bacteriocin sublancin 168 (Kouwen et al., Antimicrob Agents Chemother, 2009.)
Chapter 8. The extracellular proteome of Bacillus licheniformis grown in different media and
under different nutrient starvation conditions (Voigt et al., Proteomics, 2005)
Chapter 9. General summary and discussion
Chapter 10. Reference list
Chapter 11. Nederlandse samenvatting
Appendices:
I. Dankwoord
II. List of publications
III. Supplemental tables
7

"The greatest education in the world is watching the masters at work"
- Michael J. Jackson-
8

Chapter 1
Introduction and scope of this thesis
9

Chapter 1
Introduction
Bacteria are the oldest living organisms that inhabit this planet. During evolution some of
these prokaryotic cells have been working together to evolve into multicellular organisms.
This has resulted in the multidiversity of organisms that have existed and exist to this day.
Like the vast majority of organisms in the animal kingdom, human beings carry a huge
variety of microorganisms, including many bacterial species but also archaea and yeasts.
Collectively these organisms are known as the human microbiota. Most bacteria amongst the
human microbiota are commensal, but some of them are in fact opportunistic pathogens that
can cause a wide range of diseases. On the other hand, some bacteria seem to help the human
host by competing with opportunistic and primary pathogens. These beneficial species thus
prevent harmful bacteria to colonize and spread throughout the host. However, when such
pathogens break through the human and bacterial defenses, they can cause a wide range of
diseases which, in some cases, can be life-threatening. To conquer certain niches in the
human host, pathogenic bacteria have to overcome many stressful conditions, as imposed by
the human innate and adaptive immune systems. To accomplish this, bacteria preduce an
arsenal of virulence factors. Although one proteinaceous virulence factor can already be
sufficient to cause particular symptoms of disease, the synergistic actions of many other
proteins are needed for bacterial survival in the host. Both groups of proteins are part of the
arsenal of virulence factors that contribute to the disease-causing ability of pathogenic
bacteria. The proteins that are actively involved in the processes of colonization, invasion,
spreading, immune evasion and the triggering of excessive immune responses are all
synthesized in the cytoplasm of the bacteria, and then transported across the bacterial
membrane to an extracytoplasmic location, such as the bacterial cell wall or the host’s millieu.
These transport systems are complex systems that are embedded in the bacterial membrane
and consist of a translocation motor and a channel through which the proteins are
translocated. Like all living organisms, bacteria contain several transport systems that are
used to transport proteins across the membrane. The best-studied transport system is the
general secretion (Sec) pathway. Furthermore, several other “special purpose” transport
pathways are under investigation to understand their contribution to the transport of virulence
factors, such as the Twin-arginine translocase (Tat) and the ESX-1 or ESAT-6 secretion
system (Ess) pathway.
All proteins that are actively translocated via the Sec or Tat pathways are synthesized with Nterminal
signal peptides that lead them to the respective transport system. The signal that
directs proteins to the Ess pathway remains to be defined. During or shortly after passage of
precursor proteins through the Sec or Tat translocation channels, their signal peptides are
removed by a so-called signal peptidase. While the Tat channel has the potential to transport
fully folded proteins acrosse the membrane, the Sec channel can only handle proteins in an
unfolded state. Therefore, proteins have to fold into their active and protease-resistant
conformation after translocation through the Sec channel. This can occur spontaneously, or
with the aid of chaperones and folding catalysts. Finally, the protein is either retained in an
extracytoplasmic compartment of the cell or released into the extracellular environment. In
the case of Gram-positive bacteria, three extracytoplasmic compartments can be
distinguished: the membrane, the membrane-cell wall interface, and the cell wall. The
proteins that are exposed at the surface of bacterial cells are very important for the bacterial
10

Introduction and scope of this thesis
adherence to host tissues and evasion of the host defense systems. In addition, the virulence
factors that are released into the host milieu may damage host cells, degrade
biomacromolecules of the host, or cause strong inflammatory responses, thereby contributing
to the symptoms of disease caused by a particular bacterium (For reviews see Tjalsma et al.,
2000; Tjalsma et al., 2004; Sibbald et al., 2006; van Dijl et al., 2007; Sibbald and van Dijl,
2009).
Staphylococcus aureus is a Gram-positive bacterium that is part of the human microbiota,
residing mostly in the mucosal environment in the nose. In fact, ~20% of the human
population is a persistant carrier of S. aureus, while 60% is an intermittent carrier.
Unfortunately, S. aureus can transform from an apparently harmles commensal into a
dangerous pathogen. Once the organism crosses the defense systems of the human host, it can
spread to almost every organ and tissue, causing a wide range of diseases. These can vary
from superficial lesions, styes and furunculosis, to more serious infections such as
pneumonia, urinary tract infections, endocarditis, and in rare cases even meningitis (Cheng et
al., 2009; Dubrac et al., 2008; Fedtke et al., 2004; García-Lara et al., 2005; Novick, 2003;
van Belkum A., 2006; Wardenburg et al., 2007). Moreover, S. aureus has an amazing ability
to develop resistance against several antibiotics, which became evident already shortly after
the introduction of penicillin for clinical applications. In the mean time, S. aureus strains
exhibiting resistance to most antibiotics are known, the methicillin resistant S. aureus
(MRSA) being most notorious (annual Report EARSS 2007; http://www.rivm.nl/earss/
results/Monitoring_reports). Up till now, most MRSA infections were nosocomial (i.e.
hospital-acquired). However, recent reports indicate an increase in dangerous communityacquired
MRSA infections (Centers for Disease Control and Prevention, 2003; Grundmann et
al., 2002; Vandenesch et al., 2003). Vancomycin has been for long time a last resort antibiotic
against MRSA, but in 1996 the first vancomycin intermediate resistant strain (VISA) was
reported (Hiramatsu et al., 1997). Since then, several other cases of other VISA strains and
even strains with complete resistance (VRSA) against vancomycin have been isolated (Cui et
al., 2003; Weigel et al., 2003). Because of the anticipated rise of multiple antibiotic resistant
S. aureus strains, innovative strategies for prevention and intervention of S. aureus infections
are urgently needed. Recent studies are therefore focusing on the development of novel
antibiotics, anti-staphylococcal vaccines and therapeutic protective antibodies (Arrecubieta et
al., 2008; Glowalla et al., 2009; Middleton, 2008; Nanra et al., 2009; Otto, 2008; Schaffer
and Lee, 2009; Zweers et al., 2009).
11

Chapter 1
Scope of this thesis
Due to its large arsenal of virulence factors and the ability to adapt rapidly to externally
imposed stresses and insults, S. aureus has become one of the most “successful” human
pathogens. These virulence factors are synthesized on cytoplasmic ribosomes and transported
across the membrane to extracytoplasmic locations as introduced above. Especially for the
Gram-negative bacterium Escherichia coli, the protein translocation machinery has been
described in great detail. For studies on Gram-positive bacterial secretion systems, Bacillus
subtilis has served as the main model organism. However, B. subtilis is non-pathogenic and
relatively little is known about the precise roles of protein translocation systems in related
pathogens, such as Bacillus anthracis, Listeria monocytogenes, Mycobacterium tuberculosis,
Streptococcus pneumoniae and, last but not least, S. aureus. The present thesis studies were
aimed at defining the secretome of S. aureus. The secretome includes all proteins that are
involved in protein export processes from the cytoplasm to extracytoplasmic locations, as
well as the proteins that are translocated across the bacterial membrane. Where appropriate,
studies involved comparisons with Gram-positive bacteria that are related to S. aureus, like
Staphylococcus epidermidis, Bacillus subtilis and Bacillus licheniformis.
In the studies described in Chapter 2, the genomes of several S. aureus strains and one S.
epidermidis strain were scanned with bioinformatic tools for the presence of genes encoding
proteins that are involved in the export of extracytoplasmic proteins. Furthermore, proteins
that carry N-terminal signal peptides were identified through bioinformatics. The obtained
results were compared with each other to define the core and variant S. aureus exoproteomes.
Chapter 3 reports on a first comprehensive survey of the composition and variability of the S.
aureus exoproteome following a proteogenomics approach. Dissection of the exoproteomes
of 25 clinical isolates revealed that only seven out of 63 identified secreted proteins were
produced by all isolates, revealing a high exoproteome heterogeneity. The observed variations
were caused by both genome plasticity and an unprecedented variation in gene expression.
The data have important implications for future studies on staphylococcal virulence and the
development of protective vaccines against this pathogen.
Chapter 4 describes the construction and analysis of a collection of isogenic S. aureus
secretion mutants. The exproteomes of the mutant strains were analyzed by SDS-PAGE,
proteomics, western blotting, zymogram analysis, spreading assays, hemolysin activity
assays, electron microscopy, and a Caenorhabditis elegans killing assay. While no
phenotypes were detectable for some of the mutants, strains with mutations in dsbA, lgt or
secG genes did show clear secretion defects. Notably, in certain strains second site mutations
were observed that led to the loss of RNAIII. While this seems to be a consequence of the
natural adaptive capabilities of S. aureus, it is also an important warning for future studies on
protein secretion in this organism and it underscores the need for genetically stable model
strains.
In Chapter 5 the roles of the non-essential Sec channel components SecG and SecY2 in the
biogenesis of the extracellular proteome of S. aureus were investigated. The results show that
SecG is of major importance for protein secretion by S. aureus. No secretion defects were
observed for strains with a secY2 single mutation, but deletion of secY2 significantly
exacerbated the secretion defects of secG mutants. Furthermore, the secG secY2 double
mutant displayed a synthetic growth defect. These findings suggest that SecY2 can interact
with the Sec1 channel of S. aureus.
12

Introduction and scope of this thesis
Some of the staphylococcal virulence factors are proteins that are displayed at the cell wall
surface. In S. aureus some of these surface proteins are linked to the cell wall by so-called
sortases. In Chapter 6, the exoproteomes of S. aureus and S. epidermidis srtA mutants were
investigated and compared to the respective parental strains. Several SrtA substrates were
identified, and their final subcellular localization was found to be altered in the srtA mutants.
Among these identified proteins were the S. aureus surface protein G (SasG) and the
accumulation associated protein (Aap) from S. epidermidis. Biofilm formation was affected in
the srtA mutants. Complementation studies were performed with SrtA from S. aureus and S.
epidermidis as well as with SrtC from S. epidermidis and YhcS from B. subtilis. Only
complementation with S. aureus or S. epidermidis SrtA resulted in restoration of the parental
phenotype. In contrast, SrtC of S. epidermidis only partially seems to restore the parental
phenotype, and YhcS of B. subtilis was not able to complement for the loss of SrtA at all.
In Chapter 7, the susceptibility of bacteria towards the extremely stable and broad-spectrum
lantibiotic sublancin 168 was investigated. S. aureus is one of several important pathogens
that is susceptible towards sublancin 168. Growth inhibition and competition assays on plates
and in liquid cultures revealed that the addition of NaCl lowered the sublancin 168
susceptibility of S. aureus and B. subtilis. In addition, it was shown that the presence of the
large mechanosensitive channel of conductance MscL is important for the susceptibility of
these bacteria. Taken together, the results demonstrate that MscL is a critical and specific
determinant in bacterial sublancin 168 susceptibility that may either serve as a direct target for
this lantibiotic, or as a gate of entry to the cytoplasm.
Chapter 8 describes the exoproteome of the commercially interesting organism B.
licheniformis. With the annotated genome sequence of B. licheniformis DSM 13, a secretome
prediction analysis was performed. A total of 296 proteins were predicted to contain an Nterminal
signal peptide directing most of them into the Sec pathway. From analysis of the
extracellular proteomes of B. licheniformis it was concluded that higher amounts of protein
are secreted when cell are grown in a complex medium compared to cell grown in a minimal
medium. In addition, limitation of phosphate, carbon and nitrogen sources resulted in the
secretion of specific proteins that may be involved in counteracting the imposed starvation
conditions.
Finally, in Chapter 9 the results described in this thesis are discussed and ideas for future
research are presented.
13

“Music is a moral law
It gives soul to the universe, wings to the mind, flight to the imagination,
and charm and gaiety to life and to everything”
-Plato-
14

Chapter 2
Mapping the pathways to staphylococcal pathogenesis by
comparative secretomics
M.J.J.B. Sibbald, A.K. Ziebandt, S. Engelmann, M. Hecker, A. de Jong, H.J.M. Harmsen,
G.C. Raangs, I. Stokroos, J.P. Arends, J.Y.F. Dubois, and J.M. van Dijl
Published in Microbiology and Molecular Biology Reviews (2006) 70, 755-788
15

Chapter 2
Summary
The Gram-positive bacterium Staphylococcus aureus is a frequent component of the
human microbial flora that can turn into a dangerous pathogen. As such, this organism
is capable of infecting almost every tissue and organ system in the human body. It does
so by actively exporting a variety of virulence factors to the cell surface and
extracellular milieu. Upon reaching their respective destinations, these virulence factors
have pivotal roles in the colonization and subversion of the human host. It is therefore of
major importance to obtain a clear understanding of the protein transport pathways
that are active in S. aureus. The present review aims to provide a state-of-the-art
roadmap of staphylococcal secretomes, which include both protein transport pathways
and the extracytoplasmic proteins of these organisms. Specifically, an overview is
presented of the exported virulence factors, pathways for protein transport, signals for
cellular protein retention or secretion, and the exoproteomes of different S. aureus
isolates. The focus is on S. aureus, but comparisons with Staphylococcus epidermidis and
other Gram-positive bacteria like Bacillus subtilis are included where appropriate.
Importantly, the results of genomic and proteomic studies on S. aureus secretomes are
integrated through a comparative "secretomics" approach, resulting in a first definition
of the core and variant secretomes of this bacterium. While the core secretome seems to
be largely employed for general house-keeping functions, necessary to thrive in
particular niches provided by the human host, the variant secretome seems to contain
the “gadgets” that S. aureus needs to conquer these well-protected niches.
16

Mapping the pathways to staphylococcal pathogenesis by comparative secretomics
General introduction and scope of this review
The Gram-positive bacterium Staphylococcus aureus is a frequent component of the human
microbial flora that can turn into a dangerous pathogen. As such, this organism is capable of
infecting almost every tissue and organ system in the human body. It does so by exporting a
variety of virulence factors to the cell surface and extracellular milieu of the human host. As
in all living organisms (Wickner and Schekman, 2005), S. aureus contains several protein
transport pathways, of which the general secretory (Sec) pathway is the most well known and
best described. Proteins that need to be transported to an extracytoplasmic location contain, in
general, an N-terminal signal peptide that is needed to target the newly synthesized protein
from the ribosome to the translocation machinery in the cytoplasmic membrane. Next, the
protein is threaded through the Sec translocon in an unfolded state. During this translocation
step, or shortly thereafter, the signal peptide is removed by a so-called signal peptidase. Upon
complete membrane translocation, the protein has to fold into its correct conformation and
will then be retained in an extracytoplasmic compartment of the cell, or secreted into the
extracellular milieu. In the case of Gram-positive cocci, such as S. aureus (Figure 1), we
distinguish three extracytoplasmic subcellular compartments: the membrane, the membranecell
wall interface and the cell wall. Since surface-exposed and secreted proteins of S. aureus
play pivotal roles in the colonization and subversion of the human host, it is of major
importance to obtain a clear understanding of the protein transport pathways that are active in
this organism (Lee and Schneewind, 2001). Knowledge about the protein sorting mechanism
has become all the more relevant with the upcoming of staphylococcal resistance against lastdefence
antibiotics, such as vancomycin. The scope of this review is to provide a state-of-theart
roadmap of staphylococcal secretomes, which include both protein transport pathways and
the extracytoplasmic proteins of these organisms. The focus is on S. aureus, but comparisons
with Staphylococcus epidermidis and the best characterized Gram-positive bacterium Bacillus
subtilis are included where appropriate. Importantly, the present review aims to integrate the
results of genomic and proteomic studies on S. aureus secretomes, representing the first
documented “comparative secretomics” study. Specifically, this review deals with known and
predicted exported virulence factors, pathways for protein transport, signals for subcellular
protein sorting or secretion, and the exoproteomes of different S. aureus isolates as defined by
two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry
(Figures 2 and 3). The exoproteome is defined by all S. aureus proteins that can be identified
in the extracellular milieu of this organism and thus includes proteins actively secreted by
living cells and the remains of dead cells. For a clear appreciation of the present review, it is
important to bear in mind that the proteins exported from the cytoplasm could be directly
involved in staphylococcal virulence, whereas the respective protein export systems represent
the “pathways to pathogenesis”.
17

Chapter 2
18
A B
Figure 1. Imaging of S. aureus RN6390. (A) For scanning electron microscopy a drop of washed culture of
bacteria was fixated for 30 min with 2% glutaraldehyde in 0.1 M Cacodylate buffer, pH 7.38. Next, the fixated
bacteria were placed on a piece (1 cm 2 ) of cleaved 0.1% Poly-L Lysine coated mica sheet and washed in 0.1 M
Cacodylate buffer. This specimen was dehydrated in ethanol series consisting of 30%, 50%, 70%, 96% and
anhydrous 100% solution (3X) 10 min each, then Critical point dried with CO 2 and sputter-coated with 2-3 nm
Au/Pd (Balzers coater). The specimen was fixed on a SEM-stub-holder and observed in a JEOL FE-SEM 6301F.
(B) Micrograph of a cluster of S. aureus cells grown in blood culture medium. The cells were fixed with ethanol and
hybridized with the fluorescein labelled Peptide Nucleid Acid (PNA) probe PNA-Stau. The image was generated by
merging an epi-fluorescence image with the negative of a phase-contrast image.
Figure 2. The extracellular proteomes of different S. aureus strains
Proteins of the growth medium fractions of different staphylococcal isolates, grown in TSB medium (37 °C) to an
optical density at 540 nm (OD 540) of 10, were separated by 2D-PAGE using immobilized pH gradient (IPG) strips
in the pH range of 3 to 10 (Amersham Pharmacia Biotech, Piscataway, N. J.). Each gel was loaded with 350 µg
protein extracts and, after electrophoresis, stained with Colloidal Coomassie. Proteins were identified by MALDI-
TOF mass spectrometry. The corresponding protein spots are labelled with protein names according to the S.
aureus N315 database or NCBI entries for proteins not present in N315. The S. aureus strains that were used in
these experiments are RN6390 and COL, and four clinical isolates from the University Medical Center Groningen
named MRSA693331, 035699y/bm, 0440579/rmo, CA-MRSA021708m/rmo.

Mapping the pathways to staphylococcal pathogenesis by comparative secretomics
Figure 3. Dynamics of the amount of extracellular proteins during growth of S. aureus RN6390 in TSB
medium
(A) Individual dual channel 2D patterns of extracellular proteins during the different phases of the growth curve
of cells grown in TSB medium were assembled into a movie. The protein pattern at an OD 540 of 1 (labelled in
green) was compared with the protein pattern at the respective higher optical densities (labelled in red). As a
consequence of the dual channel labelling, spots of which the intensities do not differ in the compared gels will be
yellow; spots of different intensities will be either green or red (Bernhardt et al., 1999). (B) Growth curve of S.
aureus RN6390 grown in TSB medium as determined by OD 540 readings. The sampling points for proteomics
analyses are indicated by arrows. (C) Proteomic signatures of selected proteins representing different regulatory
groups as revealed by dual channel imaging. The relative amounts of the respective proteins at an OD 540 of 1
(spots labelled in green) of cells grown in TSB medium were compared with the relative amounts of these proteins
at higher optical densities (spots labelled in red). Proteins were stained with Sypro Ruby ® .
Exported staphylococcal virulence factors
S. aureus and S. epidermidis are organisms that occur naturally in and on the human body.
While S. epidermidis is mostly present on the human skin, S. aureus can be found on mucosal
surfaces. S. aureus is carried by 30-40% of the population (Peacock et al., 2001) and can
readily be identified in the nose, but the organism can also be detected in other moist regions
of the human body, such as axilla, perineum, vagina and rectum, thereby forming a major
reservoir for infections. Although most staphylococcal infections are nosocomial (i.e.
hospital-acquired), an increase in the number of cases of community-acquired antibiotic
(methicillin) resistant infections is currently observed world-wide (Centers for Disease
Control and Prevention, 2003; Grundmann et al., 2002; Vandenesch et al., 2003). The risk of
intravascular and systemic infection by S. aureus rises when the epithelial barrier is disrupted
by intravascular catheters, implants, mucosal damage or trauma. Interestingly, after infection,
19

Chapter 2
cells of S. aureus can persist unnoticed in the human body for long periods of time (years)
after which they can suddenly cause another infection. S. aureus is primarily an extracellular
pathogen whose colonization and invasion of human tissues and organs can lead to severe
cytotoxic effects. Nevertheless, S. aureus can also be internalized by various cells, including
non-phagocytic cells, which seems to induce apoptosis (Hauck and Ohlsen, 2006; da Silva et
al., 2004; Mempel et al., 2002). Although S. aureus has the potential to form biofilms (Götz,
2002), S. epidermidis infections are particularly notorious for the formation of thick
multilayered biofilms on indwelling catheters and other implanted devices. The formation of
such a biofilm takes place in several steps during which the bacteria first adhere rapidly to the
surface of the polymer material that has been coated with a film of proteinaceous and nonproteinaceous
organic host molecules (Escher and Characklis, 1990). Bacteria that adhere to
this film produce extracellular polymeric substances, mostly polysaccharides and proteins, in
turn resulting in a strong attachment to the polymer surface and other bacteria in the growing
biofilm. Ultimately, the biofilm is composed of multiple layers of cells, cellular debris,
polysaccharides and proteins. S. epidermidis proteins that are essential for biofilm formation
are, for example, the polysaccharide intercellular adhesin (PIA) (Mack et al., 1996), the
accumulation associated protein (AAP) (Rohde et al., 2005) and the biofilm-associated
protein (Bap) (Tormo et al., 2005). PIA is most likely identical to the polysaccharide adhesion
(PS/A).
Virulence of S. aureus
The pathogenicity of S. aureus is caused by the expression of an arsenal of virulence factors
(Table 1), which can lead to superficial skin lesions such as styes, furunculosis and
paronychia, or to more serious infections such as pneumonia, mastitis, urinary tract infections,
osteomyelitis, endocarditis and even sepsis. In very rare cases, S. aureus causes meningitis.
The virulence factors that S. aureus employs to cause these diseases are displayed at the
surface of the staphylococcal cell or secreted into the host milieu (Fedtke et al., 2004).
Specifically, these virulence factors include (a) surface proteins that promote adhesion to and
colonization of host tissues; (b) invasins that are exported to an extracytoplasmic location and
promote bacterial spread in tissues (leukocidin, kinases, hyaluronidase); (c) surface factors
that inhibit phagocytic engulfment (capsule, Protein A); (d) biochemical properties that
enhance staphylococcal survival in phagocytes (carotenoids, catalase production); (e)
immunological disguises (Protein A, coagulase, clotting factor); (f) membrane-damaging
toxins that disrupt eukaryotic cell membranes (hemolysins, leukotoxin); (g) superantigens that
contribute to the symptoms of septic shock (SEA-G, TSST, ET); and (h) determinants for
inherent and acquired resistance to antimicrobial agents. Most virulence factors are expressed
in a coordinated fashion during the growth cycle of S. aureus. The best characterized
regulators of virulence factors are the accessory gene regulator (agr) (Morfeldt et al., 1988;
Peng et al., 1988; Recsei et al., 1986) and the Staphylococcal accessory regulator (SarA)
(Cheung et al., 1992; Cheung and Projan, 1994). Ziebandt et al. (Ziebandt et al., 2004)
showed that extracellular proteins can be divided into two groups, based on the timing of their
expression in cells grown in tryptic soy broth (TSB): proteins that are mainly expressed at low
cell densities, or proteins exclusively expressed at high cell densities. Agr seems to be an
important positive regulator of proteins that are expressed at higher optical densities (e.g.
proteases, hemolysins and lipases) and a negative regulator for proteins that are expressed
during the exponential growth phase (e.g. immunodominant antigen A, secretory antigen
20

Mapping the pathways to staphylococcal pathogenesis by comparative secretomics
precursor and several proteins with unknown functions). In addition, Gill et al. (Gill et al.,
2005) identified 15 other two-component regulatory systems in the genomes of S. aureus and
S. epidermidis that are potentially involved in staphylococcal virulence. In this respect, it is
interesting to note that the antibiotic cerulenin, which is known to inhibit protein secretion by
S. aureus at sub-MIC levels, was recently reported to block transcriptional activation of at
least two regulatory determinants, agr and sae. Thus, it seems that cerulenin inhibits the
transcription of genes for secretory proteins rather than the secretion process of these proteins
(Adhikari and Novick, 2005). In contrast, it was previously believed that cerulenin would
interfere with membrane function through an inhibition of normal fatty acid synthesis.
Table 1. Virulence factors of S. aureus
Pathogenic action Virulence factors Protein or other compound Functions
Colonization of host
tissues
Lysis eukaryotic cell
membranes and
bacterial spread
Inhibition phagocytic
engulfment
Survival in
phagocytes
Immunological
disguise and
modulation
Contribution to
symptoms of septic
shock
Acquired resistance to
antimicrobial agents
Surface proteins ClfA, ClfB, FnbA, FnbB, IsdA Adhesins, fibronectin and
SdrC, SdrD, SdrE,
fibrinogen-binding proteins
Membrane- Geh, Hla, Hld, HlgA-C, HysA, Hemolysins, hyaluronidase,
damaging toxins, Lip, LukD, LukE, LukF, LukS, leukocidin, leukotoxin,
invasins
Nuc
lipases, nucleases
Surface factors CapA-P, Efb, Spa Capsule, protein A
Biochemical KatA, Staphyloxanthin Carotenoids, catalase
compounds
production
Surface proteins ClfA, ClfB, Coa, Spa Clumping factor, coagulase,
protein A
Exotoxins Eta, Etb, SEA-G, TSST-1 Enterotoxins SEA-G,
exfoliative toxin, toxic
shock syndrome toxin
TSST
Resistance proteins BlaZ, MecA, VanA MRSA, VRSA
Notably, to date relatively little information is available on the molecular nature of the stimuli
that are perceived by the major regulators of the expression of virulence factors. Overall, it
should be clear that strain-specific differences in gene regulation by agr, sae or other
regulators may dramatically influence the repetoire of produced virulence factors, thereby
having a profound impact on the disease-causing potential of different strains. Since the
interplay of different regulators and cell-to-cell communication can impact differently on the
expression of virulence factors, even the disease-causing potential of individual S. aureus
cells within a genetically identical population may vary.
Resistance of S. aureus to antibiotics
Resistance of S. aureus to antibiotics has been observed very soon after the introduction of
penicillin about sixty years ago. In the following years, the amazing ability of staphylococci
to develop resistance to antibiotics has resulted in the emergence of methicillin-resistant S.
aureus (MRSA) and S. epidermidis (MRSE) strains. In fact, methicillin resistance was
observed already in 1961 in nosocomial isolates of S. aureus, one year after the introduction
of methicillin (Jevons, 1961). The resistance towards methicillin is a result of the production
of an altered penicillin binding protein, PBP2a (or PBP2’), which has less affinity to most βlactam
antibiotics. The PBP2a protein, which is located at the membrane-cell wall interface, is
of major importance for cell wall biogenesis by mediating the cross linking of peptidoglycans.
21

Chapter 2
PBP2a is encoded by the mecA gene, which is located on a mobile genetic element, also
known as the staphylococcal cassette chromosome (SCC) mec (Chambers, 1997; Ito et al.,
2004). The SCCmec element is a basic mobile genetic element that serves as a vehicle for
gene exchange among staphylococcal species (Dobrindt et al., 2004). In addition to the mecA
gene, SCCmec carries the mecA regulatory genes mecI and mecR, an insertion sequence
element (IS431mec) and a unique cassette of recombinase genes (ccr), which are responsible
for SCCmec chromosomal integration and excision. Eight different types of SCCmec
elements, type I-V, have been identified so far, based on the classes of mecA gene and ccr
gene complexes (Ito et al., 2009). Notably, type II and III elements contain, besides mecA,
multiple determinants for resistance against non-β-lactam antibiotics. Accordingly, type II and
III SCCmec elements are responsible for multidrug resistance in nosocomial MRSA isolates.
Some SCCmec elements (e.g. type IV SCCmec), contain no other resistance gene than mecA,
and they are significantly smaller compared to for example the type II and III elements. This
might serve as an evolutionary advantage, making it easier for these mobile genetic elements
to spread across bacterial populations. Phylogenetic analyses of the genes encode by SCCmec
elements showed distant relationships with homologues in other S. aureus genomes and
suggest foreign origins for these genes.
Vancomycin resistance has been first reported for Enterococcus faecium (Leclercq et al.,
1989), and transfer of vancomycin resistance from enterococci, such as Enterococcus faecalis,
to S. aureus has been shown to occur (Noble et al., 1992). Vancomycin has long been a last
resort antibiotic for multiple resistant S. aureus strains, but already in 1996 a strain was
isolated, which showed a reduced sensitivity towards vancomycin (Hiramatsu et al., 1997).
Shortly afterwards, additional strains were isolated in different countries that were designated
as vancomycin-intermediately resistant S. aureus (VISA). These strains show a significantly
thickened cell wall, which allows them to sequester more vancomycin than non-VISA strains,
thereby preventing the detrimental effects of this antibiotic (Cui et al., 2003). A search for the
genetic basis of the lowered vancomycin sensitivity of the S. aureus Mu50 strain revealed that
important genes for cell wall biosynthesis and intermediary metabolism have mutations
compared to MRSA strains, which might lead to altered expression of genes involved in the
cell wall metabolism and a thickened cell wall (Avison et al., 2002). The first highly
vancomycin resistant strain was isolated in 2002 (Weigel et al., 2003). This strain was shown
to carry a plasmid, which contains, among other resistance genes, the vanA gene plus several
additional genes required for vancomycin resistance. The proteins encoded by these genes are
responsible for replacing the C-terminal D-alanyl-D-alanine (D-ala-D-ala) of the disaccharide
pentapeptide cell wall precursor with a depsipeptide, D-alanyl-D-lactate (D-ala-D-lac),
thereby lowering the cell wall affinity for vancomycin (Bugg et al., 1991).
Export of virulence factors from the cytoplasm
As most proteinaceous virulence factors are displayed at the surface of the staphylococcal cell
or released into the medium, it is important for our understanding of the pathogenic potential
of these organisms to map their pathways for protein transport. While specific questions
relating to surface display or secretion of particular virulence factors have been addressed for
several years, more holistic studies on the genomics and proteomics of these processes have
been documented in the scientific literature only very recently. Moreover, no systematic
analysis of pathways and cellular machinery for protein transport has thus far been performed
for staphylococci. This review is aimed at filling this knowledge gap. To do so, we have taken
22

Mapping the pathways to staphylococcal pathogenesis by comparative secretomics
full advantage of the availability of six completely sequenced and annotated S. aureus
genomes and one of the two sequenced S. epidermidis strains, as well as recently published
data on the analysis of staphylococcal cell wall- and exoproteomes. Additionally, we have
combined the published information with bioinformatics-derived data on all potential signals
for protein export from the cytoplasm and secretion into the extracellular milieu, or retention
in the membrane or cell wall. Since polytopic membrane proteins do not appear to have major
direct roles in virulence other than causing drug resistance, such membrane proteins remain
beyond the scope of this review. Furthermore, since the secretome of B. subtilis has been
characterized extensively, both at the level of the protein export machinery and the
exoproteome, we have compared the staphylococcal secretomes with that of B. subtilis. To
our knowledge this has resulted in the first “comparative secretomics” study.
S. aureus strains suitable for comparative secretomics
Fourteen sequenced and fully annotated genomes of S. aureus are available in public
databases (Table 2; http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi) and thirteen of these
genomes were used in the present study. These include one of the first hospital-acquired
MRSA isolates, S. aureus COL (Gill et al., 2005), which has been widely used in research on
staphylococcal methicillin and vancomycin resistance. The sequenced MRSA252 strain
(Holden et al., 2004) is a hospital-acquired epidemic strain, which was isolated from a patient
who died as a consequence of septicemia. The sequenced MSSA476 strain (Holden et al.,
2004) is a community-acquired invasive strain that is penicillin- and fusidic acid-resistant, but
susceptible to most commonly used antibiotics. S. aureus Mu50 and N315 (Kuroda et al.,
2001) are hospital-acquired MRSA strains isolated from Japanese patients. In addition, the
Mu50 strain displays vancomycin intermediate sensitivity. The S. aureus Mu3 strain was the
first isolated strain from a healthy carrier in Brazil that showed vancomycin-resistance
(Hiramatsu et al., 1997; Neoh et al., 2008). The community-acquired S. aureus strains MW2
(Baba et al., 2002), USA300 and USA300_TCH1516 (Diep et al., 2006) are highly virulent
MRSA strains, isolated in the USA. Both S. aureus JH1 and JH9 strains are MRSA strains
that were isolated from one patient undergoing vanconcomycin treatment on different time
points. The JH1 strain was the earliest strain isolated from the patient. The JH9 strain was
isolated at a later stage of the treatment and was diagnosed as a vancomycin-resistant strain
(Mwangi et al., 2007). Comparison between these two strains would gain insight in the
evolution of isogenic strains and the acquirement of vancomycin resistance under antibiotic
pressure. The Newman strain (Baba et al., 2008) was isolated from a human infection (Duthie
and Lorenz, 1952) and has been widely used as a research strain due to its robust virulence
phenotypes. Finally, the NCTC 8325 strain (Gillaspy et al., 2006) is generally regarded as the
prototypical strain for all genetic midifications in order to address specific gene regulatory
and virulence traits. Furthermore, the sequence of S. aureus RF122, a strain that is associated
with mastitis in cattle, is now also available in the NCBI database (Herron et al., 2002), but
has not been included in the present review which is primarily focused on staphylococcal
pathogenicity towards humans. Secretome predictions for this strain are presented in
Appendix IIIH. Using Multilocus Sequence Typing with seven housekeeping genes of the
different S. aureus strains, Holden et al. (Holden et al., 2004) showed that the MRSA252
strain is phylogenetically most distantly related to the other sequenced strains, while the
Mu50 and N315 strains are indistinguishable by MLST, and the same is true for the
MSSA476 and MW2 strains. The COL and NCTC8325 strains are relatively closely related to
23

Chapter 2
each other. However, analysis of the two major pathogenicity islands present in all these
strains shows that the distribution of these pathogenicity islands gives contradictory results on
phylogenetic relationships of the sequenced S. aureus strains (Baba et al., 2008).
Table 2. Sequenced and annotated genomes of S. aureus strains
Genome size (kbp) Nr. Of protein encoding genes
Strain Origin a Chromosome Plasmid Chromosome Plasmid
COL HA- MRSA 2809 4 2615 3
JH1 HA- MRSA 2907 30 2747 33
JH9 HA- VISA 2907 3 2697 29
MRSA252 HA- MRSA 2903 - 2656 -
MSSA476 CA- MSSA 2800 21 2579 19
Mu3 HA- VISA 2880 - 2698 -
Mu50 HA- VISA 2879 25 2697 34
MW2 CA- MRSA 2820 - 2632 -
N315 HA- MRSA 2815 25 2588 31
NCTC8325 HA- MSSA 2821 - 2892 -
Newman HA- MRSA 2879 - 2614 -
USA300 CA- MRSA 2873 3,4,37 2560 5,3,36
USA300_TCH1516 CA- MRSA 2873 27,20 2657 26,20
RF122 Bovine mastitis 2743 - 2515 -
a HA-MRSA: Hospital-acquired MRSA; CA-MRSA: Community-acquired MRSA
Sequenced and annotated genomes of other staphylococcal species, such as S. epidermidis,
Staphylococcus haemolyticus and Staphylococcus carnosus, are also publicly available.
However, with the exception of the S. epidermidis strain ATCC 12228 (Zhang et al., 2003),
these are not included in the present review, which is focused primarily on S. aureus. A
comparative genomic analysis of S. aureus COL, Mu50, MW2, N315 and the sequenced S.
epidermidis strains RP62A and ATCC 12228 has revealed that these species and strains have
a set of 1681 genes in common (Gill et al., 2005). In contrast, 454 genes are present in the S.
aureus strains, but not in S. epidermidis, whereas 286 genes are present in S. epidermidis, but
not in S. aureus. Most of the strain-specific and species-specific genes can be related to the
presence or absence of particular prophages and genomic islands.
Pathways for staphylococcal protein transport
The bacterial machinery for protein transport is currently best-described for Escherichia coli
(Gram-negative) and B. subtilis (Gram-positive) (for reviews see (de Keyzer et al., 2003;
Tjalsma et al., 2000; Tjalsma et al., 2004). Many of the known components that are involved
in the different routes for protein export from the cytoplasm and post-translocational
modification of exported proteins in these organisms are also conserved in S. aureus and S.
epidermidis (Table 3). In general, proteins that are exported are synthesized with an Nterminal
signal peptide, which directs them into a particular transport pathway. Consequently,
the presently known signal peptides are classified according to the export pathway into which
they direct the corresponding proteins, or the type of signal peptidase that is responsible for
their removal (processing) upon membrane translocation.
24

Mapping the pathways to staphylococcal pathogenesis by comparative secretomics
Table 3. Secretion machinery of S. aureus, S. epidermidis and B. subtilis
Sec-pathway S. aureus S. epidermidis B. subtilis
Chaperone Ffh + + +
FtsY + + +
FlhF - - +
CsaA - - +
Translocation motor SecA1
+
+
+
SecA2
+
+
-
Translocation channel SecY1
+
+
+
SecY2
+
+
-
SecE + + +
SecG + + +
SecDF + + +
YajC (YrbF) + + +
Lipid modification Lgt + + +
Sec-pathway S. aureus S. epidermidis B. subtilis
Signal peptidase SpsA (inactive) + + -
SpsB (SipSTUV) + + a +
SipW (ER-type) - - +
LspA + + b +
Folding catalyst PrsA + c + +
BdbC - - +
DsbA (BdbD) + + +
Cell wall anchoring SrtA + + -
SrtB + - -
SrtC - + d -
SrtD - - +
Tat-pathway
Translocase TatA
TatC
Pseudopilin pathway
Bacteriocins
Holins
Ess
25
+
+
ComGA + + +
ComGB + + +
ComC + + +
Bacteriocin-specific
ABC-transporters
-
-
? ? +
CidA (holin) + + +
LrgA (anitholin) + + +
EsaA + - +
EsaB + - +
EsaC + e - -
EssA + - -
EssB + - +
EssC + - +
Based on BLAST searches with the corresponding proteins of B. subtilis in the finished genome database
(http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi).
a
Two potentially active type I SPases are present in this strain and share homology to B. subtilis SipS and SipU
b
Two LspA proteins present in this strain
c
This protein is truncated at the C-terminus in the S. aureus JH9 strain
d
The genome of S. epidermidis RP62A only contains a srtA gene, whereas the genome of S. epidermidis
ATCC12228 also contains a srtC gene
e
This protein is missing in the S. aureus MRSA252 strain
The staphylococcal protein export pathways that have been characterized experimentally or
that can be deduced from sequenced genomes are schematically shown in Figure 4 and will be
discussed in the following sections. Since these pathways are likely to be used for the export
+
+

Chapter 2
of virulence factors to the cell surface and the milieu of the host, Figure 4 can be regarded as a
subcellular road map to staphylococcal pathogenesis.
Components of the general secretory (Sec) Pathway
26
Figure 4. The staphylococcal “pathways to
pathogenesis”. Schematic representation of a
staphylococcal cell with potential pathways for
protein sorting and secretion. (A) Proteins
without signal peptide reside in the cytoplasm.
(B) Proteins with one or more transmembrane
spanning domains can be inserted into the
membrane via the Sec, Tat or Com pathways. (C)
Lipoproteins are exported via the Sec pathway
and after lipid-modification anchored to the
membrane. (D) Proteins with cell wall retention
signals are exported via the Sec, Tat or Com
pathways and retained in the cell wall via
covalent-, or high-affinity binding to cell wall
components. (E) Exported proteins with a signal
peptide and without a membrane or cell wall
retention signal can be secreted into the
extracellular milieu via the various indicated
pathways.
The most commonly used pathway for bacterial protein transport is the general secretory
(Sec) pathway. Specifically this pathway is responsible for the secretion of the majority of the
proteins found in the exoproteome of B. subtilis and this is probably also the case for most
other Gram-positive bacteria, including S. aureus (Tjalsma et al., 2004). Unfortunately, there
are only very few published data available concerning the Sec pathway of S. aureus and,
therefore, we will fill in the current knowledge gaps with data obtained from studies in B.
subtilis or E. coli. Proteins that are exported via the Sec-pathway contain signal peptides with
recognition sites for so-called type I or type II signal peptidases (SPases). Notably, type II
SPase recognition sites overlap with the recognition sites for the diacylglyceryl transferase
Lgt. Precursor proteins with a type II SPase recognition sequence are lipid-modified prior to
processing and the resulting mature proteins are retained as lipopoteins in the cytoplasmic
membrane via their diacylglyceryl moiety. Furthermore, the Sec-dependent export of proteins
can be divided into three stages: a) targeting to the membrane translocation machinery by
export-specific or general chaperones, b) translocation across the membrane by the Sec
machinery, and c) post-translocational folding and modification. If the translocated proteins
of Gram-positive bacteria lack specific retention signals for the membrane or cell wall, they
are secreted into the growth medium.
Preprotein targeting to the membrane
In B. subtilis the only known secretion-specific chaperone is the signal recognition particle
(SRP), which consists of the small cytoplasmic RNA (scRNA), the histon-like protein HBsU
and the Ffh protein. Ffh and HBsU bind to different moieties of the scRNA. Studies in E. coli
have shown that, upon emergence from the ribosome, the signal peptide of a nascent secretory
protein can be recognized by several cytoplasmic chaperones and/or targeting factors, such as
Ffh or Trigger Factor (TF) (Eisner et al., 2003). In contrast to Ffh, which is required for co-

Mapping the pathways to staphylococcal pathogenesis by comparative secretomics
translational protein export in E. coli, the cytoplasmic chaperone SecB has mainly been
implicated in post-translational protein targeting. Notably however, SecB is absent from the
sequenced Gram-positive bacteria, including S. aureus and B. subtilis. Most likely, ribosomenascent
chain complexes of S. aureus are thus targeted to the membrane by SRP, which, by
analogy to B. subtilis and E. coli will probably involve the SRP receptor FtsY. At the
membrane, the nascent preprotein will be directed to the translocation machinery. This
process is likely to be stimulated by negatively charged phospholipids (De Leeuw et al.,
2000), the Sec translocon (Bibi, 1998; De Leeuw et al., 2000) and/or the SecA protein (Bunai
et al., 1999). In this respect SecA may not only function as the translocation motor (see
below), but also as a chaperone for preprotein targeting (Herbort et al., 1999). While it has
been shown that Ffh is essential for growth and viability in E. coli and B. subtilis, this does
not seem to be the case in all bacteria. For example, Ffh, FtsY and scRNA are not essential in
Streptococcus mutans. In this organism the SRP is merely required for growth under stressful
conditions, such as low pH (

Chapter 2
homologues are not essential for growth and viability. It is presently unknown whether SecA2
and SecY2 transport specific proteins across the membrane of S. aureus. However, it has been
shown for other pathogenic Gram-positive bacteria, which also possess a second set of SecA
and SecY, that these proteins are required for the transport of certain proteins related to
virulence. In Streptococcus gordonii, the export of GspB, a large cell-surface glycoprotein
that contributes to platelet binding, seems to be dependent on the presence of SecA2 and
SecY2 (Bensing and Sullam, 2002). This protein contains large serine-rich repeats, an LPxTG
motif for cell wall anchoring (see below), and a very large signal peptide of 90 amino acids.
In Streptococcus parasanguis two other proteins, FimA and Fap1, are known to be secreted
via SecA2-dependent membrane translocation. FimA is a (predicted) lipoprotein, which is a
major virulence factor implicated in streptococcal endocarditis. The FimA homologue in S.
aureus is a manganese-binding lipoprotein (MntA), associated with an ATP-binding cassette
(ABC) transporter. Fap1 of S. parasanguis is involved in adhesion to the surface of teeth.
Like GspB of S. gordonii, Fap1 has a long signal peptide of 50 amino acids, serine-rich
repeats and an LPxTG motif for cell wall anchoring. To date, it is not known what determines
the difference in the specificity of SecA1/SecY1 and SecA2/SecY2 translocases. However,
for S. gordonii it has been shown that Gly residues in the signal peptide are important for
directing GpsB to the SecA2/SecY2 translocon (Bensing et al., 2007). It is also not known
whether the SecA2/SecY2 shares SecE and/or SecG with the SecA1/SecY1 translocase, and
whether these translocases function completely independently from each other or whether
mixed translocases can occur. Clearly, the secE and secG genes are not duplicated in S.
aureus.
In E. coli, the heterotrimeric SecYEG complex is associated with another heterotrimeric
complex that is composed of the SecD, SecF and YajC proteins (Nouwen et al., 2005). This
complex has been shown to be involved in the cycling of SecA (Driessen et al., 1998) and
release of the translocated protein from the translocation channel (Matsuyama et al., 1993).
SecD and SecF are separate, but structurally related proteins in most bacteria, including E.
coli. Interestingly, in B. subtilis and S. aureus, natural gene fusions between the secD and
secF genes are observed. Accordingly, the corresponding SecDF proteins can be regarded as
molecular “Siamese twins” (Bolhuis et al., 1998). Unlike SecA, SecY and SecE, the SecDF
protein of B. subtilis is not essential for growth and viability and its role in protein secretion is
presently poorly understood (Bolhuis et al., 1998). B. subtilis secDF mutants only showed a
mild secretion defect under conditions of high-level synthesis of secretory proteins. The
known SecDF proteins have 12 (predicted) transmembrane domains with two large
extracytoplasmic loops between the first and second transmembrane segments, and between
the seventh and eighth transmembrane segments. For E. coli SecD it has been shown that
small deletions in the large extracytoplasmic loop result in a malfunctioning of the protein,
while the stability of the SecD/F-YajC complex is not affected (Nouwen et al., 2005). It has
therefore been proposed that this loop in SecD might provide a protective structure in which
translocated proteins can fold more efficiently. The large extracytoplasmic loop in SecF has
been proposed to interact with SecY, thereby stabilizing the translocation channel formed by
SecYEG. Homologues of the E. coli YajC protein are present in many bacteria, including S.
aureus and B. subtilis (YrbF), but their role in protein secretion has not been established yet.
It is presently not known whether the S. aureus SecDF-YajC complex associates specifically
with the SecA1/SecY1 translocase, the SecA2/SecY2 translocase, or both translocases.
28

Mapping the pathways to staphylococcal pathogenesis by comparative secretomics
Type I Signal peptidases
Signal peptides of preproteins are cleaved during or shortly after translocation by SPase I or
SPase II, depending on the nature of the signal peptide (Tjalsma et al., 2001; van Roosmalen
et al., 2004). The B. subtilis chromosome encodes five type I SPases, named SipS, SipT,
SipU, SipV and SipW (van Dijl et al., 1992; Tjalsma et al., 1997; Tjalsma et al., 1998). Two
of these, SipS and SipT, are of major importance for the processing of secretory preproteins,
growth and viability. In S. aureus only two SPase I homologues are present, SpsA and SpsB.
The catalytically active SPase I in S. aureus is SpsB, which is probably essential for growth
and viability (Cregg et al., 1996). This SPase can be used to complement an E. coli strain that
is temperature-sensitive for preprotein processing. In general, type I SPases recognize
residues at the -1 and -3 positions relative to the cleavage site (van Roosmalen et al., 2004).
For B. subtilis it has been shown that all secretory proteins identified by proteomics have Ala
at the -1 position, and 71% of these secretory proteins have Ala at the -3 position (Tjalsma et
al., 2004). In contrast, various residues are tolerated at the -2 position, including Ser, Lys,
Glu, His, Tyr, Gln, Gly, Phe, Leu, Ala, Asp, Asn, Trp and Pro. Interestingly, Bruton et al.
(Bruton et al., 2003) studied the cleavage sites in substrates of SpsB of S. aureus and showed
that this enzyme has a preference for basic residues at the -2 position and tolerance for
hydrophobic residues at this position. However, an acidic residue at the -2 position resulted in
a significantly reduced rate of processing. The second SPase I homologue of S. aureus (SpsA)
appears to be inactive, since it lacks the catalytic Ser and Lys residues that are, respectively,
replaced with Asp and Ser residues. The presence of an apparently catalytically inactive SpsA
homologue is a conserved feature of all staphylococci with sequenced genomes. Notably, in
addition to an inactive SpsA homologue, S. epidermidis contains two SpsB homologues that
respectively show the greatest similarity to SipS and SipU of B. subtilis. To date, it is not
known whether the inactive SpsA homologues contribute somehow to protein secretion in
these organisms.
Lipid-modification of lipoproteins
In E. coli, lipid-modification of prolipoproteins involves three sequential steps that are
catalyzed by cytoplasmic membrane-bound proteins. The first step involves the transfer of a
diacylglyceryl group from phosphatidylglycerol to the sulfhydryl group of the invariant Cys
residue that is present at the +1 position of the signal peptide cleavage site in lipoprotein
precursors. This step is catalyzed by a phosphatidyl glycerol diacylglyceryl transferase (Lgt)
as was shown for E. coli by Sankaran et al. (Sankaran and Wu, 1994). The recognition
sequence for Lgt, which includes the Cys residue that becomes diacylglyceryl-modified, is
known as the lipobox. The lipid-modification of the lipobox Cys residue is necessary for the
lipoprotein-specific type II signal peptidase (LspA) to recognize and cleave the signal peptide
of a prolipoprotein, which represents the second step in lipoprotein modification. The third
step involves the transfer of an N-acyl group by an N-acyl transferase (Lnt), resulting in the
formation of N-acyl diacylglycerylcysteine at the N-terminus of the mature lipoprotein.
Although Lgt and LspA are present in most, if not all bacteria, Lnt is only present in Gramnegative
bacteria (Tjalsma et al., 2001). As for other Gram-positive bacteria, no homologue
of Lnt could be detected in the genomes of S. aureus or S. epidermidis (Stoll et al., 2005),
which suggests that the lipoproteins of these organisms are not N-acylated.
29

Chapter 2
The S. aureus Lgt is a protein of 279 amino acids that contains a highly conserved HGGLIG
motif (residues 97 to 102). Although the His residue in this motif was shown to be essential
for catalytic activity of the E. coli Lgt (Sankaran et al., 1997), it is not strictly conserved in all
known Lgt proteins. On the other hand, the strictly conserved Gly at position 103 of E. coli
Lgt, which is equivalent to Gly98 of S. aureus Lgt, is required for activity of this protein.
Stoll et al. (Stoll et al., 2005) showed that a S. aureus lgt mutation has no effect on growth in
broth as was also observed for B. subtilis (Leskelä et al., 1999). Nevertheless, the absence of
Lgt has a considerable effect on the induction of an inflammatory response. Importantly, lipid
modification serves to retain exported proteins at the membrane-cell wall interface. This is
particularly relevant for Gram-positive bacteria, which lack an outer membrane that
represents a retention barrier for exported proteins. In the absence of Lgt, B. subtilis cells
release a variety of lipoproteins into the extracellular milieu, both in the form of unmodified
precursor proteins and alternatively processed mature proteins that lack the N-terminal Cys
residue (Antelmann et al., 2001). Similarly, the S. aureus lgt mutation resulted in the
shedding of certain abundant lipoproteins, such as OppA, PrsA and SitC, into the broth. These
lipoproteins are normally retained in the membrane or cell wall of S. aureus.
Type II Signal Peptidase
As described above, lipoprotein signal peptides of prolipoproteins are cleaved by type II
SPases after the Cys residue in the lipobox is modified by Lgt. Although B. subtilis and many
other bacteria contain only one copy of the lspA gene, some contain a second copy, such as S.
epidermidis, Bacillus licheniformis and Listeria monocytogenes. LspA is a membrane protein
that spans the membrane four times and both the N- and C-termini are facing the cytoplasmic
side of the membrane (Tjalsma et al., 1997; van Roosmalen et al., 2001). Six amino acid
residues are important for SPase II activity, of which two Asp residues form the active site
(Tjalsma et al., 1997). While processing of lipoproteins by LspA is essential for growth and
viability for E. coli and other Gram-negative bacteria (Wu, 1996), it is not essential for B.
subtilis (Tjalsma et al., 1999) and other Gram-positive bacteria, such as Lactococcus lactis
(Venema et al., 2003). This suggests that processing of prolipoproteins is not essential for
their functionality. The latter view is supported by the fact that PrsA, a lipoprotein required
for correct folding of translocated proteins, is essential for viability of B. subtilis (Kontinen
and Sarvas, 1993). In the absence of LspA, some of the lipoproteins of B. subtilis are
processed in an alternative way by yet unidentified proteases and activity of unprocessed
lipoproteins in lspA mutants is reduced. Also, in B. subtilis the secretion of the nonlipoprotein
AmyQ was severely reduced (Tjalsma et al., 1999). This reduction might be the
consequence of a malfunction of non-modified PrsA in AmyQ folding. Although most lspA
mutants have been studied in Gram-negative bacteria and a few non-pathogenic Grampositive
bacteria (Tjalsma et al., 1999; Venema et al., 2003), Sander et al. (Sander et al.,
2004) showed a severe attenuated phenotype of lspA mutants of the pathogen Mycobacterium
tuberculosis, which implies an important role for lipoprotein-processing by LspA during
infection of M. tuberculosis. In S. aureus both the lspA and lgt genes are present in single
copy in the genomes of all six sequenced strains. Interestingly, one of the two LspA
homologues in S. epidermidis (125 amino acids) is considerably shorter than other known
LspA proteins, including its large paralogue (177 amino acids). This is mainly the result of an
additional N-terminal transmembrane domain in the large LspA proteins. As a result the short
S. epidermidis LspA protein is predicted to have three membrane spanning domains, with the
30

Mapping the pathways to staphylococcal pathogenesis by comparative secretomics
N-terminus located on the outside of the cell, the C-terminus on the inside of the cell and the
(putative) active site Asp residues located on the outer surface of the cytoplasmic membrane.
Signal peptide peptidase
After translocation and processing of the preproteins by signal peptidases, the signal peptides
are rapidly degraded by signal peptide peptidases (SPPases). In B. subtilis two SPPases, TepA
and SppA, are known to be involved in translocation and processing of preproteins (Bolhuis
et al., 1999). While TepA is required for translocation and processing of preproteins, SppA is
only required for efficient processing of preproteins. Remarkably, no homologues of SppA or
TepA were detectable by BLAST searches in the sequenced genomes of S. aureus and S.
epidermidis. As reported by Meima and van Dijl (Meima and van Dijl, 2003), L. lactis
contains a protein that shows limited similarity to TepA of B. subtilis and ClpP of C. elegans,
suggesting that this protein might be an SPPase-analog of L. lactis. In S. aureus and S.
epidermidis this protein homologue also seems to be present and is predicted to be a
cytoplasmic membrane protein (our unpublished observations).
Folding catalysts (PrsA and BdbD)
Proteins that are transported across the membrane in a Sec-dependent manner emerge at the
extracytoplasmic membrane surface in an unfolded state. These proteins need to be rapidly
and correctly folded into their native and protease-resistant conformation, before they are
degraded by proteases in the cell wall or extracellular environment (Sarvas et al., 2004). An
important folding catalyst in B. subtilis is PrsA, which shows homology to peptidyl-prolyl
cis/trans-isomerases. PrsA is a lipoprotein (see also section “Lipoproteins”) that is essential
for efficient protein secretion and cell viability in B. subtilis (Sarvas et al., 2004; Kontinen
and Sarvas, 1993). Studies on the effects of PrsA depletion showed that the relative amounts
of extracellular proteins from PrsA-depleted cells, were significantly reduced (Vitikainen et
al., 2004). The solution structure of PrsA has been solved (Heikkinen et al., 2009), but no
data has been published on S. aureus mutants lacking PrsA and it will be interesting to
investigate whether PrsA is also essential for viability and virulence of this organism. It has
already been shown that S. aureus lacking Lgt, releases an increased amount of PrsA into the
extracellular milieu (Stoll et al., 2005), which might indicate that (most) pre-PrsA is not fully
functional, but sufficient for viability. The observation by Stoll et al. (Stoll et al., 2005) also
shows that, like in B. subtilis (Antelmann et al., 2001), the unmodified pre-PrsA is not
effectively retained in the cytoplasmic membrane.
Other proteins that are involved in proper folding of extracellular proteins in B. subtilis are the
membrane proteins BdbC and BdbD, which are involved in the formation of disulfide bonds.
Both proteins have been shown to be necessary for the stabilization of the membrane- and cell
wall-associated pseudopilin ComGC (Meima et al., 2002). This protein, which is required for
DNA binding and uptake during natural competence, contains an intramolecular disulfide
bond (Chung et al., 1998). Both BdbC and BdbD are also important for the folding of
heterologously produced E. coli PhoA, which contains two disulfide bonds, into an active and
protease-resistant conformation (Bolhuis et al., 1999; Meima et al., 2002). Though a
homologue of BdbD (named DsbA) is present in S. aureus, there is no homologue of BdbC in
this organism. The same appears to be true for S. epidermidis. Nevertheless, measurements of
the redox potential of purified DsbA indicate that this protein can act as an oxidase, and this
31

Chapter 2
view is confirmed by complementation studies in a dsbA mutant strain of E. coli (Dumoulin et
al., 2005). The absence of a BdbC homologue from staphylococci is remarkable, since B.
subtilis BdbC and BdbD are jointly required in the folding of ComGC and E. coli PhoA.
Notably, all sequenced S. aureus genomes encode homologues of ComGC, including the Cys
residues that form the disulfide bond in B. subtilis ComGC. This raises the question whether
ComGC of S. aureus does indeed contain a disulfide bond and, if so, which protein(s) are
involved in the formation of this disulfide bond. DsbA would be a candidate for this task
since it has been shown that this S. aureus protein can functionally replace BdbB, BdbC and
BdbD in the production of ComGC, E. coli PhoA and the S-S bond-containing sublancin 168
in B. subtilis (Kouwen et al., 2007). This idea is further supported by the findings of Heras et
al. (Heras et al., 2008) that the oxidized and reduced states of DsbA are energetically
equivalent, which suggests that this facilitates the reoxidation of DsbA, likely by extracellular
oxidants. Notably, S. aureus DsbA was shown to be a lipoprotein that does not seem to
contribute to the virulence of this organism as tested in mouse and Caenorhabditis elegans
models (Dumoulin et al., 2005). Furthermore, DsbA was shown to be dispensable for βhemolysin
activity, despite the fact that this protein contains a disulfide bond, which is
required for activity (Dziewanowska et al., 1996). Therefore, the biological function of DsbA
in staphylococci remains to be elucidated.
Twin-arginine translocation (Tat) pathway
The Tat-pathway exists in many bacteria, archaea, and chloroplasts. This pathway has been
named after the consensus double (twin) Arg residues that are present in the signal peptide.
The twin Arg residues are part of a motif that directs proteins specifically into the Tat
pathway. In contrast to the Sec-machinery where only unfolded proteins are translocated
across the membrane, the Tat-machinery is capable of translocating folded proteins. In Gramnegative
bacteria, streptomycetes, mycobacteria and chloroplasts, an active Tat-pathway
seems to require three core components, named TatA, TatB and TatC (Berks et al., 2005;
Dilks et al., 2003; Mori and Cline, 2001; Robinson and Bolhuis, 2001; Yen et al., 2002). In
all Gram-positive bacteria except streptomycetes and Mycobacterium smegmatis, the Tat
pathway involves only TatA and TatC (Dilks et al., 2003; Yen et al., 2002). Recent studies in
E. coli and chloroplasts have resulted in a model that proposes a key role for TatB-TatC
complexes in signal peptide reception and TatA-TatB-TatC complexes in preprotein
translocation (Cline and Mori, 2001; Alami et al., 2003). Interestingly, certain mutations in E.
coli TatA have been shown to allow this protein to compensate for the absence of TatB
(Blaudeck et al., 2005). This demonstrated that TatA is intrinsicaIly bifunctional, which is
consistent with the fact that most Gram-positive bacteria lack TatB, but have TatA (Jongbloed
et al., 2005). In B. subtilis, two minimal TatA-TatC translocases with distinct specificities are
active (Jongbloed et al., 2004). While the constitutively expressed TatAy-TatCy translocase
of B. subtilis is required for secretion of the protein with unknown function YwbN, the
TatAd-TatCd translocase seems to be expressed only under conditions of phosphate starvation
for secretion of the phosphodiesterase PhoD (Tjalsma et al., 2000; van Roosmalen et al.,
2001). Most other Gram-positive bacteria that have tatA and tatC genes, including S. aureus,
appear to have only one TatA-TatC translocase. The functionality of the S. aureus Tat
translocase was recently demonstrated (Biswas et al., 2009). In contrast to S. aureus, S.
epidermidis seems to lack a Tat pathway.
32

Mapping the pathways to staphylococcal pathogenesis by comparative secretomics
Pseudopilin export (Com) pathway
In B. subtilis four proteins, ComGC, ComGD, ComGE and ComGG, have been identified
with an N-terminal pseudopilin-like signal peptide (Tjalsma et al., 2004; Tjalsma et al.,
2000). All four of these proteins are involved in DNA binding and uptake, and are localized in
the membrane and cell wall. It is thought that these proteins form a pilus-like structure in the
cell wall or modify the cell wall to provide a passage for DNA uptake. Translocation to the
extracytoplasmic membrane surface is only possible when these proteins are processed by the
pseudopilin-specific SPase ComC in B. subtilis (Dubnau, 1999). SPases of this type are
bifunctional and do not only catalyze signal peptide cleavage, but also methylation of the Nterminus
of the mature protein (Strom et al., 1993). Furthermore, export and functionality of
the four ComG proteins depends on the integral membrane protein ComGB and the traffic
ATPase ComGA, which is located at the cytoplasmic side of the membrane (Chung and
Dubnau, 1998; Hahn et al., 2005). Homologues of ComC, ComGA, ComGB and ComGC, but
not ComGD, ComGE and ComGG, are present in the six sequenced S. aureus strains. This
suggests that the Com system of S. aureus is not involved in DNA uptake, but in another
solute transport process.
ABC transporters
Bacteriocins are peptides or proteins that inhibit the growth of other bacteria. Most of the
characterized bacteriocins can be divided into several classes, depending on specific
posttranslational modifications, the presence and processing of particular leader peptides and
the machinery for export from the cytoplasm. A well described class of bacteriocins is formed
by the lantibiotics. Members of this class are composed of short peptides that contain posttranslationally
modified amino acids, like lanthionine and β-methyllanthionine (McAuliffe et
al., 2001). The production of bacteriocins in S. aureus has been described for various strains.
S. aureus C55 produces the two lantibiotics C55α and C55β (Navaratna et al., 1998). These
lantibiotics are both encoded by a 32 kb plasmid, which is readily lost upon growth at
elevated temperatures. C55α and C55β showed antimicrobial activity towards other S. aureus
strains and Micrococcus luteus, but not towards S. epidermidis. Furthermore, the nonlantibiotics
BacR1 (Crupper et al., 1997), aureocin A53 (Netz et al., 2001) and aureocin A70
(Netz et al., 2002a; Netz et al., 2002b) have been identified as bacteriocins with activity
against a broad range of bacteria. The genes for both aureocins are located on a plasmid that is
present in S. aureus strains that were isolated from milk. By analogy with well described
bacteriocin export machinery in other organisms (Håvarstein et al., 1995; Peschel et al.,
1997), it can be anticipated that all of the afore-mentioned bacteriocins are exported to the
external staphylococcal milieu by dedicated ABC transporters. However, no experimental
evidence for this assumption has been published for S. aureus. Notably, it has been
demonstrated that the secretion of the lantibiotics epidermin and gallidermin of S. epidermidis
Tü3298 and Staphylococcus gallinarum, respectively, is facilitated by so-called onecomponent
ABC transporters. Specifically, the ABC-transporter GdmT has been implicated in
the transport of these lantibiotics (Peschel et al., 1997).
33

Chapter 2
Holins
Holins are dedicated export systems for peptidoglycan-degrading endolysins that have been
implicated in the programmed cell death of bacteria. These exporters, which are composed of
homo-oligomeric complexes, can be subdivided into two classes, depending on their number
of transmembrane segments. While class I holin subunits have three transmembrane
segments, class II holin subunits have two transmembrane segments (Young and Bläsi, 1995).
In S. aureus the lrg and cid operons are involved in murein hydrolase activity and antibiotic
tolerance (Groicher et al., 2000; Rice et al., 2003). A disrupted lrg operon leads to an increase
in murein hydrolase activity and a decrease in penicillin tolerance, and a disrupted cid operon
leads to a decrease in murein hydrolase activity and an increase in penicillin tolerance. It is
still unclear how the CidA and LrgA proteins are involved in these mechanisms, but these
proteins display significant similarity to the bacteriaphage holin protein family, suggesting
that they have a role in protein export. It has therefore been proposed that the CidA and LrgA
proteins act on the murein hydrolase activity and antibiotic tolerance analogous to holins and
antiholins, respectively (Bayles, 2000; Rice et al., 2003). Sequence similarity searches show
that the genes for LrgA and CidA are conserved in the six sequenced S. aureus strains, as well
as S. epidermidis and B. subtilis. Notably, none of the three holins of B. subtilis was shown to
be involved in the secretion of proteins to the extracellular milieu (Westers et al., 2003;
Tjalsma et al., 2004).
Ess pathway
The ESX-1 or ESAT-6 secretion system (Ess) pathway has first been described for M.
tuberculosis. It has been proposed that at least two virulence factors, ESAT-6 (early secreted
antigen target 6 kDa) and CFP-10 (culture filtrate protein 10 kDa), are secreted via this
pathway in a Sec-independent manner (Berthet et al., 1998; Sørensen et al., 1995). As this
pathway was discovered in mycobacteria, it is also known as the Snm pathway (Secretion in
mycobacteria; (Converse and Cox, 2005)). The genes for ESAT-6 en CPF-10 are located in
conserved gene clusters, which also encode proteins with domains that are conserved in FtsK-
and SpoIIIE-like transporters. These conserved FtsK/SpoIIIE domains have therefore been
termed FSDs (Burts et al., 2005). In other Gram-positive bacteria including S. aureus, B.
subtilis, Bacillus anthracis, Clostridium acetobutylicum and L. monocytogenes, homologues
of ESAT-6 have been identified (Pallen, 2002). The genes for these ESAT-6 homologues are
also found in gene clusters that contain at least one membrane protein with a FSD. In S.
aureus, two proteins named EsxA and EsxB have been identified that seem to be secreted via
the Ess pathway (Burts et al., 2005). The esxA and esxB genes are part of a cluster containing
six other genes for proteins that have been implicated in the translocation of EsxA and EsxB.
These include the cytoplasmic protein EsaB and the secreted protein EsaC (Burts et al.,
2008), as well as the predicted membrane proteins EsaA, EssA, EssB and EssC, of which
EssC contains a FSD. Mutations in essA, essB or essC result in a loss of EsxA and EsxB
production, which may relate to an inhibition of the synthesis of these proteins, or their
folding into a protease-resistant conformation. EsaB is a negative regulator of EsaC and
represses the production of EsaC in a post-transcriptional manner. EsaC, although secreted,
does not contain the WxG-motif or any other signal peptide and it is still unclear how this
protein is recognized by the Ess secretion pathway. All sequenced S. aureus strains contain
this cluster of esa, ess and esx genes, but it seems to be absent from S. epidermidis.
34

Mapping the pathways to staphylococcal pathogenesis by comparative secretomics
Interestingly, the genes for EsxB and EsaC appear to be absent from the S. aureus MRSA252
strain. This implies that the Ess machinery of this strain may be required for the transport of
only EsxA and perhaps a few other unidentified proteins. If so, EsaC would be dispensable
for an active ESAT-6 pathway and might be specifically involved in the export of EsxB. This
view is also suggested from the data published by Burts et al. (Burts et al., 2008), which show
that a S. aureus Newman strain lacking esxB does not produce EsaC. Alternatively, the Ess
pathway could be inactive in the S. aureus MRSA252 strain due to the absence of EsaC.
Lysis
Various studies have shown that certain proteins with typical cytoplasmic functions and
without known signals for protein secretion can nevertheless be detected on the extracellular
proteome of different bacteria (Tjalsma et al., 2004). Notably, many of these proteins, such as
catalase, elongation factor G, enolase, glyceraldehyde-3-phosphate dehydrogenase, GroEL
and superoxide dismutase, are amongst the most highly abundant cytoplasmic proteins. This
makes it likely that they are detectable in the extracellular proteome due to cell lysis. Perhaps,
such proteins are more resistant to extracytoplasmic degradation than other proteins that are
simultaneously released by lysis. However, the possibility that the extracellular localization of
typical cytoplasmic proteins is due to the activity of, as yet unidentified, export pathways
cannot be excluded. Clearly, until recently this possibility did still apply for the EsxA, EsxB
and EsaC proteins, which are now known to be exported via the Ess pathway. A clear
indication that the presence of certain “cytoplasmic” proteins in the extracytoplasmic milieu
of bacteria may relate to specific export processes was provided by Boël and co-workers
(Boël et al., 2004), who showed that 2-phosphoglycerate-dependent automodification of
enolase is necessary for its export from the cytoplasm.
Properties of staphylococcal signal peptides and cell
retention signals
Signal peptides
All proteins that have to be transported from the cytoplasm across the membrane to the
extracytoplasmic compartments of the cell, or the extracellular milieu, need to contain a
specific sorting signal for their distinction from resident proteins of the cytoplasm. The known
bacterial sorting signals for protein export from the cytoplasm are signal peptides (von Heijne,
1990). These signal peptides can be classified by the transport and modification pathway into
which they direct proteins. Presently, four different bacterial signal peptides are recognized
that share a common architecture, but differ in details (Figure 5). Two of these direct proteins
into the widely used Sec pathway, including the secretory (Sec type) signal peptides and the
lipoprotein signal peptides. Proteins with Sec type or lipoprotein signal peptides are processed
by different SPases (type I or type II SPases, respectively), and are targeted to different
destinations. In S. aureus the proteins with Sec type signal peptides are processed by the type
I SPase SpsB and targeted to the cell wall or extracellular milieu.
35

Chapter 2
Figure 5. General properties and classification of S. aureus signal peptides. Signal peptide properties are based
on SPase cleavage sites and the export pathways via which the preproteins are exported. Predicted signal peptides
(144) were divided into five distinct classes: secretory (Sec-type) signal peptides, twin-arginine (RR/KR) signal
peptides, lipoprotein signal peptides, pseudopilin-like signal peptides, and bacteriocin leader peptides. Most of
these signal peptides have a tripartite structure: a positively charged N-domain (N), containing lysine and/or
arginine residues (indicated by +), a hydrophobic H-domain (H, indicated by a black box), and a C-domain (C)
that specifies the cleavage site for a specific SPase. Where appropriate, the most frequently occurring amino acid
residues at particular positions in the signal peptide or mature protein are indicated. Also, the numbers of signal
peptides identified for each class and the respective SPase are indicated.
The proteins with a lipoprotein signal peptide are lipid-modified by Lgt, prior to processing
by the type II SPase LspA. In principle, these lipoproteins are retained at the membrane-cell
wall interface, but they can be liberated from this compartment by proteolytic removal of the
N-terminal Cys that contains the diacylglyceryl moiety (Antelmann et al., 2001). Proteins
with twin-arginine (RR) signal peptides appear to be processed by type I SPases, at least in B.
subtilis, and targeted to the cell wall or extracellular milieu (Tjalsma et al., 2004). The
proteins with a pseudopilin signal peptide are processed by the pseudopilin signal peptidase
ComC and most likely localized in the cytoplasmic membrane and cell wall. Finally,
bacteriocins contain a completely different sorting and modification signal that is usually
called the leader peptide. The known leader peptides show no resemblance to the aforementioned
signal peptides. The export of bacteriocins via ABC-transporters results in their
secretion into the extracellular milieu (Michiels et al., 2001; Schnell et al., 1988).
Sec type, lipoprotein and RR-signal peptides contain three distinguishable domains: the N-,
H- and C-domains. The N-terminal domain contains positively charged amino acids, which
are thought to interact with the secretion machinery and/or with negatively charged
phospholipids in the membrane. The H-domain is formed by a stretch of hydrophobic amino
acids which facilitate membrane insertion. Helix-breaking residues in the middle of the Hdomain
may facilitate H-domain looping during membrane insertion and translocation of the
precursor protein. The subsequent unlooping of the H-domain would display the SPase
recognition and cleavage site at the extracytoplasmic membrane surface where the catalytic
domains of type I and type II SPases are localized (van Roosmalen et al., 2004). Helixbreaking
residues just before the SPase recognition and cleavage site would facilitate
precursor processing by SPase I or II. In fact, these helix-breaking residues and the SPase
cleavage site, respectively, define the beginning and the end of the C-domain. Notably, the Cdomain
of pseudopilin signal peptides is located between the N- and H-domains (Chung and
Dubnau, 1995; Pugsley, 1993; Chung and Dubnau, 1998; Lory, 1998). Accordingly, processing
36

Mapping the pathways to staphylococcal pathogenesis by comparative secretomics
by pseudopilin-specific SPases, like ComC, takes place at the cytoplasmic side of the
membrane and leaves the H-domain attached to the translocated protein.
While many proteins that end up in the extracellular milieu or the cell wall of Gram-positive
bacteria have signal peptides, proteins without known export signals can also be found on
these locations. The relative numbers of proteins without known signal peptides seem to vary
per organism. While these numbers are relatively low for B. subtilis and S. aureus, they are
high for group A Streptococcus and M. tuberculosis (Tjalsma et al., 2004). As indicated
above, some of the proteins without known export signals appear to be liberated from the cell
by lysis, while others are actively exported, for example via the Ess pathway. Although the
precise export signal in proteins secreted via the Ess pathway has not yet been defined, a
WxG motif is shared by many of these proteins and may serve a function in protein targeting
(Pallen, 2002). Furthermore, the signal for specific release of lysins via holins is presently not
known.
Signal peptide predictions
Several prediction programs that are accessible through the world-wide web are useful tools
to predict whether a given protein contains some sort of sorting signal or SPase cleavage site.
The programs that we have used in this and other studies were: SignalP-NN and SignalP-
HMM version 2.0 (Nielsen et al., 1997), LipoP version 1.0 (Juncker et al., 2003), PrediSi
(Hiller et al., 2004) and Phobius (Kall et al., 2004). These programs have been designed to
identify Sec type signal peptides, N-terminal membrane anchors (Phobius), or lipoprotein
signal peptides in Gram-negative bacteria (LipoP). The TMHMM-program version 2.0
(Cserzö et al., 1997) was used to exclude proteins with (predicted) multiple membrane
spanning domains. Predictions for proteins containing a signal peptide were performed with
the SignalP program, using the Neural Network (NN) and Hidden Markov Model algorithms
(HMM). Version 2.0 of the SignalP program was preferred above Version 3.0 (Bendtsen et
al., 2004) for our signal peptide predictions in S. aureus and S. epidermidis, because the best
overall prediction accuracy was obtained with Version 2.0 in a recent proteomics-based
verification of predicted export and retention signals in B. subtilis (Tjalsma and van Dijl,
2005). Specifically, the Hidden Markov Model in SignalP 2.0 assigns probability scores to
each amino acid of a potential signal peptide and indicates whether it is likely to belong to the
N-, H-, or C-domains. Proteins with no detectable N-, H-, and C-domain were excluded from
the set. Searching for transmembrane domains was performed with the TMHMM program
and proteins with more than one (predicted) transmembrane domain were excluded from the
set, because they are most likely integral membrane proteins. All proteins with a predicted Cterminal
transmembrane segment in addition to a signal peptide were screened for the
presence of a conserved motif for covalent cell wall binding. It should be noted that this
approach does not automatically result in the exclusion of potential membrane proteins with
one N-terminal transmembrane domain. The LipoP-program was used to predict lipoproteins.
The combined results of all these programs resulted in a list of proteins, which have: a) signal
peptides with distinctive N-, H-, and C-domains, b) no additional transmembrane domains,
and c) predicted extracytoplasmic localizations. These proteins were scanned for the presence
of proteomics-based consensus motifs for type I, type II or pseudopilin-specific SPase
recognition and cleavage sites, twin-arginine motifs, and known leader peptides of
bacteriocins by BLAST searches and by use of the PATTINPROT program (http://npsapbil.ibcp.fr)
as previously described (Tjalsma and van Dijl, 2005). To define the core
37

Chapter 2
exoproteome and variant exoproteome of the S. aureus strains, the sets of proteins with
predicted signal peptides were used in multiple blasts with the freeware BLASTall from the
NCBI. The output was then filtered using Genome2D (Baerends et al., 2004).
Secretory (Sec type) signal peptides
Proteomics-based data sets of membrane, cell wall and extracellular proteins have been
extremely valuable for a recent verification of signal peptide predictions in B. subtilis
(Tjalsma and van Dijl, 2005). Such data sets are now becoming available for S. aureus, as
exemplified by studies on the membrane plus cell wall proteomes of S. aureus Phillips
(Nandakumar et al., 2005), and the extracellular proteomes of S. aureus strains that have been
derived from the recently sequenced NCTC8325 and COL strains (Ziebandt et al., 2001;
Ziebandt et al., 2004) (Figure 2). Additionally, the extracellular proteomes of several clinical
S. aureus isolates have been analyzed (Figure 2). The membrane, cell wall and extracellular
proteins of S. aureus that have been identified by proteomics (Ziebandt et al., 2001; Ziebandt
et al., 2004; Nandakumar et al., 2005; Gatlin et al., 2006; Pocsfalvi et al., 2008; Ziebandt et
al., submitted), involving 2D-PAGE and subsequent mass spectrometry, are listed in
(Supplemental tables IIIa and IIIb). These tables also show the -3 to +1 amino acid sequences
of the respective signal peptidase cleavage sites, if present.
Based on the proteomics data for membrane and extracellular proteins of B. subtilis, the
optimized -3 to +1 pattern [AVSTI] - [SEKYHQFLDGPW] – A - [AQVEKDFHLNS] for
signal peptide recognition and cleavage by type I SPases of this organism was identified
(Tjalsma and van Dijl, 2005). SPase cleavage occurs C-terminally of the invariant Ala residue
at the -1 position. The residues between square brackets in the pattern are listed in the order of
their frequency, the most frequently identified residue at each position being placed in first
position. By comparing the predicted SPase recognition and cleavage sites in signal peptides
of proteomically identified extracellular proteins of S. aureus (Supplemental table IIIa) we
defined the -3 to +1 pattern [AVST] - [KQNESDHYLFAGR] – A - [AESKDIFLQTY] for
productive recognition and cleavage by the type I SPase SpsB. Compared to the equivalent
pattern of B. subtilis it is interesting to note that the frequencies of certain residues at the -3, -
2 and +1 positions differ, as reflected by the most-frequent-first order in which they are listed
in the pattern. Moreover, Asn can be present at the -2 position, while Ile is accepted at the +1
position. The latter residues are found in the -2 and +1 positions of certain serine proteases,
hemolysins, immunoglobulin G binding protein A and aureolysin (Supplemental table IIIa). It
should also be noted that, compared to the optimized SPase recognition pattern of B. subtilis,
several residues are not found at the -3, -2 and +1 positions of potential SpsB recognition and
cleavage sites in identified extracellular proteins of S. aureus. As such residues may be
present in SPase recognition and cleavage sites of proteins that have escaped identification
through proteomics, we have included them in the -3 to +1 search pattern (printed in
lowercase) for the identification of potential secretory proteins of staphylococci: [AVSit] -
[KHNDQSYEGLRAfpw] – A - [AESDIKLTYfhnqv]. This optimized S. aureus search
pattern was used as an indicator for the quality of signal peptide predictions that were based
on the SignalP-NN, SignalP-HMM, LipoP, PrediSi, Phobius and TMHMM programs.
Proteins with potential signal peptides containing this pattern were assigned to have a high
probability for an extracytoplasmic localization and a low probability for membrane retention
(Supplemental tables IIIc-f). Proteins with potential signal peptides that do not contain this
pattern were assigned to have a high probability to be retained in the membrane (data not
38

Mapping the pathways to staphylococcal pathogenesis by comparative secretomics
shown). In this case, the uncleaved signal peptide could serve as an N-terminal membrane
anchor. Following this approach, sets of 186-211 proteins (depending on the S. aureus strain)
were identified that contain a potential signal peptide or N-terminal transmembrane segment.
Scanning for the presence of the SpsB recognition and cleavage motif [AVSit] -
[KHNDQSYEGLRAfpw] – A - [AESDIKLTYfhnqv] revealed that, depending on the S.
aureus strain investigated, 86-106 proteins carry this motif. These proteins are most likely
processed by SPase, liberated from the membrane and secreted into the extracellular milieu,
unless they contain a cell wall retention signal (see following sections). Most of the other
proteins with signal peptides that do not conform to the SpsB recognition and cleavage motif
lack the invariant Ala at the –1 position. Also, some of these preproteins contain different
residues at the –3, -2 or +1 positions. For example, Asp, Glu, Phe and Lys are highly unlikely
residues at the -3 position (van Roosmalen et al., 2004). On the other hand, some preproteins
have a Gly at the -3 position (e.g. the exotoxins 4 and 5 from S. aureus COL) or a Leu
(Supplemental tables IIIc and IIId). Since Gly and Leu residues at the -3 position of signal
peptides are accepted by the E. coli SPase, it seems likely that they are also accepted at this
position by SpsB. However, we have not included these residues in the current SpsB
recognition and cleavage motif, since we could neither identify these proteins amongst the
secreted proteins of S. aureus COL (Figure 2), nor find published evidence that these proteins
are indeed secreted. Among the proteins with predicted cleavable Sec type signal peptides
there are many known extracellular staphylococcal virulence factors, such as exotoxins,
enterotoxins (SEM, SEN and SEO), hemolysins, toxic shock syndrome toxin-1 (TSST-1),
leukotoxins (LukD, LukE), a secretory antigen SsaA homologue and the immunodominant
antigen A (IsaA). Remarkably, the lists of identified extracellular proteins of S. aureus COL
and RN6390 (Ziebandt et al., 2004) (Supplemental tables IIIa and IIIb) reveal that about 41%
of these proteins lack known signal peptides. This percentage is substantially higher than the
initial estimate of 10%, which was based on a limited proteomics-derived data set (Tjalsma et
al., 2004). It is also interesting to note that the list of identified extracellular proteins without
a signal peptide includes enolase, which may be actively exported by an unknown mechanism
(Boël et al., 2004), but lacks EsxA and EsxB, which are exported by the Ess pathway (Burts
et al., 2005).
Twin-arginine (RR-)signal peptides
The consensus RR-motif that directs proteins into the Tat pathway has previously been
defined as [KR]-R-x-#-#, where # is a hydrophobic residue (Cristóbal et al., 1999; Jongbloed
et al., 2000). Dilks et al. (Dilks et al., 2003) have used a genomic approach to identify
possible Tat substrates for 84 diverse prokaryotes using the TATFIND 1.2 program. This
study included S. aureus Mu50, MW2 and N315. Two potential Tat substrates of unknown
function were predicted for S. aureus Mu50 and MW2 and one of these was also predicted for
S. aureus N315 (Dilks et al., 2003). However, both proteins are conserved in all sequenced S.
aureus strains, including the N315 strain. One of these two predicted Tat substrates has no
known function, whereas the other was annotated as a hypothetical protein similar to a
ferrichrome ABC transporter (permease). These proteins, however, are not in our list of
proteins that have a predicted (RR-)signal peptide. Although they have signal peptides
according to the SignalP program, these proteins are localized in the cytoplasm or membrane,
respectively, according to the LipoP, PrediSi and Phobius programs. It is therefore unlikely
39

Chapter 2
that these proteins are destined for secretion. Specifically, the hypothetical permease has eight
predicted transmembrane helices.
Our own pattern searches for proteins with a possible RR-motif resulted in 24-32 positive
hits, depending on the S. aureus strain investigated. However, most of these proteins have no
detectable N-, H- or C-domains and were, therefore, discarded from our data set. Also, some
other proteins with a possible RR-motif are predicted to contain a lipoprotein signal peptide.
These predicted lipoproteins were also discarded from the list of potential S. aureus Tat
substrates, firstly, because none of the identified lipoproteins of B. subtilis that have a RRmotif
were shown to be secreted via the Tat pathway (Jongbloed et al., 2000; Jongbloed et al.,
2002), and secondly, because there is limited published evidence for other bacteria that
lipoproteins can be exported Tat-dependently (Widdick et al., 2006). Thus, it appears that
only 5-7 proteins, depending on the S. aureus strain investigated, are potentially exported by
the Tat pathway and cleaved by SpsB. However, it is noteworthy that none of the B. subtilis
proteins with a KR-motif were so far shown to be secreted Tat-dependently, even though KRmotifs
are capable of directing proteins into the Tat pathways of chloroplasts and Gramnegative
bacteria, such as E. coli and Salmonella enterica (Stanley et al., 2000; Hinsley et al.,
2001; Molik et al., 2001; Ignatova et al., 2002). If KR-motifs are also rejected by the S.
aureus Tat pathway, there would not be a single protein in any sequenced S. aureus strain that
is secreted Tat-dependently. This would be highly remarkable in view of the presence of tatA
and tatC genes in all these strains. Notably, the only known strictly Tat-dependent
extracellular proteins of B. subtilis are the phosphodiesterase PhoD (Tjalsma et al., 2000) and
the protein of unknown function YwbN (Jongbloed et al., 2004). While a homologue of PhoD
is not present in any of the six sequenced S. aureus strains, homologues of YwbN are present
in all these strains. Close inspection of the YwbN homologues of S. aureus COL, JH1, JH9,
MRSA252, MSSA476, NCTC8325, Newman, USA300 and USA300_TCH1516 revealed the
presence of an N-terminal RR-motif, but a potential signal peptide was not identified as such
by the SignalP program. In contrast, the YwbN homologues of S. aureus Mu3, Mu50, MW2
and N315 appeared to lack this RR-motif. According to comparisons of the deduced amino
acid sequences, the latter three YwbN homologues would miss the first 40 residues of B.
subtilis YwbN. Most likely, this is not the case since the sequences upstream of the annotated
S. aureus Mu3, Mu50, MW2 and N315 ywbN genes encode for a peptide with a RR-motif in
the same open reading frame as the ywbN structural gene (Supplemental table IIIc). Thus, the
RR-motif of S. aureus Mu3, Mu30, MW2 and N315 YwbN has escaped identification due to
a systematic difference in sequence annotation. Recent studies by Biswas et al. (Biswas et al.,
2009) have shown that the YwbN homologue of S. aureus is an iron-dependent peroxidase
(now named FepB). Tat mutant S. aureus strains did no longer export active FepB, and the
RR-signal peptide of FepB was able to direct Tat-dependent secretion of prolipase or protein
A. Interestingly, FepB is needed for iron-acquisition in S. aureus, and in a mouse kidney
abscess model the bacterial loads of tat or fepB mutant strains were substantially reduced.
Pseudopilin signal peptides
The signal peptides of pseudopilins differ from the Sec type signal peptides in the location of
SPase cleavage sites. In pseudopilin signal peptides, this cleavage site is located between the
N- and H-domains (Lory, 1994). The consensus recognition and cleavage motif for
pseudopilin SPases, such as ComC, is K-G-F-x-x-x-E. Cleavage by pseudopilin SPases occurs
within this motif, between the Gly and Phe residues. Upon cleavage the Phe residue is
40

Mapping the pathways to staphylococcal pathogenesis by comparative secretomics
methylated. In all sequenced S. aureus strains, three proteins were found, which have the
canonical pseudopilin SPase recognition and cleavage motif. These proteins are homologues
of the cold shock proteins CspB, CspC and CspD of B. subtilis. However, even though these
proteins do contain the pseudopilin SPase recognition and cleavage pattern, they lack the Hdomain.
Since the active site of pseudopilin SPases is located in the cytoplasm, cleavage of
the CspBCD homologues of S. aureus would be possible, but their export via the Com
pathway is unlikely. Nevertheless, it should be noted that one of the CspBCD homologues of
S. aureus, which is known as CspA, was found in the extracellular proteome of a clinical
isolate (Figure 2 and Supplemental table IIIb). To verify the absence or presence of
pseudopilins in S. aureus, BLAST searches with the known ComGC, ComGD, ComGE or
ComGG proteins of B. subtilis were performed. This revealed the presence of only one
potential pseudopilin, which is a homologue of B. subtilis ComGC. Although the consensus
pseudopilin SPase recognition and cleavage site is absent from S. aureus ComGC, a putative
cleavage pattern (Q-A-F-T-L-I-E) is present at the position in the ComGC signal peptide
where a pseudopilin SPase recognition and cleavage site would be expected. Further analyses
revealed that similar observations can be made for ComGC homologues in other Grampositive
bacteria, such as Bacillus cereus, B. anthracis, L. monocytogenes, S. haemolyticus
and Oceanobacillus iheyensis. By comparing the ComGC homologues of these organisms, an
expanded search pattern for Gram-positive bacterial pseudopilin SPase recognition and
cleavage sites can be defined as [KEQRS]-[GA]-F-x-x-x-E. Interestingly, using this expanded
search pattern, two additional potential pseudopilins of S. aureus were identified. These
potential pseudopilins show similarity to ComGD of B. cereus and B. anthracis, and ComGF
of Bacillus halodurans and L. lactis. It remains to be shown whether the three identified
potential pseudopilins of S. aureus are indeed able to assemble into pilin-like structures after
processing by the ComC homologue. If so, it will be even more interesting to identify their
biological function, for example in adhesion to surfaces, motility, or export of proteins. Such
functions could play a role in virulence and have been attributed to type IV pili and
pseudopilins of Gram-negative bacteria (Lory, 1998).
Bacteriocin leader peptides
Bacteriocins form a distinct group of proteins with cleavable N-terminal signal peptides,
which are often called leader peptides. These leader peptides only have N- and C-domains,
and thus completely lack the hydrophobic H-domain. The bacteriocin leader peptides are
invoked in posttranslational modification and prevention of premature antimicrobial activity,
which would be deleterious to the producing organism. Of the sequenced S. aureus
bacteriocins, C55α and C55β contain a leader peptide (Navaratna et al., 1999), whereas
leader peptides are absent from aureocin A53 (Netz et al., 2001) and aureocin A70 (Netz et
al., 2001). Two potential lantibiotics with leader peptides were identified by sequencing the
genomes of S. aureus MW2 (Baba et al., 2002) (GI numbers 49486642 and 49486641) and
MSSA476. In both strains, the corresponding genes are located on the genomic island νSAβ.
Both S. aureus proteins show similarity to the lantibiotic gallidermin precursor GdmA of
Staphylococcus gallinarum, and to the lantibiotic epidermin precursor EpiA of S. epidermidis.
Notably, the S. aureus COL strain contains only one of these two potential lantibiotics, which
is most similar to the potential MW2 lantibiotic with the accession number 49486641. Two
additional putative bacteriocins that were identified by genome sequencing seem to be
homologous to L. lactis lactococcin 972. The hypothetical protein SAP019 (N315 annotation)
41

Chapter 2
is plasmid-encoded in S. aureus N315 and MSSA476, and chromosomally encoded in S.
aureus MRSA252. The other hypothetical bacteriocin SAS029 is chromosomally encoded in
all sequenced S. aureus strains. Recently, a program for detecting potential bacteriocins in
bacterial genomes (BAGEL) has been released (de Jong et al., 2006). This program is based
on the properties of known bacteriocins, including genes that lie close to these bacteriocin
genes. Such genes may encode proteins involved in the processing or transport of the
bacteriocin. Using the BAGEL program with standard settings to detect potential bacteriocins
in the sequenced and annotated S. aureus strains, 2-6 proteins were identified as significant.
No published data is presently available on the charateristics of these proteins, so it remains to
be seen whether they are genuine bacteriocins.
A potential Ess export signal?
As described above, the EsxA, EsxB and EsaC proteins are secreted by S. aureus via the Ess
route (Burts et al., 2005). All three proteins lack a known signal peptide, but are specifically
transported across the membrane nonetheless. This implies that these two proteins must
contain an export signal that is recognized by one or more Ess pathway components. The
nature of this signal is presently unknown. The most common feature of proteins that are
known (or predicted) to be translocated across the membrane via the Ess pathway is a WxGmotif,
which is located at ~100 amino acids from the N-terminus of the protein (Pallen, 2002).
In particular since the WxG motif appears to be absent from EsaC, the involvement of the
WxG-motif in Ess targeting remains to be demonstrated.
Retention signals
Lipoproteins
Lipoproteins appear to be exported via the Sec-pathway. During, or shortly after
translocation, the invariant Cys in the lipobox is diacylglyceryl-modified by Lgt. This results
in signal peptide cleavage by SPase II and retention of the mature lipoprotein in the
membrane. Based on the cleavage sites of lipoproteins that have been identified in various
Gram-positive bacteria, Sutcliffe et al. (Sutcliffe and Harrington, 2002) have reported the -4
to +2 lipobox pattern [LIVMFESTAG] - [LVIAMGT] - [IVMSTAFG] - [AG] – C -
[SGANERQTL]. Furthermore, they reported that neither the charged residues Asp, Glu, Arg,
or Lys, nor Gln are present in the region between six and twenty residues N-terminal of the
lipobox. Searching the translated proteins encoded by the thirteen S. aureus genomes with the
pattern shown above using the PATTINPROT program, revealed about 50 proteins with this
motif (Supplemental table IIIe and IIIf). A comparison of the PATTINPROT results to the
results obtained with the LipoP program shows that 12-18 more potential lipoproteins may be
present in S. aureus. Most of these extra predicted lipoproteins contain an amino acid at the -
1-position (mostly Ser) or the +2-position (mostly Asp) that differs from the lipobox pattern
of Sutcliffe et al. (Sutcliffe and Harrington, 2002). Recently, Tjalsma and van Dijl (Tjalsma
and van Dijl, 2005) have proposed the lipobox search pattern [LITAGMV] - [ASGTIMVF] -
[AG] – C - [SGENTAQR] for potential lipoproteins of B. subtilis on the basis of published
proteomics data. The only difference compared to the pattern by Sutcliffe et al. (Sutcliffe and
Harrington, 2002) is that Leu absent from the +2 position, which is due to the fact that no
42

Mapping the pathways to staphylococcal pathogenesis by comparative secretomics
potential B. subtilis lipoprotein with Leu at this position was identified by proteomics.
Consistently, none of the predicted S. aureus lipoproteins has a Leu at the +2 position
(Supplemental tables IIIe and IIIf). It is also noteworthy that some lipoproteins contain a
[KR]-R-x-#-# motif in their signal peptides, although it has not been shown yet that
lipoproteins can be transported via the Tat-pathway. Finally, the hypothetical protein Lpl2 of
S. aureus N315 and Mu50 was excluded from our lipoprotein predictions, because Asp does
not seem to occur at the +2 position of lipoproteins from Gram-positive bacteria (Juncker et
al., 2003; Tjalsma and van Dijl, 2005). Nevertheless, the homologues of Lpl2 of the other
sequenced S. aureus strains are classified as lipoproteins, because they have residues at the +2
position that conform to the lipobox consensus.
Lipoprotein Release Determinant
Although lipoproteins were generally believed to be retained at the membrane-cell wall
interface, the presence of lipoproteins in the growth medium of B. subtilis was documented by
Antelmann et al. (Antelmann et al., 2001). This unexpected finding is correlated to the
proteolytic removal of the amino-terminal, lipid-modified Cys, which suggests that the
observed lipoprotein release into the growth medium is caused by proteolytic “shaving” after
processing by LspA. In most of these lipoproteins Tjalsma and van Dijl (Tjalsma and van
Dijl, 2005) identified the +1 to +10 consensus sequence C – G - [NSTF] – x - [SGN] – x -
[SGKAE] – x – x - [SGA] that might represent the recognition site for a yet unidentified
shaving protease. Most probably, a Gly at the +2 position is of major importance for
lipoprotein release into the growth medium, while a Ser at this position seems to inhibit this
proces. In the sequenced genomes of S. aureus only one lipoprotein could be found with the
exact motif described above. By searching for patterns with 80% similarity to the consensus
sequence (i.e. one different residue), five to seven additional lipoproteins can be found,
depending on the S. aureus strain. In none of these proteins, Thr was found at the +3 position.
Instead, a Lys was identified at this position in one or two predicted lipoproteins with a
potential release motif, depending on the S. aureus strain. In other predicted lipoproteins with
a potential release motif, no Gly or Ala residues were found at the +7 position. However, one
of these lipoproteins contains a predicted release motif with a Gln at the +7 position. To date,
a total number of four potential lipoproteins have been identified in the extracellular milieu of
S. aureus. The first one was identified by Ziebandt et al. (Ziebandt et al., 2004). This protein
has been annotated as a thioredoxin reductase (Supplemental table IIIa), but it shows no
similarity to known thioredoxin reductases. Instead, it is highly similar to phosphate-binding
lipoproteins, such as PstS of B. subtilis. It should be noted that this protein was not predicted
as a lipoprotein, because the signal peptide contains a Gln residue in the N-domain.
According to the search profile of Sutcliffe et al. (Sutcliffe and Harrington, 2002) lipoproteins
would not contain Gln residues at this position. On the other hand, PstS of B. subtilis is a
lipoprotein and it would seem quite likely that this is also true for its S. aureus homologue.
The other three potential lipoproteins were identified in the growth medium of clinical
isolates (Tabel 4). Remarkably, none of these four lipoproteins with an extracellular
localization contain the complete lipoprotein release motif that was identified in extracellular
lipoproteins of B. subtilis. However, they do contain a Gly residue at the +2 position, which
strengthens the idea that this amino acid residue is probably important for lipoprotein release.
It is interesting to note that in lipoproteins of Gram-negative bacteria, an Asp, Gly, Phe, or
Trp residue at the +2 position prevents the transport of the mature lipoprotein to the outer
43

Chapter 2
membrane (Tokuda and Matsuyama, 2004; Narita and Tokuda, 2006). In Gram-positive
bacteria no outer membrane is present and it is currently not known whether the residues at
the +2 position have a role in subcellular protein sorting. However, a Gly at this position does
seem to promote lipoprotein release into the extracellular milieu, not only in B. subtilis, but
also in S. aureus.
Cell wall binding domains
Proteins that have to be displayed on the bacterial surface must be retained by non-covalent or
covalent binding to the peptidoglycan moiety of the cell wall. In B. subtilis, several proteins
involved in cell wall turnover contain repeated domains in the C-terminal part of the protein,
which have affinity for cell wall components (Ghuysen et al., 1994; Margot and Karamata,
1996; Rashid et al., 1995). Specifically, the B. subtilis proteins LytD, WapA, YocH, YvcE,
and YwtD have been reported to bind to the cell wall (Ghuysen et al., 1994; Margot and
Karamata, 1996; Rashid et al., 1995). While WapA is not conserved in staphylococci, various
S. aureus proteins with regions that show amino acid sequence similarity to LytD, YocH,
YvcE, and YwtD of B. subtilis can be found by BLAST searches. Accordingly, these S.
aureus proteins may be cell wall-bound, but this remains to be shown.
One of the domains that have affinity for cell wall components is the “Lysin Motif”, or LysM
domain, which has first been described for bacterial lysins (Ponting et al., 1999). The number
of LysM domains can differ for wall-bound proteins from different Gram-positive bacterial
species (Steen, 2005). For example, XlyA of B. subtilis contains only one LysM domain,
whereas three domains can be detected in AcmA of L. lactis, or even five or six domains in
muramidases from Enterococcus species (Joris et al., 1992). Using the LysM domain of
AcmA from L. lactis in BLAST searches against the six sequenced and annotated S. aureus
genomes, four proteins with one or more LysM domains were detected. These proteins
include a hypothetical protein similar to autolysins (SA0423), the secretory antigen SsaA
homologue (SA0620), a conserved hypothetical protein (SA0710), and the LytN protein.
A different domain that can facilitate protein binding to the cell wall is the GW domain. In L.
monocytogenes, the surface-exposed InlB protein contains three C-terminal GW domains.
Each domain consists of ~80 amino acids and starts with a Gly and Trp residue (Braun et al.,
1997). This domain specifically binds to lipoteichoic acids in the cell wall (Jonquieres et al.,
1999), thereby facilitating the interaction of L. monocytogenes with components of human
host cells. The only protein found in the sequenced S. aureus strains with GW domains is the
autolysin protein Atl (Baba and Schneewind, 1998; Baba and Schneewind, 1996). This
bifunctional autolysin contains three GW repeats of ~97 amino acids. The protein is exported
as a prepro-Atl precursor of 1256 amino acids. Subsequent processing steps result in the
removal of the signal peptide and the propeptide, and the separation of the mature region into
an amidase and a glucosaminidase (Oshida et al., 1995). A similar separation of the mature
region into an amidase and glucosaminidase has been reported for the AtlE protein of S.
epidermidis (Heilmann et al., 1997). The GW repeats are both necessary and sufficient to
direct reporter proteins to the equatorial surface ring of S. aureus cells where cell division
starts.
Other S. aureus wall proteins that contain repeated domains with potential wall binding
properties have been described. These include the clumping factors A and B (ClfAB)
(Hartford et al., 1997; Ní Eidhin et al., 1998), several serine aspartate repeat proteins
(SdrCDE) (Josefsson et al., 1998), the homologue of S. gordonii GspB (SasA) (Siboo et al.,
44

Mapping the pathways to staphylococcal pathogenesis by comparative secretomics
2005) (see also the section on covalent cell wall binding below), and the ECM-binding
protein homologue (Clarke et al., 2002). Although not documented in the literature, additional
proteins with Sec type signal peptides and potential cell wall binding repeats that can be
recognized readily. These are the cell wall surface anchor family protein SACOL2505, and
the methicillin-resistant surface protein SACOL0050, which both contain C-terminal repeated
regions of ~130 amino acids. The latter protein, which shows a high degree of sequence
similarity to the SACOL2505 protein, is only found in S. aureus COL, but not in the five
other sequenced strains. This is due to the fact that the gene for SACOL0050 is localized on
the mec cassette 1 and therefore not present in the other strains. Notably, the SACOL2505
homologues in S. aureus Mu50 and N315 seem to lack the C-terminal part of the protein with
the repeats. A close inspection of the sequence of the corresponding genes in these strains
revealed that there is a frameshift mutation or sequencing error in these genes, resulting in an
apparent or real C-terminal truncation of the corresponding proteins. Thus, the C-terminal cell
wall binding repeats are absent or appear to be absent. Interestingly, most of the aforementioned
proteins with cell wall binding motifs also contain the motif (LPxTG) for covalent
attachment to the cell wall by sortase A or sortase B (see below).
It should be noted that a variety of known cell wall binding domains, such as the choline
binding domain (Yother and White, 1994), the Cpl-7 cell wall binding domain (Garcia et al.,
1990) and the fructosyltransferase cell wall binding domain (Huard et al., 2003; Milward and
Jacques, 1990; Rathsam et al., 1993) appear to be absent from staphylococcal proteins.
Covalent attachment to the cell wall
Cell wall sorting proteins, known as sortases, exist in many Gram-positive bacteria and serve
to anchor proteins that are destined for cell surface display to the cell wall (Dramsi et al.,
2005; Ton-That et al., 2004a). In almost all Gram-positive bacteria there is at least one sortase
present and often genes for more than one sortase-like protein can be detected in a single
genome. These transpeptidases catalyze the formation of an amidebond between the
carboxylgroup of a Thr and the freed amino end of pentaglycine cross-bridges in
peptidoglycan precursors. Subsequently, the peptidoglycan precursors with covalently bound
proteins are incorporated into the cell wall. More recently, it has been shown that sortases can
also be involved in protein polymerization leading to the assembly of pili on the surface of
Gram-positive bacteria, such as Corynebacterium diphtheriae (Ton-That et al., 2004b; Gaspar
and Ton-That, 2006). The 3D-structure of sortase A (SrtA) of S. aureus revealed that this
protein has a unique β-barrel structure in which a catalytic Cys residue is positioned close to a
His residue. This suggests that sortase A forms a thiolate-imidazolium ion pair for catalysis
(Ilangovan et al., 2001; Ton-That et al., 2002). Furthermore, it has been shown, that a
conserved Arg residue is needed for efficient catalysis (Marraffini et al., 2004). The catalytic
cysteine is part of a conserved motif, TLxTC, which can be found in the C-terminal part of
the protein (x is usually Val, Thr or Ile). Recently, a classification of sortases has been
proposed by Dramsi et al. (Dramsi et al., 2005) based on phylogenetic analyses of 61 sortases
that are encoded by the genomes of 22 Gram-positive bacteria. These analyses showed that
sortases can be grouped into four different classes (A-D). Class A consists of sortases from
many low GC% Gram-positive bacteria, including L. monocytogenes, Streptococcus pyogenes
and S. aureus. The second class (Class B) is present in only a few low GC% Gram-positive
bacteria, including, L. monocytogenes, B. anthracis and S. aureus. Sortases of this class
contain three amino acid segments that are not present in the sortases of class A. These
45

Chapter 2
sortases recognize a different motif (NPQTN in S. aureus). The genes for substrates of class B
sortases are often found at the same locus as the sortase gene. The largest class (Class C)
consists of sortases from high GC% and low GC% Gram-positive bacteria. The genes for
class C sortases are often present in multiple copies per genome. Characteristic for this class
of sortases is a C-terminal hydrophobic domain that might serve as a membrane anchor, and a
conserved proline residue behind the catalytic site. Finally, class D sortases are present in
high and low GC% Gram-positive bacteria. This class can be divided into three subclusters,
depending on whether they are present in bacilli, clostridia or actinomycetales. Since class C
and D sortases are absent from S. aureus, the (potential) substrates of these enzymes will not
be reviewed here.
Sortase A recognition signal
For interaction with host cells during infection, many proteins are anchored to the cell wall of
staphylococcal cells, thereby enabling the cells to adhere and invade the host cells, or to evade
the immune system. Many of these proteins contain an LPxTG-motif in their C-terminal part,
which is recognized by the cell wall sorting protein sortase A. In each of the six sequenced S.
aureus strains there is only one sortase gene present, which encodes a class A sortase. The
LPxTG motif of sortase A substrates is followed by a stretch of hydrophobic amino acids and
at least one positively charged amino acid (Lys or Arg) at the C-terminus. After translocation
across the membrane, the LPxTG motif is recognized by SrtA and subsequently cleaved
between the Thr and Gly residues (Mazmanian et al., 2001; Navarre and Schneewind, 1994).
A transpeptidation is then mediated by SrtA through amide-linkage of the C-terminal Thr of
the protein to pentaglycine cross-bridges. It has been suggested that SrtA actually uses lipid II
as a peptidoglycan substrate and that the proteins that are linked to lipid II are subsequently
incorporated into the cell wall. In addition to the canonical LPxTG motif, a LPxAG motif can
also be recognized and cleaved by SrtA (Roche et al., 2003). It has been reported that S.
aureus has 19 proteins that carry the LPxTG motif and 2 proteins that carry a LPxAG motif at
their C-termini (Roche et al., 2003) (Table 4). Many of these proteins have been shown to be
expressed. These include: protein A (Spa), two clumping factors (ClfA and ClfB; also contain
potential wall binding repeats), a collagen-binding protein (Cna), three serine aspartate repeat
proteins (SdrC, SdrD and SdrE; also contain potential wall binding repeats), two fibronectinbinding
protein (FnbpA and FnbpB) (reviewed by Foster and Hook, (Foster and Hook,
1998)), a plasmin-sensitive protein (Pls) (Savolainen et al., 2001), FmtB (Komatsuzawa et al.,
2000), and several S. aureus surface (Sas) proteins (Roche et al., 2003). A recent study on the
cell wall and membrane proteome by Nandakumar et al. (Nandakumar et al., 2005) resulted
in the identification of two proteins with an LPxTG cell wall sorting signal. Many of the
LPxTG proteins contain in their N-terminal signal peptide a conserved motif, [YF]-SIRK
(with some variance) that has also been observed in other proteins that are substrates for SrtA
in several Gram-positive bacteria (Bae and Schneewind, 2003). However, this sequence is not
found in all of the SrtA substrates and it can also be found in non-cell wall proteins. This
suggests that [YF]-SIRK is not a specific SrtA targeting sequence. Interestingly, proteins that
contain an LPxTG-motif and a [YF]-SIRK motif in their signal peptides seem to be
distributed along the staphyolococcal cell surface in a different manner than those proteins
that have an LPxTG-motif, but lack the [YF]-SIRK motif. Those proteins that do contain the
[YF]-SIRK motif seems to be positioned in a ring-like structure that forms during cell
division for the separation of cells, while the proteins that lack this motif are directed to the
cell pole (Dedent et al., 2008). One of the sas genes, sasA also known as sraP, is situated in
46

Mapping the pathways to staphylococcal pathogenesis by comparative secretomics
the secA2/secY2 cluster and has an unusually long signal peptide (90 residues). Similar to
what has been reported for the cell wall-bound GspB protein in S. gordonii (Bensing and
Sullam, 2002), it was recently shown that the accessory SecA2/SecY2 system is needed for
the transport of SasA/SraP across the membrane (Siboo et al., 2008). Depending on the
sequenced S. aureus strain, 10-13 proteins with an LPxTG cell wall sorting signal, followed
by a hydrophobic stretch of residues and a positively charged C-terminus can be found (Table
4). Among these proteins are the fibrinogen-binding protein A (ClfA), immunoglobulin G
binding protein A precursor (Spa), and the Ser-Asp rich fibrinogen-binding, bone
sialoprotein-binding protein (SdrC).
Table 4. Staphylococcal proteins with (potential) Sec type signal peptides and (potential)
signals for covalent cell wall binding
PID Protein Function Signal
15925728 AsdA Adenosine synthase A LPKTG
15925815 Spa Immunoglobulin G binding protein A precursor LPETG
15925838 a SasD hypothetical protein LPAAG
15926239 b SdrC Ser-Asp rich fibrinogen-binding, bone sialoprotein-binding protein LPETG
15926240 c,d SdrD Ser-Asp rich fibrinogen-binding, bone sialoprotein-binding protein LPETG
15926241 b,d,e SdrE Ser-Asp rich fibrinogen-binding, bone sialoprotein-binding protein LPETG
15926464 ClfA fibrinogen-binding protein A, clumping factor LPDTG
15926713 IsdB conserved hypothetical protein LPKTG
15926714 IsdA cell surface protein LPKTG
15926715 IsdC conserved hypothetical protein NPQTN
15927308 c HarA hypothetical protein LPKTG
15927333 d,f SasC hypothetical protein, similar to FmtB protein LPNTG
15927741 b,g SasB FmtB protein LPDTG
15928076 b,c,h Aap hypothetical protein, similar to accumulation-associated protein LPKTG
15928081 c,d FnbB fibronectin-binding protein homolog LPETG
15928082 d,i FnbA fibronectin-binding protein homolog LPETG
15928174 j SasK hypothetical protein LPKTG
15928216 ClfB Clumping factor B LPETG
15928232 b SasF conserved hypothetical protein LPKAG
15928240 k SasA hypothetical protein, similar to streptococcal hemagglutinin protein LPDTG
57652419 l Pls methicillin-resistant surface protein LPDTG
21284341 m Cna collagen adhesin precursor LPKTG
27467746 n SE0828 Lipoprotein VsaC LPETG
27468418 n SE1500 hypothetical protein LPKTG
27468419 n SE1501 hypothetical protein LPNTG
27468546 n SE1628 hypothetical protein LPETG
27469070 n SE2152 hypothetical protein LPNTG
a Truncated in S. aureus NCTC 8325, thereby missing the C-terminal part containing the LPxTG motif
b Proteins that are also present in S. epidermidis
c The genes encoding SdrC, HarA and FnbB are not present in S. aureus MRSA252
d These proteins have a lower SignalP score than our threshold score
e The gene encoding SdrE is not present in S. aureus NCTC8325
f Truncated in S. aureus Mu50, thereby missing the C-terminal part containing the LPxTG motif
g The gene encoding SasB is not present in S. aureus MRSA252, MSSA476 and USA300_TCHC1516; truncated
in S. aureus MW2
h Truncated in S. aureus Mu50, N315 and Newman, thereby missing the C-terminal part containing the LPxTG
motif
i Truncated in S. aureus Newman, thereby missing the C-terminal part containing the LPxTG motif (Grundmeier
et al., 2004)
j The gene encoding SasK is not present in S. aureus COL, MRSA252, MSSA476, NCTC 8325, Newman, USA300
and USA300_TCHC1516; truncated in S. aureus MW2, thereby missing the N-terminal signal peptide
k This protein has an unusually long signal peptide (90 amino acids)
l The gene encoding Pls is only present in S. aureus COL
m The gene encoding Cna is not present in S. aureus COL, JH1, JH9, Mu50, N315, NCTC 8325, Newman,
USA300 and USA300_TCHC1516
n Only present in S. epidermidis
47

Chapter 2
Five additional proteins (SdrD, SdrE, SasC, FnbA and FnbB) with an LPxTG motif can be
found among the S. aureus strains (Table 4). These five proteins were excluded from our
initial list, because the corresponding SignalP scores were lower than our (high) score criteria,
or, as was shown for the FnbA and FnbB proteins of the S. aureus Newman strain, the LPxTG
motif is missing due to a premature stop codon (Grundmeier et al., 2004). However, since
some of the domains present in these proteins (besides the LPxTG motif) are conserved in
well-described cell wall proteins, they have been included in Table 4. The remaining proteins
with a cell wall sorting signal are either missing in one or more S. aureus strains, or have been
annotated wrongly. Interestingly, S. epidermidis ATCC 12228 contains a gene for a class C
sortase (srtC), which seems to be absent from other staphylococci. This SrtC is most closely
related to sortases of L. lactis and Streptococcus suis. Two proteins with LPxTG motifs,
which are encoded by the same genomic island as SrtC, also seem to be strain-specific (Gill et
al., 2005).
Sortase B recognition signal
All sequenced S. aureus strains contain sortase B (SrtB) in addition to SrtA. The gene for
SrtB is situated at a locus, which is involved in the uptake of haeme iron (Mazmanian et al.,
2002). This locus also contains the gene for the cell wall protein IsdC, which contains the
SrtB recognition sequence NPQTN. In addition, this locus contains the genes for the SrtA
substrates IsdA and IsdB that both contain LPxTG motifs. Notably, IsdC is so far the only
known protein known to be anchored to the cell wall by sortase B. IsdC is cleaved by SrtB
between the Thr and Asn residues of the NPQTN motif. The only other S. aureus protein with
a motif that resembles NPQTN is the DNA-binding protein II, but this protein is probably not
cell wall bound, because it lacks a signal peptide for export from the cytoplasm.
Comparative secretome analysis
Comparing the predicted secretomes of S. aureus and S. epidermidis with those of B. subtilis
and other Gram-positive bacteria revealed that most of the known components of the
translocation machinery are present in S. aureus. The most notable differences are the second
set of secA and secY genes in S. aureus, the absence of known signal peptide peptidases from
S. aureus and S. epidermidis, the absence of a BdbC homologue from S. aureus and S.
epidermidis, the presence of a second lspA gene in S. epidermidis, the absence of a Tat system
from S. epidermidis, and the absence of two potential components in the Ess pathway from S.
aureus MRSA252 (Table 3).. However, it has been shown that the second secA/secY set is
involved in the export of virulence factors in other pathogens (Takamatsu et al., 2005).
Though most known determinants for protein export, processing and post-translocational
modification in other Gram-positive bacteria are also present in S. aureus, in many cases it
remains to be investigated to what extent they are necessary for protein export in general and
the export of virulence factors in particular.
As shown by multiple BLAST comparisons, the core exoproteome of the sequenced S. aureus
strains consists of 68 proteins (Supplemental table IIIc). All of these proteins have a signal
peptide with a potential SpsB recognition and cleavage site. 46 of these core exoproteins have
already been identified in the extracellular milieu and/or membrane/cell wall proteome of
different S. aureus isolates (Ziebandt et al., 2001; Ziebandt et al., 2004; Nandakumar et al.,
2005; Gatlin et al., 2006; Pocsfalvi et al., 2008; Ziebandt et al., submitted). Interestingly, 31
core exoproteins of S. aureus are also conserved in S. epidermidis, suggesting that they
48

Mapping the pathways to staphylococcal pathogenesis by comparative secretomics
belong to a core staphylococcal exoproteome, which is presently still poorly defined.
Interestingly, the core exoproteome of S. aureus seems to be largely composed of enzymes,
like proteases, and other factors, like fibrinogen- and IgG-binding proteins, that are required
for life in the ecological niches provided by the human host (Supplemental table IIIc). This is
particularly true also for the proteins that have the potential to be covalently bound to the cell
wall (Table 4). In contrast, the variant exoproteome of S. aureus contains most of the known
staphylococcal toxins and immunomodulating factors (Supplemental tables IIId and IIIg).
This suggests that the components of the variant exoproteome should be regarded as specific
gadgets of S. aureus that help this organism to conquer certain protected niches of the human
host, thereby causing disease. If this idea is correct, proteins of unknown function that belong
to the variant exoproteome should be regarded as potentially important virulence factors.
The (predicted) extracellular toxins of S. aureus are not present in S. epidermidis. This is
mainly due to the fact that these toxins are encoded by pathogenicity islands in the genomes
of S. aureus strains that have, so far, not been observed in S. epidermidis genomes. Proteins
with predicted signal peptides that are specific for S. epidermidis are listed in Supplemental
table IIIi. Notably, the majority (i.e. 31 out of 37) of predicted S. epidermidis exoproteins that
have homologues in S. aureus share this homology with components of the core exoproteome
of S. aureus (Supplemental tables IIIc and IIId). This suggests that also in S. epidermidis the
core exoproteome is involved in housekeeping functions. In contrast to the exoproteome, it is
presently difficult to speculate about housekeeping- and disease-causing roles of the constant
and variant lipoproteomes of S. aureus. This is due to the fact that the function of only few S.
aureus lipoproteins is known (Supplemental tables IIIe, IIIf and IIIh). In general terms, it is
presently not clear why S. epidermidis seems to export a lower number of different proteins
(94 in total) than S. aureus (~165 in total). This difference is all the more remarkable since the
total number of proteins encoded by the genomes of S. aureus (~2600) and S. epidermidis
(~2500) are comparable.
Compared to B. subtilis and B. licheniformis (Voigt et al., 2005), S. aureus is also predicted to
export a relatively higher number of proteins from the cytoplasm to the membrane-cell wall
interface, the cell wall and the extracellular milieu. The genomes of B. subtilis and B.
licheniformis contain ~4100 protein-encoding genes, while the S. aureus genomes contain
significantly less genes (~2600). Using the most recent prediction protocols (Tjalsma and van
Dijl, 2005), B. subtilis is predicted to export 190 proteins to an extracytoplasmic location
whereas, depending on the strain investigated, S. aureus is predicted to export 145-168
proteins (this review). Accordingly, as judged by the relative numbers of protein-encoding
genes, S. aureus strains appear to export 1-2% more proteins to an extracytoplasmic location
than the afore-mentioned bacilli. Most probably, this is related to the fact that S. aureus needs
an arsenal of virulence factors, such as toxins and surface proteins, for colonization of and
survival in its preferred niches in the human host. Such proteins are of less importance for soil
bacteria, such as B. subtilis and B. licheniformis, which thrive predominantly on dead organic
matter.
Perspectives
The present review provides a survey of possible protein transport pathways to staphylococcal
pathogenesis. In many cases the knowledge gathered from protein secretion studies in other
organisms has been projected on S. aureus, assuming that similar pathways or pathway
components have similar functions in different organisms. Clearly, this leaves room for
49

Chapter 2
surprises when such pathways are investigated thoroughly in S. aureus. The same was true for
studies on protein secretion in B. subtilis. These studies showed, for example, that the absence
of SecDF has barely any consequences for protein secretion by B. subtilis, whereas SecD and
SecF are of key importance for protein translocation in E. coli, the organism in which SecD/F
was first discovered (Bolhuis et al., 1998). Likewise, LspA was shown to be dispensable in B.
subtilis, but not in E. coli (Tjalsma et al., 1999; Prágai et al., 1997). Thus the relative
importance of different secretion machinery components of S. aureus needs to be assessed in
a systematic manner, preferably in an isogenic background. Such studies would need to
address the importance of secretion machinery components for in vitro growth on different
substrates (e.g. broth or blood), and virulence in in vivo model systems (e.g. C. elegans,
Drosophila melanogaster, mice and rats) (Bae et al., 2004; García-Lara et al., 2005;
Tarkowski et al., 2001). These studies should be complemented with a proteomic verification
of our present lipoproteome, wall proteome and exoproteome predictions. Such a verification
could involve both gel-based proteomics approaches as outlined in this review, and more
sophisticated gel-free proteomics approaches (Völker and Hecker, 2005). This would lead to
an improved understanding of the contribution of each protein transport pathway and its
substrate proteins to staphylococcal cell physiology and virulence. Since the virulence of
different S. aureus strains will not only depend on the presence (or absence) of particular
genes for virulence factors, but also on their expression, such proteomic studies should also
include experiments on the impact of major regulatory systems, such as agr, sae and sarA. On
this basis, it should be possible to identify the most promising candidate drug targets in the
staphylococcal secretome. Alternatively, secretome components thus identified could
represent promising candidates for novel vaccines. For all these efforts, comparative
secretomics approaches will provide the essential information on those potential drug targets
that are most important for both bacterial housekeeping functions and virulence. Novel drugs
and vaccines designed against these targets are likely to have the highest impact (Götz, 2004).
Acknowledgements
We thank Harold Tjalsma and members of the Groningen and European Bacillus Secretion
Groups and the BACELL Health, Tat machine and StaphDynamics consortia for stimulating
discussions. M.J.J.B.S., A.K.Z., S.E., M.H., J.-Y.F.D., and J.M.v.D. were supported by Grants
LSHC-CT-2004-503468, LSHG-CT-2004-005257 and LSHM-CT-2006-019064 from the
European Union. M.H. was supported from grants of the “Deutsche Forschungsgemeinschaft”,
the “Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie”, and the
“Fonds der Chemischen Industrie”.
50

“Music is the medicine of the mind”
-John A. Logan-
52

Chapter 3
Proteogenomics uncovers extreme heterogeneity in the
Staphylococcus aureus exoproteome due to genomic plasticity
and variant gene regulation
A.K. Ziebandt, M. Degner, M.J.J.B. Sibbald, J.P. Arends, M.A. Chlebowicz,
H. Kusch, D. Albrecht, R. Pantuček, J. Doškar, W. Ziebuhr, B.M. Bröker, M. Hecker,
J.M. van Dijl, and S. Engelmann
Submitted for publication, in revision
53

Chapter 3
Summary
Sequencing of at least thirteen S. aureus isolates has shown that genomic plasticity
impacts significantly on the repertoire of virulence factors. However, genome
sequencing does not reveal which genes are de facto expressed by individual isolates.
Here, we have therefore performed a first comprehensive survey of the composition and
variability of the S. aureus exoproteome following a proteogenomics approach. This
involved multi locus sequence typing, virulence gene and prophage profiling by
multiplex PCR, and proteomic analyses of secreted proteins using two-dimensional
protein gel electrophoresis. Dissection of the exoproteomes of 25 clinical isolates revealed
that only seven out of 63 identified secreted proteins were produced by all isolates,
indicating a remarkably high exoproteome heterogeneity within one bacterial species.
The observed variations were caused by both genome plasticity and an unprecedented
variation in gene expression. Our data imply that genomic studies focussing on virulence
gene conservation patterns need to be complemented by protein expression analyses to
assess the full virulence potential of bacterial pathogens like S. aureus. Importantly, the
extensive variability of secreted virulence factors in S. aureus also suggests that the
development of protective vaccines against this pathogen requires a carefully selected
combination of invariably expressed antigens.
54

Proteogenomics uncovers extreme heterogeneity in the Staphylococcus aureus
exoproteome due to genomic plasticity and variant gene regulation
Introduction
Staphylococcus aureus causes a wide variety of human infections ranging from superficial
lesions to severe systemic diseases. Up to one third of the human population carries S. aureus
as a commensal bacterium without developing any clinical symptoms. Nevertheless, the
colonizing strain can serve as an endogenous reservoir for infection, the incidence of
bacteremia being less than 0.02% (von Eiff et al., 2001). Individuals colonized with S. aureus
have thus a higher risk to develop an S. aureus infection, but they are also more likely to
overcome the disease (Wertheim et al., 2004). It is undisputed that the incidence and outcome
of invasive staphylococcal diseases strongly depend on host factors, in particular the immune
status of a patient. Yet, the species S. aureus is highly diverse and up to 30% of the genomes
of different isolates consist of variable regions, such as pathogenicity islands, lysogenic
bacteriophages, and plasmids (Witney et al., 2005). Since these genetic elements encode the
majority of virulence factors, it is generally thought that their presence critically determines
the clinical symptoms and outcome of an S. aureus infection. However, virulence-associated
genes may also be located in the core genome as illustrated by the spa, aur, hla, lip, clfAB,
map/eap, fnbA, and coa genes (Holden et al., 2004; Peacock et al., 2002). It is generally
accepted that certain strains are more virulent than others (Melles et al., 2004), although,
under certain conditions, any S. aureus genotype may have the potential to induce lifethreatening
infections. This suggests that there is no simple relationship between bacterial
genotype and clinical outcome. Apart from several well-defined toxin-mediated diseases (e.g.
toxic shock and scalded skin syndrome, necrotizing pneumonia) (Musser et al., 1990;
Gemmell, 1995; Labandeira-Rey et al., 2007), it remains difficult to predict the onset and
course of an S. aureus infection in a given patient solely on the basis of the virulence gene
repertoire of the strain involved. In fact, the vast majority of severe S. aureus infections are
probably caused by the concerted action of multiple virulence factors. Molecular typing and
genome analyses of clinical isolates focussing on virulence gene distribution have been major
steps towards a thorough understanding of these complex phenomena. Also, there is growing
evidence that the presence and activities of certain regulators play a role in the variation of
virulence gene expression in clinical isolates (Blevins et al., 2002; Karlsson and Arvidson,
2002).
Here, we complement the genetic identification of virulence factors in different S. aureus
isolates with proteome analyses of secreted proteins, which represent an important reservoir
of virulence factors. More specifically, in this proteogenomics approach we have addressed
the questions (i) of whether particular virulence genes are expressed in different isolates and,
if so, (ii) in what quantities? The results reveal an unprecedented heterogeneity in the
analyzed S. aureus exoproteomes, which is caused by both genomic plasticity and an amazing
variability in the levels of gene expression.
Materials and Methods
Bacterial strains
Twenty five S. aureus isolates derived from a variety of human infections were collected by the
university hospital in Groningen. All strains were identified as S. aureus by plating and coagulase tests.
Resistance against antibiotics was determined by routine disk diffusion assays. For RNAIII
55

Chapter 3
transcription analyses, S. aureus RN6390, COL, and Newman were used as reference strains (Shafer
and Iandolo, 1979; Duthie and Lorenz, 1952; Novick, 1967).
Multilocus sequence typing (MLST)
MLST was performed according to the protocol described by Enright et al. (Enright et al., 2000). The
obtained sequences for each locus were submitted to the Internet database (www.mlst.net) and the
resulting allelic profiles were assigned to particular sequence types (ST) for each isolate. The eBURST
(Based upon related sequences) algorithm software was used to classify different sequence types into
clusters of clonal complexes (CC).
Detection of prophages and virulence genes using multiplex PCR
Genomic DNA was extracted from S. aureus clinical isolates and used as a template in multiplex PCR
assays targeting structural prophage genes of head-tail modules described previously (Pantucek et al.,
2004). Three different phage types (A-like, B-like, and F-like) corresponding to putative phage species
3A, 11, and 77, respectively, were thus identified. The F-like phages include clearly distinguishable
subgroups Fa and Fb, while the B-like phages include 5 subgroups Ba – Be of different phages, in
which the packaging, head, and tail genes belong to different modules. Therefore, three PCR assays
were used for phage identification (Table 1).
Table 1. Oligonucleotides used in this study
Target Primer Sequence 5´- 3´ Reference
S. aureus, positive SAU1
control
SAU2
A-like phage, tail SGA1
SGA2
B-like phage (all SGB1
subgroups), tail SGB2
F-like phage (both SGF1
subgroups), tail SGF2
B-like phage, SGBa1
subgroup Ba portal SGBa2
B-like phage, SGBb1
subgroup Bb portal SGBb2
B-like phage, SGBc1
subgroup Bc portal SGBc2
B-like phage, SGBd1
subgroup Bd portal SGBd2
B-like phage, SGBe1
subgroup Be portal SGBe2
F-like phage, SGFa1
subgroup Fa portal SGFa2
F-like phage, SGFb1
subgroup Fb portal SGFb2
Phage integrase phiSa1-F
ФSa1 (phage ETA) phiSa1-R
Phage integrase phiSa2-F
ФSa2 (phage 12) phiSa2-R
Phage integrase phiSa3-F
ФSa3 (phage 13) phiSa3-R
Phage integrase phiSa4-F
ФSa4
phiSa4-R
Phage integrase phiSa5-F
ФSa5 (phage 11) phiSa5-R
Phage integrase phiSa6-F
ФSa6 (phage L54a) phiSa6-R
Phage integrase phiSa7-F
ФSa7 (phage 96) phiSa7-R
Phage integrase phiSa8-F
ФSa8 (phage 53) phiSa8-R
GACGGCTTTGATGGCTAGTGG
AGTTAATTCACGCCCTAGTG
TATCAGGCGAGAATTAAGGG
CTTTGACATGACATCCGCTTGAC
ACTTATCCAGGTGGYGTTATTG
TGTATTTAATTTCGCCGTTAGTG
CGATGGACGGCTACACAGA
TTGTTCAGAAACTTCCCAACCTG
AAGATGATAACTTTAGTGGCAC
TCATTGATGTYTCTAGGGTC
CTGATTATGTGTACGCAGAG
TTCCGTTAAACTCGTCAGA
TTGTTAAGGAACCYAAGCC
GCCTCTAATTCTTCGTGCTC
AAGTTACGTCGCTGGC
GCTTGTTCTGCTGGCACTCT
AAATGAAACTATTCCGTGTT
AAYGCTATAAAYGGYACTCT
TACGGGAAAATATTCGGAAG
ATAATCCGCACCTCATTCCT
AGACACATTAAGTCGCACGATAG
TCTTCTCTGGCACGGTCTCTT
AAGCTAAGTTCGGGCACA
GTAATGTTTGGGAGCCAT
TCAAGTAACCCGTCAACTC
ATGTCTAAATGTGTGCGTG
GAAAAACAAACGGTGCTAT
TTATTGACTCTACAGGCTGA
ATTGATATTAACGGAACTC
TAAACTTATATGCGTGTGT
AAAGATGCCAAACTAGCTG
CTTGTGGTTTTGTTCTGG
GCCATCAATTCAAGGATAG
TCTGCAGCTGAGGACAAT
AAACTAAAGCTGAGGCAAC
TCATTAGTACGACCTCGAC
GTCCGGTAGCTAGAGGTC
GGCGTATGCTTGACTGTGT
56
(Pantucek et al., 2004)
(Pantucek et al., 2004)
(Pantucek et al., 2004)
(Pantucek et al., 2004)
this study
this study
this study
this study
this study
(Pantucek et al., 2004)
(Pantucek et al., 2004)
this study
this study
this study
this study
this study
this study
this study
this study

Proteogenomics uncovers extreme heterogeneity in the Staphylococcus aureus
exoproteome due to genomic plasticity and variant gene regulation
Target Primer Sequence 5´- 3´ Reference
hlb hlb-2
AGCTTCAAACTTAAATGTCA (Goerke et al., 2006)
hlb-527
CCGAGTACAGGTGTTTGGTA
sea Sea-1
AGATCATTCGTGGTATAACG (Van Wamel et al., 2006)
Sea-2
TTAACCGAAGGTTCTGTAGA
sep Sep-1
AATCATAACCAACCGAATCA (Van Wamel et al., 2006)
Sep-2
TCATAATGGAAGTGCTATAA
sak Sak-1
AAGGCGATGACGCGAGTTAT (Van Wamel et al., 2006)
Sak-2
GCGCTTGGATCTAATTCAAC
chp Chip-up CACCATCATTCAGCGAAAG this study
Chip-low GATATATAAAGGTTTGGCAAG
scn Scin-up
AGTCTTTTGACTTAAGAGC this study
Scin-low GTTTTAGCATCACCACTAGTA
geh gehSA-up TTCTTAATGGCTACAACGAGT this study
gehSA-low GATAAAATCGACATGATCCC
hlgA hlgA_F
GTCAAGGTGCAGAAATCATC this study
hlgA_R GAAATAGTCTCTTGCTGCTG
splD splD_F
CCAAAGCAGAAAATAGTGTG this study
splD_R
CATAACACCAATTGCTTCTC
splE splE_F
GTGTCGTTTCGATAGGATCT this study
splE_F
GTTGCCTTTAACTGACAGCA
24636604 24636604_F GACAGCATCTCACTGTAAAG this study
24636604_R TCACGCTTTTGACAGATAAG
SAR0422 SAR0422_F CAAGTTTAGCATTGGGAATG this study
SAR0422_R CGTCGATAGAATCACCCATAC
SAR0435 SAR0435_F GTTTAGCACTAGGGATTTTG this study
SAR0435_R GGCTCTGTCTTATAAAGTTC
SAR1905 SAR1905_F CGATTGCAACTCTTTCATTC this study
SAR1905_R CCATACAAAGGCATACTAAG
SA1755 SA1755_F CTTTTGAACCGTTTCCTACA this study
SA1755_R CAGTATTTTTTGTGCCTTTC
SA0092 SA0092_F GTAAAGAAGCGGAAGTTAAG this study
SA0092_R GCTTTATTCGTCGGTATATC
SA1001 SA1001_F CTGCAGGTCTTTTAACTCAA this study
SA1001_R GCTTTCTTCACATCACCTTG
SACOL0478 SACOL0478_F GTTAGATCACAAGCTACTCA this study
SACOL0478_R GACGTTGATGTAACACTATC
RNAIII RNAIII_F AGGAAGGAGTGATTTCAATG this study
RNAIII_R CTAATACGACTCACTATAGGGAG
AACTCATCCCTTCTTCATTAC
Notably, the nomenclature of the prophage genomes based only on the genes for virion proteins is not
absolute, because of their mosaic structure. Therefore, a novel PCR-based molecular assay for
identification and classification of S. aureus phage integrase genes was also applied for prophage
characterization. For designation of bacteriophage integrase gene families, the updated classification
scheme was used which denotes the previously sequenced phages and S. aureus genomes ФSa1 - ФSa5
(Lindsay and Holden, 2004). Since so far unclassified integrases were identified, the new designations
ФSa6, ФSa7 and ФSa8 were introduced in this work for integrases of phages L54a (GenBank
Accession no. M27965), Ф96 (NC_007057) and Ф53 (NC_007049), respectively. For multiplex PCR,
the reaction mixtures (25 µl) consisted of 75 mM Tris-HCl pH 9.0, 50 mM KCl, 2 mM MgCl2, 250 µM
each of dNTPs, primer pairs phiSa1 and phiSa2 (0.25 µM each), phiSa3 (0.2 µM each), phiSa4 and
phiSa5 (0.2 µM each), phiSa6, phiSa7 and phiSa8 (0.1 µM each). Primer sequences are listed in Table
1. To each reaction, 1.5 unit Taq DNA polymerase (Invitrogen) and template DNA (50 ng) in a 3 µl
volume were added. PCR was performed using 30 cycles of amplification consisting of denaturation (1
min, 94°C), annealing (1 min 30 s, 56°C) and DNA chain extension (1 min, 72°C). Phage associated
virulence genes sea, sep, sak, chp, and scn as well as geh and hlb were detected using standard PCR
with the primers listed in Table 1.
For the analysis of genes encoding superantigens and other extracellular proteins as well as for agr
typing, primer pairs were used as shown in Table 1 or described previously (Holtfreter et al., 2004;
57

Chapter 3
Lina et al., 2003). The amplifications were performed with Taq polymerase in a thermocycler with the
following conditions: initial denaturation at 95°C for 5 min, followed by 30 stringent cycles (1 min of
denaturation at 95°C, 1 min of annealing at the temperature indicated in Table 1, and 1 min of
extension at 72°C), and a final extension step at 72°C for 5 min. The quality of the DNA extracts and
the absence of PCR inhibitors were confirmed by amplification of glyceraldehyde-3-phosphate
dehydrogenase or 16S rRNA. The PCR products were then analyzed by electrophoresis through a 1%
agarose gel. At least two independent experiments were performed for each determination.
Extracellular proteins
For the preparation of extracellular protein extracts, bacteria were grown in Tryptic Soy Broth (TSB).
At optical densities (OD540) of 10, the extracellular proteins from 100 ml supernatant were precipitated,
washed, dried, and resolved as described previously (Ziebandt et al., 2004). The protein concentration
was determined using Roti ® -Nanoquant according to manufacturer´s instructions (Carl Roth GmbH &
Co, Karlsruhe, Germany).
Analytical and preparative 2D polyacrylamide gel electrophoresis (PAGE)
Protein extracts (350 µg) were loaded onto commercially available IPG strips (pH 3-10, GE-
Healthcare, Uppsala, Sweden). 2D PAGE was performed as described previously (Bernhardt et al.,
1999; Eymann et al., 2004). The resulting protein gels were stained with colloidal Coomassie Blue G-
250 (Candiano et al., 2004) and scanned with the light scanner.
Protein identification
For identification of proteins by Matrix-assisted Laser Desorption Ionization-Time of Flight mass
spectrometry (MALDI-TOF MS), Coomassie stained protein spots were excised from gels using a spot
cutter (Proteome Work TM ) with a picker head of 2 mm and transferred into 96-well microtiter plates.
Digestion with trypsin and subsequent spotting of peptide solutions onto the MALDI targets were
performed automatically in an Ettan Spot Handling Workstation (GE-Healthcare, Little Chalfont,
United Kingdom) using a modified standard protocol (Eymann et al., 2004). MALDI-TOF MS
analyses of spotted peptide solutions were carried out on a Proteome-Analyzer 4700/4800 (Applied
Biosystems, Foster City, CA as described previously (Eymann et al., 2004). MALDI-TOF-TOF
analysis was performed for the three highest peaks of the TOF spectrum as described previously
(Eymann et al., 2004; Wolf et al., 2008). Database searches were performed using the GPS explorer
software version 3.6 (build 329) with the organism-specific databases.
By using the MASCOT search engine version 2.1.0.4. (Matrix Science, London, UK) the combined MS
and MS/MS peak lists for each protein spot were searched against a database containing protein
sequences derived from the genome sequences of all sequenced S. aureus strains and, moreover, all
additional protein sequences of S. aureus that have been found in publically available databases. Search
parameters were as described previously (Wolf et al., 2008).
RNA-isolation and dot blot analysis
Total RNA from S. aureus was isolated using the acid-phenol method with some modifications (Fuchs
et al., 2007). Digoxigenin-labeled RNA probe for RNAIII was prepared by in vitro transcription with
T7 RNA polymerase by using a PCR fragment as template. The PCR fragment was generated by using
chromosomal DNA of S. aureus N315 and the respective oligonucleotides (Table 1). Dot blot analyses
were carried out by using serial dilutions of total RNA prepared from S. aureus isolates and reference
strains grown in TSB medium to an optical density (OD540) of 10. The digoxigenin-labeled RNA probe
was used for hybridization. The hybridization signals were detected using a Lumi-Imager and analyzed
using the software package Lumi-Analyst (Roche Diagnostics, Mannheim, Germany).
58

Proteogenomics uncovers extreme heterogeneity in the Staphylococcus aureus
exoproteome due to genomic plasticity and variant gene regulation
Results and Discussion
Genetic characterization of clinical isolates
A total of 25 clinical isolates, including eight nosocomial methicillin-resistant S. aureus
(MRSA) and three community-acquired MRSA (caMRSA) isolates, were used in this study.
The isolates were obtained from 19 patients with different clinical symptoms during a 4.5
year period (from February 2001 to September 2005). Strains were isolated from blood, nose,
feet, perineum, fingers, throat, pleural fluid, and umbilicus. Five of these isolates were
colonizing strains while 12 isolates came from septic patients, five isolates induced wound
infections, four isolates arthritis, three isolates pneumonia, two isolates cholangitis, and three
individual isolates were involved in abscess formation, panaritium, or endocarditis. On the
basis of strain typing data obtained by Pulsed-Field-Gel-Electrophoresis (PFGE) and Multi-
Locus-Sequence-Typing (MLST) the isolates were grouped into eleven different sequence
types (Supplemental table IVa). Three new sequence types were detected, i.e. ST869, ST870,
and ST903. Moreover, the agr types and the prevalence of enterotoxin genes and other
clinically relevant genes (e.g. mecA, pvl, etd, eta) were determined for all isolates by using
multiplex PCR (Table 2, Supplemental table IVb).
The sea gene (72%) as well as genes belonging to the enterotoxin gene cluster (egc) (44%)
were found to be the most prevalent enterotoxin genes, while none of the isolates carried tst-1,
sec, see, seh, or sel. Moreover, pvl was identified in four and etd in two isolates, respectively.
Typing of agr revealed that six isolates encoded agr1, thirteen agr2, three agr3 and none of
the isolates encoded agr4. Additionally, we analyzed the prophage content of these isolates by
a multiplex PCR assay. Altogether we identified 11 different prophages. While most of the
isolates contained three prophages, no known prophage was detected in two isolates (N, W)
(Table 3). The hlb converting phage Sa3 was the most prevalent phage: in 20 isolates we
identified either the Fa-type (16 isolates) or the Fb-type of the Sa3 prophage. This prophage is
common among human isolates and often encodes immune evasion molecules (SAK, SCIN,
and CHIP) as well as enterotoxins (SEA or SEP) and has previously been detected in
collections of clinical strains (Goerke et al., 2006). Moreover, we identified the Fa-type phage
Sa1 (1 isolate), the A-type phages Sa2 (7 isolates), Sa4 (1 isolate), and Sa6 (6 isolates), as
well as the B- type phages Sa1 (2 isolates), Sa5 (1 isolate), Sa6 (3 isolates), Sa8 (9 isolates),
and Sa9 (2 isolates).
According to our genetic characterization, the 25 isolates represent 17 clonally divergent
strains. Five strains were isolated more than once, either in the same patient or in different
patients (Supplemental table IVa). Interestingly, isolates C, D, G, H, I, J, and K might have
evolved from just one clone (=G228) which was responsible for a hospital outbreak and
caused wound infections in three of the patients included in this study.
The exoproteomes of clinical isolates are highly heterogeneous
Proteomics is an extremely powerful tool for analyzing virulence gene expression in multiple
clinical isolates. Since the extracellular proteome of a pathogenic bacterium represents a key
reservoir of virulence factors, the present study focused on this particular subproteome. All 25
isolates were cultivated in TSB medium and extracellular proteins were prepared from the
supernatants at an optical density (OD) at 540 nm of 10. Proteins were separated on 2D gels
and protein spots were identified by MALDI-TOF MS/MS or N-terminal sequencing
59

Chapter 3
(Supplemental tables IVb). Comparison of extracellular protein patterns revealed an
unanticipated degree of exoproteome heterogeneity among the 17 clonally different strains
(Figure 1).
Table 2. Virulence genes identified by multiplex PCR in different clinical S. aureus isolates
Isolates
Gene A B C D E F G H I J K L M N O P Q R S T U V W X Y
eta
etd + +
hlgA + + + + + + + + + + + + + + + + + + + + + + + + +
pvl + + + +
SA0092 + + + + + + + + + + + + + + + + + + + + + + +
SA1001 + + + + + + + + + + + + + + + + + + + + + + +
SA1755 + + + + + + + + +
SACOL0478 + + + + + +
SAR0422 + + + + + + + + + + + + + + + + + + + + + + + + +
SAR0435 +
SAR1905 + + +
sea + + + + + + + + + + + + + + + +
seb + + + + + + + + +
sec
sed + +
see
seg + + + + + + + + + + +
seh
sei + + + + + + + + + + +
sej + +
sek + + + +
sel
sem + + + + + + + + + + +
sen + + + + + + + + + + +
seo + + + + + + + + + + +
sep + + +
seq + + + +
ser + +
seu +
splD + + + + + + + + + + + + + + + + + + + + + + + + +
splE + + + + + + + + + + +
tst-1
24636604 + +
60

Proteogenomics uncovers extreme heterogeneity in the Staphylococcus aureus exoproteome due to genomic plasticity and variant gene
regulation
Table 3. Prophages identified in different clinical S. aureus isolates
Phage integrase Classification of prophages according to Innate immune evasion cluster # of
Assumed lysogenic pattern
genes for virion proteins
pattern of F-like phages phages
(type of capsid a /integrase class)
Strain Sa1 Sa2 Sa3 Sa4 Sa5 Sa6 Sa7 Sa8 Alike
B-
like
F-
like Ba Bb Bc Bd Be Fa Fb hlb sea sep sak chp scn
A + + + + + + + + 3 A-phage/Sa2; B-phage/Sa6; Fa-phage/Sa3
B + + + + + + + + + + + 3 A-phage/Sa2; B-phage/Sa6; Fa-phage/Sa3
C + + + + + + + + + + + 3 A-phage/Sa6; B-phage/Sa8; Fa-phage/Sa3
D + + + + + + + + + + + 3 A-phage/Sa6; B-phage/Sa8; Fa-phage/Sa3
E + + + + + + + + + + + 3 A-phage/Sa2; B-phage/Sa8; Fb-phage/Sa3
F + + + + + + + + + + + 3 A-phage/Sa2; B-phage/Sa8; Fb-phage/Sa3
G + + + + + + 2 A-phage/Sa6; B-phage/Sa8
H + + + + + + + + + + + 3 A-phage/Sa6; B-phage/Sa8; Fa-phage/Sa3
I + + + + + + + + + + + 3 A-phage/Sa6; B-phage/Sa8; Fa-phage/Sa3
J + + + + + + + + + + + 3 A-phage/Sa6; B-phage/Sa8; Fa-phage/Sa3
K + + + + + + + + + + + 3 A-phage/Sa6; B-phage/Sa8; Fa-phage/Sa3
L + + + + + + + + 2 A-phage/Sa2;Fb-phage/Sa3
M + + + + + + + 2 A-phage/Sa2;Fb-phage/Sa3
N + + 0
O + + + + + + + 1 Fa-phage/Sa3
P + + + + + + + + + 2 B-phage/Sa1; Fa-phage/Sa3
Q + + + + + + + + + + + + + 4 A-phage/Sa2; B-phage/Sa5; B-phage/Sa6; Faphage/Sa3
R + + + + + 1 B-phage/Sa8
S + + + + + + 1 Fa-phage/Sa3
T + + + + + + 1 Fa-phage/Sa1
U + + + + + + + + + + 3 A-phage/Sa4; B-phage/not identified; Fa-phage/Sa3
V + + + + + + + + + + 2 B-phage/Sa1; Fa-phage/Sa3
W + 0
X + + + + + + 1 Fa-phage/Sa3
Y + + + + + + 1 Fa-phage/Sa3
a According to R. Pantuček et al. (Pantucek et al., 2004)
61

Chapter 3
Figure 1. Characterization of extracellular proteomes of different clinical S. aureus isolates. Cells were grown
in TSB medium to an OD 540 of 10. Proteins in culture supernatants were collected by TCA precipitation. 350 µg of
the protein extract of each strain was separated on 2D gels, using commercially available IPG strips (pH 3-10,
GE-Healthcare, Sweden) for the first dimension. Protein spots were detected by staining with colloidal Coomassie
Brillant Blue. For protein identification, individual protein spots were excised from the gel and digested with
trypsin. The resulting peptide solution was analyzed by tandem mass spectrometry on a Proteome Analyzer
4700/4800 (Applied Biosystems, USA). The respective strain is indicated in the upper left corner of each gel.
Altogether 206 distinct proteins were identified (Supplemental table IVb). 107 of these
proteins showed signal peptides typical for Sec-translocated proteins. Using PSORTb
software (http://www.psort.org/psortb), 63 of the Sec-translocated proteins were predicted to
62

Proteogenomics uncovers extreme heterogeneity in the Staphylococcus aureus
exoproteome due to genomic plasticity and variant gene regulation
be extracellular, 19 were predicted to be cell wall-bound proteins (Figure 2) and the
localization of a further 25 proteins is currently unknown (Sibbald et al., 2006). Moreover, we
identified 72 cytoplasmic proteins and 5 membrane proteins. The N-terminal sequences
(AEANSSMVSKK and GTTPAAA) of two protein spots did not match any of the protein
sequences present in the NCBI and other databases, indicating that our knowledge of
extracellular proteins produced by S. aureus is not yet complete.
Membrane; 5
Unknown; 47
Cell wall; 19
Extracellular; 63
Cytosolic; 72
Cytosolic
Extracellular
Cell wall
Membrane
Unknown
63
Figure 2. Predicted localization of extracellular
proteins of 25 clinical S. aureus isolates. A total of
206 distinct proteins were identified in the growth
medium fractions of 25 clinical isolates. For the
prediction of protein localization PSORT software
was used.
Interestingly, only seven out of 63 predicted Sec-dependent extracellular proteins were found
to be produced by all 17 clonally different strains (i.e. Aly, IsaA, Lip, LytM, Nuc, SA0620,
and SA2097). A further nine proteins (i.e. Aur, Geh, GlpQ, Hla, HlgB, SA0570, SA1812,
SspA, SspB) were identified in at least 80% of these strains (Supplemental table IVc).
Interestingly, four of the invariant proteins (i.e. IsaA, LytM, SA0620, and SA2097) were
recently shown to be regulated by the WalK/WalR two-component system which is essential
for cell viability and cell wall metabolism (Dubrac et al., 2007). IsaA and LytM share a
conserved transglycosylase/muramidase domain, and SA0620 and SA2097 contain a CHAP
amidase domain. All these proteins have an N-terminal cell wall-binding domain in common,
indicating that they are exported and possibly non-covalently associated to the cell wall. It is
worth noting in this context that IsaA-specific antibodies have previously been detected in
healthy adults, colonized patients and patients suffering from sepsis, suggesting that IsaA is
also expressed in vivo (Lorenz et al., 2000; Clarke et al., 2006). Most strikingly, the amounts
of the invariant proteins varied significantly between individual strains (Figure 3).
Expression heterogeneity probably triggered by varying SigB and SarA activities has been
observed before for extracellular proteases (Karlsson and Arvidson, 2002). Notably, our
present data indicate that this phenomenon is by no means restricted to proteases, but applies
to secreted virulence factors in general.
31 proteins were found to be unique for one or two strains under the conditions tested. While
the functions of some of these proteins, such as Hlb, LukE, and SEB, in S. aureus-associated
virulence are well characterized, the functions of other proteins are less clear and, in many
cases, remain to be elucidated. Why were those proteins missing from the exoproteome of the
remaining strains? There are at least three possible explanations for this phenomenon: the
respective genes (i) are absent, (ii) represent pseudo genes or (iii) are not expressed or
expressed in very low amounts. The lack of detection of SED, SEK, SEP, SEQ, SER, Etd,
24636604, and SAR0435 correlated with the absence of their coding genes. By contrast, while
HlgA, SplD, SAR0422, SA0092, and SA1001 were encoded by at least 80% of the strains,
these proteins were synthesized in detectable amounts only in one or two strains (Table 4).

Chapter 3
Figure 3. Relative amounts of extracellular proteins detected in at least 80% of investigated S. aureus isolates.
The respective sector on the 2D gel of each isolate is shown for the proteins indicated. The proteins were stained
with colloidal Coomassie Brillant Blue as described for Figure 1.
Table 4. Identification of genes for which gene products were detected in 20% of the
investigated S. aureus isolates a
Gene A B C D E F G H I J K L M N O P Q R S T U V W X Y
sea + + + + + + + + + + + + + + + +
seb + + + + + + + + +
sed + +
sek + + + +
sep + + +
seq + + + +
ser + +
etd + +
hlgA + + + + + + + + + + + + + + + + + + + + + + + + +
splD + + + + + + + + + + + + + + + + + + + + + + + + +
splE + + + + + + + + + + +
24636604 + +
SAR0422 + + + + + + + + + + + + + + + + + + + + + + + + +
SAR0435 +
SAR1905 + + +
SA1755 + + + + + + + + +
SA0092 + + + + + + + + + + + + + + + + + + + + + + +
SACOL0478 + + + + + +
SA1001 + + + + + + + + + + + + + + + + + + + + + + +
a genes whose gene product was detected on 2D gels are shaded grey for the respective strains
64

Proteogenomics uncovers extreme heterogeneity in the Staphylococcus aureus
exoproteome due to genomic plasticity and variant gene regulation
Variations in the expression levels of virulence genes may relate to differential activities of
specific S. aureus gene regulators. Especially RNAIII has previously been identified as a
main regulator of virulence gene expression (Novick, 2003), and the loss of RNAIII was
shown to affect the extracellular protein pattern of S. aureus dramatically (Ziebandt et al.,
2004). To analyse RNAIII levels, we performed dot blot analyses using RNA prepared from
cells grown to an OD540 of 10. This revealed that RNAIII was not produced at detectable
levels in 11 isolates (C, D, G, H, I, J, K, T, W, X, Y) (Figure 4). With the exception of isolate
W this correlates very well with a diminished expression level of late virulence factors
(Figure 1), confirming the general importance of RNAIII in virulence gene expression. At the
same time this shows that exceptions are possible as was the case for isolate W. Interestingly,
these 11 isolates were involved in wound infection, arthritis or cholangitis, respectively
(Supplemental table IVa). In mice, agr mutants were shown to have a growth advantage
within a mixed population of S. aureus residing in abscesses and wounds (Schwan et al.,
2003). Possibly, reduced RNAIII levels and the resulting decreased expression of RNAIIIdependent
virulence genes might be correlated to the induction of some of the observed
clinical symptoms. In the 14 remaining isolates, RNAIII transcripts have been detected in
varying amounts, which may, at least in part, account for the observed differences in
virulence factor production.
Similar exoprotein patterns in closely related isolates
While virulence gene expression of clonally different isolates was highly variable, we
observed very similar protein expression patterns in closely related isolates (i.e. B398, E8,
L80, G228, and X8) (Supplemental table IVa, Figure 1). Isolates L and M (=L80), which
belong to the clonal group ST80, displayed all the genetic characteristics (pvl, hlgA, etd,
edinB) as well as characteristic resistance patterns typically found in caMRSA strains with
epidemic spread in the European population (Supplemental table IVa, Tables 2 and 3)
(Monecke et al., 2006; Vandenesch et al., 2003). The isolates were derived from two patients
suffering from panaritium and abscesses, respectively. Interestingly, the extracellular protein
pattern of isolates L and M was identical, suggesting that the virulence gene expression
pattern may be very stable over extended periods of time (April 2002 - April 2004) (Figure 1).
The protein expression profiles of three other isolate pairs, A/B (=B398), E/F (=E8), and X/Y
(=X8) were also very similar. The strain pair A/B was identified by MLST as sequence type
398. This clonal lineage of S. aureus is frequently found on pigs in the Netherlands and was
recently described to spread from animals to humans (Armand-Lefevre et al., 2005; van
Belkum A. et al., 2008; Witte et al., 2007). In the present study the isolates came from a
mother and her daughter and were involved in pneumonia and phlegmone, respectively. They
encode pvl but none of the known superantigens. As described for other isolates of this
lineage, strain typing by PFGE failed, which was possibly due to the activity of a
restriction/methylation system typical for this lineage (Bens et al., 2006). Notably, the two
isolates of strains B398 differed with respect to mecA gene conservation (Supplemental table
IVa), suggesting that the acquisition or loss of methicillin resistance had no significant
influence on virulence gene expression (Figure 1). This difference in mecA conservation was
also observed for the two X8 strains.
As indicated above, one strain (G228), which was responsible for a hospital outbreak, was
identified seven times in four different patients. The first isolate (G) was obtained at the end
of 2003 and the most recent isolate (I) was identified early in 2005 as the colonizing S. aureus
65

Chapter 3
strain of a patient who suffered from a wound infection induced by this strain (isolate H) one
year before. We found the hlb converting phage FaSa3 in all these isolates, except for isolate
G, as might be expected from the phage dynamics during infection (Goerke et al., 2006;
Goerke et al., 2004). In accordance with this, the phenotype of strain G288 changed from Hlb
positive (G) to Hlb negative (C, D, H, I, J, K) (Table 3). However, the prophage did not
significantly alter the expression of other virulence-associated genes (Figure 1). This might be
mainly due to the fact that RNAIII was not expressed in these isolates under our experimental
conditions (Figure 4) and, accordingly, only a few virulence factors were produced.
Figure 4. Transcription of RNAIII in different clinical S. aureus isolates. RNA was prepared from cells grown in
TSB to an optical density of 10. Serial dilutions of total RNA of clinical isolates and reference strains (RN6390,
COL, Newman) were blotted and crosslinked onto the same positively charged nylon membrane. The membranebound
RNA was hybridized with a digoxigenin labelled RNA probe complementary to RNAIII. Chemiluminescence
signals were detected with a LumiImager (Roche Diagnostics, Mannheim, Germany).
Concluding remarks
In conclusion, our data show that in the species S. aureus, genome plasticity is only one of
several factors involved in exoproteome profile heterogeneity. Expression regulation as well
as protein secretion and modification processes add further dimensions to the heterogeneity of
the virulence potential of S. aureus. Such effects might be further enhanced and fine-tuned by
promoter mutations, differential activities of regulatory molecules and translation regulation
mechanisms. Most probably, the profoundly heterogeneous expression pattern of virulence
genes observed under identical in vitro conditions reflects a very high degree of variability in
vivo. It seems reasonable to suggest that these different virulence protein patterns are linked to
different clinical symptoms in the host. We are currently comparing virulence gene
expression profiles of S. aureus isolates that induced very similar symptoms. In this way, we
hope to identify symptom-/disease-related proteomic signatures that may help in elucidating
specific pathogenic mechanisms. It has been shown that S. aureus carriers mount a very
selective and protective antibody response against superantigens of their colonizing strains
(Holtfreter et al., 2006). Similarly, specific adaptive immune responses might also be raised
against other virulence factors, which vary between strains to a similar extent. These and
other immune mechanisms might explain why S. aureus carriers with bacteremia generally
have a better outcome than non-carriers (Wertheim et al., 2004). Finally, given the extensive
variability of virulence factors and mechanisms in S. aureus, our study has important
implications for vaccine development. The data strongly suggest that the development of
protective vaccines will require a very careful selection and combination of bacterial antigens.
66

Proteogenomics uncovers extreme heterogeneity in the Staphylococcus aureus
exoproteome due to genomic plasticity and variant gene regulation
Acknowledgements
We are indebted to J. Ziebuhr for critical comments on the manuscript and S. Holtfreter and
S. Kozitskaya for assistance in some experiments. Financial support was provided by CEU
(StaphDynamics, LSHM-CT-2006-019064; BaSysBio, LSHG-CT-2006-037469), BMBF
(031U107A/-207A; 031U213B), DFG (GK212/3-00, SFB/TR34, FOR 585), and Top Institute
Pharma (T4-213).
67

“Music is my religion”
-Johnny A. Hendrix-
68

Chapter 4
Characterization of Staphylococcus aureus secretion mutants
M.J.J.B. Sibbald, T. Winter, M. ten Brinke, G. Buist, D.G.A.M. Koedijk, M.M. van der Kooi-
Pol, T. Msadek, O. Poupel, V. Rühmling, I. Stokroos, E. Tsompanidou,
H. Antelmann, M. Hecker, S. Engelmann, J.M. van Dijl
To be submitted
69

Chapter 4
Summary
Staphylococcus aureus is a dangerous human pathogen that can cause a range of
diseases. The genome of this bacterium encodes an arsenal of virulence factors that are
exported to extracytoplasmic locations. Once translocated across the membrane, these
proteins are either retained at the surface of the cell or released into the environment. S.
aureus contains several protein secretion pathways of which the general Sec pathway
seems to be most frequently used. Other potential secretion pathways include the Twinarginine
translocation (Tat), Com and Ess pathways. This study reports on the analysis
of S. aureus secretion mutants. Sixteen isogenic S. aureus secretion mutants were
created, and the influence of the respective mutations on the exoproteome composition
was analyzed. Furthermore, the strains were tested for hemolysin production, and
Caenorhabditis elegans killing. Several S. aureus mutants showed significantly altered
exoproteomes, such as the secG or lgt mutants. In contrast, no obvious secretion defects
were observed for the other mutant strains lacking factors required for protein
translocation, precursor processing, and attachment of proteins to the cell wall. Notably,
in various mutant strains second-site mutations were observed that led to the loss of
hemolysin production, suggesting the acquisition of agr mutations. While this seems to
be a consequence of the natural adaptive capabilities of S. aureus, it is also an important
caveat for future studies on protein secretion in this organism that underscores the need
for genetically stable model strains.
70

Introduction
Characterization of Staphylococcus aureus secretion mutants
Staphylococcus aureus is part of the normal human microbiota. In its ecological niches such
as the skin and the nose it is no major threat to the human host. About 20% of the population
carries S. aureus permanently, ~60% can be a transient carrier and ~20% is apparently free of
S. aureus (Peacock et al., 2001). However, S. aureus can also turn into a dangerous pathogen
that can cause many different diseases, ranging from superficial skin lesions such as styes and
furunculosis, to more serious infections such as pneumonia, urinary tract infections and
endocarditis, and in rare cases even meningitis. Soon after the introduction of penicillin in the
1960s, resistant S. aureus strains were isolated in hospitals and ever since multidrug resistant
strains have been a major problem in hospitals. Vancomycin has long been a last resort
antibiotic. However, in 1996 the first vancomycin intermediate resistant strain (VISA) was
reported and soon after this date vancomycin resistant strains (VRSA) were isolated from
hospital patients (Hiramatsu et al., 1997;Weigel et al., 2003).
One of the prime reasons why S. aureus is able to infect almost every organ and tissue in the
human body is that S. aureus cells can produce a very diverse arsenal of virulence factors that
are exported to the cell surface and host milieu. These factors include proteins that are
necessary for: 1, adherence to cells (e.g. clumping factors, fibronectin and fibrinogen binding
proteins); 2, invasion and spreading throughout the host (e.g. hyaluronidase, leukocidin); 3,
evasion of the immune system (e.g. protein A and other IgG-binding proteins, capsuleforming
proteins); and 4, damaging host cells, thereby contributing to the symptoms of septic
shock (e.g. exotoxins, exfoliative toxin, toxic shock syndrome toxin TSST).
Figure 1 Protein secretion pathways in S. aureus. (A) Schematic representation of a S. aureus cell with Sec, Tat,
Com, and Ess pathways for protein secretion. Furthermore, potential protein secretion pathways involving ABC
transporters or holins are shown. Proteins synthesized with an appropriate secretion signal are directed to one of
the pathways and translocated across the membrane. The final destination of a translocated protein depends on
various other features, such as the presence of a membrane anchor, a lipobox, or a cell wall binding motif. (B)
Characteristics of signal peptides. Secretory signal peptides consist of three domains known as the N-, H-, and Cregions
(Sibbald et al., 2006). Signal peptide cleavage by signal peptidase occurs in the C-region. The signal or
leader peptides of bacteriocins do not contain a specific H-domain. Ess-dependent proteins lack a cleavable Nterminal
signal peptide, but often contain a WXG-motif in the middle of the protein that may serve as a targeting
signal.
Bacteria have acquired several pathways to transport proteins to extracytoplasmic locations.
Proteins that are translocated across the membrane of Gram-positive bacteria are either
retained at the surface of the cell via special mechanisms or, if no retention signal is present,
these proteins are secreted into the environment. In S. aureus, at least six different protein
71

Chapter 4
secretion pathways have been identified (Figure 1; (Sibbald et al., 2006)). The majority of
secreted proteins are synthesized with a signal peptide, which directs them to a specific
secretion pathway. During or after translocation across the membrane the signal peptide is
cleaved from the mature protein by a signal peptidase. The protein is then folded into the right
conformation (with or without the help of folding catalysts) and either released into the
environment or retained at the surface of the cell. Most proteins (and virulence factors) are
translocated across the membrane via the (general) Sec pathway. These proteins are
synthesized in the cytoplasm with a typical Sec-type signal peptide and then directed to the
membrane-embedded Sec machinery. The most important components of the Sec machinery
are the cytoplasmic translocation motor SecA and the membrane-embedded translocation
channel, which is composed of SecY, SecE, and SecG (Hanada et al., 1996;Veenendaal et al.,
2004). SecA binds and hydrolyzes ATP, and through the accompanying conformational
changes it pushes the preprotein through the SecYEG channel. It has been shown for S.
aureus that both SecA, SecE and SecY are essential for growth and viability (Chaudhuri et
al., 2009). In addition, S. aureus has a second set of secA and secY genes in its genome (here
referred to as secA2 and secY2). These genes lie in an operon together with the gene for the
only known SecA2-SecY2 substrate, SraP, and several other genes that are probably involved
in the glycosylation of SraP (Siboo et al., 2005;Siboo et al., 2008). The secA2 secY2 operon is
also found in several other pathogenic Gram-positive bacteria, such as Streptococcus
gordonnii (Bensing and Sullam, 2002). SecDF is a protein that is associated with the Sec
channel, together with a protein named YajC in E. coli or YrbF in B. subtilis (Tjalsma et al.,
2000;Sibbald et al., 2006). In S. aureus and several other Gram-positive bacteria, the SecDF
protein seems to be a natural fusion of homologues of the Escherichia coli SecD and SecF
proteins, and it is thus regarded as a “Siamese Twin protein” (Bolhuis et al., 1998). The
secDF gene of Bacillus subtilis is not required for cell growth and viability, and its deletion
has a relatively mild negative effect on protein translocation (Bolhuis et al., 1998). By
contrast, secDF seems to be essential in S. aureus (Chaudhuri et al., 2009). So far, the precise
role of SecDF in the translocation process has remained unclear.
Type I signal peptidases remove signal peptides from secretory precursor proteins to liberate
these proteins from the membrane upon translocation (van Roosmalen et al., 2004). The
genome of S. aureus contains two genes encoding for the signal peptidases SpsA and SpsB of
which the latter is essential in S. aureus (Chaudhuri et al., 2009;Cregg et al., 1996). SpsA
seems to be an inactive signal peptidase since the catalytic Ser and Lys residues are replaced
with Asp and Ser residues, respectively (van Roosmalen et al., 2004;Dalbey et al., 1997).
SpsB recognizes an -3 A-X-A -1 motif (with a few other amino acids permitted at the -3
position) and cleaves after the Ala residue at the -1 position (Sibbald et al., 2006).
It should be noted that the Sec pathway can only facilitate membrane passage of proteins in an
unfolded state (Driessen and Nouwen, 2008;Papanikou et al., 2007;Yuan et al., 2009). While
most translocated proteins seem to have an intrinsic capability to fold into their native
conformation, their post-translocational folding in vivo is usually catalyzed by chaperones and
other folding catalysts, such as DsbA and PrsA (Tjalsma et al., 2000;Tjalsma et al.,
2004;Sibbald et al., 2006). PrsA is a general folding catalyst with peptidyl-prolyl cis/trans
isomerase activity that was shown to be involved in the correct folding of AmyQ in B. subtilis
(Kontinen and Sarvas, 1993) and the protective antigen of B. anthracis (Williams et al.,
2003). DsbA is a homologue of the E. coli DsbA and B. subtilis BdbD proteins, which are
both oxidases involved in the formation of disulfide bonds in exported proteins (Kouwen and
van Dijl, 2009a). However, unlike its homologues in E. coli and B. subtilis, the S. aureus
72

Characterization of Staphylococcus aureus secretion mutants
DsbA does not seem to require a membrane-embedded partner protein for its re-oxidation
during catalysis. Instead, this protein is re-oxidized by components in the extracellular
environment (Heras et al., 2008;Kouwen et al., 2007).
In addition to the Sec pathway, Gram-positive bacteria can use several other special purpose
pathways for protein export from the cytoplasm. These include the YidC pathway, the Twinarginine
translocation (Tat) pathway, the Com pathway, secretion via holins or ABC
transporters, and the Ess pathway (Sibbald et al., 2006;Driessen and Nouwen, 2008;Yuan et
al., 2009).
YidC functions as a membrane insertase to mediate membrane protein insertion either by
itself or in concert with SecYEG. In the Sec pathway, YidC is linked to the Sec translocase
via SecDF (see (Yuan et al., 2009)). The YidC homologues in B. subtilis have been
designated SpoIIIJ and YqjG (Murakami et al., 2002;Tjalsma et al., 2003;Saller et al., 2009).
The Tat translocase consists of the TatA and TatC subunits. This translocase recognizes a
twin-arginine motif that consists of two adjacent (twin) Arg residues followed by another
amino acid and two hydrophobic amino acids (R-R-x-φ-φ, where φ is a hydrophobic amino
acid) (Cristóbal et al., 1999;Jongbloed et al., 2000). In contrast to the Sec pathway, this
translocase is able to transport folded proteins across the membrane, which seems of
particular relevance for the export of proteins with bound co-factors. The Tat pathway has
been studied very well in E. coli and B. subtilis (Dilks et al., 2003;Berks et al.,
2005;Robinson and Bolhuis, 2001), but fairly little is known about the roles of this pathway in
S. aureus. Recently, the Tat pathway of S. aureus has been studied using biochemical and
proteomics approaches, but this has led to the identification of only one substrate so far,
which is FepB (Yamada et al., 2007;Schmaler et al., 2009).
The Com pathway of B. subtilis is used for the export and assembly of pseudopili that are
necessary for DNA binding and uptake during natural genetic competence development
(Tjalsma et al., 2004;Tjalsma et al., 2000). Homologues of some of the components and the
secreted proteins are also present in S. aureus. So far, limited information is available about
this pathway in S. aureus. Morikawa and colleagues have shown that the expression of several
competence genes is under control of the SigH factor (Morikawa et al., 2003), but whether
these genes are involved in DNA uptake has so far remained unclear.
For S. aureus it has been shown that at least three proteins (i.e. EsxA, EsxB and EsaC) are
secreted via the ESX-1 or ESAT-6 secretion system (Ess) pathway (Burts et al., 2005;Burts et
al., 2008). First discovered in Mycobacterium tuberculosis, this pathway seems to be
conserved in several Gram-positive pathogens (Pallen, 2002). The EssA, EssB, and EssC
components are required for a functional Ess secretion machinery. Notably, proteins secreted
via the Ess pathway do not contain a classical signal peptide, but comparison of many of these
secreted proteins revealed a conserved WXG motif in the middle of these proteins (Pallen,
2002). Whether this motif represents an Ess targeting signal remains to be elucidated.
S. aureus and related Gram-positive bacteria have several mechanisms for the retention of
proteins that have been exported from the cytoplasm. One of these mechanisms is to bind the
protein to the membrane via a lipid anchor. Such lipoproteins are synthesized with a Sec type
signal peptide containing the so-called lipobox (von Heijne, 1989). The lipobox contains an
invariant Cys residue at the +1 position and the consensus sequence -3 L-x-x-C +1 . The lipobox
is recognized by the diacylglyceryl transferase Lgt and the invariant Cys is then modified by
transferring a diacylglyceryl group from phosphatidylglycerol to the sulfhydryl group of this
Cys residue (Tjalsma et al., 2000). Upon cleavage by the lipoprotein-specific signal peptidase
II, the lipid-modified Cys residue serves as the membrane anchor for the mature lipoprotein. It
73

Chapter 4
has been shown that S. aureus Lgt is important for virulence (Stoll et al., 2005;Bubeck et al.,
2006;Hashimoto et al., 2006), and that a mutant lacking the lgt gene is attenuated in the
induction of an inflammatory response (Stoll et al., 2005). Likewise, Lgt is important for the
virulence in other Gram-positive pathogens, such as Listeria monocytogenes (Baumgärtner et
al., 2007;Machata et al., 2008). Paradoxically, it has also been reported than an lgt mutant of
S. aureus can be hyper-virulent (Stoll et al., 2005;Bubeck et al., 2006). The molecular basis
for these different observations is presently not clear.
Another mechanism employed for the specific retention of proteins in the cell envelope is
their covalent attachment to the cell wall. In S. aureus and many other Gram-positive
pathogens, this reaction is catalyzed by sortases (Dramsi et al., 2005;Ton-That et al., 2004).
Based on phylogenetic analyses of 61 sortases, at least four classes of sortase (A-D) can be
distinguished (Dramsi et al., 2005). In the genome of S. aureus two genes encoding sortases
are present. These enzymes recognize proteins with a C-terminal signal, which consists of the
recognition motif (LPxTG), followed by a stretch of hydrophobic residues and several
positively charged residues (Arg or Lys). SrtA recognizes proteins with an LPxTG or LPxAG
motif and cleaves N-terminally of the Gly residue. At the same time, SrtA couples the mature
protein through its new C-terminal Thr or Ala residue to the peptidoglycan in the cell wall
(Mazmanian et al., 2001;Navarre and Schneewind, 1994). In S. aureus 21 proteins have been
identified with SrtA signals. For several of these proteins (e.g. protein A, ClfA, SasG) it has
been shown that they are indeed attached to the cell wall by SrtA, and that they are involved
in virulence (Corrigan et al., 2007;Josefsson et al., 2001). The gene encoding the second
sortase of S. aureus, SrtB, lies in an operon that also encodes the SrtB substrate IsdC and
several other genes involved in iron acquisition (Mazmanian et al., 2003). The structure of
this operon is conserved in other Gram-positive pathogens, such as Bacillus anthracis,
Bacillus cereus and L. monocytogenes (Dramsi et al., 2005). IsdC contains a C-terminal
NPQTN SrtB recognition motif instead of the SrtA recognition motifs LPxTG or LPxAG
(Mazmanian et al., 2002). These different substrate specificities of SrtA and SrtB relate to
differences in their substrate binding pockets (Bentley et al., 2007).
In addition to covalent binding of proteins to the cell wall, there are also several proteins that
bind to components of the cell wall via non-covalent interactions. This non-covalent cell wall
binding can involve several different domains with repeated motifs, such as the LysM domain
(Buist et al., 2008), the GW domain (Braun et al., 1997), or other domains as encountered in
the clumping factors A and B (Hartford et al., 1997;Ní Eidhin et al., 1998) and the serine
aspartate repeat proteins SdrC, SdrD, and SdrE (Josefsson et al., 1998).
Most studies in the area of protein secretion by S. aureus were focused on the organism’s
virulence factors and their modes of action. Relatively little attention has been attributed to
the export and folding mechanisms for these virulence factors and other exported proteins. In
this chapter, the construction and analysis of S. aureus protein secretion mutants is described.
As such it represents a first step to define the functions of secretion machinery components of
this pathogen in a systematic manner. Genes for several secretion machinery components
were deleted from the chromosome (Table 1) and the exoproteomes of the resulting mutant
strains were analyzed with functional assays, molecular biological approaches and
proteomics.
74

Characterization of Staphylococcus aureus secretion mutants
Table 1 Relevant components of S. aureus secretion pathways a
Protein Essential b
Sec-pathway
Chaperone Ffh Y n.a.
FtsY Y n.a.
Translocation motor SecA1
Y
n.a.
SecA2
N
N
Translocation channel SecY1
Y
n.a.
SecY2
N
Y
SecE Y n.a.
SecG N Y
SecDF Y N
YrbF ? n.a.
Membrane insertase SpoIIIJ/YqjG Y N
Lipid modification Lgt N Y
Signal peptidase SpsA (inactive)
N
SpsB
Y c
Y
N
LspA N Y
Folding catalyst PrsA N Y
DsbA N Y
Cell wall attachment SrtA N Y
SrtB N Y
Tat-pathway
Pseudopilin pathway
Holins
Ess
TatA
TatC
ComGA
N
Y
ComGC
N
Y
ComC N Y
CidA N Y
LrgA N Y
EsaA
EssA
EssB
EssC
75
N
N
Deletion Mutant
a Table adapted from Sibbald et al. (2006); b Chaudhuri et al. (2009); c Cregg et al. (1996); for proteins marked
“n.a.” we did not attempt to delete the corresponding genes, because they were known to be essential (ffh,
ftsY, secA1, secY1, secE), or of seemingly minor relevance (yrbF).
Material & Methods
Bacterial strains and growth
Strains and plasmids used in this study are listed in Table 2. E. coli and S. aureus strains were grown at
37°C under aerobic conditions. E. coli strains were grown in Luria-Bertani broth (LB). Unless stated
otherwise, S. aureus strains were grown at 37°C in tryptic soy broth (TSB) under vigorous shaking or
on trypic soy agar (TSA) plates. Antibiotics for E. coli strains were added in the following final
concentrations: ampicillin 100 µg/ml, kanamycin 20 µg/ml, and erythromycin 100 µg/ml. For S. aureus
the following final concentrations were used: kanamycin 20 µg/ml, and erythromycin 5 µg/ml. To
monitor β-galactosidase activity in S. aureus, 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (Xgal)
was added to the plates at a final concentration of 80 µg/ml.
N
N
N
N
Y
Y
N
N
N
N

Chapter 4
Table 2 Plasmids and bacterial strains used
Plasmids Relevant Properties Reference
TOPO Cloning vector pCR®-Blunt II-TOPO® vector; Km R Invitrogen
technologies
Life
pMAD E. coli / S. aureus shuttle vector that is temperature-sensitive
in S. aureus and contains the bgaB gene, Ery R , Amp R
(Arnaud et al.,
2004)
pDG783 1.5-kb kanamycin resistance cassette in pSB118; Amp R , Km R (Guérout-Fleury
et al., 1995)
“cidA”::kan-pMAD pMAD plasmid containing the flanking regions of S. aureus
cidA, also contains the kanamycin gene, Ery R , Kan R
This work
pMUTIN4 Insertion vector with Pspac promoter, Ery R (Vagner
1998)
et al.,
“comC”-pMAD pMAD with flanking regions of S. aureus comC, Ery R This work
“comGA”-pMAD pMAD with flanking regions of S. aureus comGA, Ery R This work
“comGC”-pMAD pMAD with flanking regions of S. aureus comGC, Ery R This work
“dsbA”-pMAD pMAD with flanking regions of S. aureus dsbA, Ery R This work
“lgt”-pMAD pMAD with flanking regions of S. aureus lgt, Ery R This work
“lrgA”::kan-pMAD pMAD with flanking regions of S. aureus lrgA, also contains
the kanamycin gene, Ery R , Kan R
This work
“lspA”-pMAD pMAD with flanking regions of S. aureus lspA, Ery R This work
“prsA”-pMAD pMAD with flanking regions of S. aureus prsA, Ery R This work
“secDF”-pMAD pMAD with flanking regions of S. aureus secDF, Ery R This work
“secA2”-pMAD pMAD with flanking regions of S. aureus secA2, Ery R This work
“secG”-pMAD pMAD with flanking regions of S. aureus secG, Ery R This work
“secY2”-pMAD pMAD with flanking regions of S. aureus secY2, Ery R This work
“spsA”-pMAD pMAD with flanking regions of S. aureus spsA, Ery R This work
“spsB”-pMAD pMAD with flanking regions of S. aureus spsB, Ery R This work
“srtA”-pMAD pMAD with flanking regions of S. aureus srtA, Ery R This work
“srtB”-pMAD pMAD with flanking regions of S. aureus srtB, Ery R This work
“tatA”-pMAD pMAD with flanking regions of S. aureus tatA, Ery R This work
“tatC”::kan-pMAD pMAD with flanking regions of S. aureus tatC, also contains
the kanamycin gene, Ery R , Km R
This work
“tatAC”-pMAD pMAD with flanking regions of S. aureus tatAC, Ery R This work
secA2-pMUTIN4 Controlled expression of secA2 This work
Strains
E. coli
Genotype Reference
DH5α supE44; hsdR17; recA1; gyrA96; thi-1; relA1 (Hanahan, 1983)
TOP10 Cloning host for TOPO vector; F - mcrA ∆(mrr-hsdRMSmcrBC)
Φ80lacZ∆M15 ∆lacX74 recA1 araD139 ∆(araleu)7697
galU galK rpsL (Str R ) endA1 nupG
S. aureus RN4220
Parental strain Restriction-deficient derivative of NCTC 8325, cured of all
known prophages, rsbU-, agr-
76
Invitrogen Life
technologies
∆cidA::kan
(Kreiswirth et al.,
1983)
b
cidA, Kan R This work
∆comC b comC This work
∆comGA b comGA This work
∆comGC b comGC This work
∆dsbA b dsbA This work
∆lgt b lgt This work
∆lrgA::kan lrgA, Km R This work
∆lspA b lspA This work
∆prsA b prsA This work
∆secG b secG This work
∆secY2 b secY2 This work
∆spsA b spsA This work

Characterization of Staphylococcus aureus secretion mutants
Strains
S. aureus RN4220
Genotype Reference
∆srtA b srtA This work
∆srtB b srtB This work
∆tatA tatA This work
∆tatC::kan tatC, Km R This work
∆tatAC b S. aureus SH1000
tatA tatC This work
Parental strain rsbU+ derivative of 8325-4 (rsbU+, agr+) (Horsburgh et al.,
2002)
sasG-pMUTIN4 Overexpression of SasG from the pMUTIN4 plasmid; Ery R (Corrigan et al.,
2007)
sasG-pMUTIN4 ∆srtA Overexpression of SasG from the pMUTIN4 plasmid; srtA;
Ery R
This Work
Construction of mutant strains
Mutants were constructed with the temperature-sensitive pMAD plasmid as described by Arnaud and
colleagues (Arnaud et al., 2004) (Figure 2). Primers used to PCR amplify the flanking regions of genes
of interest are listed in Table 3. The flanking regions of ~500 bp were obtained by PCR using the
primer pairs F1/R1 and F2/R2.
Figure 2 Schematic representation of the pMAD-based strategy for deletion of genes from the S. aureus
chromosome. The flanking regions upstream and downstream of a target gene that is to be deleted are represented
as grey boxes marked “front” and “back”. These front and back regions are PCR-amplified, merged by PCR, and
cloned in the plasmid pMAD. Integration of pMAD with the merged front and back regions (step 1) can occur via
single cross-over recombination upstream or downstream of the target gene (the diagram shows integration
upstream of the target gene). A second recombination event (step 2), leading to pMAD excision from the
chromosome, can take place either at the front or back region. Depending on the place where this second
recombination event occurs, the target gene will either be excised from the chromosome (right), or remain intact
on the chromosome (left).
77

Chapter 4
Table 3 Primers used in this study
Primer Sequence (5’→3’)
cidA-F1 AATAAAGACTTTTACTTGAAT
cidA-R1 a TTAGAACTCCAATTCACCCATGGCCCCCGCGCCATCCCTTTCTAAATA
cidA-F2 a CCGCAACTGTCCATACCATGGCCCCCTGATTACGTGCAAGCCTTATTAAT
cidA-R2 GATAGAAGATTCAAATCTTCC
comC-F1 CGAGATGGTCAAACATTTAAG
comC-R1 TCACGTCAGTCAGTCACCATGGCAATGACAACCTCCTTATGTAAA
comC-F2 TGCCATGGTGACTGACTGACGTGAAAATTAAAGAAATGGTAA
comC-R2 AACTGCGATGATTGCATTGGC
comGA-F1 ATATCGGAGCAGTCGATGATA
comGA-R1 TTACGTCAGTCAGTCACCATGGCAAAAAACACCTCCTACATA
comGA-F2 TGCCATGGTGACTGACTGACGTAAACTACATTCTAAGAAGCG
comGA-R2 GAGCATTACTACAATTATAGT
comGC-F1 GCTCAATAAGATAAACTTTGT
comGC-R1 CTACGTCAGTCAGTCACCATGGCAATATTAACCTCCATTATTTTA
comGC-F2 TGCCATGGTGACTGACTGACGTAGAAAGCAGTCAGCATTTAC
comGC-R2 GATTCATCATTGGTATCAATA
dsbA-F1 ATTTCTTTGGATATTTATATT
dsbA-R1 CTACGTCAGTCAGTCACCATGGCAAATAACTCCTATTCATAT
dsbA-F2 TGCCATGGTGACTGACTGACGTAGTCTTAATTGTTGAGATCA
dsbA-R2 CTTTCGTTATAGTTTTCCCAC
lgt-F1 GGTGTTGGTGTACTAATTACC
lgt-R1 CTACGTCAGTCAGTCACCATGGCATCAACCTACTCCTCACTCTTA
lgt-F2 TGCCATGGTGACTGACTGACGTAGTGATAGTTTGAGGAAATTTTT
lgt-R2 ACATTATTATTCTTTTGCGCC
lrgA-F1 TAAAGCCAAAGATGATAATAA
lrgA-R1 a TTAGAACTCCAATTCACCCATGGCCCCCGCCTCCTACGTTTGATTTAA
lrgA-F2 a CCGCAACTGTCCATACCATGGCCCCCTAACCACTTAGCACTAAACACACC
lrgA-R2 GTAATTCGGAAAAGCTTTAAG
lspA-F1 CCAATTAAGTGTAGACGATTC
lspA-R1 TTACGTCAGTCAGTCACCATGGCATTTCGTTCCTCCAATCAATCG
lspA-F2 TGCCATGGTGACTGACTGACGTAATGGAGACTTATGAATTTAACA
lspA-R2 CGATATATTTTCTTTTAACAG
prsA-F1 GAAAATGGCTTATATTCTATA
prsA-R1 TTACGTCAGTCAGTCACCATGGCAAGTTGAAACTCCTTTGTAAGT
prsA-F2 TGCCATGGTGACTGACTGACGTAACACAAAACCGAGCGACCGTGG
prsA-R2 TTTGTTATATAGTGGTATTAT
secA2-F1 GTATAAAAGCATGCGGGTGAC
secA2-R1 a TTAGAACTCCAATTCACCCATGGCCCCCTTACTTCCCCACCATTCAGTT
secA2-F2 a CGCAACTGTCCATACCATGGCCCCCTAAATGAAAAGGGGTAGCGCATGA
secA2-R2 GTCGCATATATAATTTCGCTT
secDF-F1 TTTTGCGGTTATGTATTTCTT
secDF-R1 a TTAGAACTCCAATTCACCCATGGCCCCC TGAACACCTCATTATTTACG
secDF-F2 a CCGCAACTGTCCATACCATGGCCCCCTAAAATGAATTAAGCGGTATGTGA
secDF-R2 ATCACTAAAATTGTAGTTGCG
secG-F1 TTAAAACAGGACGCTTTATTG
secG-R1 TTACGTCAGTCAGTCACCATGGCAAAATTGTCCTCCGTTCCTTAT
secG-F2 TGCCATGGTGACTGACTGACGTAAGGTCCGGCGATGTAAATGTCG-
secG-R2 GCGTGCATATTCTAAAAAGCC
secY2-F1 TGTCTGGTTCACAAAGCATTT
secY2-R1 TTACGTCAGTCAGTCACCATGGCAGTTGCACCTCTTTTATATCAA
secY2-F2 TGCCATGGTGACTGACTGACGTAAGGAGGTAATTATGAAATACTT
secY2-R2 GCCTCTCCCTGATCATCAAAA
78

Characterization of Staphylococcus aureus secretion mutants
Primer Sequence (5’→3’)
spsA-F1 TAGAGCTATAATTCCAGTATT
spsA-R1 TTACGTCAGTCAGTCACCATGGCAGATGTCACTCCTTTTTCGATC
spsA-F2 TGCCATGGTGACTGACTGACGTAAAAAGAGGTGTCAAAATTGAAA
spsA-R2 CCAACAATTTGGTCTTCATCA
spsB-F1 ATTTGATTTCATTGATACTTG
spsB-R1 a TTAGAACTCCAATTCACCCATGGCCCCCTCTTTTTAAGATTTGAACTG
spsB-F2 a CCGCAACTGTCCATACCATGGCCCCCTAATATGAAACAAATACAACATCG
spsB-R2 CCCATAATATTTTGCTTGTGA
srtA-F1 AATGGTGTAGTAATTGACTAG
srtA-R1 TTACGTCAGTCAGTCACCATGGCAACGTTAAGGCTCCTTTTATAC
srtA-F2 TGCCATGGTGACTGACTGACGTAATCTATTACGCTAATGGATGAA-
srtA-R2 CTCACATTACTTACTATTAAT
srtB-F1 TGAAAATATGGAGCGACGTAT
srtB-R1 TTACGTCAGTCAGTCACCATGGCAAAAAATCCTCTTTTATTAACG
srtB-F2 TGCCATGGTGACTGACTGACGTAAACAGAAAAGAGGATAATTATG
srtB-R2 ATCAAAATGATATAATTGATG
tatA-F1 TTATGGCATTTACATTATCTG
tatA-R1 CTACGTCAGTCAGTCACCATGGCAGATAATCAACCTCACTCATAA
tatA-F2 TGCCATGGTGACTGACTGACGTAGCACTGACCACACCTTACTGGT
tatA-R2 GACCCATAAATAATATTGGTA
tatC-F1 TGATGAAATGGCTGAAGCTGG
tatC-R1 a TTAGAACTCCAATTCACCCATGGCCCCCAAAATTTTTACTAACCGATG
tatC-F2 a CCGCAACTGTCCATACCATGGCCCCCTAACCTTATACGAATCAATGCTGT
tatC-R2 CGATTAGTAATGGTAATTTGG
kan-F1 GGGGGCCATGGGTGAATTGGAGTTCGTCTTG
kan-R1 GGGGGCCATGGTATGGACAGTTGCGGATGTA
secA2-F3 CGGAATTCGAATCCAGTACGATTTTTAG
secA2-R3 CGGGATCCTCCCGGTAACATACGACCTG
RNAIII-F AGGAAGGAGTGATTTCAATG
RNAIII-R CTAATACGACTCACTATAGGGAGAACTCATCCCTTCTTCATTAC
Overlapping nucleotides are shown in bold; restriction sites in primers are underlined
a These primers have an overlap with the kanamycin resistance cassette from pDG783
Primers R1 and F2 contained an overlap of 21 nucleotides (seven codons), which served to fuse the
amplified “front” and “back” flanking regions by PCR. For the deletion of some genes (cidA and lrgA),
a kanamycin resistance cassette was introduced between the respective amplified flanking regions. To
this end, overlap was created between the 5’ and 3’sequences of the kanamycin resistance cassette and
the front and back regions. The kanamycin resistance cassette was obtained by PCR using plasmid
pDG783 as a template. In the case of the tatAC mutant, the upstream region of tatA was linked with the
downstream region of tatC. The linked fragments were purified either from gel or using the High Pure
PCR Product Purification Kit (Roche). Next, they were cloned in the TOPO vector. The constructs thus
obtained were cut with EcoRI and the merged flanking regions were ligated to pMAD cut with EcoRI.
Upon transformation of E. coli, cells containing pMAD plasmids with the appropriate merged flanking
regions were identified through colony PCR with specific primers. Correct “gene”-pMAD constructs
were used for electro-transformation of competent S. aureus RN4220 cells. Transformants were
selected at 30°C on TSA plates containing erythromycin and X-gal. A blue colony was transferred to
10 ml Brain Heart Infusion (BHI) and grown at 30°C without shaking. From the overnight culture 100
µl was transferred to 10 ml fresh BHI, grown for one hour at 30°C and then transferred to 42°C and
grown for six hours without shaking. Dilutions of the culture were transferred to plates containing
erythromycin and X-GAL and grown for 48 hours at 42°C. A blue colony was transferred to 10 ml BHI
broth and grown at 42°C. From the overnight culture 10 µl was transferred to 10 ml fresh BHI and
growth was continued at 30°C for 6 hours. Dilutions of the culture were transferred to TSA plates
79

Chapter 4
containing X-gal and incubated for 48 hours at 30°C. White colonies were selected and tested for
erythromycin sensitivity on TSA plates containing X-gal with or without erythromycin. Colonies were
screened for particular gene deletions by colony PCR. Genomic DNA of seemingly correct mutants
was isolated using the GenElute Bacterial Genomic DNA Kit (Sigma) for further verification of gene
deletions by PCR. To delete particular genes from the S. aureus SH1000 genome, the respective pMAD
constructs were transferred from the S. aureus RN4220 strain to the SH1000 strain by transduction with
phage φ85 (Novick, 1991).
To place the secA2 gene under control of the IPTG-inducible Pspac promoter, a 600 bp fragment,
starting before the ribosome-binding site of secA2, was cloned into the pMUTIN4 plasmid using the
EcoRI and BamHI restriction sites. This construct was then introduced into S. aureus RN4220 by
electro-transformation.
DNA sequence analyses were performed at ServiceXS, Leiden, the Netherlands.
Scanning electron microscopy
For scanning electron microscopy, bacteria were fixed for 30 min with 2% glutaraldehyde in 0.1 M
Cacodylate buffer, pH 7.38. The fixated bacteria were placed on a piece (1 cm 2 ) of cleaved 0.1% Poly-
L Lysine coated mica sheet and washed in 0.1 M Cacodylate buffer. This specimen was dehydrated in
ethanol series consisting of 30%, 50%, 70%, 96% and anhydrous 100% solution (3X) 10 min each,
then Critical point dried with CO2, and sputter-coated with 2-3 nm Au/Pd (Balzers coater). The
specimen was fixed on a SEM-stub-holder and observed in a JEOL FE-SEM 6301F microscope.
RNA-isolation and dot blot analysis
Total RNA from S. aureus was isolated using the acid-phenol method with some modifications (Fuchs
et al., 2007). A digoxigenin-labeled RNA probe for RNAIII was prepared by in vitro transcription with
T7 RNA polymerase by using a PCR fragment as template. The PCR fragment was generated by using
chromosomal DNA of S. aureus and the respective oligonucleotides (Table 3). Dot blot analyses were
carried out by using serial dilutions of total RNA prepared from S. aureus isolates and reference strains
grown in TSB medium to an optical density (OD540) of 10. The digoxigenin-labeled RNA probe was
used for hybridization. The hybridization signals were detected using a Lumi-Imager and analyzed
using the Lumi-Analyst software package (Roche Diagnostics, Mannheim, Germany).
Hemolysin activity
To test the hemolysin activity, blood agar plates containing 5% sheep blood (Mediaproducts B.V.)
were inoculated with overnight cultures and incubated for 24 hours at 37°C.
Cell fractionation, SDS-PAGE, and Western blotting
Overnight cultures of S. aureus strains were diluted in TSB to an OD540 of 0.05 and grown at 37°C
under aerobic conditions. To monitor growth, samples were taken every hour and the OD540 was
measured. Samples for subsequent experiments were taken after six hours. Cells were separated from
the medium by centrifugation (2 min, 14.000 rpm). Proteins in the medium fraction were precipitated
with 10% trichloroacetic acid (TCA), washed with acetone, and dissolved in 100 µl 1x Loading Buffer
(Invitrogen). Cells were resuspended in 300 µl 1x Loading Buffer (Invitrogen) and disrupted with glass
beads using the Precellys ® 24 bead beating homogenizer (Bertin Technologies). Non-covalently cell
wall bound proteins were obtained using KSCN treatment. Cells from 20 ml cultures were collected by
centrifugation (5 min, 6.000 rpm), washed with 5 ml PBS, and incubated for 10 min with 1,5 ml 1M
KSCN on ice. After centrifugation (15 min, 8000 rpm) the non-covalently cell wall bound proteins
were precipitated from the supernatant fraction with 10% TCA, washed with acetone and dissolved in
100 µl 1x Loading Buffer (Invitrogen). Upon addition of Reducing Agent (Invitrogen), the samples
were incubated at 95ºC for 5 min. Proteins were separated by SDS-PAGE using precast NuPage gels
(Invitrogen). The amounts of protein used for SDS-PAGE were corrected for the OD540 of the
respective culture. Coomassie staining of gels was performed using the SimplyBlue TM SafeStain
80

Characterization of Staphylococcus aureus secretion mutants
(Invitrogen). For Western blotting, proteins separated by SDS-PAGE were blotted onto a nitrocellulose
membrane (Protran®, Schleicher & Schuell). Immunodetection of particular proteins was performed
with specific rabbit antibodies raised against TrxA (Miller, submitted) or DsbA (Kouwen et al., 2007),
or mouse antibodies against SspB (Shaw et al., 2004). Bound primary antibodies were visualized using
fluorescent IgG secondary antibodies (IRDye 680 or 800 CW goat anti-mouse/anti-rabbit from LiCor
Biosciences). Membranes were scanned for fluorescence at 700 or 800 nm using the Odyssey Infrared
Imaging System (LiCor Biosciences).
Zymography
Zymography was used to test whether the secretion of staphylococcal cell wall hydrolases was affected
in mutant strains. Micrococcus luteus cell wall fragments were from Sigma. S. aureus RN4220 cell
wall fragments were isolated as described previously (Steen et al., 2003) with minor adaptations. After
breaking the cells by bead-beating, the cell walls were boiled in 4% SDS for 30 min and washed with
PBS. This procedure was repeated three times. 12.5% PAA gels contained 10% cell wall fragments
from either M. luteus or S. aureus RN4220. All sample amounts were corrected for OD540 of the
respective cultures. Upon electrophoresis, the gels were incubated overnight at room temperature in a
buffer containing 25 mM Tris (pH 8.0) and 1% Triton X-100. After washing with water, the gels were
stained with a 1% methylene blue solution in potassium hydroxide (Valence and Lortal, 1995).
Analytical and preparative two-dimensional (2-D) PAGE
Extracellular proteins from 100 ml culture supernatant were precipitated, washed, dried, and resolved
as described previously (Ziebandt et al., 2004). The protein concentration was determined using Roti®-
Nanoquant (Carl Roth GmbH & Co, Karlsruhe, Germany). Preparative 2-D PAGE was performed by
using the immobilized pH gradient technique (Bernhardt et al., 1999;Eymann et al., 2004). The protein
samples (350 µg) were separated on immobilized pH gradient strips (Amersham Pharmacia Biotech,
Piscataway, NJ) with a pH range of 3-10. The resulting protein gels were stained with colloidal
Coomassie Blue G-250G (Candiano et al., 2004) and scanned with the light scanner. Each experiment
was performed at least three times.
For identification of proteins by MALDI-TOF MS, Coomassie-stained protein spots were excised from
gels using a spot cutter (Proteome WorkTM) with a picker head of 2 mm and transferred into 96-well
microtiter plates. Digestion with trypsin and subsequent spotting of peptide solutions onto the MALDI
targets were performed automatically in an Ettan Spot Handling Workstation (GE-Healthcare, Little
Chalfont, United Kingdom) using a modified standard protocol. MALDI-TOF MS analyses of spotted
peptide solutions were carried out on a Proteome-Analyzer 4700/4800 (Applied Biosystems, Foster
City, CA) as described previously (Eymann et al., 2004). MALDI-TOF-TOF analysis was performed
for the three highest peaks of the TOF spectrum as described previously (Eymann et al., 2004;Wolff et
al., 2007). Database searches were performed using the GPS explorer software version 3.6 (build 329)
with the organism-specific databases.
By using the MASCOT search engine version 2.1.0.4. (Matrix Science, London, UK) the combined MS
and MS/MS peak lists for each protein spot were searched against a database containing protein
sequences derived from the genome sequences of S. aureus NCTC8325. Search parameters were as
described previously (Wolff et al., 2007). For comparison of protein spot volumes, the Delta 2D
software package was used (Decodon GmbH Germany). The induction ratio of mutant to parental
strain was calculated for each spot (normalized intensity of a spot on the mutant image/normalized
intensity of the corresponding spot on the parental image). The significance of spot volume differences
of two-fold or higher was assessed by the Student´s t test (α

Chapter 4
assay, 25 L4-stage nematodes were transferred to the plate containing the S. aureus spots, and each
assay was performed in triplicate. The plates were incubated at 25°C and the numbers of living
nematodes were counted at 24 hour intervals. Nematodes that were not moving after plate tapping or
gentle touching with a platinum wire were counted as dead. Statistical analysis of nematode survival
was performed using the StatView program version 5.0 (SAS Institute Inc.) to create the cumulative
survival plots by the Kaplan-Meier method.
Results and Discussion
Construction of an S. aureus secretion mutant collection
Judged by previous studies in organisms like E. coli and B. subtilis, at least 30 proteins can
fulfill potential roles in protein secretion by S. aureus (Table 1). Of these 30 proteins, the Ffh,
FtsY, SecA1, SecY1, SecE and SpsB proteins are known to be essential for growth of E. coli,
B. subtilis and most likely also S. aureus (Ji et al., 2001;Cregg et al., 1996;Sibbald et al.,
2006). Furthermore, in these organisms the role of YrbF in protein secretion seemed so far of
minor importance. We therefore focused our attention on the potential roles of the remaining
23 proteins in protein secretion. To this purpose, we tried to delete the corresponding genes
with the pMAD chromosomal integration-excision system (Figure 2). In this manner, we were
able to completely delete the secY2, secG, lgt, spsA, lspA, prsA, dsbA, srtA, srtB, tatA, tatC,
comGA, comGC, comC, cidA, or lrgA genes from the S. aureus RN4220 chromosome. In
contrast, several attempts to delete the secA2, secDF, spoIIIJ/yqjG, esaA, essA, essB and essC
genes remained unsuccessful, as was the case for a control experiment in which we tried to
delete spsB. The observation that secDF and spoIIIJ/yqjG could not be deleted is consistent
with the results of a recent Transposon-Mediated Differential Hybridisation (TMDH) analysis
in which about a million transposon mutants were screened using a microarray approach
(Chaudhuri et al., 2009). This analysis revealed 351 S. aureus genes that are of major
importance for growth and cell viability, among which the secDF and spoIIIJ/yqjG genes.
This finding points towards an important difference in the secretion machinery of B. subtilis
and S. aureus since secDF is completely dispensable for growth, cell viability and protein
secretion in B. subtilis (Bolhuis et al., 1998), and the same is true for the individual spoIIIJ
and yqjG genes (Tjalsma et al., 2003). However, consistent with the situation in S. aureus, the
B. subtilis spoIIIJ and yqjG genes cannot be deleted simultaneously, which shows that YidC
function is essential for this organism (Tjalsma et al., 2003). Furthermore, the studies by
Chaudhuri et al. confirmed the essentiality of the ffh, ftsY, secA1, secE, secY1 and spsB genes
(Chaudhuri et al., 2009). Interestingly, the secA2, esaA, essA, essB and essC genes were not
identified as potentially essential in the TMDH analysis. Studies by Burts et al. (Burts et al.,
2005;Burts et al., 2008) have shown that the esaA, essA, essB and essC genes can be mutated,
which suggests that our attempts to delete these genes were unsuccessful due to an unknown
technical problem. However, we can presently not exclude the possibility that these genes are
essential in S. aureus RN4220 while being dispensable in other strains. Direct evidence that
secA2 is dispensable for S. aureus was provided by Siboo et al. (Siboo et al., 2008), who
reported the deletion of the secA2 gene from S. aureus ISP479C. This suggests that secA2 is
either essential in S. aureus RN4220 or that we were unlucky in our attempts to delete this
gene. As a final approach, we therefore attempted to deplete the cells of SecA2 by replacing
the original promoter sequences with the Pspac promoter through a single cross-over
integration of plasmid pMUTIN4 in front of the secA2 gene. As can be expected for a non-
82

Characterization of Staphylococcus aureus secretion mutants
essential gene, cells with the correctly integrated pMUTIN4 construct were able to grow in
the absence of IPTG.
Growth properties of S. aureus secretion mutants
For all obtained S. aureus secretion mutants, the growth in TSB under vigorous shaking at
37°C was monitored through optical density readings at 540 nm (OD540). All mutants derived
from strain RN4220 displayed highly comparable growth rates, reaching OD540 values of ~15
in the post-exponential growth phase. Consistent with this observation, the cells of these
secretion mutants showed no detectable morphological differences (Figure 3).
Figure 3 Scanning electron microscopy of S. aureus secretion mutants. S. aureus RN4220-derived secretion
mutants were grown in TSB and fixated for scanning electron microscopy as described in the Materials and
Methods section. (A) S. aureus RN4220 parental strain, (B) ∆comGA, (C) ∆lgt, (D) ∆secG, (E) ∆srtA, and (F)
∆tatAC.
Remarkably, the comC, comGA, comGC, and prsA mutants derived from S. aureus SH1000
reached significantly higher OD540 values in the post-exponential growth phase (OD540 of
~15-20) than the parental strain SH1000 or the corresponding dsbA, lgt, or lsp mutants (OD540
of ~8-10; Figure 4A). Furthermore, some obtained secY2 mutants in S. aureus SH1000 were
able to grow to high density, whereas others reached optical densities comparable to that of
the parental strain (Figure 4A). Upon close inspection, we observed that also different isolates
of S. aureus SH1000 were able to reach high OD540 values of ~15-20 (Figure 4A), whereas
this was not the case for the strain as it was originally obtained from Dr. Simon Foster
(University of Sheffield, UK).
83

Chapter 4
D
O
540
18
16
14
12
10
8
6
4
2
0
0 5 10 15 20 25
Time (h)
SH1000+
SH1000-
secY2+
secY2-
comGA
dsbA
lgt
prsA
Figure 4 Growth properties of mutant S. aureus strains and RNAIII production. (A) Growth curves of several
different S. aureus SH1000 secretion mutants. Strains were grown in TSB at 37 o C under vigorous shaking and the
OD 540 was measured at hourly intervals. The S. aureus SH1000+ and ∆secY2+ strains show hemolysin activity on
blood agar plates, while the SH1000- and ∆secY2- strains do not show this feature. (B) RNA was prepared from S.
aureus cells grown in TSB to optical densities at 540 nm of 1, 10 or 15. Serial dilutions of total RNA of S. aureus
RN4220 wild-type (WT) or ∆prsA (left panels), or S. aureus SH1000 wild-type (WT) or ∆prsA (right panels) were
blotted and cross linked onto a positively charged nylon membrane. The membrane-bound RNA was hybridized
with a digoxigenin labeled RNA probe complementary to RNAIII. Chemiluminescence signals were detected with a
LumiImager (Roche Diagnostics, Mannheim, Germany).
Since the growth to high density coincided with a non-hemolytic phenotype on blood agar
plates, we tested whether these strains might have accumulated agr mutations, leading to the
loss of the regulatory RNAIII. Indeed, dot blot analyses performed for the S. aureus SH1000
prsA mutant strain, for which this high density growth phenotype was first observed, revealed
the loss of RNAIII production (Figure 4B). In contrast, the S. aureus RN4220 prsA mutant
produced RNAIII at levels comparable to those of the parental strain (Figure 4B).
Figure 5. Agr-like phenotypes of S. aureus SH1000-derived secretion mutants. (A) The S. aureus SH1000
parental strain (WT), an SH1000 ∆cidA strain, an SH1000 ∆comGC strain, an SH1000 ∆dsbA strain, and an
SH1000 ∆prsA strain were grown on blood agar plates and incubated overnight at 37ºC. Hemolytic activity is
detectable as a halo around the streaked cells. (B) S. aureus SH1000 (WT), S. aureus SH1000 ∆cidA, S. aureus
SH1000 ∆comC, S. aureus SH1000 ∆comGA, S. aureus SH1000 ∆comGC, and S. aureus SH1000 ∆dsbA were
grown in TSB medium at 37 o C till the early stationary phase. Samples of extracellular proteins isolated from the
growth medium (M), non-covalently cell wall-bound proteins (CW) and total cells (C) were used for Western
blotting and immunodetection with specific antibodies against TrxA or SspB. Note that the antibodies against TrxA
are also bound by the IgG-binding proteins protein A (Spa) and Sbi. Bands corresponding to Spa, Sbi, SspB and
TrxA are marked with arrows.
84

RN4220 OD 540 of 20
RN4220 ∆comGAOD 540 of 20
Characterization of Staphylococcus aureus secretion mutants
RN4220 ∆comGA
SH1000 OD 540 of 15
SH1000 ∆srtA OD 540 of 15
RN4220 ∆srtA
Geh
SAOUHSC_02241
Hlb
1
HlgB 2
HlgC
3
1 2
1 2 4
Sle1
YfnI
1 2
2
1
3
SssA
HlY
SAOUHSC_00094
Plc
RN4220 OD 540 of 15
RN4220 ∆secG OD 540 of 15
RN4220 OD 540 of 20
RN4220 ∆tatAC OD 540 of 20
RN4220 ∆secG
4
5
6
SAOUHSC_02979
1 2 3 4
1
2 3
IsaA
RN4220 ∆tatAC
85
2 Spa
1
SdrD
RN4220 OD 540 of 15
RN4220 ∆prsA OD 540 of 15
SH1000 OD 540 of 8
SH1000 ∆prsA OD 540 of 15
RN4220 ∆prsA
SH1000 ∆prsA
Figure 6. Extracellular proteomes of S. aureus secretion mutants. (A) Coomassie stained gel of extracellular
proteins of S. aureus RN4220 secretion mutants and the corresponding parental strain. Arrows indicate the major
changes in the banding patterns of extracellular proteins of particular secretion mutants. (B) False-colored dualchannel
images of 2-D gels of extracellular proteins of S. aureus RN4220 ∆comGA, ∆prsA, ∆secG, ∆srtA or
∆tatAC (labeled red) and S. aureus RN4220 (labeled green). Additionally, a false-colored dual-channel image of
2-D gels of extracellular proteins of S. aureus SH1000 ∆prsA (labeled red) and S. aureus SH1000 (labeled green)
is shown. Proteins (350 µg) isolated from the supernatants of S. aureus strains grown in TSB medium were
separated on 2-D gels by using immobilized pH gradient strips in the pH range of 3-10. Proteins were stained with
colloidal Coomassie Brilliant Blue. Spots of proteins present in equal amounts in the media of the mutant and
parental strains appear in yellow, spots of proteins present in higher amounts in media of the mutant strains
appear in red, and spots of proteins present in higher amounts in the medium of the parental strain appear in
green.
B
A

Chapter 4
These findings suggested that several of the secretion mutants obtained in S. aureus SH1000
have accumulated agr mutations, which would impact on the production of various secreted
virulence factors (Novick, 2003). This view was confirmed by a systematic analysis of
hemolysin secretion on blood agar plates (Figure 5A) and Western blotting analyses (Figure
5B), which revealed that all mutants growing to high cell density no longer secreted
hemolysin or the cysteine protease SspB. Instead, these strains secreted protein A at strongly
elevated levels (Figure 5). As shown by Western blotting with antibodies against the
cytoplasmic protein TrxA, the mutations causing the agr phenotype did not affect the
secretion of the second IgG-binding protein Sbi, nor did they result in increased levels of lysis
and subsequent release of TrxA from the cells. Since the secretion of many proteins was
suppressed by the mutations leading to the agr phenotype, as was shown by proteomics
analyses for the S. aureus SH1000 prsA mutant (Figure 6), mutants with such a phenotype
were excluded from all further protein secretion studies.
In addition to hemolysin, protein A and sspB, it has been shown that agr regulates several
proteases, such as serine proteases SplA-F (Saïd-Salim et al., 2003), the metalloprotease Aur
(Arvidson and Tegmark, 2001;Chan and Foster, 1998), and the cysteine protease SspB
(Arvidson and Tegmark, 2001;Saïd-Salim et al., 2003). Furthermore, by zymography we
observed various differences between wild-type strains and strains with an agr phenotype
with respect to the cellular and extracellular accumulation of as yet unidentified cell wall
hydrolases (data not shown). At present the precise nature of the mutations leading to the agr
phenotype of S. aureus SH1000-derived secretion mutants is not clear. PCR on genomic DNA
of these strains and subsequent sequencing revealed that there are no mutations present in the
RNAIII region. This suggests that the mutations are located in other regions of the agr
regulon that control the expression of RNAIII. Though the S. aureus strains used in this study
seem to be suitable for mutagenesis studies, the present results underscore the view that
caution must be taken when deciding on what S. aureus strain to use for mutagenesis studies.
S. aureus SH1000 is clearly suitable for studying many processes, but for mutagenesis with
pMAD-based plasmids, it may be not the best choice due to the high frequency at which the
obtained mutants display an agr-like phenotype. We can only speculate about the possible
reasons. Conceivably, the temperature changes that are needed to promote chromosomal
integration and excision of pMAD may give a competitive growth advantage to SH1000
derivatives with an agr phenotype. However, also the introduction of pMAD itself may
represent a stressful event for the cells that could lead to the selection for mutations that cause
the agr phenotype. It has already been shown that mutations in the agr locus can easily occur
during re-culturing of S. aureus strains (Somerville et al., 2002), and this also seems to
happen in patients (Traber et al., 2008). Our present results indicate that the same can occur
with S. aureus SH1000, especially when cells are subjected to our pMAD-based mutagenesis
regime.
Exoproteome analysis by SDS-PAGE and proteomics
To study the effects of the different deletions of secretion machinery genes, the exoproteomes
of the mutant S. aureus strains were analyzed by SDS-PAGE or 2D-gelelectrophoresis. This
revealed that the exoproteomes of some mutants were severely changed due to the respective
gene deletions, while the exoproteomes of other deletion mutants had remained unaltered
(Figure 6). Especially, the secG and lgt mutants displayed a substantially different
exoproteome composition compared to the parental strain RN4220 (Figure 6A).
86

Characterization of Staphylococcus aureus secretion mutants
The significant differences in the exoproteome of the secG mutant were highly unexpected
since deletion of secG in E. coli and B. subtilis does not seem to have a strong impact on
protein export in these bacteria (Nishiyama et al., 1994;Van Wely et al., 1999). As further
detailed in Chapter 5, many proteins are present at increased or decreased levels in the
exoproteome of the S. aureus RN4220 secG mutant and this effect was reversed by ectopic
expression of secG. Proteins that were detected in reduced amounts include well-known
secreted virulence factors, such as the lipase Geh, the alpha and beta hemolysins and the
leukocidins F and S, as well as the cell surface proteins SdrC and SdrD. Conversely, the
extracellular levels of other virulence factors, like protein A, the immunodominant antigen A
(IsaA) and the secretory antigen precursor SsaA were significantly increased. These
observations suggest that the absence of SecG changes the translocation efficiency of
different exoproteins via the SecYE channel to different extents. However, we cannot
completely rule out indirect effects on the transcription of the genes for certain exoproteins,
their translation, or their post-translocational folding or degradation. In contrast to the secG
mutation, no secretion defect was observed upon the deletion of the gene for the accessory
Sec channel component SecY2. This confirmed the results obtained by Siboo et al., who
showed that a secY2 mutation does not alter the S. aureus exoproteome (Siboo et al., 2008).
Till now, the only known SecY2-dependent protein of S. aureus is SraP, and it seems as if the
SecA2/SecY2 translocon is devoted to the translocation of this protein alone. Whether SecG
and/or SecE interact with this accessory SecA2/SecY2 translocon remains to be determined.
Similar to the secY2 deletion strain, no secretion defects were observed for strains lacking the
tatA and/or tatC genes, the comGA, comGC or comC genes, or the genes for the holin CidA
and antiholin LrgA (Figure 6B). This suggests that the Tat, Comand holing-antiholin
pathways serve very specific roles in the translocation of particular non-abundantly expressed
proteins, or proteins that are not detectable by 2-D PAGE due to their particular pI and size
properties. This view was recently confirmed for the S. aureus Tat pathway, which seems to
be required for the translocation of only one protein, namely FepB (Biswas et al.,
2009;Yamada et al., 2007). So far, we have not been able to detect FepB by our 2-D PAGE
approach.
The spsA gene encodes a type I signal peptidase that seems to lack the active site serine and
lysine residues (van Roosmalen et al., 2004). Interestingly, the occurrence of this type of an
apparently inactive type I signal peptidase is wide-spread amongst the Firmicutes.
Unfortunately, our proteomics studies did not shed light on the biological function of SpsA as
no differences were observed between the exoproteomes of our spsA deletion mutant and the
parental strain RN4220 (Figure 6).
The exoproteome of the lgt mutant showed the most pronounced differences compared to the
parental strain (Figure 6A). This result was not completely unexpected as previous studies of
Stoll et al. (Stoll et al., 2005) had shown that an S. aureus lgt mutant released high amounts of
certain unmodified prelipoproteins, such as the oligopeptide-binding protein OppA, the
peptidyl-prolyl cis-trans isomerase PrsA, and the staphylococcal iron transporter SitC, into the
growth medium. As shown by Western blotting, the same was true for the unmodified pre-
DsbA precursor, which was completely released into the growth medium of lgt mutant cells.
In contrast, in the parental strain, the mature DsbA fractionated with cells and non-covalently
cell wall-associated proteins (Figure 7). The latter finding seems to suggest that some mature
DsbA is not retained at the membrane but in the cell wall. These findings confirm earlier
observations of pre-lipoprotein release by lgt mutant strains of B. subtilis (Antelmann et al.,
2001;Tjalsma et al., 1999) and L. lactis (Venema et al., 2003). The release of lipoprotein
87

Chapter 4
precursors by lgt mutant cells indicates that lipoprotein signal peptides are too short or
insufficiently hydrophobic to retain the unmodified lipoprotein precursors in the membrane.
The fact that the exoproteome of the lspA mutant strain, lacking the lipoprotein-specific signal
peptidase II, remained unaltered confirms the notion that correctly lipid-modified but
immature lipoprotein precursors do remain anchored to the membrane (Figure 6A).
Figure 7. Localization of the lipoprotein DsbA in different S. aureus secretion mutants. S. aureus SH1000 (WT),
S. aureus SH1000 ∆dsbA, and S. aureus SH1000 ∆lgt were grown in TSB medium at 37 o C till the early stationary
phase. Samples of extracellular proteins isolated from the growth medium (M), non-covalently cell wall-bound
proteins (CW) and total cells (C) were used for Western blotting and immunodetection with specific antibodies
against DsbA. Bands corresponding to the precursor (preDsbA) and mature forms of DsbA are marked with
arrows.
Remarkably, deletion of either of the two known extracytoplasmic catalysts for protein
folding, namely DsbA and PrsA, had no detectable effects on the composition of the S. aureus
exoproteome (Figure. 6, A and B). This suggests that PrsA is required for the folding of only
a very limited number of extracytoplasmic proteins of S. aureus that are not readily detectable
by 2-D PAGE or that are not expressed under the tested experimental conditions. Likewise,
our results indicate that DsbA has a very specific function, as was previously proposed on the
basis of genetic and biochemical analyses (Kouwen et al., 2007;Dumoulin et al., 2005).
Unexpectedly, 2-D PAGE revealed no clear differences in the exoproteomes of the srtA or
srtB mutants compared to the parental strain (Figure 6). Proteins with an LPxTG, LPxAG or
NPQTN motif are not covalently anchored to the cell wall in the srtA or srtB mutants.
Therefore, one might expect the release of these proteins into the growth medium of srtA or
srtB mutant strains. However, it is well conceivable that some proteins released by srtA or
srtB mutant cells escaped detection by 2-D PAGE, because of their physical properties,
because they are expressed at very low levels, or because they are not expressed at all under
the tested conditions. The latter is most likely true for the srtB mutant since the only known
substrate, IsdC, is only expressed under iron-limiting conditions (Mazmanian et al., 2003).
Furthermore, it has been shown that the sortase A substrate SasG is not detectable in wildtype
strains of S. aureus (Corrigan et al., 2007). Another possible explanation for the fact that
no LPxTG proteins were identified in the exoproteome of the srtA mutant is that these
proteins may remain linked to the cell wall in other ways. For example, all LPxTG proteins
have a C-terminal membrane anchor that is proteolytically removed during covalent cell wall
attachment of these proteins by sortase A. This anchor has the potential to retain the Cterminally
uncleaved proteins in the membrane. In addition, a range of LPxTG proteins also
contain other cell wall binding motifs (repeats) that would maintain protein linkage to the cell
wall even in absence of sortase A. These issues have been addressed in more detail in
Chapter 6.
88

Characterization of Staphylococcus aureus secretion mutants
Caenorhabditis elegans killing assay
Many S. aureus proteins that are transported to the cell surface or extracellular milieu have a
role in virulence (Sibbald et al., 2006). This raises the question to what extent the generated
mutations influence staphylococcal virulence. In most published studies this is addressed by
experiments with animal models such as mice or rats. However, such analyses are timeconsuming
and require special facilities. Therefore, we explored the use of a Caenorhabditis
elegans killing assay to screen for possible virulence defects of our secretion mutants.
Importantly, this assay was previously used by Sifri and colleagues to study the effects of
several S. aureus strains on the survival of nematodes (Sifri et al., 2003), including strains
that carried mutations in regulators of virulence factors, such as agr and sarA, or strains that
did not produce α-hemolysin. The S. aureus strains that carried these mutations were
attenuated in killing the nematodes and this showed that the assay could be used to probe the
virulence of particular S. aureus strains. The results of the C. elegans killing assays for a
selection of our S. aureus secretion mutants are shown in Figure 8.
Figure 8. Kaplan-Meier survival plots of C. elegans infected with S. aureus mutants. S. aureus strains were
spotted on TSA plates and nematodes were placed onto these plates. Live nematodes were counted every 24 hours
and transferred to fresh TSA plates containing S. aureus spots. The assay for each strain was performed three
times. The plots were drawn with the StatView version 5.0 program (SAS Institute, Inc.).
The tested S. aureus dsbA, spsA srtA, secG, secY2 and secG-secY2 mutants displayed C.
elegans killing rates, which were comparable to that of the parental strain. There were,
however, a few mutants that showed attenuated killing rates, such as the lspA and srtB
mutants. Conversely, the results in Figure 8 imply that lgt and tatAC mutant strains are
slightly more virulent. The effect of the lspA mutation indicates that uncleaved lipoproteins,
retained at the membrane in the absence of signal peptidase II, are insufficiently active for full
virulence of S. aureus towards C. elegans. On the other hand, the increased release of
lipoproteins by the lgt mutant may make S. aureus more virulent towards C. elegans. It has
been shown previously that lipoproteins are responsible for activation of TLR2 and that such
proteins are an important factor for the pathogenesis of S. aureus (Hashimoto et al.,
2006;Kurokawa et al., 2009;Schmaler et al., 2009). Conversely, it has been reported that S.
89

Chapter 4
aureus lgt mutants may be hypervirulent in vivo (Stoll et al., 2005;Bubeck et al., 2006), which
would be in line with our present findings. The observation that the srtB mutant is attenuated
in C. elegans killing suggests that the IsdC protein is required for C. elegans killing and that
S. aureus cells are exposed to iron-limiting conditions during C. elegans infection
(Mazmanian et al., 2003). This would in fact suggest that the reason why the lspA mutant is
attenuated might be related to the malfunction of certain lipoproteins involved in iron uptake
(Schmaler et al., 2009). Remarkably, the tatAC mutant showed a higher killing rate compared
to the parental strain. This finding is somewhat difficult to reconcile with the observed C.
elegans killing phenotypes of the srtB and lspA mutants, because the only confirmed substrate
for the S. aureus Tat pathway is the FepB protein, which is an iron-dependent peroxidase
needed for iron uptake (Biswas et al., 2009). Moreover, the bacterial loads of tatAC and tatfep
mutant strains in a mouse kidney abscess model were decreased, suggesting a requirement
of the Tat pathway for virulence. At present, it remains unfortunately unclear why the effects
of the srtB and lspA mutations on the one hand and the tatAC mutation on the other hand are
so different. Finally, it is important to note that no C. elegans killing phenotype was observed
for the srtA mutant. Clearly, the proteins anchored by sortase A to the cell surface are very
important for the virulence of S. aureus towards mammals as they are, for example, involved
in the binding of fibrinogen (McDevitt et al., 1997), binding to nasal epithelial cells (Corrigan
et al., 2009), evasion of the immune system (Sasso et al., 1991) or biofilm formation
(Corrigan et al., 2007). Possibly, these traits are less relevant for S. aureus virulence towards
much simpler host organisms such as nematodes.
Outlook
In conclusion, our present studies show that many of the non-essential determinants for
protein secretion by S. aureus have only a limited impact on general protein secretion by this
organism. This implies that these factors are mainly required for special purposes that may,
however, be relevant for the virulence of S. aureus. Defining the precise roles of such
secretion factors will require more in-depth studies, especially under infection mimicking
conditions. An important lesson that was learned from the studies with the S. aureus strain
SH1000 is that this strain quite rapidly accumulates mutations causing an agr-like phenotype.
This underscores the need for genetically stable model strains for molecular genetics and
proteomics approaches to fully define the roles of the secretome in staphylococcal virulence.
Acknowledgements
We like to thank W. Baas for technical assistance, and colleagues from the StaphDynamics
and TIPharma programs for helpful discussions. Financial support was provided by CEU
(StaphDynamics, LSHM-CT-2006-019064; DFG (GK212/3-00, SFB/TR34, FOR 585), and
Top Institute Pharma (T4-213).
90

“Besides being a guitar player, I'm a big fan of the guitar
I love that damn instrument”
-Steven S. Vai-
92

Chapter 5
Synthetic effects of secG and secY2 mutations on exoproteome
biogenesis in Staphylococcus aureus
M.J.J.B. Sibbald # , T. Winter # , M.M. van der Kooi-Pol, T. Bosma, T. Schäfer, K. Ohlsen,
M. Hecker, H. Antelmann, S. Engelmann, and J.M. van Dijl
# both authors contributed equally to this work
Submitted for publication, in revision
93

Chapter 5
Summary
The Gram-positive pathogen Staphylococcus aureus secretes various proteins into its
extracellular milieu. Bioinformatics analyses have indicated that most of these proteins
are directed to the canonical Sec pathway, which consists of the translocation motor
SecA and a membrane-embedded channel composed of the SecY, SecE and SecG
proteins. In addition, S. aureus contains an accessory Sec2 pathway involving the SecA2
and SecY2 proteins. Here we have addressed the roles of the non-essential channel
components SecG and SecY2 in the biogenesis of the extracellular proteome of S. aureus.
The results show that SecG is of major importance for protein secretion by S. aureus.
Specifically, the extracellular accumulation of eight abundant exoproteins and seven cell
wall-bound proteins was significantly affected in the secG mutant. No secretion defects
were detected for strains with a secY2 single mutation. However, deletion of secY2
exacerbated the secretion defects of secG mutants, affecting the extracellular
accumulation of one additional exoprotein and one cell wall protein. Furthermore, the
secG secY2 double mutant displayed a synthetic growth defect. These findings suggest
that SecY2 can interact with the Sec1 channel of S. aureus. Such an interaction would be
consistent with the presence of a single set of secE and secG genes in S. aureus.
94

Synthetic effects of secG and secY2 mutations on exoproteome biogenesis in
Staphylococcus aureus
Introduction
Staphylococcus aureus is a well-represented component of the human microbiota as nasal
carriage of this Gram-positive bacterium has been shown for 30-40% of the population
(Peacock et al., 2001). This organism can, however, turn into a dangerous pathogen that is
able to infect almost every tissue in the human body. S. aureus has become particularly
notorious for its high potential to develop resistance against commonly used antibiotics
(Hiramatsu et al., 1997; Weigel et al., 2003). Accordingly, the S. aureus genome encodes an
arsenal of virulence factors that can be expressed when needed at different stages of growth.
These include surface proteins and invasins that are necessary for colonization of host tissues,
surface-exposed factors for evasion of the immune system, exotoxins for the subversion of
protective host barriers, and resistance proteins for protection against antimicrobial agents
(Schneewind et al., 1992).
Most proteinaceous virulence factors of S. aureus are synthesized with an N-terminal
signal peptide to direct their transport from the cytoplasm across the membrane to an
extracytoplasmic location, such as the cell wall or the extracellular milieu (Schneewind et al.,
1992; Sibbald et al., 2006). Based on signal peptide predictions using a variety of algorithms,
it is believed that most exoproteins of S. aureus are exported to extracytoplasmic locations via
the general Secretory (Sec) Pathway (Sibbald et al., 2006). These pre-proteins are bound by
the translocation motor protein SecA. Through repeated cycles of ATP binding and
hydrolysis, SecA pushes the protein in an unfolded state through the membrane-embedded
SecYEG translocation channel (Driessen and Nouwen, 2008; Papanikou et al., 2007). This
channel is homologous to the eukaryotic Sec61αγβ channel and it can be found in all three
kingdoms of life (Pohlschröder et al., 1997; Yuan et al., 2009). Upon initiation of the
translocation process, the proton-motive force is thought to accelerate pre-protein
translocation through the Sec channel (Nishiyama et al., 1993). Recently, the structure of the
SecA/SecYEG complex from the Gram-negative bacterium Thermotoga maritima was solved
at 4.5 A resolution (Zimmer et al., 2008). In this structure, one SecA molecule is bound to
one set of SecYEG channel proteins. The core of the Sec translocon consists of the SecA,
SecY and SecE proteins, which are essential for growth and viability of bacteria, such as
Escherichia coli and Bacillus subtilis (Cabelli et al., 1988; Brundage et al., 1990; Kobayashi
et al., 2000). In contrast, the channel component SecG is dispensable for growth, cell viability
and protein translocation (Nishiyama et al., 1993). Nevertheless, SecG does enhance the
efficiency of pre-protein translocation through the SecYE channel. This is of particular
relevance at low temperatures and in the absence of a proton-motive force (Hanada et al.,
1996). Several studies suggest that E. coli SecG undergoes topology inversion during preprotein
translocation (Nishiyama et al., 1993; Nagamori et al., 2000; Sugai et al., 2007). Even
so, van der Sluis et al. reported that SecG cross-linked to SecY is fully functional despite its
fixed topology (van der Sluis et al., 2006). During or shortly after membrane translocation of
a pre-protein through the Sec channel, the signal peptide is removed by signal peptidase. This
is a prerequisite for the release of the translocated protein from the membrane (Antelmann et
al., 2001; van Roosmalen et al., 2004).
Several pathogens, including Streptococcus gordonii, Streptococcus pneumonia,
Bacillus anthracis, Bacillus cereus, and S. aureus contain a second set of chromosomal secA
and secY genes named secA2 and secY2, respectively (Sibbald and van Dijl, 2009).
Comparison of the amino acid sequences of the SecY1 and SecY2 proteins shows that their
similarity is low (about 20% identity), and that the conserved regions are mainly restricted to
95

Chapter 5
the membrane spanning domains. It has been shown for S. gordonii that the transport of at
least one protein is dependent on the presence of SecA2 and SecY2. This protein, GspB, is a
large cell-surface glycoprotein that is involved in platelet binding (Bensing and Sullam,
2002). The protein contains an unusually long N-terminal signal peptide of 90 amino acids,
large serine-rich repeats, and a C-terminal LPxTG motif for covalent cell wall binding. The
gspB gene is located in a gene cluster with the secA2 and secY2 genes. Two other genes in
this cluster encode for the glycosylation proteins GftA and GftB, which seem to be necessary
for stabilization of pre-GspB. Furthermore, the asp4 and asp5 genes in the secA2 secY2 gene
cluster show similarity to secE and secG, and they are important for GspB export by S.
gordonii (Takamatsu et al., 2005). Despite this similarity, SecE and SecG cannot complement
for the absence of Asp4 and Asp5, respectively. The secA2/secY2 gene cluster is also present
in S. aureus, but homologues of the asp4 and asp5 genes are lacking. This seems to suggest
that SecA2 and SecY2 of S. aureus share the SecE and SecG proteins with SecA1 and SecY1.
The sraP gene in the secA2/secY2 gene cluster of S. aureus encodes a protein with similar
features as described for GspB. Siboo and colleagues (Siboo et al., 2005) have shown that
SraP is glycosylated and capable of binding to platelets. Importantly, the disruption of sraP
resulted in a decreased ability to initiate infective endocarditis in a rabbit model. Consistent
with the findings in S. gordonii, SraP export was shown to depend on SecA2/SecY2 (Siboo et
al., 2008). However, it has remained unclear whether other S. aureus proteins are also
translocated across the membrane in a SecA2/SecY2-dependent manner.
The present studies were aimed at defining the roles of two Sec channel components,
SecG and SecY2, in protein secretion by S. aureus. The results show that secG and secY2 are
not essential for growth and viability of S. aureus. While the absence of SecY2 by itself had
no detectable effect, the absence of SecG had a profound impact on the composition of the
exoproteome of a S. aureus. Various extracellular proteins were present in decreased amounts
in the growth medium of secG mutant strains, which is consistent with impaired Sec channel
function. However, a few proteins were present in increased amounts. Furthermore, the
absence of secG caused a serious decrease in the amounts of the cell wall-bound Sbi protein.
Most notable, a secG secY2 double mutant strain displayed synthetic growth and secretion
defects.
Material & Methods
Bacterial strains and plasmids
All strains used in this study are listed in Table 1. Unless stated otherwise, E. coli strains were grown in
Luria-Bertani broth (LB). S. aureus strains were grown at 37°C in tryptic soy broth (TSB) under
vigorous shaking or on trypic soy agar (TSA) plates. If appropriate, media for E. coli were
supplemented with 100 µg/ml ampicillin or 100 µg/ml erythromycin, and media for S. aureus with 5
µg/ml erythromycin, 5 µg/ml tetracyclin or 20 µg/ml kanamycin. To monitor β-galactosidase activity in
cells of E. coli and S. aureus, 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) was added to
the plates at a final concentration of 80 µg/ml.
96

Synthetic effects of secG and secY2 mutations on exoproteome biogenesis in
Staphylococcus aureus
Table 1. Plasmids and bacterial strains used
Plasmids Properties Reference
TOPO pCR®-Blunt II-TOPO® vector; Km R Invitrogen Life
technologies
pCN51 E. coli / S. aureus shuttle vector that contains a cadmium-inducible (Charpentier et
promoter
al., 2004)
pMAD E. coli / S. aureus shuttle vector that is temperature-sensitive in S.
aureus and contains the bgaB gene, Ery R , Amp R
(Arnaud et al.,
2004)
pUC18 Amp R , ColE1, F80dLacZ, lac promoter (Norrander
al., 1983)
et
pDG783 1.5-kb kanamycin resistance cassette in pSB118; Amp R (Guérout-Fleury
et al., 1995)
secG-pCN51 pCN51 with S. aureus secG gene, Amp R ; Ery R This work
secY2-pCN51 pCN51 with S. aureus secY2 gene, Amp R ; Ery R This work
Strains
E. coli
Genotype Reference
DH5α supE44; hsdR17; recA1; gyrA96; thi-1; relA1 (Hanahan,
1983)
TOP10 Cloning host for TOPO vector; F - mcrA ∆(mrr-hsdRMS-mcrBC)
Φ80lacZ∆M15 ∆lacX74 recA1 araD139 ∆(ara-leu)7697 galU
galK rpsL (Str R ) endA1 nupG
S. aureus RN4220
Parental strain Restriction-deficient derivative of NCTC 8325, cured of all known
prophages, partial defect in agr
97
Invitrogen Life
technologies
(Kreiswirth et
al., 1983)
∆secG secG This work
∆secY2 secY2 This work
∆secG ∆secY2 secG secY2 This work
S. aureus SH1000
WT Functional rsbU+ derivative of 8325-4 rsbU+, agr+ (Horsburgh et
al., 2002)
∆secG rsbU+, agr+, secG This work
∆secY2 rsbU+, agr+, secY2 This work
∆secG ∆secY2 rsbU+, agr+, secG secY2 This work
S. aureus Newman
∆spa spa (Patel et al.,
1987)
∆spa ∆sbi spa sbi This work
Construction of S. aureus mutant strains
Mutants of S. aureus were constructed using the temperature-sensitive plasmid pMAD (Arnaud et al.,
2004) and previously described procedures (Kouwen et al., 2009c). Primers (Table 2) were designed
using the genome sequence of S. aureus NCTC8325 (http://www.ncbi.nlm.nih.gov/nuccore/
NC_007795). All mutant strains were checked by isolation of genomic DNA using the GenElute
Bacterial Genomic DNA Kit (Sigma) and PCR with specific primers.
To delete the secG or secY2 genes, primer pairs with the designations F1/R1 and F2/R2 were
used for PCR amplification of the respective upstream and downstream regions (each ~500 bp), and
their fusion with a 21 bp linker. The fused flanking regions were cloned in pMAD, and the resulting
plasmids were used to delete the chromosomal secG or secY2 genes of S. aureus RN4220. To delete the
secG or secY2 genes from the S. aureus SH1000 genome, the respective pMAD constructs were
transferred from the RN4220 strain to the SH1000 strain by transduction with phage φ85 (Novick,
1991).

Chapter 5
Table 2. Primers used in this study
Primer Sequence (5’→3’)
secG-F1 TTAAAACAGGACGCTTTATTG
secG-R1 TTACGTCAGTCAGTCACCATGGCA AAATTGTCCTCCGTTCCTTAT
secG-F2 TGCCATGGTGACTGACTGACGTAA GGTCCGGCGATGTAAATGTCG
secG-R2 GCGTGCATATTCTAAAAAGCC
secY2-F1 TGTCTGGTTCACAAAGCATTT
secY2-R1 TTACGTCAGTCAGTCACCATGGCA GTTGCACCTCTTTTATATCAA
secY2-F2 TGCCATGGTGACTGACTGACGTAA GGAGGTAATTATGAAATACTT
secY2-R2 GCCTCTCCCTGATCATCAAAA
sbi-F1 TGTGTTCCTTTATTTTCTGCG
sbi-R1 GAACTCCAATTCACCCATGGCCCCC CCCCAACTAGCAACTTCGAG
sbi-F2 CCGCAACTGTCCATACCATGGCCCCC GGAAATAATCAATCAAAAATATCTTCTC
sbi-R2 CTATTAAACCAACTGCTAAAGTTGC
kan-F1 GGGGGCCATGGGTGAATTGGAGTTCGTCTTG
kan-R1 GGGGGCCATGGTATGGACAGTTGCGGATGTA
secG-F3 GGGGGGTCGACGGGATATACTACTTGTCGTATATA
secG-R3 GGGGGGAATTCCCTTACATACCAAGATAACTTATGCA
secY2-F3 GGGGGGTCGACGTCTTTTTAATGTTTTTGATA
secY2-R3 GGGGGGAATTCCCTTACCAATACTGGTTTAAAAATGG
spa_for ACCTGCTGCAAATGCTGCGC
spa_rev a CTAATACGACTCACTATAGGGAGA GGTTAGCACTTTGGCTTGGG
geh_for CACATCAAATGCAGTCAGG
geh_rev a CTAATACGACTCACTATAGGGAGA AATCGACATGATCCCATCC
hlb_for ATCAAACACCTGTACTCGG
hlb_rev a CTAATACGACTCACTATAGGGAGA CGTAGTAATATGGGAACGC
Overlap in primers are in bold; restriction sites are underlined
a Oligonucleotides containing the recognition sequence for T7 polymerase at the 5’ end (shown in italic)
To create the spa sbi double mutant of S. aureus Newman, the sbi gene was deleted from a spa
mutant strain kindly provided by T. Foster (Patel et al., 1987). For this purpose, the kanamycin
resistance marker encoded by pDG783 was introduced between the sbi flanking regions via PCR with
the primer pairs sbi-F1/sbi-R1, sbi-F2/sbi-R2 and kan-F1/kan-R1. The obtained ~1000 bp fragment
was ligated into pMAD, and the resulting plasmid was used to transform competent S. aureus Newman
spa cells. Blue colonies were selected on TSA plates with erythromycin and kanamycin, and the spa
sbi double mutant was subsequently identified following the previously described protocol (Kouwen et
al., 2009c).
For complementation studies, the secG or secY2 genes were cloned into plasmid pCN51
(Charpentier et al., 2004). Expression of genes cloned in this plasmid is directed by a cadmiuminducible
promoter. Primer pairs with the F3/R3 designation (Table 2) were used to amplify the secG
or secY2 genes. These primers contain an EcoRI restriction site at the 5’ end and a SalI restriction site
at the 3’end of the amplified gene. PCR products were purified using the PCR Purification Kit (Roche),
and ligated into the TOPO-vector (Invitrogen). The resulting constructs were then cut with EcoRI and
SalI, and the secG or secY2 genes (284 and 1233 bp, respectively) were isolated from an agarose gel
and ligated into pCN51 cut with EcoRI and SalI. This resulted in the secG- and secY2-pCN51
plasmids. Competent S. aureus RN4220 ∆secG, ∆secY2 or ∆secG ∆secY2 cells were transformed with
these plasmids by electroporation and colonies were selected on TSA plates containing erythromycin.
The plasmids were then transferred to S. aureus SH1000 by transduction as described above.
Analytical and preparative two-dimensional (2-D) PAGE
Extracellular proteins from 100 ml culture supernatant were precipitated, washed, dried, and resolved
as described previously (Ziebandt et al., 2004). The protein concentration was determined using Roti ® -
Nanoquant (Carl Roth GmbH & Co, Karlsruhe, Germany). Preparative 2-D PAGE was performed by
98

Synthetic effects of secG and secY2 mutations on exoproteome biogenesis in
Staphylococcus aureus
using the immobilized pH gradient technique (Bernhardt et al., 1999; Eymann et al., 2004). The protein
samples (350 µg) were separated on immobilized pH gradient strips (Amersham Pharmacia Biotech,
Piscataway, NJ) with a pH range of 3-10. The resulting protein gels were stained with colloidal
Coomassie Blue G-250G (Candiano et al., 2004) and scanned with the light scanner. Each experiment
was performed at least three times.
For identification of proteins by MALDI-TOF MS, Coomassie-stained protein spots were
excised from gels using a spot cutter (Proteome Work TM ) with a picker head of 2 mm and transferred
into 96-well microtiter plates. Digestion with trypsin and subsequent spotting of peptide solutions onto
the MALDI targets were performed automatically in an Ettan Spot Handling Workstation (GE-
Healthcare, Little Chalfont, United Kingdom) using a modified standard protocol. MALDI-TOF MS
analyses of spotted peptide solutions were carried out on a Proteome-Analyzer 4700/4800 (Applied
Biosystems, Foster City, CA) as described previously (Eymann et al., 2004). MALDI-TOF-TOF
analysis was performed for the three highest peaks of the TOF spectrum as described previously
(Eymann et al., 2004; Wolff et al., 2007). Database searches were performed using the GPS explorer
software version 3.6 (build 329) with the organism-specific databases.
By using the MASCOT search engine version 2.1.0.4. (Matrix Science, London, UK) the
combined MS and MS/MS peak lists for each protein spot were searched against a database containing
protein sequences derived from the genome sequences of S. aureus NCTC8325. Search parameters
were as described previously (Wolff et al., 2007). For comparison of protein spot volumes, the Delta
2D software package was used (Decodon GmbH Germany). The induction ratio of parental strain to
mutant was calculated for each spot (normalized intensity of a spot on the parental image/normalized
intensity of the corresponding spot on the mutant image). The significance of spot volume differences
of two-fold or higher was assessed by the Student´s t test (α

Chapter 5
incubated at 95ºC. Proteins were separated by SDS-PAGE using precast NuPage gels (Invitrogen) and
subsequently blotted onto a nitrocellulose membrane (Protran ® , Schleicher & Schuell). The presence of
IsaA, Aly or DsbA was monitored by immunodetection with specific polyclonal antibodies raised in
mice (IsaA, Aly) or rabbits (DsbA (Kouwen et al., 2007)) at 1:10.000 dilution. Bound primary
antibodies were visualized using fluorescent IgG secondary antibodies (IRDye 800 CW goat antimouse/anti-rabbit
from LiCor Biosciences). Membranes were scanned for fluorescence at 800 nm using
the Odyssey Infrared Imaging System (LiCor Biosciences).
Results
The exoproteomes of secG and secY2 mutant S. aureus strains
To investigate the roles of SecG and SecY2 in the biogenesis of the S. aureus exoproteome,
the respective genes were completely deleted from the chromosome of S. aureus strain
RN4220. This resulted in the single mutant strains ∆secG and ∆secY2, and the double mutant
∆secG ∆secY2. Next, cells of these mutants were grown in TSB medium until they reached
the stationary phase (Figure 1; not shown for the ∆secY2 strain).
Figure 1 Growth of S. aureus secG and secG secY2 mutants. The S. aureus strains RN4220 ∆secG (A), ∆secG
∆secY2 (B), and the parental strain RN4220 were grown in 100 ml TSB medium under vigorous shaking at 37 o C.
Sampling points for the preparation of extracellular proteins are indicated in the growth curve by arrows.
All three mutants displayed similar exponential growth rates as the parental strain. However,
the secG secY2 double mutant entered the stationary phase at a lower optical density
(OD540=8) than the parental strain and the ∆secG mutant (OD540=15). Extracellular proteins
were collected from the supernatant of the cell cultures that had reached stationary phase for
analysis by 2-D PAGE (Figures 1 and 2). Comparison of the exoproteomes of the secG
mutant and its parental strain revealed that eleven proteins with Sec-type signal peptides and
type I signal peptidase cleavage sites (i.e. SAOUHSC-00094, SdrD, Sle1, Geh, Hlb, HlY,
HlgB, HlgC, Plc, SAOUHC-02241 and SAOUHSC-02979) were present in significantly
decreased amounts when SecG was absent from the cells. This was also true for the secreted
moiety of the polytopic membrane protein YfnI, which is processed by signal peptidase I as
was previously shown for the YfnI homologue of B. subtilis (Antelmann et al., 2001). In
contrast, the amounts of three other exoproteins (i.e. IsaA, SsaA, and Spa) were considerably
increased due to the secG deletion (Figure 2A; Table 3A). These effects of the secG mutation
were fully compensated when secG was ectopically expressed from plasmid secG-pCN51
(Figure 2C). Northern blot analyses revealed similar transcript levels for geh, hlb and spa in
the secG mutant and the parental strain RN4220. This shows that the changes in the amounts
100

Synthetic effects of secG and secY2 mutations on exoproteome biogenesis in
Staphylococcus aureus
of the respective exoproteins in the secG mutant were not caused by a decreased transcription
of the corresponding genes (Figure 3).
Geh
HlgB HlgC
Sle1
SAOUHSC_02241
1
2
2
1
3
1 2 4
SssA
3
HlY
Hlb
SAOUHSC_00094
Plc
RN4220 OD 540 of 15
RN4220 ∆secG OD 540 of 15
4
1 2
5
6
SAOUHSC_02979
1 2 3 4
RN4220 OD 540 of 15
RN4220 ∆secG secG-pCN51OD 540 of 15
1
2
IsaA
3
2 Spa
1
SdrD
A
C
HlgC
101
Geh
1
LipA
SAOUHSC_02979
1 2 3
2
2
SAOUHSC_02241
1
2
3
HlY
Hlb
1 2
1 2
3 4
SssA
SAOUHSC_00094
4
Plc
LytM
RN4220 OD 540 of 15
RN4220 ∆secG ∆secY2 OD 540 of 8
Figure 2. The extracellular proteomes of S. aureus secG and secG secY2 mutants. (A) False-colored dualchannel
image of 2D gels of extracellular proteins of S. aureus RN4220 (green) and S. aureus RN4220 ∆secG
(red). Proteins (350 µg) isolated from the supernatant of S. aureus RN4220 and S. aureus RN4220 ∆secG grown in
TSB medium to an OD 540 of 15 were separated on 2D gels by using immobilized pH gradient strips in the pH
range of 3-10. Proteins were stained with colloidal Coomassie Brilliant Blue. Protein spots present in equal
amounts in both strains appear in yellow, protein spots present in higher amounts in the secG mutant appear in
red, and protein spots present in higher amounts in the parental strain appear in green. (B) False-colored dualchannel
image of 2D gels of extracellular proteins of S. aureus RN4220 (green) and S. aureus RN4220 ∆secG
∆secY2 (red). For experimental details see (A). Protein spots present in equal amounts in both strains appear in
yellow, protein spots present in higher amounts in the secG secY2 mutant appear in red, and protein spots present
in higher amounts in the parental strain appear in green. (C) False-colored dual-channel image of 2D gels of
extracellular proteins of S. aureus RN4220 (green) and S. aureus RN4220 ∆secG secG-pCN51 (red). For
experimental details see (A). All protein spots are yellow, indicating that both strains secreted the respective
proteins in equal amounts.
6
1 2
IsaA
4
3
2
1
Spa
B

Chapter 5
Table 3A: Cell wall proteins with altered secretion patterns in S. aureus ∆secG and ∆secG∆secY2
Protein a Function Mr/pI
mature
ORFID S. aureus
NCTC8325
102
Accession
NCBI
Relative level compared to parental strain c
Predicted
location b ∆secG/WT ∆secG ∆secY2/WT
IsaA 1 immunodominant antigen A 24.2/6.6 SAOUHSC_02887 88196515 cell wall 2.0 4.2
IsaA 2 immunodominant antigen A 24.2/6.6 SAOUHSC_02887 88196515 cell wall 2.4 8.6
IsaA 3 immunodominant antigen A 24.2/6.6 SAOUHSC_02887 88196515 cell wall 2.9 4.8
IsaA 4 immunodominant antigen A 24.2/6.6 SAOUHSC_02887 88196515 cell wall 12.4
LytM peptidoglycan hydrolase, putative 34.3/6.7 SAOUHSC_00248 88194055 cell wall 4.3
SAOUHSC_00094 1 hypothetical protein 21.8/9.4 SAOUHSC_00094 88193909 cell wall 0.2
SAOUHSC_00094 2 hypothetical protein 21.8/9.4 SAOUHSC_00094 88193909 cell wall 0.3
SAOUHSC_00094 3 hypothetical protein 21.8/9.4 SAOUHSC_00094 88193909 cell wall 0.4
SAOUHSC_00094 4 hypothetical protein 21.8/9.4 SAOUHSC_00094 88193909 cell wall 0.3 0.3
SAOUHSC_00094 5 hypothetical protein 21.8/9.4 SAOUHSC_00094 88193909 cell wall 0.5
SAOUHSC_00094 6 hypothetical protein 21.8/9.4 SAOUHSC_00094 88193909 cell wall 0.2 0.3
SdrD 1 SdrD protein, putative 14.6/3.9 SAOUHSC_00545 88194324 cell wall 0.3
Sle1 (Aaa) autolysin precursor, putative 35.8/9.9 SAOUHSC_00427 88194219 cell wall 0.4
Spa 1 protein A 55.6/5.4 SAOUHSC_00069 88193885 cell wall 3.7 3.3
Spa 2 protein A 55.6/5.4 SAOUHSC_00069 88193885 cell wall 5.0 2.9
SsaA secretory antigen precursor, putative 29.3/9.1 SAOUHSC_02571 88196215 cell wall 5.0 9.9
a
Several proteins are detectable as multiple spots. The spot numbers as marked in Figure 2 are indicated in superscript.
b
Protein localization was predicted as described in Sibbald et al. (Sibbald et al., 2006); SceD, SsaA and IsaA were shown to be bound ionically to the cell wall by Stapleton et al.
(Stapleton et al., 2007); Sle1 (Aaa) has two LysM domains that can bind to peptidoglycan.
c
The induction ratio of mutant to parental strain was calculated for each spot (normalized intensity of a spot on the mutant image/normalized intensity of the corresponding spot on
the parental image). The significance of spot volume differences of two-fold or higher was assessed by the Student´s t test (α

Synthetic effects of secG and secY2 mutations on exoproteome biogenesis in Staphylococcus aureus
Table 3B: Extracellular proteins with altered secretion patterns in S. aureus ∆secG and ∆secG∆secY2
Protein a Function Mr/pI
mature
ORFID S. aureus
NCTC8325
103
Accession
NCBI
Relative level compared to parental strain c
Predicted
location b ∆secG/WT ∆secG ∆secY2/WT
Geh lipase precursor 76.4/9.6 SAOUHSC_00300 88194101 extracellular 0.4 0.2
Hlb 1 truncated β-hemolysin 31.3/8.2 SAOUHSC_02240 88195913 extracellular 0.1 0.2
Hlb 2 truncated β-hemolysin 31.3/8.2 SAOUHSC_02240 88195913 extracellular 0.4 0.4
HlY 1 α-hemolysin precursor 35.9/9.1 SAOUHSC_01121 88194865 extracellular 0.3 0.5
HlY 2 α-hemolysin precursor 35.9/9.1 SAOUHSC_01121 88194865 extracellular 0.4 0.2
HlY 3 α-hemolysin precursor 35.9/9.1 SAOUHSC_01121 88194865 extracellular 0.2
HlY 4 α-hemolysin precursor 35.9/9.1 SAOUHSC_01121 88194865 extracellular 0.4 0.2
HlgB leukocidin F subunit precursor 36.7/9.8 SAOUHSC_02710 88196350 extracellular 0.3
HlgC leukocidin S subunit precursor, putative 35.7/9.7 SAOUHSC_02709 88196349 extracellular 0.3 0.4
LipA 1 Lipase 76.7/7.7 SAOUHSC_03006 88196625 extracellular 0.5
LipA 2 Lipase 76.7/7.7 SAOUHSC_03006 88196625 extracellular 0.3
LipA 3 Lipase 76.7/7.7 SAOUHSC_03006 88196625 extracellular 0.4
Plc 1-phosphatidylinositol phosphodiesterase
precursor
37.1/8.6 SAOUHSC_00051 88193871 extracellular
0.3 0.3
SAOUHSC_02241 1 hypothetical protein 38.7/9.1 SAOUHSC_02241 88195914 extracellular 0.1 0.2
SAOUHSC_02241 2 hypothetical protein 38.7/9.1 SAOUHSC_02241 88195914 extracellular 0.3 0.4
SAOUHSC_02241 3 hypothetical protein 38.7/9.1 SAOUHSC_02241 88195914 extracellular 0.2 0.4
SAOUHSC_02979 1 hypothetical protein 69.3/6.3 SAOUHSC_02979 88196599 extracellular 0.4
SAOUHSC_02979 2 hypothetical protein 69.3/6.3 SAOUHSC_02979 88196599 extracellular 0.3 0.3
SAOUHSC_02979 3 hypothetical protein 69.3/6.3 SAOUHSC_02979 88196599 extracellular 0.3
SAOUHSC_02979 4 hypothetical protein 69.3/6.3 SAOUHSC_02979 88196599 extracellular 0.5
YfnI 1 polytopic membrane protein, signal
peptidase I substrate
74.4/9.5 SAOUHSC_00728 88194493 extracellular 0,4 0,2
YfnI 2 polytopic membrane protein, signal
peptidase I substrate
74.4/9.5 SAOUHSC_00728 88194493 extracellular 0,4 0,2
a
Several proteins are detectable as multiple spots. The spot numbers as marked in Figure 2 are indicated in superscript.
b
Protein localization was predicted as described in Sibbald et al. (Sibbald et al., 2006); SceD, SsaA and IsaA were shown to be bound ionically to the cell wall by Stapleton et al.
(Stapleton et al., 2007); Sle1 (Aaa) has two LysM domains that can bind to peptidoglycan.
c
The induction ratio of mutant to parental strain was calculated for each spot (normalized intensity of a spot on the mutant image/normalized intensity of the corresponding spot on
the parental image). The significance of spot volume differences of two-fold or higher was assessed by the Student´s t test (α

Chapter 5
geh
RN4220 RN4220∆secG
OD1 OD10 OD15 OD1 OD10 hlb
OD15
RN4220 RN4220∆secG
OD 1 OD10 OD 15
spa
OD1 OD10 OD15
RN4220 RN4220∆secG
OD 1 OD10 OD15
OD1 OD10
OD 15
OD 1
OD1
OD1
OD10
104
OD 10
OD 10
OD 15
Dual image
RN4220
RN4220 ∆secG
OD 15
OD 15
Dual image
RN4220
RN4220 ∆secG
Dual image
RN4220
RN4220 ∆secG
Figure 3. Expression of SecG-dependent exoproteins. RNA and exoproteins were collected from S. aureus
RN4220 and S. aureus RN4220 ∆secG grown in TSB medium at 37°C. Samples were collected at three different
points during growth (OD 540 of 1, 10 and 15). In the Northern blotting experiments, membranes were hybridized
with digoxigenin-labeled RNA probes specific for geh, hlb or spa. Protein spots from 2-D PAGE analyses of the
respective proteins collected at OD 540 of 1, 10, and 15 are shown for the secG mutant and its parental strain both
separately and as dual-channel images.
Deletion of the secY2 gene encoding a channel component of the accessory Sec
system in S. aureus, did not affect the extracellular protein pattern (data not shown).
However, the deletion of both secG and secY2 caused additional changes in the extracellular
proteome compared to the secG single mutant (Figure 2B). Specifically, one additional
exoprotein was identified in decreased amounts (i.e. LipA) and one additional exoprotein (i.e.
LytM) was identified in increased amounts (Table 3A and 3B). Furthermore, proteins such as
Spa, IsaA, and SsaA were secreted in higher amounts not only by the secG mutant, but also
by the secG secY2 double mutant. This effect was significantly exacerbated for IsaA and
SsaA in the secG secY2 double mutant. It is interesting to note that IsaA, LytM, Spa, and
SsaA represent cell surface-associated proteins (Schneewind et al., 1992; Ramadurai et al.,
1999; Stapleton et al., 2007).
In contrast, most proteins that were secreted in reduced amounts in the secG or secG secY2
mutants are secretory proteins without retention signals, except for SAOUHSC-00094 (Table
3A and 3B). Importantly, also the secretion and growth defects of the secG secY2 mutant
strain could be fully reversed by ectopic expression of secG from the plasmid secG-pCN51,
and the synthetic effects of the secG and secY2 mutations could be reversed by plasmid
secY2-pCN51 (data not shown).

Synthetic effects of secG and secY2 mutations on exoproteome biogenesis in
Staphylococcus aureus
Impaired export of cell wall-bound Sbi in secG mutant cells
Western blotting experiments were performed to investigate whether particular protein export
defects of the secG and secY2 mutants had remained unnoticed in the proteomic analyses.
These analyses included secreted proteins in the growth medium, non-covalently cell wall
attached and cellular proteins of S. aureus strains RN4220 and S. aureus SH1000.
Furthermore, we used specific antibodies against membrane proteins, lipoproteins, cell wall
proteins and exoproteins. For most tested proteins no differences were detectable between the
secG and/or secY2 mutant strains and their parental strain. However, these analyses showed
that a band of ~50 kDa, which was cross-reactive with all tested sera, had disappeared from
the fraction of non-covalently bound cell wall proteins of the secG mutant. It is known that
proteins, such as protein A (Sasso et al., 1991) and Sbi (Zhang et al., 1998) have IgG-binding
properties. To investigate whether the missing band would relate to protein A or Sbi, protein
fractions from a spa mutant, and a spa sbi double mutant, were included in the Western
blotting analyses.
Figure 4. Sbi localization to the cell wall of S. aureus depends on SecG. (A) S. aureus SH1000 (WT), S. aureus
SH1000 ∆secG, and S. aureus SH1000 ∆secG secG-pCN51 were grown in TSB medium at 37 o C till the early
stationary phase. Samples of extracellular proteins isolated from the growth medium (M), non-covalently cell wallbound
proteins (CW) and total cells (C) were used for Western blotting and immunodetection with serum of mice
immunized with IsaA. As a contol for Sbi production, the strains S. aureus Newman ∆spa and S. aureus Newman
∆spa ∆sbi were included in the analyses. (B) Proteins of S. aureus SH1000 (wt), S. aureus SH1000 ∆secG, and S.
aureus SH1000 ∆secY2 were used for Western blotting and immunodetection as in (A). The position of Sbi is
marked with an arrow.
As shown in Figure 4A, the band of ~50 kDa that was missing from the non-covalently bound
cell wall proteins in the secG mutant was also missing from these proteins in the spa sbi
double mutant, but not in the spa single mutant (only the results for S. aureus SH1000 are
shown but essentially the same results were obtained for S. aureus RN4220). Taken together,
these findings show that Sbi is non-covalently bound to the cell wall of S. aureus RN4220 and
SH1000, and that SecG is required for export of Sbi from the cytoplasm to the cell wall. As
was the case for the secreted S. aureus proteins detected by proteomics, Sbi export to the cell
wall was not affected by the absence of SecY2 (Figure 4B). Finally, it is noteworthy that Sbi
is only detectable amongst the non-covalently bound cell wall proteins of S. aureus RN4220
and SH1000, whereas it is detectable both in a cell wall-bound and a secreted state in S.
aureus Newman.
105

Chapter 5
Discussion
The extracellular and surface-associated proteins of bacterial pathogens, such as S. aureus,
represent an important reservoir of virulence factors. Accordingly, protein export mechanisms
will contribute to the virulence of these organisms. While protein export has been well
characterized in model organisms, such as E. coli and B. subtilis, relatively few functional
studies have addressed the protein export pathways of S. aureus. Notably, the Sec pathway is
generally regarded as the main pathway for protein export but, to date, this has not been
verified experimentally in S. aureus. Therefore, the present studies were aimed at assessing
the role of the Sec pathway in establishing the extracellular proteome of S. aureus. We
focused attention on the non-essential channel component SecG as this allowed a facile coassessment
of the non-essential accessory Sec channel component SecY2. Our results show
that the extracellular accumulation of proteins is affected to different extents by the absence
of SecG: some proteins are present in reduced amounts, some are not affected and some are
present in elevated amounts. Furthermore, the effects of the absence of SecG are exacerbated
by deletion of SecY2, suggesting that SecY2 directly or indirectly influences the functionality
of the general Sec pathway. This is all the more remarkable since the absence of SecY2 by
itself had no detectable effects on the composition of the extracellular proteome of S. aureus.
The observation that the secretion of a wide range of proteins was affected by the
absence of SecG is consistent with the fact that all of these proteins contain Sec-type signal
peptides.On the other hand, this finding is remarkable since studies in other organisms, such
as E. coli (Nishiyama et al., 1993) and B. subtilis (Van Wely et al., 1999) have shown that
deletion of secG had fairly moderate effects on protein secretion in vivo. In B. subtilis, a
phenotype of the secG mutation was only observed under conditions of high overproduction
of secretory proteins (Van Wely et al., 1999). Clearly, our present data show that SecG is
more important for Sec-dependent protein secretion in S. aureus than in B. subtilis or E. coli.
Importantly, the transcription of genes for three proteins (Geh, Hlb and Spa) that were
affected in major ways by the absence of SecG was not changed, and all observed effects of
the secG mutation could be reversed by ectopic expression of secG. This suggests that the
observed changes in the exoproteome composition of the S. aureus secG mutant strain relate
to changes in the translocation efficiency of proteins through the Sec channel rather than
regulatory responses at the gene expression level. This could be due to altered recognition of
the respective signal peptides or mature proteins by the SecG-less Sec channel, or
combinations thereof. However, some indirect effects, for example at the level of translation
of exported proteins or cell wall binding of proteins like IsaA, LytM, Spa and SsaA, can
currently not be excluded especially since no proteins were found to accumulate inside the
secG mutant cells (data not shown). It remains to be shown why the extracellular
accumulation of particular proteins is affected by the absence of SecG, while that of other
proteins remains unaffected.
Unexpectedly, our studies revealed that export of the IgG-binding protein Sbi to the
cell wall was almost completely blocked in secG mutant strains. The reason why this export
defect was not detected by 2-D PAGE relates to the fact that Sbi is predominantly cell wallbound
in the tested S. aureus strains under the experimental conditions used. It has been
proposed previously that Sbi would remain cell wall-attached through a proline-rich wallbinding
domain and electrostatic interactions (Zhang et al., 1998). Nevertheless, Burman and
colleagues showed that Sbi is extracellular and they suggested that cell surface-bound Sbi
might be disadvantageous for the bacterium due to its role in modulating the complement
106

Synthetic effects of secG and secY2 mutations on exoproteome biogenesis in
Staphylococcus aureus
system (Burman et al., 2008). On the other hand, cell surface localization of Sbi would be
appropriate for interference with the adaptive immune system through IgG binding (Atkins et
al., 2008). Irrespective of these previously reported findings, our Western blotting analyses
show that Sbi is non-covalently bound to the cell wall, not only in S. aureus SH1000 and S.
aureus RN4220, but also in S. aureus Newman. However, consistent with the findings of
Burman et al., Sbi was also detected in the growth medium of S. aureus Newman, which
indicates that the location of Sbi in the cell wall or extracellular milieu may differ for different
S. aureus strains. In case of the Newman strain, the release of Sbi into the growth medium
could be due to the fact that this strain produces Sbi at increased levels compared to the
RN4220 and SH1000 strains (Rogasch et al., 2006). Conceivably, this increased production
might lead to a saturation of available cell wall binding sites for Sbi.
Many of the proteins of which the extracellular amounts are changed due to the
absence of SecG are considered to be important virulence factors of S. aureus. These proteins
are involved host colonization (e.g. the serine-aspartic acid repeat proteins SdrC and SdrD),
invasion of host tissues (e.g. hemolysins and leukocidins), cell wall turnover (LytM), and
evasion of the immune system (Spa, Sbi). The altered amounts of these proteins suggest that
S. aureus strains depleted of SecG might perhaps be less virulent. However, in a mouse
infection model no changes in virulence of the S. aureus secG mutant strain could be detected
(data not shown). This implies that the presence or absence of SecG is not critical for S.
aureus virulence, at least under the conditions tested.
Since we were unable to detect secretion defects for secY2 single mutant strains, our
studies confirm that only very few proteins are translocated across the membrane in a
SecA2/SecY2-dependent manner as has previously been suggested by Siboo et al. (Siboo et
al., 2008). Furthermore, we did not detect differences in the export of glycosylated proteins
by the secY2 mutants (data not shown), which is in line with the suggestion that glycosylated
proteins are not strictly dependent on the accessory Sec pathway for export (Siboo et al.,
2008). It was therefore quite surprising that the secY2 mutation exacerbated the secretion
defect of the S. aureus secG mutant. In fact, the secretion of two additional proteins was
found to be affected in the secG secY2 double mutant. Moreover, a synthetic growth defect
was observed for this double mutant. At this stage, it seems most likely that both the growth
defect and the secretion defects are consequences of an impaired Sec channel function.
However, it is also possible that the exacerbated secretion defects are, to some extent, a
secondary consequence of the growth defect of the double mutant. Irrespective of their
primary cause, these synthetic effects of the secG and secY2 mutations imply that the regular
Sec channel can somehow interact with the Sec2 channel. Whether this means that mixed Sec
channels with both SecY and SecY2 exist remains to be determined. However, this possibility
would be consistent with the observation that S. aureus lacks a second set of secE and secG
genes. It would thus be important to focus future research activities in this area on possible
interactions between the regular Sec channel components and SecY2.
107

Chapter 5
Acknowledgements
We like to thank W. Baas and M. ten Brinke for technical assistance, S. Dubrac for providing
the pCN51 plasmid, T. Foster for the spa mutant of S. aureus Newman, Decodon GmbH
(Greifswald, Germany) for providing Delta2D software, and T. Msadek and other colleagues
from the StaphDynamics and AntiStaph programs for advice and stimulating discussions.
M.J.J.B.S, T.W., M.M.v.d.K.-P., T.B., T.S., K.O., M.H., H.A., S.E. and J.M.vD. were in parts
supported by the CEU projects LSHM-CT-2006-019064, LSHG-CT-2006-037469 and PITN-
GA-2008-215524, the Top Institute Pharma project T4-213, and the DFG research grants
GK212/3-00, SFB/TR34 and FOR585.
108

109

“One good thing about music:when it hits you feel no pain”
-Robert N. Marley-
110

Chapter 6
Partially overlapping substrate specificities of sortases A and C of
staphylococci
M.J.J.B. Sibbald # , X.M. Yang # , E. Tsompanidou, M. Hecker, D. Becher,
G. Buist, J.M. van Dijl
# both authors contributed equally to this work
Submitted for publication
111

Chapter 6
Summary
Staphylococcus aureus and Staphylococcus epidermidis display many proteins on their
cell surface that are covalently linked to the peptidoglycan moiety by sortases. The class
A sortase (SrtA) of S. aureus and S. epidermidis attaches proteins with an LPxTG motif
to the cell wall. Deletion of the srtA genes of these bacteria resulted in the dislocation of
several LPxTG proteins, such as ClfA, SasG, SdrC, SdrD, and protein A of S. aureus,
and Aap of S. epidermidis 1457, to the growth medium. Nevertheless, substantial
amounts of these proteins remained cell wall-bound through non-covalent interactions.
The protein dislocation phenotypes of srtA mutations in S. aureus and S. epidermidis
could be fully reverted by ectopic expression of srtA genes of either species.
Interestingly, the class C sortase (SrtC) from S. epidermidis 12228 was able to revert the
dislocation of ClfA, SasG and Aap to significant extents, showing that the substrate
specificities of SrtA and SrtC overlap at least partially. Interestingly, biofilm formation
was affected in srtA mutants of S. aureus, but not in the S. epidermidis srtA mutant. This
difference can be correlated to the expression levels of particular covalently cell wallbound
proteins involved in protein-dependent biofilm formation, such as S. aureus SasG
and its S. epidermidis homologue Aap. Remarkably, SrtA activity was a limiting
determinant for protein-dependent biofilm formation in S. aureus and S. epidermidis,
whereas SrtC expression interfered with biofilm formation in S. epidermidis 1457. Taken
together, these findings imply that sortases can have modulating roles in staphylococcal
biofilm formation.
112

Partially overlapping substrate specificities of sortases A and C of staphylococci
Introduction
Staphylococcus aureus and Staphylococcus epidermidis are both part of the normal human
microbiota (Peacock et al., 2001;Wertheim et al., 2004;Bibel et al., 1976). However, both
organisms have the potential to cause life-threatening infections, especially when they
become invasive and reach the blood stream. In addition, S. aureus and especially S.
epidermidis are notorious for their ability to form biofilms on medical devices and implants
(Escher and Characklis, 1990). Cell surface-associated proteins play crucial roles in the
colonization and invasion of host tissues by staphylococci, and such proteins also have
important roles in biofilm formation. The surface-exposed proteins can either be covalently or
non-covalently linked to the bacterial cell wall. Covalent protein linkage to the peptidoglycan
moiety of the cell wall is catalyzed by specific enzymes known as sortases.
Gram-positive bacteria, such as S. aureus and S. epidermidis, but also various bacilli,
streptococci and corynebacteria have one or more sortase-encoding genes (Dramsi et al.,
2005;Sibbald and van Dijl, 2009). For several pathogens, such as S. aureus, Listeria
monocytogenes and Streptococcus pneumoniae, it has been shown that the deletion of sortase
genes results in decreased virulence (Mazmanian et al., 2000;Garandeau et al., 2002;Bierne et
al., 2002;Hava and Camilli, 2002). This underscores the importance of covalent cell wall
attachment of particular proteins in the pathogenicity of Gram-positive bacteria. Based on
structural and functional criteria, Dramsi and colleagues (Dramsi et al., 2005) classified
sortases into four different groups. Sortase A (SrtA) enzymes link several proteins with
LPxTG or LPxAG motifs to the cell wall. Such proteins are mainly involved in adhesion to
specific organ tissue, survival during phagocytosis, and invasion of host cells. The LPxTG or
LPxAG motifs are cleaved by sortase between the Thr/Ala and Gly residues. The free Thr
residue is then linked to the active site Cys residue of sortase A (Marraffini et al., 2006;Scott
and Barnett, 2006). Formation of an amide bond between the Thr residue of the surface
protein and the pentaglycine cross-bridge of branched lipid II completes the covalent protein
attachment to the cell wall. For S. aureus and other Gram-positive pathogens it has been
shown that srtA mutants have a decreased ability to establish a successful infection in animal
models (Bierne et al., 2002;Mazmanian et al., 2000). In contrast to sortase A, most sortase B
(SrtB) enzymes are involved in iron metabolism. In S. aureus the srtB gene is part of the isd
operon, which contains several genes (isdA-G) that are important for iron-acquisition
(Mazmanian et al., 2003). SrtB recognizes the NPQTN motif in the C-terminus of the IsdC
protein, and cleaves this motif C-terminally of the Thr residue thereby linking IsdC to the cell
wall (Mazmanian et al., 2002). Class C sortases (SrtC) are found in several Gram-positive
bacteria and these sortases seem to be involved in the formation of pili (Mandlik et al.,
2008;Ton-That and Schneewind, 2004;Scott and Barnett, 2006). In S. pneumoniae three class
C sortases and their substrates were shown to be important for pilus formation and virulence
in animal models (Hava and Camilli, 2002). Notably, the genes encoding class C sortases and
their substrates are not part of the core genomes of particular species. This suggests that these
genes have been acquired through horizontal gene transfer thereby giving the cells an
adaptive advantage to certain host niches. Several Gram-positive bacteria have a class D
sortase (Dramsi et al., 2005) but, so far, relatively little is known about this class of sortases.
The class D sortase of Streptomyces coelicolor links several proteins to the surface of the cell
wall. These proteins contain so-called chaplin domains and have been shown to coat the
surfaces of aerial hyphae and spores (Claessen et al., 2003;Elliot et al., 2003). Bacillus
subtilis 168 also has a gene for a SrtD homologue named yhcS. However, no clear phenotype
113

Chapter 6
for yhcS mutant strains could be identified, and the only two B. subtilis proteins with typical
sortase recognition sites, YhcR and YfkN, were found to be secreted both by the yhcS mutant
and the parental strain 168 (H. Westers, doctoral thesis, University of Groningen).
The crystal structure of SrtA revealed that the active site Cys residue is in close proximity of a
His residue (Ilangovan et al., 2001;Zong et al., 2004;Suree et al., 2009;Race et al., 2009).
Furthermore, an Arg residue in the C-terminus of the protein also seems to be important for
efficient catalysis (Marraffini et al., 2004;Frankel et al., 2007;Bentley et al., 2007), probably
by stabilizing the transition state. The Cys residue is part of the TLxTC motif that is
conserved in all sortases (Figure 1). While the Thr-180, Leu-181 and Ile-182 of SrtA seem to
determine the conformation of the substrate binding pocket, Thr-183 is most likely involved
in positioning the Cys-184 and Arg-197 residues. Other residues that might play a role in
efficiently binding surface proteins to the cell wall include Val-168 and Leu-169, which are
involved in substrate recognition. These residues recognize the Leu-Pro residues in the Cterminal
LPxTG motif by hydrophobic interactions (Bentley et al., 2007). Also, Glu-171
seems to be important for the function of SrtA and it was proposed that this residue binds
Ca 2+ , thereby stabilizing the β6/β7 loop region of SrtA (Naik et al., 2006).
Figure 1. Alignment of staphylococcal sortases A, B and C. Sortases of S. aureus NCTC8325 and S. epidermidis
ATCC12228 were aligned using the ClustalW (EMBL-EBI) algorithm with standard settings (Thompson et al.,
1994). The database accession codes of the aligned sortase sequences are: S. aureus SrtA (gi 88196468); S. aureus
SrtB (gi 88194835); S. epidermidis SrtA (gi 27468994); and S. epidermidis SrtC (gi 27468398). Residues
conserved in all sortases are marked with black shading, and residues present in three of the four sequences are
marked with grey shading. Active site residues are marked with #.
All sequenced Staphylococcus species contain multiple genes for proteins with cell wall
sorting motifs. In total, twenty-one proteins with this motif have been identified in S. aureus
and twelve in S. epidermidis (Sibbald et al., 2006). One of the proteins with a C-terminal
LPxTG motif is the Staphylococcus aureus surface protein G (SasG; (Roche et al., 2003)). It
has been shown that this protein is involved in adhesion to nasal epithelial cells. Interestingly,
SasG is also involved in protein-based biofilm formation, independent of the ica-encoded
polysaccharide for intercellular adhesion (PIA) or poly-N-acetyl glucosamine (PNAG)
(Corrigan et al., 2007). The homologue of SasG in S. epidermidis is the accumulation
associated protein (Aap). This protein has been implicated in the formation of both
polysaccharide- (Hussain et al., 1997) and protein-based biofilms (Rohde et al., 2005;Rohde
et al., 2007). SasG and Aap contain several functional domains. Both SasG and Aap are
synthesized with a classical signal peptide containing the YSIRK/GS motif in the N-terminal
part of the signal peptide. Dedent et al. (Dedent et al., 2008) have provided evidence that the
YSIRK/GS motif directs site-specific secretion at the cross wall, which is the peptidoglycan
layer that is formed during cell division to separate new daughter cells. Proteins without this
motif are addressed to the cell pole of S. aureus. The N-terminal A domain of SasG is
114

Partially overlapping substrate specificities of sortases A and C of staphylococci
unrelated to the equivalent domain in Aap, but it is followed by a stretch of amino acids that
is 59% identical to the equivalent domain in Aap. Next, several repeats of 128 residues are
present that are known as G5 domains. These domains have been shown to bind N-acetyl
glucosamine (Bateman et al., 2005). It has been shown that these domains are necessary for
SasG- or Aap-dependent biofilm formation, and that they need to be proteolytically separated
from the respective mature proteins to fulfill their roles in intercellular adherence (Rohde et
al., 2005;Corrigan et al., 2007). Furthermore, it has been shown for S. aureus SasG that at
least five G5 domains are necessary for biofilm formation (Corrigan et al., 2007). The G5
domains of both SasG and Aap promote the intercellular interactions in a Zn 2+ -dependent
manner (Conrady et al., 2008).
In contrast to S. epidermidis RP62A, the S. epidermidis ATCC12228 strain contains a srtC
gene (Comfort and Clubb, 2004). This srtC gene lies on a genomic island (νSe2) that also
contains the genes for the surface proteins SesJ and SesK (Gill et al., 2005). Both proteins
contain an LPxTG motif in their C-termini. Unfortunately, the biological roles of SesJ and
SesK are presently unclear. Class C sortases have also been found in a few other Grampositive
bacteria where they recognize proteins with a C-terminal LPxTG motif for covalent
cell wall binding (Dramsi et al., 2005). In C. diphtheriae it has been shown that SrtC is
necessary for the formation of pili (Ton-That and Schneewind, 2003). Notably, the three pilus
subunits SpaA, SpaB and SpaC are synthesized with a C-terminal LPxTG motif. SrtC is
responsible for the processing of these subunits and their linkage to a neighboring subunit.
Homologues of these proteins have also been identified in other bacteria, such as S.
pneumoniae, Streptococcus agalactiae and Enterococcus faecalis (Dramsi et al., 2005). It
seems unlikely that SrtC fulfils a similar role in S. epidermidis as pilus formation has not been
observed in this bacterium. Importantly, whereas SrtA seems to handle a wide range of
different substrates with LPxTG motifs, SrtC seems to be dedicated to the processing of only
a few specific substrates, in some cases even only one substrate (Barnett et al., 2004;Hava and
Camilli, 2002;Hava et al., 2003;Ton-That and Schneewind, 2003).
The present studies were aimed at addressing the question to what extent sortases of different
classes and from different Firmicutes, like S. aureus, S. epidermidis and B. subtilis, can
functionally replace each other. While the class D sortase of B. subtilis does not seem to
complement for the absence of SrtA in S. aureus or S. epidermidis, the SrtA proteins from S.
aureus and S. epidermidis can complement for each other. Remarkably, our results show that
SrtC of S. epidermidis can partially complement for the absence of SrtA in both
Staphylococcus species. This implies that class A and class C type sortases of staphylococci
have partially overlapping substrate specificities.
Material & Methods
Bacterial strains
All strains and plasmids used in this study are listed in Table 1. Escherichia coli strains were grown in
Luria-Bertani broth (LB) at 37°C and under vigorous shaking. Unless stated otherwise, S. aureus and S.
epidermidis strains were grown on tryptic soy broth (TSB), tryptic soy agar (TSA) or brain heart
infusion (BHI) at 37°C and under vigorous shaking. Where necessary, antibiotics were added in the
following concentrations: ampicillin (100 µg/ml), erythromycin for E. coli (100 µg/ml), erythromycin
for S. aureus and S. epidermidis (5 µg/ml), kanamycin (20 µg/ml), chloramphenicol (15 µg/ml). To
monitor β-galactosidase activity in cells of E. coli, S. aureus or S. epidermidis, 5-bromo-4-chloro-3indolyl-β-D-galactopyranoside
(X-gal) was added to the plates at a final concentration of 80 µg/ml.
115

Chapter 6
Table 1. Bacterial strains and plasmids used
Plasmids Properties Reference
TOPO pCR®-Blunt II-TOPO® vector; Km R Invitrogen Life technologies
“srtA”-TOPO TOPO plasmid containing the flanking regions of S.
aureus srtA, Kan R
This work
srtC-pUC18 pUC18 plasmid with the S. epidermidis srtC gene, Amp R This work
“srtA”SE-TOPO TOPO plasmid containing the flanking regions of S.
epidermidis srtA, Kan R
This work
pCN51 E. coli - S. aureus shuttle vector that contains a cadmiuminducible
promoter
(Charpentier et al., 2004)
pMAD E. coli - S. aureus shuttle vector that is temperaturesensitive
in S. aureus and contains the bgaB gene, Ery R ,
(Arnaud et al., 2004)
Amp R
pUC18 Amp R , ColE1, F80dLacZ, lac promoter (Norrander et al., 1983)
Sa-srtA-pCN51 pCN51 with S. aureus srtA gene, Amp R ; Ery R This work
Se-srtA-pCN51 pCN51 with S. epidermidis srtA gene, Amp R ; Ery R This work
Se-srtC-pCN51 pCN51 with S. epidermidis ATCC12228 srtC gene,
Amp R ; Ery R
This work
Bs-yhcS-pCN51 pCN51 with B. subtilis yhcS gene, Amp R ; Ery R This work
Strains
E. coli
Genotype Reference
DH5α supE44; hsdR17; recA1; gyrA96; thi-1; relA1 (Hanahan, 1983)
TOP10 Cloning host for TOPO vector; F - mcrA ∆(mrr-hsdRMSmcrBC)
Φ80lacZ∆M15 ∆lacX74 recA1 araD139 ∆(araleu)7697
galU galK rpsL (Str R S. aureus RN4220
) endA1 nupG
Invitrogen Life technologies
Parental strain Restriction-deficient derivative of NCTC 8325, cured of
all known prophages
(Kreiswirth et al., 1983)
∆srtA srtA This work
∆srtA ∆spa srtA spa; replacement of spa by kanamycin resistance
marker; Kan R
This work
∆srtA-comp-Sa ∆srtA strain complemented with S. aureus srtA through
Sa-srtA-pCN51
This work
∆srtA-comp-Se ∆srtA strain complemented with S. epidermidis srtA
through Se-srtA-pCN51
This work
∆yhcS-comp-Bs ∆srtA strain complemented with B. subtilis yhcS through
Bs-yhcS-pCN51
This work
∆srtA-comp-srtC
S. aureus SH1000
∆srtA strain complemented with S. epidermidis srtC
throught Se-srtC-pCN51
This work
Parental strain Functional rsbU+ derivative of 8325-4 rsbU+, agr+ (Horsburgh et al., 2002)
∆srtA rsbU+, agr+, srtA This work
∆srtA ∆spa rsbU+, agr+, srtA spa; replacement of spa by kanamycin
resistance marker; Kan R
This work
∆srtA-comp-Sa ∆srtA complemented with S. aureus srtA through SasrtA-pCN51
This work
∆srtA-comp-Se ∆srtA complemented with S. epidermidis srtA through
Se-srtA-pCN51
This work
∆yhcS-comp-Bs ∆srtA complemented with B. subtilis yhcS through BsyhcS-pCN51
This work
∆srtA-comp-srtC ∆srtA complemented with S. epidermidis srtC through
Se-srtC-pCN51
This work
sasG-pMUTIN4 Overexpression of SasG due to integrated pMUTIN4
plasmid; Ery R
(Corrigan et al., 2007)
sasG-pMUTIN4
∆srtA
∆srtA; overexpression of SasG due to integrated
pMUTIN4 plasmid; Ery R
116
This Work

Partially overlapping substrate specificities of sortases A and C of staphylococci
Strains Genotype Reference
sasG-pMUTIN4 ∆srtA; overexpression of SasG due to integrated
∆srtA comp-srtA pMUTIN4 plasmid; complemented with S. aureus srtA
through Sa-srtA-pRIT5H; Ery R , Cm R
This Work
sasG-pMUTIN4 ∆srtA; overexpression of SasG due to integrated
∆srtA comp-srtC pMUTIN4 plasmid; complemented with S. epidermidis
srtC through Se-srtC-pRIT5H; Ery R , Cm R
This Work
S. epidermidis 1457
Parental Biofilm positive strain (Mack et al., 1992)
∆srtA srtA This work
∆srtA-comp-Sa ∆srtA; complemented with S. aureus srtA through SasrtA-pCN51
This work
∆srtA-comp-Se ∆srtA; complemented with S. epidermidis srtA through
Se-srtA-pCN51
This work
∆yhcS-comp-Bs ∆srtA; complemented with B. subtilis yhcS through BsyhcS-pCN51
This work
∆srtA-comp-srtC ∆srtA; complemented with S. epidermidis srtC through
Se-srtC-pCN51
This work
Construction of sortase mutants
Mutants of S. aureus and S. epidermidis were constructed using the temperature-sensitive plasmid
pMAD (Arnaud et al., 2004) and previously described procedures (Kouwen et al., 2009). Primers
(Table 2) were designed using the genome sequences of S. aureus NCTC8325 and S. epidermidis
RP62A (http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi). All mutant strains were checked by
isolation of genomic DNA using the GenElute Bacterial Genomic DNA Kit (Sigma) and PCR with
specific primers.
To delete the srtA genes, primer pairs with the designations F1/R1 and F2/R2 were used for
PCR amplification of the respective upstream and downstream regions (each ~500 bp), and their fusion
with a 21 bp linker. The fused flanking regions were cloned in pMAD, and the resulting plasmids were
used to delete the chromosomal srtA genes of S. aureus RN4220 and S. epidermidis 1457. To delete the
srtA gene from the S. aureus SH1000 genome, the respective pMAD constructs were transferred from
the RN4220 strain to the SH1000 strain by transduction with phage φ85 (Novick, 1991).
Complementation of srtA mutations
Primer pairs for PCR amplification of srtA from S. aureus, srtA from S. epidermidis, yhcS from B.
subtilis and srtC from S. epidermidis were based on the genome sequences of the S. aureus
NCTC8325, S. epidermidis RP62A and B. subtilis 168 strains (Table 2). For expression in S. aureus the
RBS and start codon of S. aureus srtA was used and for expression in S. epidermidis the RBS and start
codon of S. epidermidis srtA was used. PCR products were purified using the PCR Purification Kit
(Roche), and ligated into the pUC18 plasmid (Norrander et al., 1983). The cloned sortase genes were
then excised from the resulting constructs with restriction enzymes as specified in Table 2 and ligated
into plasmids pCN51 or pRIT5H that were cut with the same enzymes. Competent S. aureus RN4220
∆srtA cells were transformed with these plasmids by electro-transformation and colonies were selected
on TSA plates containing erythromycin. Subsequently, the plasmids were transferred from S. aureus
RN4220 to S. aureus SH1000 strains via transduction as described above, or to S. epidermidis 1457
strains via electro-transformation of competent cells with the purified plasmids.
Under standard laboratory growing conditions, no SasG production is detectable in S. aureus (Roche et
al., 2003). To study the localization of SasG, we used strains in which the expression of SasG is
directed by the IPTG-inducible Pspac-promoter of plasmid pMUTIN4 (kindly provided by T. Foster
(Corrigan et al., 2007)). The sasG-pMUTIN4 plasmid was transferred to the S. aureus SH1000 ∆srtA
strain by transduction as described above. Since sasG-pMUTIN4 carries an erythromycin resistance
marker, strains containing this plasmid cannot be transformed with derivatives of plasmid pCN51.
Therefore, the pRIT5H plasmid (Morikawa et al., 2003) was used for complementation experiments
117

Chapter 6
with S. aureus srtA or S. epidermidis srtC in the S. aureus ∆srtA sasG-pMUTIN4 strain. Genes cloned
in pRIT5H are transcribed from the S. aureus spa promoter. The srtA and srtC genes were PCR
amplified with primer pairs srtA-F4/R4 and srtC-F2/R2, respectively, and cloned into the pRIT5H
plasmid using restriction enzymes specified in Table 2. The resulting plasmids were introduced into S.
aureus RN4220 strains containing sasG-pMUTIN4 via electro-transformation, and they were
subsequently transferred to sasG-pMUTIN4-containing S. aureus SH1000 strains via transduction.
Transformants and transductants were selected on TSA plates with erythromycin and chloramphenicol.
Table 2. Primers used in this study
Primer Sequence (5’→3’)
Construction of S. aureus srtA mutant
srtA-F1 AATGGTGTAGTAATTGACTAG
srtA-R1 TTACGTCAGTCAGTCACCATGGCAACGTTAAGGCTCCTTTTATAC
srtA-F2 TGCCATGGTGACTGACTGACGTAATCTATTACGCTAATGGATGAA
srtA-R2 CTCACATTACTTACTATTAAT
Construction of S. epidermidis srtA mutant
esrtA-F1 AACTTTGTTCTTTAGCGTAACGAAT
esrtA-R1 TGCCATGGTGACTGACTGACGTAATTATGTTACTCCTTTATATTTATT
esrtA-F2 TTACGTCAGTCAGTCACCATGGCATATTCTTATAAGTGAAAGATACGTA
esrtA-R2 CTTTATAGATGACTGCTCCAT
Complementation in S. aureus
srtA-F3 CAGCCGGATCCAATGTATAAAAGGAGCCTTAACGT (BamHI)
srtA-R3 CGGAATTCTTATTTGACTTCTGTAGCTACAAA (EcoRI)
srtA-F4 GGGGGGGATCCTTAACAGGCATTGTGAAATGT (BamHI)
srtA-R4 GGGGGGTCGACCCTTATTTGACTTCTGTAGCT (SalI)
esrtA-F3 CAGCCGGATCCAATGTATAAAAGGAGCCTTAACGTATGAAGCAGTGGATGAATAGA
(BamHI)
esrtA-R3 CG GAATTCTTAGTTAATTTGTGTAGCTATGAA (EcoRI)
yhcS-F1 AAAACTGCAGAATGTATAAAAGGAGCCTTAACGTATGAAAAAAGTTATTCCACTA (PstI)
yhcS-R1 CAGCCGGATCCTTAAGTCACTCGTTTTCCATATAT (BamHI)
esrtC-F1 GGGGGGTCGACTGAGGAGGTACATATGAGTGC (SalI)
esrtC-R1 GGGGGGGATCCATTTATAATTTGAAAATACCA (BamHI)
esrtC-F2 GGGGGGGATCCTGAGGAGGTACATATGAGTGC (BamHI)
esrtC-R2 GGGGGGTCGACATTTATAATTTGAAAATACCA (SalI)
Complementation in S. epidermidis
srtA-F5 CAGCCGGATCCAAATAAATATAAAGGAGTAACATAAATGAAAAAATGGACAAATCG (BamHI)
srtA-R5 CGGAATTCTTATTTGACTTCTGTAGCTACAAA (EcoRI)
esrtA-F4 GGGGGGGATCCAAATAAATATAAAGGAGTAACATAA (BamHI)
esrtA-R4 GGGGGGAATTCTTAGTTAATTTGTGTAGCTATGA (EcoRI)
yhcS-F2 AAAACTGCAG AAATAAATATAAAGGAGTAACATAAATGAAAAAAGTTATTCCACTA (PstI)
yhcS-R2 CAGCCGGATCCTTAAGTCACTCGTTTTCCATATAT (BamHI)
Overlapping parts are shown in bold
Restriction sites are underlined and shown in parentheses
Cell fractionation, SDS-PAGE, and Western blotting
Overnight cultures were diluted to an OD540 of 0.05 and grown in 25 ml TSB under vigorous shaking.
For complementation of mutant strains with pCN51-based plasmids, CdSO4 was added after three
hours of growth to a final concentration of 0.25 µM. For complementation of mutant strains with
pRIT5H-based plasmids, IPTG was added after three hours of growth to a final concentration of 1 mM.
Samples were taken after six hours of growth and separated in growth medium, whole cell and noncovalently
cell wall-bound protein fractions. Cells were separated from the growth medium by
centrifugation of 1 ml of the culture. The proteins in the growth medium were precipitated with 250 µl
50% trichloroacetic acid (TCA), washed with acetone and dissolved in 100 µl Loading Buffer
118

Partially overlapping substrate specificities of sortases A and C of staphylococci
(Invitrogen). Cells were resuspended in 300 µl Loading Buffer (Invitrogen) and disrupted with glass
beads using the Precellys ® 24 bead beating homogenizer (Bertin Technologies). From the same culture
20 ml was used for the extraction of non-covalently bound cell wall proteins using KSCN. Cells were
collected by centrifugation, washed with PBS, and incubated for 10 min with 1M KSCN on ice. After
centrifugation the non-covalently cell wall bound proteins were precipitated from the supernatant
fraction with TCA, washed with acetone and dissolved in 100 µl Loading Buffer (Invitrogen). Upon
addition of Reducing Agent (Invitrogen), the samples were incubated at 95ºC. Proteins were separated
by SDS-PAGE using precast NuPage gels (Invitrogen) and subsequently blotted onto a nitrocellulose
membrane (Protran ® , Schleicher & Schuell). The presence of Aap, SasG or Clumping factor A (ClfA)
was monitored by immunodetection with specific polyclonal antibodies raised in rabbits at 1:10.000
dilution. These antibodies were kind gifts from D. Mack (Aap) (Rohde et al., 2005) and T. Foster
(SasG and ClfA) (Roche et al., 2003;McDevitt et al., 1995). Bound primary antibodies were visualized
using fluorescent IgG secondary antibodies (IRDye 800 CW goat anti-rabbit from LiCor Biosciences).
Membranes were scanned for fluorescence at 800 nm using the Odyssey Infrared Imaging System
(LiCor Biosciences).
Protein identification by mass spectrometry
Proteins were separated by SDS-PAGE as described before and gels were stained with SimplyBlue TM
SafeStain (Invitrogen). Protein bands were cut from the gels and in-gel digestion of the proteins was
performed as described by Eymann et al. 2004 (Eymann et al., 2004). All peptides obtained from an ingel
digestion were separated by liquid chromatography and measured online by ESI mass spectrometry.
LC-MS/MS analyses were performed using a nanoACQUITY UPLC system (Waters) coupled to an
LTQ Orbitrap or LTQ-FTICR mass spectrometer (Thermo Fisher Scientific,Waltham, MA) creating
an electro spray by the application of 1.5 kV between Picotip Emitter (SilicaTip, FS360-20-10
Coating P200P, New Objective) and transfer capillary. Peptides were loaded onto a trap column
(nanoAcquity UPLC TM column, Symmetry® C18, 5 µm, 180 µm inner diameter x 20 mm, Waters) and
washed 3 min with 99% buffer A (0.1% (v/v) acetic acid) with a flow rate of 10 µl/min. Elution was
performed onto an analytical column (nanoAcquity UPLC TM column, BEH130 C18 1.7 µm, 100 µm
inner diameter x 100 mm, Waters) by a binary gradient of buffer A and B (100% (v/v) acetonitrile,
0.1% (v/v) acetic acid) over a period of 80 min with a flow rate of 400 nl/ min.
For MS/MS analysis full survey scans were performed in the Orbitrap or FTICR (m/z 300–2000) with
resolutions of 30,000 in the Orbitrap or 50,000 for the FTICR respectively. The full scan was followed
by MS/MS experiments of the five most abundant precursor ions acquired in the LTQ via CID.
Precursors were dynamically excluded for 30 sec, and unassigned charge states as well as singly
charged ions were rejected.
For protein identification tandem mass spectra were extracted using Sorcerer TM v3.5 (Sage-N Research,
Inc. Milpitas, CA). Charge state deconvolution and deisotoping were not performed. All MS/MS
samples were analyzed using Sequest (ThermoFinnigan, San Jose, CA; version v.27, rev. 11), applying
the following search parameters: peptide tolerance, 10 ppm; tolerance for fragment ions, 1 amu; b- and
y-ion series; an oxidation of methionine (15.99 Da) was considered as variable modification (maximal
three modifications per peptide). Sequest was set up to search the S. epidermidis RP62A database
(extracted from NCBI, including concatenated reverse database, 4600 entries) assuming the digestion
enzyme trypsin. For S. aureus RN4220 and S. aureus SH1000 samples, the S. aureus NCTC 8325-4
database (extracted from NCBI, including concatenated reverse database, 5784 entries) was used.
Scaffold (version Scaffold_2_04_00, Proteome Software Inc., Portland, OR) was used to validate
MS/MS based peptide and protein identifications. Peptide identifications were accepted if they
exceeded specific database search engine thresholds. Sequest identifications required at least deltaCn
scores of greater than 0.10 and XCorr scores of greater than 2.2, 3.3 and 3.8 for doubly, triply and
quadruply charged peptides. Protein identifications were accepted if they contained at least 2 identified
peptides. With these filter parameter no false positive hit was obtained.
119

Chapter 6
Biofilm formation
Biofilm formation by S. aureus and S. epidermidis strains was monitored with crystal-violet
(Christensen et al., 1985). S. aureus overnight cultures were diluted 100-fold in TSB containing 5%
glucose and 100 µl aliquots of the diluted cultures were transferred to a 96-well microtiter plate. After
four hours incubation at 37ºC, non-adhered cells were removed and the wells were rinsed with PBS
(pH 7,0). Fresh medium was added to the wells and the cultures were incubated for 24 hours at 37ºC.
The supernatant was removed from the wells and non-adhered cells were removed by rinsing with PBS.
Biofilms were fixated with 100 µl 99% methanol for 15 min. The supernatant was removed and after
air-drying for 20 min, 100 µl 0,4% crystal-violet solution was added to the wells. Upon 20 min
incubation, the supernatant was removed and the wells were rinsed three times with MilliQ. Bound
crystal-violet was released by adding 150 µl 33% acetic acid, and the absorbance of the released
crystal-violet was measured at 590 nm with the BIO-TEK ® ELx800 TM Universal Microplate Reader
(BioTek Instruments, Inc.). For each strain, the assay was repeated sixteen times.
Results
Protein export in srtA mutants of S. aureus
In the absence of functional SrtA, proteins with an LPxTG or LPxAG motifs will not be
covalently anchored to the cell wall of S. aureus. Therefore, one might expect at least a partial
release of these proteins into the growth medium of srtA mutant cells, especially if they are
subject to the “shaving activity” of exported proteases. To investigate how a srtA mutation
impacts on the localization of exported proteins of S. aureus RN4220, the srtA gene of this
strain was deleted. As shown by SDS-PAGE, the banding pattern of extracellular proteins of
the srtA mutant strain displayed major differences compared to the parental strain since the
intensity of several bands was strongly increased (Figure 2A; compare lanes 1 and 2). In
contrast, the differences observed for the non-covalently cell wall-bound proteins of both
strains were much less pronounced (Figure 2B, lanes 1 and 2). Importantly, deletion of srtA
affected neither the growth rate of S. aureus RN4220 nor the optical density reached in the
stationary phase, and no obvious morphological differences between cells of the srtA mutant
and the parental strain were detectable by electron microscopy (chapter 4). To identify the
proteins in bands of which the relative amounts were increased in the medium or cell wall
fractions of the srtA mutant (Figure 2), these bands were cut from gels and analyzed by mass
spectrometry. Of the seven identified proteins, only SdrC, SdrD and protein A contain an
LPxTG motif (Table 3). Interestingly, all three of these proteins also contain a YSIRK motif
in their signal peptides, as does the LipA protein, which was also secreted in higher amounts
by the srtA mutant strain. A very prominent effect of the srtA deletion was observed for
protein A (Figure 2A). Moreover, SdrC- and SdrD-specific bands were identified several
times in the medium fraction of the S. aureus srtA mutant, whereas these proteins were not at
all detectable in the growth medium of the parental strain (Figure 2A and data not shown).
The absence of srtA also had some clear effects on the composition of the non-covalently cell
wall-bound proteins, which contained increased amounts of the MHC class II analog protein
Map, and decreased amounts of the 5’-nucleotidase SA0295 (Figure 2B).
Since several well-studied LPxTG proteins like SasG (Corrigan et al., 2007) and the clumping
factor A (ClfA; (Josefsson et al., 2001;Siboo et al., 2001;Sullam et al., 1996)) are not
detectable in the Coomassie-stained gels, we studied the localization of these proteins by
Western blotting and subsequent immunodetection with specific antibodies.
120

Partially overlapping substrate specificities of sortases A and C of staphylococci
Figure 2. SDS-PAGE analyses of proteins secreted by S. aureus and S. epidermidis. Sortase mutants of two S.
aureus strains (RN4220 and SH1000) and one S. epidermidis strain (1457) were grown in TSB medium at 37 o C till
the early stationary growth phase. Proteins in the growth medium (A) and non-covalently cell wall-bound proteins
(B) were collected and separated by SDS-PAGE. Gels were stained with Coomassie. Samples were loaded as
follows: lane 1, parental strain; lane 2, srtA mutant; lane 3, srtA mutant complemented with plasmid-borne S.
aureus srtA; lane 4, srtA mutant complemented with plasmid-borne S. epidermidis srtA; lane 5, srtA mutant
complemented with plasmid-borne B. subtilis yhcS; and lane 6, srtA mutant complemented with plasmid-borne S.
epidermidis srtC. Protein bands marked with arrows were cut from the gel and identified by mass spectrometry.
121

Chapter 6
Table 3. Extracellular proteins identified in sortase mutants of S. aureus and S.
epidermidis
Protein GI Accession # Function Mw mature
protein (kD)
122
Cell Wall
binding domain
S. aureus
SdrC (YSIRK) 88194324 SdrC protein, putative 98.8 LPETG
SdrD (YSIRK) 88194325 SdrD protein, putative 136.9 LPETG
LipA (YSIRK) 88194101 Lipase a 72.3 -
Spa (YSIRK) 88193885 protein A 49.7 LPETG, LysM (1x)
Hla 88194865 hemolysin A 33.3 -
Map 88195840 also known as Eap or p70;
MHC class II analog protein
62.5
b
SA0295
S. epidermidis
88194087 5'-nucleotidase, lipoprotein
e(P4) family
30.2 -
Aap (YSIRK) 57865793 accumulation associated
protein
246.3 LPDTG, G5 (7x)
AtlE 57866522 bifunctional autolysin 145.2 GW-repeats (3x)
GehC
(YSIRK)
57865740 lipase 73.7 -
SERP0100 57866082 LysM domain protein c 32.5 LysM (3x)
SERP0270 57866259 hypothetical protein 16.2 -
a
cell wall binding has been shown, but no particular wall-binding domain has been identified (Bowden et al.,
2002).
b
Cell wall binding depends on acetylation of lipoteichoic acid (Harraghy et al., 2003)
c
also known as ScaA or AaE; autolysin with a C-terminal cysteine, histidine-dependent amidohydrolase/
peptidase (CHAP) domain; binding to fibrinogen, fibronectin and vitronectin (Heilmann et al., 2003)
To reduce background levels of IgG that was bound by protein A, we used S. aureus SH1000
for all Western blotting experiments as this strain produces less protein A than the RN4220
strain. Furthermore, to detect SasG, we used strains that expressed sasG from the pMUTIN4
plasmid upon induction with IPTG (Corrigan et al., 2007). In cells of the parental strain
SH1000, SasG was detected predominantly as a band of very high molecular weight (Figure
3A). Furthermore, the growth medium of the parental strain contained multiple SasG-specific
protein species that were detectable in the Western blots as a ladder of discrete protein bands
with molecular weights that were much lower than the molecular weight of the SasG detected
in cells (Figure 3A). In contrast, the high molecular weight band of SasG was not detectable
in cells of the srtA mutant, and much higher amounts of the low molecular weight bands were
detectable not only in the growth medium fraction, but also in the fraction of non-covalently
cell wall-bound proteins (Figure 3A). Similar to what we observed for SasG, a high molecular
mass species of the ClfA protein was detectable in cells of the parental strain SH1000,
whereas the growth medium contained two low molecular ClfA-specific protein species
(Figure 3B). The high molecular weight species of ClfA was not detectable in the srtA mutant
cells, and substantial amounts of at least three low molecular weight species of ClfA were
detectable in the cell wall-bound protein fractions of these cells (Figure 3B). Furthermore, the
ratio between the two low molecular weight extracellular ClfA species was changed in the
srtA mutant. Taken together, these findings show that SrtA is indispensable for covalent cell
wall attachment of proteins with LPxTG motifs, and that these proteins are to greater or lesser
extents released into the growth medium of cells lacking SrtA.

Partially overlapping substrate specificities of sortases A and C of staphylococci
Figure 3. Localization of LPxTG proteins in srtA mutants of S. aureus and S. epidermidis complemented with
different srtA or srtC genes. Sortase A mutants of S. aureus SH1000 and S. epidermidis 1457 were grown in TSB
medium at 37 o C till the early stationary growth phase. Samples of extracellular proteins isolated from the growth
medium (M), non-covalently cell wall-bound proteins (CW) and total cells (C) were used for Western blotting and
immunodetection with antibodies against S. aureus SasG (panel A), S. aureus ClfA (panel B) and S. epidermidis
Aap (panel C). The positions of SasG, ClfA, and Aap are marked with arrows. Constructs used for
complementation of ∆srtA mutations are indicated on top of each gel.
Protein export in a srtA mutant of S. epidermidis
To investigate how a srtA mutation influences the localization of proteins in S. epidermidis,
the extracellular and non-covalently cell wall-bound proteins of S. epidermidis 1457 and a
srtA mutant derivative of this strain were analyzed by SDS-PAGE. Bands of different
intensities were excised and analyzed by mass spectrometry. A major difference in
localization was observed for the Aap protein, which was dislocated to the growth medium of
the srtA mutant in much higher amounts than was the case for the parental strain 1457 (Figure
2A). Furthermore, while two Aap-specific bands were detectable in the growth medium of the
parental strain, which probably correspond to the 220 kD and 180 kD forms of Aap described
by Rohde et al. (Rohde et al., 2005), a third Aap-specific band was detectable in the medium
of the srtA mutant. This might be the 140 kD Aap-derived band that is necessary for biofilm
formation. Also, in the non-covalently cell wall-bound protein fraction of the S. epidermidis
srtA mutant (Figure 2B), three bands for Aap were identified, which seem to correspond to
the previously described 220 kD, 180 kD and 140 kD isoforms. Notably, these three isoforms
are not present amongst the non-covalently cell wall-bound proteins of the parental strain.
Furthermore, the fraction of non-covalently cell wall-bound proteins of the srtA mutant
contained somewhat lower amounts of the bifunctional autolysin AtlE. The subcellular
localization of Aap was further examined by Western blotting and immunodetection with
specific antibodies. As shown in Figure 3C, two dominant Aap-specific bands of ~220 kD and
~180 kD, and a faint band of ~140 kDa, were detectable in growth medium samples of the
parental strain 1457. These bands probably correspond to the 220 kDa, 180 kDa and 140 kDa
bands identified by Rohde et al (Rohde et al., 2005). Remarkably, no Aap was detectable in
the total cell fraction. In this respect, the behavior of Aap seems to differ from that of its S.
aureus homologue SasG, which was detectable as a high molecular weight band (Figure 3A).
Due to the srtA mutation, the extracellular amounts of the 220 kDa, 180 kDa and 140 kDa
forms of Aap were significantly increased, and substantial amounts of the 220 kDa and 180
kDa forms were also detectable in the fraction of non-covalently cell wall-bound proteins. To
a lesser extent the 220 kDa and 180 kDa forms were also detectable in the cellular fraction of
the srtA mutant. These findings imply that, in the absence of SrtA, substantial amounts of Aap
remain bound to the cell wall, but in a non-covalent manner.
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Chapter 6
Complementation analysis of srtA mutant strains with Class A, C and D sortases
Deletion of the srtA genes of S. aureus and S. epidermidis resulted for both organisms in clear
changes in the localization of several proteins to the cell wall and growth medium. To
distinguish between the effects of the srtA mutation and possible unwanted second site
mutations, a complementation analysis was performed with the homologous srtA gene
expressed from a plasmid. As shown in Figures 2 and 3, full reversion of the observed protein
localization phenotypes was achieved by ectopic expression of the S. aureus srtA gene in the
S. aureus ∆srtA mutants, and by ectopic expression of the S. epidermidis srtA gene in the S.
epidermidis ∆srtA mutant. Interestingly, full reversion of the observed phenotypes was also
achieved when the S. aureus srtA gene was expressed in the S. epidermidis ∆srtA mutant, or
when the S. epidermidis srtA gene was expressed in the S. aureus ∆srtA mutant (Figure 2,
Figure 3C). This raised the question to what extents the phenotypes of srtA mutant strains
could also be reverted by ectopic expression of sortases of other classes that also seem to
recognize the LPxTG motif, like SrtC or SrtD. To address this question, the S. epidermidis
srtC gene and the B. subtilis yhcS gene (encoding a class D sortase) were expressed from the
same plasmid-borne promoters that were used for the complementation analyses with srtA
genes. Furthermore, for expression in the S. aureus srtA mutant, all heterologous sortase
genes were provided with the ribosome-binding site and start codon from S. aureus srtA to
minimize any possible differences in translation of the different sortases. Conversely, for
expression in the S. epidermidis srtA mutant, all heterologous sortase genes were provided
with the ribosome-binding site and start codon from S. epidermidis srtA. As shown in Figure
2 and Figure 3C, none of the phenotypes observed in S. aureus or S. epidermidis srtA mutant
strains were reversed by expression of yhcS from B. subtilis. In contrast, expression of srtC
did lead to the complementation of some, but not all phenotypes of srtA mutant strains. As
shown in Figure 2, expression of srtC in the S. aureus srtA mutant did not result in lowered
extracellular levels of the LPxTG proteins SdrC, sdrD and protein A as would be expected if
srtC could complement for the absence of srtA. However, srtC expression did result in a
partial complementation of the localization defect observed for SasG in the S. aureus srtA
mutant, where clearly lowered amounts of non-covalently cell wall-bound forms of SasG
were observed, as well as an increased amount of the high molecular weight cellular form of
SasG (Figure 3A). Similarly, srtC expression in the S. aureus srtA mutant resulted in a partial
restoration of the localization of ClfA, the most prominent effect being the re-appearance of
the high molecular weight form of ClfA in the cellular fraction (Figure 4B). Furthermore, srtC
expression substantially reduced the amounts of low molecular weight forms of ClfA in the
fraction of non-covalently cell wall-bound proteins, but not to the extent that was observed
when the srtA mutant was complemented with srtA of S. aureus (Figure 3B).
Consistent with the observations for SasG in S. aureus (Figure 3A), expression of srtC in the
S. epidermidis srtA mutant resulted in a significant reversion of the dislocation phenotype of
the SasG homologue Aap (Figure 2 and Figure 3C). Upon srtC expression, the amounts of the
~220 kDa and ~140 kDa forms were clearly reduced in the growth medium, as well as the
non-covalently cell wall-bound protein fractions of S. epidermidis ∆srtA. In both fractions, the
strongest effects of srtC expression were observed for the ~220 kDa species of Aap, which
virtually disappeared. Furthermore, the expression of srtC resulted in increased amounts of
AtlE in the fraction of non-covalently cell wall-bound proteins. Taken together, these findings
show that SrtA and SrtC have at least partially overlapping substrate specificities.
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Partially overlapping substrate specificities of sortases A and C of staphylococci
Biofilm formation by complemented srtA mutants of S. aureus and S. epidermidis
Surface proteins like SasG and protein A of S. aureus (Corrigan et al., 2007;Merino et al.,
2009) and Aap in S. epidermidis (Hussain et al., 1997;Rohde et al., 2005;Rohde et al., 2007)
have been implicated in biofilm formation. Therefore, we analyzed the biofilm-forming
capacity of complemented srtA mutants of S. aureus and S. epidermidis using a crystal-violetbinding
assay. The results are summarized in Figure 4.
A 590
2,5
2
1,5
1
0,5
0
1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6
RN4220 SH1000 1457
A
125
A 590
2,5
2
1,5
1
0,5
0
WT srtA Sa.srtA-pRIT5H Se.srtC-pRIT5H
Figure 4. Biofilm formation by srtA mutants of S. aureus and S. epidermidis complemented with different srtA
or srtC genes. Biofilm formation in 96-well microtiter plates by cells grown for 24 hours at 37ºC in TSB medium
with 5% glucose was measured using crystal-violet staining. The amounts of crystal-violet liberated from the
biofilms upon treatment with 33% acetic acid were determined by measuring the absorbance at 590 nm (A 590). For
each strain, biofilm formation was measured sixteen times. (A) A 590 measurements for ∆srtA mutants of S. aureus
RN4220, S. aureus SH1000 and S. epidermidis 1457. 1, parental strain; 2, ∆srtA mutant; 3, ∆srtA mutant
complemented with srtA from S. aureus; 4, ∆srtA mutant complemented with srtA from S. epidermidis; 5, ∆srtA
mutant complemented with yhcS of B. subtilis; 6. ∆srtA mutant complemented with srtC of S. epidermidis. (B) A 590
measurements for the srtA mutant of S. aureus SH1000 overproducing SasG and complemented with srtA from S.
aureus, or srtC of S. epidermidis.
Interestingly, the biofilm-forming capacity of srtA mutants of the S. aureus strains RN4220
and SH1000 was significantly reduced compared to the respective parental strains. Biofilm
formation by the srtA mutant of S. aureus RN4220 was largely complemented by expression
of the srtA genes from S. aureus or S. epidermidis, whereas biofilm formation by the srtA
mutant of S. aureus SH1000 was complemented by the S. aureus srtA gene, but not by the S.
epidermidis srtA gene. This suggests that at least one covalently cell wall-bound protein of S.
aureus is not properly attached to the cell wall by the heterologously produced SrtA of S.
epidermidis. Furthermore, neither yhcS of B. subtilis, nor srtC of S. epidermidis were able to
restore biofilm formation by S. aureus srtA mutant strains. Interestingly, the negative effect of
the srtA mutation on biofilm formation by the S. aureus SH1000 ∆srtA strain was largely
suppressed by SasG overproduction (Figure 4B), which indicates that, despite the absence of
SrtA, sufficient SasG was correctly localized to have a stimulating effect on biofilm
formation. Notably, biofilm formation was enhanced to levels that exceeded the biofilm
formation by the SasG-overproducing parental strain SH1000 when the S. aureus srtA gene or
the S. epidermidis srtC gene were ectopically expressed. This shows that SrtA is a limiting
factor for the correct localization and functionality of overproduced SasG and that this
particular function of SrtA can also be fulfilled by SrtC.
In contrast to what was observed in srtA mutants of S. aureus RN4220 and SH1000, but
similar to the SasG overproducing S. aureus srtA mutant, the srtA mutant of S. epidermidis
1457 did not display a clear defect in biofilm formation. Interestingly however, the ectopic
expression of srtA from S. aureus or S. epidermidis in the S. epidermidis srtA mutant resulted
in a significant increase in the biofilm-forming capacity of S. epidermidis, similar to what was
B

Chapter 6
observed for SasG-overproducing S. aureus SH1000 strains. In contrast, the ectopic
expression of srtC interfered with biofilm formation by S. epidermidis (Figure 4A). Taken
together, our present findings show that the activities of SrtA and SrtC can be a limiting
factors in biofilm formation by S. aureus and S. epidermidis, and that this feature can be
correlated with the level of SasG production in S. aureus.
Discussion
Surface proteins of the Gram-positive bacterial pathogens S. aureus and S. epidermidis serve
important roles in virulence and biofilm formation. Several of these surface proteins are
linked covalently to the cell wall by the transpeptidase SrtA, which recognizes a C-terminal
LPxTG motif for cell wall attachment of its substrates. Previous studies have already shown
that srtA mutants of S. aureus are attenuated in their ability to cause infections in animal
models (Mazmanian et al., 2001). In the present studies we report for the first time the
construction of a srtA mutant of S. epidermidis. This mutant and equivalent srtA mutants of S.
aureus were used to address three questions. First, we investigated to what extent srtA
mutations affect the localization of cell wall-associated proteins. The results show that the
absence of SrtA causes substantial changes in the composition of the S. aureus and S.
epidermidis exoproteomes, mainly due to the secretion of normally cell wall-attached
proteins. Nevertheless, substantial amounts of the different LPxTG proteins remain attached
to the cell wall in a non-covalent manner. Secondly, the srtA mutants were used to study
whether there is any overlap in the substrate specificities of class A, C and D sortases, all of
which recognize proteins with LPxTG motifs. Our results show that the substrate specificities
of the staphylococcal class A and C sortases overlap only partially. Thirdly, we addressed the
roles of sortases in biofilm formation by S. aureus and S. epidermidis, which revealed that
sortase activity can be a limiting factor in this process.
Three possible phenotypes can be expected when SrtA is not expressed. Firstly, the LPxTG
proteins may remain anchored to the cell surface through their C-terminal transmembrane
domain. Secondly, the LPxTG proteins may be released into the growth medium through
proteolytic “shaving” by extracellular proteases, a phenomenon that was previously observed
for many unprocessed lipoproteins (Antelmann et al 2001; Venema et al 2003; Stoll et al
2005). Thirdly, the LPxTG proteins may remain attached to the cell surface via non-covalent
interaction with components of the cell wall. Clearly, all LPxTG proteins investigated in the
present studies were released into the growth medium of srtA mutant strains, which implies
that they had lost their C-terminal transmembrane domains. Nevertheless, we cannot exclude
the possibility that a subfraction of these proteins remained attached to the membrane via an
uncleaved C-terminal transmembrane domain. Furthermore, substantial amounts of the
LPxTG proteins remained attached to the cell wall in a non-covalent manner. This is not
altogether surprising since several of these proteins have repeated cell wall-binding domains.
For example, LysM domains for peptidoglycan-binding are present in the protein A of S.
aureus (Buist et al., 2008), and G5 repeats for N-acetylglucosamine-binding are present in
SasG of S. aureus and Aap of S. epidermidis (Bateman et al., 2005). Interestingly, high
molecular weight species of SasG were observed in srtA-proficient cells of S. aureus. These
might represent SasG proteins interacting with each other through their G5 domains as was
previously proposed for the homologous Aap protein of S. epidermidis (Conrady et al., 2008).
However, the high molecular weight species may also represent SasG molecules covalently
attached to the cell wall of S. aureus. Similar explanations can be entertained for the presence
126

Partially overlapping substrate specificities of sortases A and C of staphylococci
of a high molecular weight form of ClfA in srtA-proficient cells of S. aureus. Clearly, in
absence of srtA these proteins are not linked covalently to the cell wall and, in agreement with
this notion, no high molecular weight species of SasG and ClfA were detectable in srtA
mutant cells. It is in this context remarkable that we did not detect a high molecular weight
species of Aap in srtA-proficient S. epidermidis cells. Possibly, such a species remained
undetectable due to a poor mobility in SDS-PAGE.
Interestingly, all LPxTG proteins of S. aureus and S. epidermidis that were found to be
dislocated in the respective srtA mutant strains contain a YSIRK/GS domain within their
signal peptide. It has been shown that the proteins with this YSIRK/GS motif, such as ClfA,
protein A, fibronectin-binding protein B (FnbpB), and the serine-aspartate repeat proteins
SdrC and SdrD display a ring-like distribution on the S. aureus cell surface (Dedent et al.,
2008). This has led to the proposal that proteins with the YSIRK/GS motif are sitespecifically
translocated to the cross wall, which is the peptidoglycan layer that forms during
cell division to separate new daughter cells. Our finding that in particular proteins with the
YSIRK/GS motif in their signal peptides are dislocated to the growth medium when SrtA is
absent could suggest that the release of these proteins from the cell wall is related to the site
of secretion or surface display. This could also be a possible explanation for the observed
increased release of the lipase LipA by the srtA mutant of S. aureus. However, it has to be
noted that secretion of the lipase GehC of S. epidermidis, which also has the YSIRK/GC
motif in its signal peptide, was not detectably influenced by the srtA mutation. Most likely,
the observed effects of srtA mutations on the localization of non-LPxTG proteins, such as
LipA, Hla and Map of S. aureus, or AtlE of S. epidermidis are indirectly caused by the
absence of SrtA. This could relate to as yet unidentified alterations in the cell wall
composition of srtA mutant strains, or perhaps even to precluded interactions with LPxTG
proteins that are dislocated due to the srtA mutations.
To date, little information is available on the class C and D sortases in Gram-positive bacteria.
Since these sortases also recognize the LPxTG motif, we studied the complementation of the
S. aureus and S. epidermidis srtA mutants with srtC from S. epidermidis ATCC12228 or yhcS
(srtD) from B. subtilis 168. No complementation was observed upon introduction of yhcS in
any of the srtA mutant strains tested. This may either mean that YhcS does not recognize the
LPxTG proteins that we monitored in the present studies, or that the cells contained
insufficient amounts of YhcS which could, for example, be due to inefficient translation or
post-translational degradation. In contrast, partial complementation was observed for the
localization of SasG and ClfA in srtA mutant strains of S. aureus expressing srtC, and for Aap
and AtlE in the srtA mutant strain of S. epidermidis expressing srtC. The molecular basis for
this partial overlap in the specificities of SrtA and SrtC is presently not completely clear.
Firstly, the “LPxTG” sites of SasG (LPKTG), ClfA (LPDTG) and Aap (LPDTG) differ only
in the non-conserved central “x residue” with the LPxTG sites of SdrC, SdrD and protein A
(LPETG). This could mean that a Glu residue at the x position is not acceptable for SrtC,
whereas Lys or Asp residues at this position are acceptable both for SrtA and SrtC. Based on
bioinformatics analyses, Comfort and Club (Comfort and Clubb, 2004) have classified
various LPxTG recognition sites for different sortases, which suggests that a central Lys
residue in the LPxTG motif, as encountered in SasG, would be acceptable to several different
groups of sortases. This would be consistent with our present findings that SasG is a substrate
for SrtA and SrtC. In contrast, this bioinformatics-based classification did not indicate LPxTG
motifs with an Asp residue at the central x position as SrtA substrates. Even so, our present
analyses indicate that proteins, like ClfA and Aap, which have an LPDTG motif are SrtA
127

Chapter 6
substrates that are also recognized by SrtC. Clearly, at this stage we cannot rule out the
possibility that other features of SasG, ClfA and Aap are probably responsible for the fact that
these LPxTG proteins are substrates for SrtA and SrtC, while SdrC, SdrD and protein A are
only substrates for SrtA. Furthermore, SrtC displays several structural differences to class A
sortases (Figure 1). Specifically, SrtA of S. aureus and SrtC of S. epidermidis 12228 merely
share 34% amino acid sequence identity. Importantly, the key residues involved in catalysis
(His-120, Cys-184 and Arg-197) and substrate recognition (Val-168 and Leu-169) are
conserved in both sortases, but the differences between both proteins are large enough to
allow for specific differences in the geometry of their active sites. Similarly, a stretch of
amino acids in the β6/β7 loop was shown to determine the substrate specificity of SrtB
(Bentley et al., 2007). Intriguingly, Aap appears to be conserved also in S. epidermidis 12228
from which the srtC gene was derived. This suggests that the SrtC of this S. epidermidis strain
may not only be dedicated to cell wall attachment of the LPxTG proteins SesJ and SesK as
was previously suggested (Gill et al., 2005), but it may also be involved in covalent cell wall
attachment of Aap.
The results of our comparative analyses on the roles of sortases in biofilm formation by S.
aureus and S. epidermidis are intriguing. Clearly, SrtA is important for biofilm formation by
S. aureus, whereas this sortase is dispensable for biofilm formation by S. epidermidis. At this
stage, it is difficult to say which LPxTG protein is responsible for the SrtA-dependence of
biofilm formation by S. aureus, but protein A is clearly an attractive candidate. Firstly,
Merino et al. have recently shown the involvement of protein A in the formation of proteindependent
biofilms in S. aureus (Merino et al., 2009) and, secondly, a major dislocating effect
of the srtA mutation was observed for protein A in the present studies. However, also other
LPxTG proteins may be involved in this phenomenon. Furthermore, SasG overexpression was
able to compensate for the absence of SrtA, underpinning the importance of SasG for proteindependent
biofilm formation. However, in this case, the levels of biofilm formation were even
further increased upon ectopic expression of SrtA or SrtC, which indicates that sortase
activity is a limiting factor for biofilm formation under the conditions tested. The finding that
StrC production in SasG-overproducing cells did also stimulate biofilm formation is
consistent with the finding that SrtC was able to revert the SasG dislocation phenotype of srtA
mutant cells at least in part. In the light of these findings, the observation that S. epidermidis
srtA mutant cells were not impaired in biofilm formation is not really surprising since these
cells produced readily detectable amounts of the SasG homologue Aap. Also consistent with
the findings in S. aureus cells producing SasG, the ectopic expression of SrtA in S.
epidermidis had a strongly stimulating effect on biofilm formation. This shows that at least in
S. epidermidis strain 1457, SrtA is a limiting determinant for biofilm formation. Whether this
is also the case in other S. epidermidis strains remains to be shown. Quite unexpectedly, srtC
expression resulted in impaired biofilm formation by S. epidermidis 1457. However, this
observation can be reconciled with the fact that the S. epidermidis strain 12228 from which
the srtC gene was isolated is unable to form biofilms (Zhang et al., 2003), despite the fact that
this strain has an Aap-encoding gene. It is thus tempting to speculate that protein-dependent
biofilm formation by S. epidermidis 12228 is suppressed by SrtC. Possibly, the SrtCdependent
suppression of biofilm formation in S. epidermidis 1457 is correlated to the
strongly suppressed extracellular accumulation of the ~220 kDa and ~140 kDa forms of Aap,
which are actually present in lower amounts than observed in the growth medium of the
parental strain 1457. However, this needs to be investigated in more detail in future studies
128

Partially overlapping substrate specificities of sortases A and C of staphylococci
with particular attention for the role of srtC in a possible suppression of biofilm formation in
S. epidermidis 12228.
Acknowledgements
We like to thank W. Baas and M. ten Brinke for technical assistance, S. Dubrac for providing
the pCN51 plasmid, and colleagues from the StaphDynamics and TIPharma programs for
helpful discussions. Financial support was provided by the CEU (LSHM-CT-2006-019064,
LSHG-CT-2006-037469 and PITN-GA-2008-215524), DFG (GK212/3-00, SFB/TR34, FOR
585), and Top Institute Pharma (T4-213).
129

“Without music, life would be a mistake”
-Friedrich W. Nietzsche-
130

Chapter 7
The large mechanosensitive channel MscL determines bacterial
susceptibility to the bacteriocin sublancin 168
R.H.M. Kouwen, E.N. Trip, E.L. Denham, M.J.J.B. Sibbald,
J.-Y.F. Dubois, and J.M. van Dijl #
Published in Antimicrobial Agents and Chemotherapy (2009) 53, 4702-4711
131

Chapter 7
Summary
Bacillus subtilis strain 168 produces the extremely stable and broad-spectrum lantibiotic
sublancin 168. Known sublancin 168 susceptible organisms include important
pathogens, such as Staphylococcus aureus. Nevertheless, since its discovery, the mode of
action of sublancin 168 has remained elusive. The present studies were, therefore, aimed
at the identification of cellular determinants for bacterial susceptibility towards
sublancin 168. Growth inhibition and competition assays on plates and in liquid cultures
revealed that sublancin 168-mediated growth inhibition of susceptible B. subtilis and S.
aureus cells is affected by the NaCl concentration in the growth medium. Added NaCl
did not influence the production, activity or stability of sublancin 168 but, instead,
lowered the susceptibility of sensitive cells towards this lantibiotic. Importantly, the
susceptibility of B. subtilis and S. aureus cells towards sublancin 168 was shown to
depend on the presence of the large mechanosensitive channel of conductance MscL. In
contrast, MscL was not involved in susceptibility towards the bacteriocins nisin or Pep5.
Taken together, our unprecedented results demonstrate that MscL is a critical and
specific determinant in bacterial sublancin 168 susceptibility that may either serve as a
direct target for this lantibiotic, or as a gate of entry to the cytoplasm.
132

The large mechanosensitive channel MscL determines bacterial susceptibility to
the bacteriocin sublancin 168
Introduction
Lantibiotics are small post-translationally modified peptides with antimicrobial activity that
are produced by Gram-positive bacteria (Chatterjee et al., 2005; McAuliffe et al., 2001; Sahl
and Bierbaum, 1998). The Bacillus subtilis strain 168 is known to produce an extremely
stable lantibiotic named sublancin 168. This lantibiotic exhibits bactericidal activity against
other Gram-positive bacteria, including important pathogens, such as Bacillus cereus,
Streptococcus pyogenes, and Staphylococcus aureus (Paik et al., 1998; Stein, 2005).
Sublancin 168 is encoded by the sunA gene, which is located within the SPβ prophage region
of the B. subtilis 168 chromosome (Hemphill et al., 1980; Lazarevic et al., 1999; Paik et al.,
1998). Newly synthesized sublancin 168 is exported from the cytoplasm by the ABC
transporter SunT, the gene of which is located immediately downstream of sunA (Serizawa et
al., 2005; Dorenbos et al., 2002). SunT also contains a proteolytic domain (McAuliffe et al.,
2001) and is, therefore, thought to be required both for sublancin 168 export and concomitant
removal of the leader peptide (Dorenbos et al., 2002; Paik et al., 1998). It is noteworthy that
sublancin 168 displays the extraordinary characteristic for lantibiotics of having two disulfide
bonds in addition to a β-methyllanthionine bridge (Paik et al., 1998). The thiol-disulfide
oxidoreductase BdbB, which is encoded by a gene downstream of sunA and sunT, appears to
be of major importance for the formation of the disulfide bonds (Kouwen et al., 2007;
Dorenbos et al., 2002).
The cellular target(s) of sublancin 168 and the determinant(s) for producer immunity
against this lantibiotic have remained elusive for a long time. Very recently however, we have
identified the SunI protein (also known as YolF) as the immunity protein that protects
producer cells against sublancin 168 (Dubois et al., 2009). SunI was found to be both required
and sufficient for sublancin 168 immunity, even when produced in a heterologous sublancinsensitive
host organism, such as S. aureus. Interestingly, localization studies showed that the
SunI protein is anchored to the membrane through a single N-terminal membrane-spanning
domain with the bulk of the protein facing the cytoplasm. This is a topology that has not been
reported before for other known bacteriocin immunity proteins (Dubois et al., 2009).
The present studies were aimed at identifying bacterial determinants that confer
susceptibility to sublancin 168. To date, two major mechanisms for bactericidal lantibiotic
activity have been reported. Type A lantibiotics, such as nisin (Kuipers et al., 1993; Siegers et
al., 1996), epidermin (Peschel and Götz, 1996), and Pep5 (Meyer et al., 1995) usually act by
forming pores in the cytoplasmic membrane of sensitive target organisms in processes that
may involve specific molecules, such as the cell wall precursor lipid II (Breukink et al., 1999;
Wiedemann et al., 2001). In contrast, type B lantibiotics, such as cinnamycin (Fredenhagen et
al., 1990) and mersacidin (Chatterjee et al., 1992), inhibit particular enzyme functions. On the
basis of its leader peptide sequence, sublancin 168 was previously classified as a type A
lantibiotic (Paik et al., 1998). Nevertheless, sublancin 168 does not display the usual flexible,
elongated and amphipathic molecular shape that is so characteristic for other type A
lantibiotics (Nagao et al., 2006), suggesting that sublancin 168 might have a specific mode of
action. Consistent with this idea, our present results show that the sublancin 168 susceptibility
of B. subtilis and S. aureus is determined by the presence of large-conductance
mechanosensitive channels, which is an unprecedented finding.
133

Chapter 7
Materials and Methods
Bacterial strains, plasmids and growth media
The bacterial strains and plasmids used in this study are listed in Table 1.
Table 1. Strains and plasmids used in this study
Plasmids Relevant properties Reference
pDG783 pSB118 derivative; contains the kanamycin resistance marker
from Streptococcus faecalis; Amp R ; Km R
(Guérout-Fleury et
al., 1995)
pUC18 Ap R , ColE1, φ80lacZ, lac promoter (Sambrook et al.,
1989)
pX Vector for the integration of genes in the amyE locus of B.
subtilis; integrated genes will be transcribed from the xylose
inducible xylA promoter; carries the xylR gene; Amp R ; Cm R
(Kim et al., 1996)
pXTC pX derivative in which the chloramphenicol resistance marker
has been replaced with a tetracycline resistance marker; Amp R (Darmon et al.,
; 2006)
Tet R
TOPO pCR®-Blunt II-TOPO® vector; Km R Invitrogen Life
Technologies, Inc.
pMAD shuttle vector for E. coli and S. aureus with thermosensitive ori
in S. aureus; contains the bgaB gene; Em R ; Amp R
(Arnaud et al., 2004)
pMAD-mscL pMAD plasmid with the flanking regions of S. aureus mscL;
Em R ; Amp R
This work
BaSysBio II Ligation-independent cloning vector based on pDG1727 for
Promoter-GFP activity analysis; Amp R , Spec R
Stéphane Aymerich
et al., unpublished
Strains
E. coli
Genotype Reference
DH5α F - , Ф80dlacZ ∆M15 endA1 recA1 gyrA96 thi-1 hsdR17 (rk- Invitrogen Life
mk+) supE44 relA1 deoR ∆(lacZYA-argF) U169
Technologies, Inc.
TOP10 Cloning host for TOPO vector; F - mcrA ∆(mrr-hsdRMS-mcrBC)
Φ80lacZ∆M15 ∆lacX74 recA1 araD139 ∆(ara-leu)7697 galU
galK rpsL (Str R Invitrogen Life
B. subtilis
) endA1 nupG
Technologies, Inc.
168 trpC2 (Kunst et al., 1997)
168 Cm trpC2; amyE::pX; Cm R
(Dubois et al., 2009)
∆SPβ trpC2; ∆SPβ; sublancin 168 sensitive (Dorenbos et al.,
2002)
∆SPβ Tc trpC2; ∆SPβ; amyE::pXTC; sublancin 168 sensitive; Tet R
(Dubois et al., 2009)
∆mscL trpC2; pMUTIN4mcs::mscL; double crossover deletion of mscL;
Em R
BSFA collection
strain BFS1257
∆SPβ∆mscL trpC2; ∆SPβ; pMUTIN4mcs::mscL; Em R This work
Strains
B. subtilis
Genotype Reference
∆sunA-
∆yolF
trpC2; ∆yolF; ∆sunA; Km R (Dubois et al., 2009)
∆sunA trpC2; ∆sunA; Km R S. aureus
(Dubois et al., 2009)
RN4220 Restriction-deficient derivative of NCTC 8325, cured of all (Kreiswirth et al.,
known prophages; rsbU-; agr-
1983)
RN4220 RN4220 that contains the pMAD vector for erythromycin
Em resistance; Em R
This work
RN4220
∆mscL
RN4220 derivative; rsbU-; agr-; ∆mscL This work
134

The large mechanosensitive channel MscL determines bacterial susceptibility to
the bacteriocin sublancin 168
The standard LB medium consisted of 1% trypton, 0.5% yeast extract and 1% NaCl, pH 7.4. Where
appropriate, the NaCl content of the LB medium was adjusted to concentrations ranging from 0 to 5%.
S. aureus strains were grown in brain-heart infusion broth (BHI) or tryptone soya broth (TSB). Where
necessary, media were supplemented with antibiotics at the following concentrations: ampicillin, 100
µg/ml (Escherichia coli); kanamycin, 20 µg/ml (E. coli, B. subtilis, S. aureus); chloramphenicol, 5
µg/ml (E. coli and B. subtilis); tetracycline, 10 µg/ml (E. coli and B. subtilis); erythromycin, 100 µg/ml
(E. coli), 2 µg/ml (B. subtilis) or 5 µg/ml (S. aureus); spectinomycin, 100 µg/ml (B. subtilis). To
visualize α-amylase activity (specified by the amyE gene), LB plates were supplemented with 1%
starch. To visualize β-galactosidase activity, plates contained 5-bromo-4-chloro-3-indolyl-β-Dgalactopyranoside
(X-GAL) at a final concentration of 80 µg/ml.
DNA techniques
Procedures for DNA amplification, restriction, ligation and transformation of E. coli DH5α were
carried out according to standard laboratory procedures (Sambrook et al., 1989). Chromosomal DNA
of B. subtilis was isolated according to Bron and Venema (Bron and Venema, 1972). B. subtilis was
transformed as described by Kunst and Rapoport (Kunst and Rapoport, 1995). S. aureus was
transformed by electroporation as described by Kreiswirth et al. (Kreiswirth et al., 1983). All primers
used for PCR are listed in Table 2. PCR products were purified using the High Pure PCR Purification
Kit (Roche Applied Science).
Table 2. Primers used in this study
Primer Sequence (5’→3’)
pSU1 ATATATACCATCATTGAATCGAGA
pSU2 CAAGACGAACTCCAATTCAC AAAACTATCGTCAATTCTGCAGA
pYF1 TACATCCGCAACTGTCCATA GATTATCATAACTACATATTCCAT
pYF2 GCTACTCAGTAAGCTTGCACT
Kana1 GTGAATTGGAGTTCGTCTTG
Kana2 TATGGACAGTTGCGGATGTA
mscL-F1 CATAGCAAACCATGAAATTGT
mscL-R1 TCACGTCAGTCAGTCACCATGGCATTACACTCAACCTCTCTTTTT
mscL-F2 TGCCATGGTGACTGACTGACGTGATTTTTAAATAAAAAGAGATGG
mscL-R2 GCAAATCTGAGATTAGCAACA
sunA Fwd CCGCGGGCTTTCCCAGCCAAATAGTTAGTATTAAAGAGTCAGAC
sunA Rev GTTCCTCCTTCCCACCGTTTGCAATCCGGATTACATT
mscL Fwd CCGCGGGCTTTCCCAGCTTAAAGAAATTATTCCTCACC
mscL Rev GTTCCTCCTTCCCACCTATATTTGTAAAGAAAAAAAGAC
Note: The 5' sequences of primer pSU2 (printed in italics) is complementary to the Kana1 primer. The 5'
sequences of primer pYF1 (printed in italics) are complementary to the Kana2 primer. Sequences for linkage of
amino acids are shown in bold. Sequences for ligation-independent cloning are shown underlined.
Construction of B. subtilis and S. aureus mscL mutant strains
A B. subtilis 168 strain lacking the mscL gene (originally named ywpC) was obtained from the B.
subtilis functional analysis (BSFA) program. This ∆mscL strain was constructed in the laboratory of
Prof. G. Rapoport according to a protocol described in detail on the Micado website
(http://genome.jouy.inra.fr/cgi-bin/micado/index.cgi). Briefly, a PCR fragment containing the 500 bp
region upstream of the mscL gene was fused to a fragment containing the 500 bp region downstream of
the mscL gene and cloned into a pMUTIN4mcs vector using the BamHI and HindIII sites. This plasmid
was subsequently used to transform B. subtilis 168, thereby replacing the mscL gene with the pMUTIN
vector in a double crossover recombination event, which yielded strain ∆mscL. Strain ∆SPβ∆mscL was
constructed by transformation of strain ∆SPβ with genomic DNA of strain ∆mscL and selection for
erythromycin resistant transformants. Correct deletion of mscL from the genomes of the ∆mscL and
∆SPβ∆mscL strains was verified by PCR.
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Chapter 7
Mutants of S. aureus were constructed using the chromosomal integration-excision approach
described by Arnaud et al. (Arnaud et al., 2004). Primers for the downstream (mscL-F2/mscL-R2) and
upstream (mscL-F1/mscL-R1) regions of mscL (Table 2) were designed to obtain PCR products of 524
and 522 bp, respectively, including a 24 bp linker. These PCR products were linked in 10 PCR cycles.
The resulting 1025 bp product was directly ligated into the TOPO-vector (Invitrogen) according to the
manufacturer’s protocol. This construct was digested with BamHI and the 1030 bp product was ligated
into the chromosomal integration-excision vector pMAD, resulting in pMAD-mscL. S. aureus RN4220
cells were transformed with the pMAD-mscL plasmid by electroporation, and grown on TSA-plates
containing erythromycin and X-GAL for 48 hours at 30°C. To obtain cells with a chromosomally
integrated copy of pMAD-mscL, blue colonies were used to inoculate overnight cultures in BHI
medium. Next, 10 ml BHI was inoculated with 100 µl overnight culture, grown for 1 hour at 30°C and
then transferred to 42°C for 6 hours. To select cells with a chromosomally integrated copy of pMADmscL,
dilutions (1000x) of the culture were plated on TSA plates with erythromycin and X-GAL and
incubated for 48 hours at 42°C. To subsequently obtain cells that had excised pMAD-mscL from the
chromosome, blue colonies with integrated pMAD-mscL were used to inoculate overnight cultures in
BHI medium at 42°C. Next, 10 ml BHI was inoculated with 10 µl of the overnight culture and growth
was continued for 6 hours at 30°C. Dilutions (1000x) of the cultures were plated on TSA plates with X-
GAL and incubated at 42°C for 48 hours. White colonies were tested for erythromycin sensitivity and
checked for the presence or absence of mscL by colony PCR. The correct deletion of mscL was
confirmed by PCR on isolated genomic DNA using the Bacterial Genomic DNA Isolation Kit (Sigma).
Lantibiotic activity assays
A sublancin 168-induced B. subtilis growth inhibition assay was performed on plates essentially as
described by Dorenbos et al. (Dorenbos et al., 2002). Briefly, indicator strains and strains to be tested
for sublancin 168 production were grown overnight in LB broth containing the appropriate
antibiotic(s). Overnight cultures of the indicator strains were then diluted 100-fold in LB, and 100 µl
aliquots of the diluted cultures were plated on LB agar. After drying of the plates, 2 µl aliquots of
undiluted overnight cultures of strains to be tested for sublancin 168 production were spotted.
Alternatively, aliquots of nisin, Pep5, or concentrated spent medium with sublancin 168 were spotted.
The plates were then incubated overnight at 37 o C, and growth inhibition of the indicator strain was
analyzed the next day. Nisin was obtained from Sigma, and Pep5 was kindly provided by Dr. Hans
Georg Sahl.
Lyophilization of spent medium
The concentration of sublancin 168 in spent medium of B. subtilis 168 cells was increased by
lyophilization. For this purpose, B. subtilis 168 or the negative control strain ∆sunA were grown in 25
ml of LB broth containing 0% NaCl. At an OD600 of 2.5, cells were separated from the growth medium
by centrifugation and medium fractions were frozen in liquid nitrogen. The frozen growth media were
lyophilized for 4 days under vacuum using a freeze dryer. After this period, the lyophilized medium
was resuspended in 500 µl demineralized water and filtered (0.22 µm) before use. 2 µl of the
concentrate was spotted on agar plates.
Co-culturing of B. subtilis and S. aureus strains
B. subtilis 168 and S. aureus strains were grown separately overnight in LB medium. In the morning,
cultures were diluted to an OD600 of 0.05 in fresh LB medium. Next, specific strains to be tested
together were mixed in a 1:1 ratio, resulting in co-cultures consisting of 50% sublancin producing cells
(B. subtilis 168 Cm) and 50% non-producing cells (either B. subtilis ∆SPβ Tc, B. subtilis ∆SPβ∆mscL,
S. aureus RN4220 Em, or S. aureus RN4220 ∆mscL). Upon mixing, growth was continued for eight or
nine hours. Samples for plating were taken at hourly intervals during growth. The samples thus
obtained were diluted 10 4 or 10 6 fold, and plated on LB agar containing specific antibiotics that permit
growth of only one of the two co-cultured strains. After overnight incubation at 37 o C, resistant colonies
136

The large mechanosensitive channel MscL determines bacterial susceptibility to
the bacteriocin sublancin 168
were counted, and numbers of colony forming units (CFU) per ml of culture of each strain at the time
of sampling were calculated. In case of the S. aureus RN4220 ∆mscL strain, which does not contain an
antibiotic resistance marker, no antibiotics were present in the plates used to determine the CFU
numbers of this strain. Specifically, the CFU number of the ∆mscL strain was calculated by subtraction
of the CFU number of the co-cultivated antibiotic resistant strain from the total CFU number of the two
co-cultivated strains. The presence of B. subtilis and/or S. aureus in samples used for plating was
inspected by light microscopy.
Promoter activity assay
The promoter regions of mscL and sunA were amplified by PCR from genomic DNA prepared from B.
subtilis 168 using the primers described in Table 2. The resulting PCR fragments were prepared for
ligation-independent cloning using T4 DNA Polymerase (Novagen) and dTTP (Aslanidis and de Jong,
1990). The BaSysBioII cloning vector was digested with SmaI, purified from an agarose gel and
prepared for ligation-independent cloning using T4 DNA polymerase (Novagen) and dATP. 1 µl of the
treated vector and 3 µl of the treated insert were mixed and left to anneal at room temperature before
transformation of E. coli. The resulting constructs PmscL-GFP and PsunA-GFP were used to transform
B. subtilis 168 and transformants were selected on LB plates containing spectinomycin. Strains with
mscL-GFP or sunA-GFP fusions were pre-cultured in LB containing 1% or 5% NaCl and diluted 1:100
in the same medium three hours before the start of the growth experiments. Next, the cells were diluted
in the respective medium to an optical density at 600 nm (OD600) of 0.01. Growth was continued in
triplicate wells of a 96-well black optical bottom microtitre plate (Nunc) that was placed in a Biotek
Synergy 2 plate reader (37°C, variable shaking). The OD600 and fluorescence (excitation 485/20,
emission 528/20) of the strains were measured for 14 hours. Fluorescence measurements were
processed essentially as described by Ronen et al. (Ronen et al., 2002). Before starting the experiment
the OD’s of the wells at 977 nm and 900 nm were measured to allow light path correction to 1 cm. For
each of the recorded fluorescence data points the blank of neat LB (average of 3 wells) was removed
and each point was corrected for a light path of 1 cm using the following equation: (0.18/(OD977 -
OD900)). The average of the three samples was then calculated. The fluorescence data was further
processed by calculating the average of the three samples and subtracting the background fluorescence
of the parental strain 168 (without GFP) for each data point. The promoter activity was calculated using
the following equation: (GFP(t) – GFP (t-1))/O.D. (t). We acknowledge the collaborative effort with
the teams of Stéphane Aymerich, Kevin Devine, Vincent Fromion, and Tony Wilkinson in
standardising the fluorescence measurements. Each experiment was repeated at least three times.
Results
Sublancin 168 activity is salt dependent.
To study the activity of sublancin 168 under different growth conditions we made use of a
previously developed sublancin growth inhibition plate assay. The essence of this assay, in
which strains potentially producing sublancin 168 were spotted onto a lawn of sensitive or
immune indicator cells, is demonstrated in Figure 1A. This Figure shows that the ∆SPβ strain,
that lacks all genes of the SPβ prophage (including those for sublancin production and
immunity), is not able to grow in the vicinity of the sublancin 168-producing parental strain
168. This growth inhibition is strictly dependent on the presence of an intact copy of the sunA
gene for sublancin 168 since no zone of growth inhibition is formed around ∆sunA spotted
cells on a plated ∆SPβ cell layer (Figure 1A). As previously demonstrated (Dubois et al.,
2009), the sublancin 168 susceptibility of the ∆SPβ cells is solely due to the absence of the
sunI (yolF) immunity gene, since full sublancin 168 immunity can be restored by ectopic
expression of sunI (Figure 1A). To screen for possible sublancin 168 activity determinants,
137

Chapter 7
we deployed this assay to monitor the effects of growth medium composition on the activity
of sublancin 168. It was thus noticed that the sublancin 168 activity was dependent on the
type of growth medium used in the plate assays (data not shown). Upon inspection of the
composition of the tested media, it was found that, in particular, the NaCl content seemed to
differ.
Figure 1. Sublancin 168-mediated growth inhibition of B. subtilis. (A) sublancin 168 growth inhibition assay.
Strains to be tested for sublancin 168 production were spotted on a lawn of indicator cells. The names of strains
spotted to test for sublancin production are listed above the plate images. Names of the strains plated as indicators
for sublancin sensitivity/immunity are listed below the plate images. (B) NaCl-dependent growth inhibition by
sublancin 168. Strain 168 was used as the spotted sublancin 168 producer, strain ∆SPβ was used as the plated
indicator for sublancin activity. The percentage of NaCl listed below each image indicates the amount of NaCl that
was present in the LB agar plate and in the liquid LB cultures from which the plated or spotted cells were derived.
(C) Growth inhibition by sublancin 168 depends on the presence of MscL. The sublancin 168 producer B. subtilis
168 was spotted on a lawn of plated ∆SPβ ∆mscL indicator cells as described for panel B. (D) Growth inhibition
by nisin or Pep5 does not depend on the presence of MscL. B. subtilis 168 or 2µl aliquots of either 2.5 mg/ml nisin
or 10 mg/ml Pep5 dissolved in water were spotted on a lawn of plated ∆SPβ or ∆SPβ∆mscL indicator cells on LB
agar as in panel A.
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The large mechanosensitive channel MscL determines bacterial susceptibility to
the bacteriocin sublancin 168
To investigate whether the NaCl concentration might influence the outcome of our sublancin
growth inhibition assay, we performed a series of sublancin 168 activity assays in which the
LB broth and agar media used for growth of the 168 and ∆SPβ cells contained increasing
concentrations of NaCl, ranging from 0 to 5%. The results of these experiments showed that
the size of the growth inhibition zone of the ∆SPβ cells is inversely correlated with the NaCl
content of the growth medium (Figure 1B). Next, we investigated whether this effect of NaCl
was related to the plate assay or whether this also occurred in liquid media. For this purpose,
we performed co-culturing and competition experiments in liquid medium by inoculation of
the sublancin 168 producing B. subtilis strain 168 amyE::pX (chloramphenicol R ) in growth
medium in a 1:1 ratio with the non-producing B. subtilis strain ∆SPβ amyE::pXTC
(tetracycline R ). This was done both in LB medium containing the standard concentration of
1% NaCl and in LB medium containing 5% NaCl. The results of co-cultivation and
subsequent transfer of samples to plates containing either chloramphenicol or tetracycline
showed that the ∆SPβ strain was able to survive only for a few hours in the presence of the
sublancin 168 producing strain when these strains were grown in standard LB medium
(Figure 2A). In contrast, when the co-cultures were grown in LB containing 5% NaCl, the
∆SPβ strain was not inhibited by the presence of B. subtilis 168 (Figure 2B). As expected, the
deleterious effect of the 168 strain on the ∆SPβ strain was not observed when the sunA gene
was deleted from the 168 strain (Figure 2C). These findings were fully consistent with those
obtained by the sublancin 168 growth inhibition assays on LB agar (Figure 1B). Taken
together, the results show that the NaCl concentration in the growth medium influences the
outcome of sublancin 168 growth inhibition assays. This suggests that either the production of
sublancin 168, the activity of sublancin 168 or the susceptibility of the target cells are
influenced by the concentration of NaCl in the growth medium.
Figure 2. Assessment of salt-dependent sublancin 168 activity, and MscL-determined sublancin 168
susceptibility, by co-cultivation in liquid cultures. Co-cultures of B. subtilis 168 Cm (white bars) together with B.
subtilis ∆SPβ Tet or ∆SPβ∆mscL Tet (black bars) in LB containing 1% NaCl (A/D) or 5% NaCl (B/E). Likewise,
B. subtilis 168 ∆sunA (white bars) was co-cultured together with B. subtilis ∆SPβ Tet (black bars) in normal LB
containing 1% NaCl (C). The overnight grown strains 168 Cm, 168 ∆sunA, ∆SPβ Tet or ∆SPβ ∆mscL Tet were
diluted to an OD 600 of 0.05 in fresh LB medium containing the required amounts of NaCl and mixed in a 1:1 ratio,
resulting in co-cultures consisting of 50% B. subtilis 168 Cm or B. subtilis 168 ∆sunA plus 50% of B. subtilis
∆SPβ Tet or B. subtilis ∆SPβ∆mscL Tet. Growth was continued and monitored by OD 600 measurements (depicted
as a black line) and samples were plated at hourly intervals. Chloramphenicol, kanamycin and tetracycline
resistant colonies were counted and used to calculate the number of colony forming units (CFU) per ml of culture
for each strain at each time point of sampling.
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Chapter 7
NaCl affects the sublancin 168 susceptibility of B. subtilis
To explore the nature of the effect of NaCl, as observed in the sublancin 168 activity assays,
we first investigated a possible influence of NaCl on the production of sublancin 168. As an
indication for this, we measured the activity of the sunA promoter in the B. subtilis 168 strain
grown in LB medium with either 1% or 5% NaCl, using a transcriptional fusion between the
sunA promoter and the GFP gene (PsunA-GFP). The results of this analysis show that the
sunA promoter displayed similar average promoter activities in both media, although the 168
cells grew somewhat slower in LB medium with 5% NaCl than in LB medium with 1% NaCl
(Figure 3A). This suggests that the different outcomes in the sublancin 168 growth inhibition
assays in the presence of 1% or 5% NaCl are not due to differences in the levels of sublancin
168 production by B. subtilis 168. Interestingly, expression of sunA is rather suppressed until
late exponential phase, which seems to coincide with the onset of growth inhibition as
observed in Figure 2A.
A
5000
4000
3000
2000
Promoter
B
Promoter
Activity
140
2500
2000
1500
1000
500
-500
-1000
-1500
0
1
0 100 200 300 400 500 600
Figure 3. Promoter activities of sunA and mscL. Promoter activities of sunA (A) and mscL (B) were measured
with a B. subtilis 168 strain expressing GFP from the native sunA or mscL promoter. Promoter activity is
calculated as (GFP(t)-GFP(t-1)) / OD 600. Cells were grown in LB containing 1% NaCl (black lines) or 5% NaCl
(grey lines). Promoter activity is shown with continuous lines; OD 600 is shown with interrupted lines.
As an alternative, we investigated the possible direct effects of NaCl on the stability or
activity of sublancin 168. For this purpose, we concentrated sublancin 168 by lyophilization
of spent medium (without NaCl) from the sublancin producer B. subtilis 168. By doing so, we
increased the sublancin 168 concentration 50-fold. Figure 4A shows that a spotted concentrate
of this spent medium inhibits the growth of plated ∆SPβ cells, whereas a control concentrate
from the spent medium of a ∆sunA strain does not inhibit the growth of plated ∆SPβ cells.
Next, we used the concentrate from the 168 spent medium to establish whether NaCl might
directly affect the stability or activity of sublancin 168. For this purpose, the concentrated
spent media were incubated overnight with or without 5% NaCl, prior to spotting. The very
similar sizes of the resulting growth inhibition zones of ∆SPβ cells on plates without NaCl
(Figure 4B) revealed that the activity of the concentrated sublancin 168 was not affected by
overnight incubation with 5% NaCl. This indicated that the absence of sublancin 168-directed
growth inhibition during growth on LB with 5% NaCl is not due to an irreversible
inactivating effect of NaCl on sublancin 168.
To investigate the possible effect of NaCl on the ∆SPβ indicator cells, we also spotted
the concentrated 168 medium on ∆SPβ cells that were plated on LB agar plates containing 5%
NaCl. The results show that ∆SPβ cells plated on LB agar containing 5% NaCl were no
longer sensitive to the spotted sublancin 168, in contrast to ∆SPβ cells plated on LB agar
containing 0% NaCl (Figure 4B). Taken together, these findings indicate that NaCl does not
Time
2,5
2
1,5
0,5
0
OD 600nm

The large mechanosensitive channel MscL determines bacterial susceptibility to
the bacteriocin sublancin 168
influence the production, activity or stability of sublancin 168. Instead NaCl seems to
influence the sublancin 168 susceptibility of B. subtilis.
Figure 4. NaCl influences the sublancin 168 susceptibility of B. subtilis. (A) Sublancin 168 growth inhibition
assays were performed with concentrated spent medium fractions (“concentrate”) of B. subtilis 168 (contains
sublancin 168) or B. subtilis 168 ∆sunA (no sublancin present), which were spotted on a lawn of plated ∆SPβ
cells. The concentrates were obtained by lyophilization of spent media. (B) Before spotting, the concentrated
sublancin 168 was incubated overnight with 0 or 5% salt, indicated by “NaCl in concentrate”. The NaCl content
in the LB agar plates is indicated below the images.
Sublancin 168 activity depends on the presence of MscL
In search for possible cellular mechanisms that could be involved in the NaCl-dependent
susceptibility of target cells towards sublancin 168, we explored the available literature for
known effects of NaCl on bacterial growth and survival. This drew our attention to a possible
role of the mechanosensitive channels of ion conductance. These channels are located in the
cytoplasmic membrane and catalyze the efflux of ions and osmolytes from the cytoplasm
when cells encounter a downshift in the osmolarity of their environment (Blount et al., 1999;
Martinac, 2001; Pivetti et al., 2003; Sukharev et al., 1997). The opening or closing of these
channels is dependent on the osmolarity (salt content) of the media in which cells are
growing. At high osmolarity of the medium the channels will mostly be closed, while at low
osmolarity they may be open more frequently due to a constantly increasing turgor pressure in
the cell. We therefore investigated a possible involvement of these channels in the observed
NaCl-dependent sublancin 168 susceptibility of B. subtilis. For this purpose, the gene
encoding the largest mechanosensitive channel, named MscL, was deleted from the genome
of the sublancin 168 sensitive strain ∆SPβ. Then, sublancin 168 growth inhibition assays were
performed with this ∆SPβ∆mscL strain to assess its susceptibility to sublancin 168 in the
presence of different concentrations of NaCl. The results, shown in figure 1C, were striking
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Chapter 7
since the growth of B. subtilis ∆SPβ∆mscL was not at all inhibited by the sublancin 168
producer strain, irrespective of the absence or presence of NaCl (Figure 1C). These results
were confirmed by co-culturing experiments with the 168 and ∆SPβ∆mscL strains in liquid
media, which showed that growth of the ∆SPβ∆mscL strain was not inhibited by the parental
strain producing sublancin 168, irrespective of the NaCl concentration (Figure 2, D and E).
This shows that the susceptibility of the ∆SPβ cells to sublancin 168 depends on the presence
of the mscL gene. To investigate a possible effect of NaCl on the production level of MscL,
we monitored the mscL promoter activity in B. subtilis cells during growth in LB medium
containing 1% or 5% NaCl. This was done with a transcriptional PmscL-GFP fusion
construct. The results of this analysis show that the mscL promoter activity profile of cells
grown in LB medium with 5% NaCl is comparable to that of cells grown in LB with 1% NaCl
(Figure 3B). Growth in LB medium with 5% NaCl does therefore not seem to prevent MscL
production, which is consistent with our previously published results on the effects of hypoosmotic
shock on MscL proficient and deficient cells (Kouwen et al., 2009b). Taken together,
these observations imply that the susceptibility of sensitive B. subtilis cells toward sublancin
168 is dependent on the production of MscL.
To investigate whether MscL is also involved in the susceptibility of B. subtilis to
lantibiotics other than sublancin 168, we tested the sensitivity of ∆SPβ∆mscL cells for the
type A lantibiotics Nisin and Pep5 using a plate assay (Figure 1D). The results showed that
∆SPβ∆mscL cells have no altered sensitivity towards 2.5 mg/ml Nisin or 10 mg/ml Pep5 as
compared to the ∆SPβ strain. These results therefore indicate that MscL is a specific
determinant for sublancin 168 susceptibility in B. subtilis.
MscL-dependent sublancin 168 susceptibility is conserved in Staphylococcus aureus
Sublancin 168 has antimicrobial activity against a broad range of Gram-positive bacteria. The
observed MscL-dependent sublancin 168 susceptibility of B. subtilis prompted us to
investigate whether this phenomenon is specific for B. subtilis 168, or whether MscL is also
involved in the sublancin 168 susceptibility of other bacteria. Therefore, the sublancin 168
activity against the pathogenic Gram-positive bacterium S. aureus was also investigated. As a
first approach, the sublancin 168 producing B. subtilis strain 168 amyE::pX
(chloramphenicol R ) was used to inoculate LB growth medium in a 1:1 ratio with the S. aureus
strain RN4220 Em (erythromycin R ). NaCl was present at concentrations of either 1% or 5%.
The results of this co-cultivation and subsequent transfer of samples to plates (Figure 5, A and
B) were comparable to those obtained with the B. subtilis ∆SPβ strain (Figure 2, A and B).
When grown in normal LB containing 1% NaCl, the S. aureus strain was only able to survive
for a few hours in the presence of the sublancin 168 producing B. subtilis strain (Figure 5A),
whereas the S. aureus strain was hardly inhibited by the sublancin 168 producing B. subtilis
strain when grown in LB medium containing 5% NaCl (Figure 5B). To confirm that this
inhibitory effect was indeed due to the sublancin 168 produced by the B. subtilis 168 strain,
we also co-cultured the ∆sunA strain with the S. aureus strain RN4220 Em. Indeed, the results
show that the S. aureus strain was hardly inhibited by the ∆sunA strain, although a slight
inhibitory effect of B. subtilis on the growth of S. aureus was still detectable (Figure 5,
compare A and C). Notably, when we performed sublancin 168 growth inhibition assays on
agar plates during which the ∆sunA strain was spotted on a lawn of plated RN4220 Em cells,
a growth inhibition zone around the ∆sunA strain was still observed (data not shown).
142

The large mechanosensitive channel MscL determines bacterial susceptibility to
the bacteriocin sublancin 168
Figure 5. Sublancin 168 susceptibility of S. aureus is determined by MscL. (A) Co-cultivation of B. subtilis 168
Cm (white bars) together with S. aureus RN4220 Em (black bars) in LB containing 1% NaCl. (B) Co-cultivation of
B. subtilis 168 Cm (white bars) together with S. aureus RN4220 Em (black bars) in LB containing 5% NaCl. (C)
Co-cultivation of B. subtilis 168 ∆sunA (white bars) together with S. aureus RN4220 Em (black bars) in LB
containing 1% NaCl. (D) Co-cultivation of B. subtilis 168 Cm (white bars) together with S. aureus RN4220 ∆mscL
Em (black bars) in LB containing 1% NaCl. All strains were grown overnight and diluted to an OD 600 of 0.05 in
fresh LB medium containing the indicated amounts of NaCl and mixed at a 1:1 ratio. The number of colony
forming units (CFU) per ml was calculated as described in the legend to Figure 2 and the “Materials and
Methods” section. OD 600 measurements are depicted as a black line.
This indicated that B. subtilis also produces other antimicrobial factors to which S. aureus is
sensitive. However, sublancin 168 seems to represent the most effective anti-staphylococcal
activity of B. subtilis, as can be concluded from the strong inhibitory effect on the growth of
S. aureus shown in Figure 5. Finally, we tested whether sublancin 168 susceptibility of S.
aureus was also dependent of MscL. For this purpose, we constructed an S. aureus RN4220
strain lacking the mscL gene, and performed subsequent co-culturing experiments of this
strain together with the sublancin producing strain B. subtilis 168. The results of this coculturing
showed that growth of the RN4220 ∆mscL strain was no longer inhibited by the
deleterious effects of B. subtilis 168 producing sublancin (Figure 5D). Also in this case, the
RN4220 ∆mscL S. aureus strain was still slightly inhibited by the B. subtilis 168 strain, but
this effect was comparable to the effect observed when the S. aureus RN4220 strain was cocultured
with the B. subtilis ∆sunA strain. This clearly shows that the S. aureus RN4220
∆mscL strain is not sensitive to sublancin 168. Taken together, these observations demonstrate
that the susceptibility of S. aureus to sublancin 168 is dependent on the presence of an MscL
channel and that this feature is conserved in B. subtilis and S. aureus.
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Chapter 7
Discussion
In the present manuscript we report on bacterial and environmental factors that determine
bacterial susceptibility to the lantibiotic sublancin 168. Since its discovery in 1980, the
antimicrobial mechanism of this broad-range and highly stable lantibiotic has never been so
much as hinted at. We now show that the sublancin 168 susceptibility of cells lacking the
immunity protein SunI is dependent on the NaCl content of the growth medium. By contrast,
the production, stability and activity of the sublancin 168 seem to remain largely unaffected
by NaCl. Furthermore, we show that MscL plays a critical and unprecedented role in the
mode of action of sublancin 168, since this channel is indispensable for sublancin 168
susceptibility. As MscL determines sublancin 168 susceptibility both in B. subtilis and S.
aureus, it is well conceivable that the respective mechanism is conserved in many sublancin
168 susceptible bacteria.
Mechanosensitive ion channels are membrane-embedded channels that are present in
all three domains of life, but are especially widespread among bacteria (Blount et al., 1999;
Martinac, 2001; Pivetti et al., 2003; Sukharev et al., 1997). So far, three families of
mechanosensitive channels have been distinguished, named MscM (Mini), MscS (Small), and
MscL (Large). These channels are activated at different levels of applied pressure (Berrier et
al., 1996). The MscL channel opens at the highest applied pressure and also has the highest
pore diameter (30 Å). Mechanosensitive channels catalyze the efflux of osmolytes or
osmoprotectants upon hypo-osmotic shock (Berrier et al., 1992). When cells encounter a
sudden downshift in osmolarity of their environment they respond by excreting ions and
osmolytes from the cytoplasm in order to maintain an appropriate turgor pressure. The cells
thereby protect themselves from death by lysis due to overpressure. Possession of effective
osmo-regulatory protection mechanisms seems, therefore, of vital importance, in particular
for soil-dwelling organisms that are frequently exposed to hypo-osmotic shocks, such as rain.
In fact, it has been reported that B. subtilis cells lacking functional mechanosensitive
channels, especially MscL, are severely compromised in their ability to survive a hypoosmotic
shock (Hoffmann et al., 2008; Wahome and Setlow, 2008). Notably, in the present
studies we did not expose the cells to hypo-osmotic shock, but merely grew them in LB media
with varying concentrations of NaCl. Furthermore, we also did not observe any difference in
viability between the B. subtilis or S. aureus parental strains and their ∆mscL derivative
strains under the applied conditions as long as sublancin 168 was absent. It seems therefore
unlikely that sublancin 168 exerts its bactericidal activity by blocking the MscL channels of
sensitive organisms. Yet, the sublancin 168 susceptibility was clearly shown to depend on the
presence of MscL. Although MscL channels are vital for lowering the turgor pressure during
osmotic shock, it is not unlikely that these channels can also occasionally open during a
constant environmental osmolarity, especially if cells are grown in media of low osmolarity.
Since an open state of the MscL channel is potentially hazardous for cells due to ion loss, and
since a closed state of the MscL channel is more or less equivalent to the absence of MscL, it
is very well conceivable that sublancin 168 susceptibility relates to an open state of the MscL
pore. This would fit with the observed protective effect of salt against the detrimental effects
of sublancin 168. In fact, this protective effect of salt was the prime reason for us to
investigate a possible involvement of MscL in bacterial susceptibility to sublancin 168. The
MscL channels open at a certain level of pressure on the membrane, which is usually the
result of cell swelling due to a difference in the osmolarity (or salt content) between the
cytoplasm and the environment. The higher the salt content of the media, the less likely it is
144

The large mechanosensitive channel MscL determines bacterial susceptibility to
the bacteriocin sublancin 168
that mechanosensitive channels are open. Therefore, the observation that addition of salt to
the media lowers the susceptibility to sublancin 168, apparently without affecting sublancin
168 production or activity, seems to indicate that the MscL channel needs to be in an open
state to allow for bactericidal activity of sublancin 168. If so, sublancin 168 might prevent the
MscL pore from closing, which would result in cell death by leakage of ions from the
cytoplasm. This putative mechanism would in fact be consistent with the classification of
sublancin 168 as a group A lantibiotic, since lantibiotics of this class are known to create
pores in the membrane. In this case, however, sublancin 168 would keep an already existing
pore in an open state. Clearly, several alternative hypotheses can still be entertained. For
example, MscL might function as a gate for sublancin 168 import into the cell, allowing it to
perform bactericidal activity by interacting with an unidentified essential cytoplasmic target.
Furthermore, MscL might be involved in an indirect process that is required to promote the
bactericidal activity of sublancin 168.
Interestingly, even though MscL is conserved in all Gram-positive organisms, not all
of these organisms are inhibited by sublancin 168. This might be due to the existence of
systems for lantibiotic producer immunity or natural lantibiotic resistance. Recently, we have
identified the B. subtilis sublancin 168 producer immunity protein. This protein, SunI (YolF),
was also found to give S. aureus immunity against sublancin 168 when heterologously
expressed in this organism. SunI was shown to be a membrane protein with an Nout-Cin
topology, the bulk of the protein facing the cytoplasm (Dubois et al., 2009). This topology
was unprecedented for known lantibiotic producer immunity proteins but, notably, it seems
compatible with all above-mentioned mechanisms by which MscL might confer susceptibility
to sublancin 168. For example, SunI might close an MscL pore that is kept in an open state by
sublancin 168, it might prevent interactions between sublancin 168 and MscL (or other
compounds) in the membrane, or it might block the entry of sublancin 168 into the cytoplasm.
If producer immunity proteins analogous to SunI are active in other Gram-positive bacteria,
they may perhaps also be able to counteract the bactericidal effects of sublancin 168. A
second and perhaps more likely possibility is the existence of effective resistance
mechanisms. For example, it was shown that certain genes of the σ W regulon confer sublancin
168 resistance to B. subtilis (Butcher and Helmann, 2006). Furthermore, an altered membrane
or cell wall with an increased net positive charge might protect against the bactericidal effects
of cationic lantibiotics, like sublancin 168 (Peschel et al., 1999; Peschel and Collins, 2001).
Such a resistance mechanism has been shown to exist in Staphylococcus epidermidis which,
indeed, is resistant to sublancin 168 (Paik et al., 1998).
In conclusion, the present studies have focused a general interest on mechanosensitive
channels as potential determinants for bacterial susceptibility towards bacteriocins.
Specifically, we have identified a critical role of the MscL channel in the susceptibility of B.
subtilis and S. aureus towards the lantibiotic sublancin 168. Our findings suggest that this
may be a conserved phenomenon in sublancin 168 sensitive organisms. Therefore, ongoing
and future studies, including electrophysiology and interaction studies, are aimed at
identifying the precise mechanisms by which (i) sublancin 168 exerts its bactericidal activity
and (ii) MscL confers susceptibility to sublancin 168. Such studies will also further increase
our knowledge of mechanosensitive channels in general, which is important as these channels
serve important functions in all domains of life, including humans.
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Acknowledgements
We thank Magda van der Kooi-Pol for technical assistance, Hans-Georg Sahl for the gift of
purified Pep5, Leslie Aïchaoui-Denève, Stéphane Aymerich, Eric Botella, Kevin Devine,
Mark Fogg, Vincent Fromion, Annette Hansen, Matthieu Jules, Pascal Neveu, Sjouke
Piersma, Patrick Veiga and Tony Wilkinson for their collaboration in developing tools and
protocols for pBaSysBioII-based fluorescence measurements, and other colleagues from the
BaSysBio program for helpful discussions. Funding for this project was provided by the CEU
projects LSHG-CT-2004-503468, LSHG-CT-2004-005257, LSHM-CT-2006-019064 and
LSHG-CT-2006-037469, the transnational SysMO initiative through project BACELL
SysMO, the European Science Foundation under the EUROCORES Programme EuroSCOPE,
and grant 04-EScope 01-011 from the Research Council for Earth and Life Sciences of the
Netherlands Organization for Scientific Research.
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147

“Although one can get very clever at home, progress comes a lot quicker
when you step into a room with other people and start playing”
-Stephen J. Howe-
148

Chapter 8
The extracellular proteome of Bacillus licheniformis grown in
different media and under different nutrient starvation
conditions
B. Voigt, T. Schweder, M.J.J.B. Sibbald, D. Albrecht, A. Ehrenreich, J. Bernhardt,
J. Feesche, K.H. Maurer, G. Gottschalk, J.M. van Dijl, M. Hecker
Published in Proteomics (2005) 6, 268-281
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Summary
The now finished genome sequence of Bacillus licheniformis DSM 13 allows the
prediction of the genes involved in protein secretion into the extracellular environment
as well as the prediction of the proteins which are translocated. From the sequence 296
proteins were predicted to contain an N-terminal signal peptide directing most of them
to the Sec system, the main transport system in Gram-positive bacteria. Using 2-DE the
extracellular proteome of B. licheniformis grown in different media was studied. From
the approximately 200 spots visible on the gels, 89 were identified that either contain an
N-terminal signal sequence or are known to be secreted by other mechanisms than the
Sec pathway. The extracellular proteome of B. licheniformis includes proteins from
different functional classes, like enzymes for the degradation of various macromolecules,
proteins involved in cell wall turnover, flagellum- and phage-related proteins and some
proteins of yet unknown function. Protein secretion is highest during stationary growth
phase. Furthermore, cells grown in complex medium secrete considerably higher protein
amounts than cells grown in minimal medium. Limitation of phosphate, carbon and
nitrogen sources results in the secretion of specific proteins that may be involved in
counteracting the starvation.
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and under different nutrient starvation conditions
Introduction
Export of proteins from the cytoplasm to the extracellular environment is a common
phenomenon for all kinds of cells, including bacteria. Exported bacterial proteins can fulfil
different functions, either in the cell wall, like proteins involved in cell wall synthesis and
turnover and in folding and quality control of exported proteins, or in the surrounding
environment, like proteins involved in the usage of nutrient sources, in the communication
between cells, proteins with antimicrobial activity and virulence factors (Antelmann et al.,
2001; Gohar et al., 2002; Nouwens et al., 2003; Ziebandt et al., 2004). The entirety of the
secreted proteins and the components of the secretory protein machinery have been defined as
the secretome (Tjalsma et al., 2004).
To direct proteins to the transport machineries located in the cytoplasmic membrane, they are
usually synthesised as precursors with an N-terminal signal peptide. These signal peptides are
recognised by targeting factors in the cytoplasm which target the proteins to the transport
machinery in the membrane. The protein is then transported through the membrane via this
translocation machinery in an energy dependent manner. Finally, the signal peptide is cleaved
by specific signal peptidases. Bacteria have developed different pathways to secrete proteins.
The most important one is the general secretory or Sec pathway. The twin-arginine
translocation pathway (Tat), ABC transporters and the pseudopilin pathway are secretion
pathways for special purposes and have been found to transport only few proteins in Bacillus
subtilis (Tjalsma et al., 2004; Jongbloed et al., 2002; Jongbloed et al., 2004).
The highest level of protein secretion in B. subtilis was observed when cells were grown in a
complex medium (Antelmann et al., 2001). Cells grown in minimal medium (MM) secrete
considerable lower amounts of protein (Hirose et al., 2000). Most extracellular proteins have
been found after entry into the stationary growth phase.
B. licheniformis is known to secrete a number of different proteins into the extracellular
medium and this ability has been used in the fermentation industry for a long time, especially
for the production of industrial enzymes. The availability of the genome sequence of B.
licheniformis allows the prediction of the composition of its secretome. For example, all
genes for the Sec machinery have been found in the B. licheniformis sequence (Veith et al.,
2004; Veith et al., 2004). In addition, there are also two tatA genes (i.e., tatAd and tatAy) and
two tatC genes (i.e., tatCd and tatCy), which encode subunits of the Tat secretion machinery.
The nature of many of the extracellular enzymes which have been deduced from the sequence
of B. licheniformis suggests that they have functions in utilisation of alternative nutrient
sources that might be present in the environment. There are numerous hydrolases for the
degradation of various high molecular weight carbohydrates (e.g., α-amylase, cellulase,
chitinase) as well as several extracellular proteases and other degradative enzymes (Veith et
al., 2004; Rey et al., 2004). Despite of its biotechnological significance there has been no
investigation of the protein secretion process in B. licheniformis at a proteome-wide scale so
far. The recently finished genome sequence of B. licheniformis DSM 13 opened the chance to
establish proteomics of this organism. The cytoplasmic proteome and the regulation of central
carbon metabolic pathways were in the focus of our first study (Voigt et al., 2004). In the
study presented here, we analysed the secretion of proteins of B. licheniformis cells grown in
different media and under different nutrient starvation conditions by 2-DE.
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Materials and methods
Strains and growth conditions
B. licheniformis DSM 13 (equivalent to ATCC 14580, type strain from the German Collection of
Microorganisms and Cell Cultures, DSMZ GmbH Braunschweig, Germany) was cultivated at 37ºC
under vigorous agitation either in a complex medium (Luria Broth, LB) or in MM containing 15mM
(NH4)2SO4, 8mM MgSO4 x 7 H2O, 27mM KCl, 7mM sodium citrate (2 H2O), 50mM Tris-HCl pH 7.5,
supplemented with 0.6mM KH2PO4, 2mM CaCl2 (2 H2O), 1 mM FeSO4 x 7 H2O, 10 mM MnSO4 x 4
H2O and 0.2% glucose. For the starvation experiments, the concentrations of glucose, phosphate and
nitrogen were reduced to 0.08%, 0.15mM and 1.0mM, respectively. The concentration of the limiting
nutrient was adjusted in such a way that the cultures grew to a maximum OD of about 1.0 (growth
curve in LB and MM see Voigt et al. (Voigt et al., 2004)).
Preparation of the extracellular protein fraction
Bacteria were harvested during exponential growth (OD500 0.4 in MM, OD540 2.0 in LB), at the onset of
the stationary growth phase (OD500 1.0 in MM, OD540 4.0 in LB) and during the stationary phase (MM
1 h after transition into the stationary phase, OD540 6.0 in LB). PMSF (3mM) was added when the
cultures were harvested to prevent proteolysis during sample preparation. The cells were removed by
centrifugation at 4ºC (8000 rpm, 10 min). TCA was then added to the medium to a final concentration
of 10%. The extracellular proteins were precipitated at 47C overnight and collected by centrifugation
(4ºC, 10 000 rpm, 60 min). The protein pellet was washed eight times with 96% ethanol, dried and
dissolved in a solution containing 8 M urea and 2 M thiourea. The protein concentration of the extract
was determined with the RotiNanoquant Kit (Roth).
2-DE
For the IEF protein extracts (500 mg protein) were loaded onto commercially available IPG strips (pH
3–10 NL, Amersham Biosciences) according to Büttner et al. (Büttner et al., 2001). In the second
dimension, polyacrylamide gels of 12.5% acrylamide and 2.6% bisacrylamide were used. The resulting
2-D gels were stained with colloidal CBB as described by Voigt et al. (Voigt et al., 2004).
Protein identification
Proteins were identified by MS. Protein spots were excised from stained gels with the Proteome
Works Spot Cutter System (Bio-Rad). In-gel digestion with trypsin and the extraction of the peptides
were done in the Ettan Spot HandlingWorkstation (Amersham Biosciences) according to a modified
protocol of the manufacturer. Peptide masses were determined in the Proteomics Analyser 4700
(Applied Biosystems). The 4700 Explorer Software V2.0 was used for spectrum calibration and
analysis. After calibration, peak lists within a mass range of 900–3700 Da were created using the ’peak
to mascot’ script of the 4700 Explorer Software. For the peak search, filters were set to a peak
density of 20 peaks per 200 Da, a minimal peak area of 350 and maximal 60 peaks per spot. Peak lists
were created with a signal to noise (S/N) ratio of 10 to 15. When necessary, TOF-TOF measurements
for the three highest peaks in a spectrum were carried out. The internal calibration was automatically
performed as one-point-calibration either with the mono- isotopic arginine (M+H) + peak, m/z at
175.119, or with the lysine (M+H) + peak, m/z at 147.107, when the peaks reached at least an S/N ratio
of 5. Peak lists were created as described above with the following settings: a mass range from 60 to
precursor ion mass minus 20 Da, a peak density of five peaks per 200 Da, a minimal peak area of 100
and maximal 20 peaks per precursor. Peak lists were created with an S/N ratio of 5. For database search
the Mascot search engine (Matrix Science Ltd, London, UK) with a specific B. licheniformis sequence
database was used. If this search did not result in the identification of the protein, PMF were reanalysed
using the gpmaw software (Peri et al., 2001).
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and under different nutrient starvation conditions
Signal peptide prediction
For prediction of signal peptides the SignalP 2.0 software (http://www.cbs.dtu.dk/services/SignalP-
2.0/) (Nielsen et al., 1997) and the LipoP 1.0 software (http://www.cbs.dtu.dk/services/LipoP/)
(Juncker et al., 2003) were used with the settings for Gram-positive organisms. Signal peptides were
predicted by a Hidden Markov Model as well as by a neural networks method. Only proteins
recognized by both methods to contain a signal peptide are classified as such. Proteins that are
predicted to contain one or more membrane spanning domains in addition to an N-terminal signal
peptide were excluded from the list (note that proteins with one membrane spanning domain in the Nterminal
part of the protein might be wrongly identified as containing a signal peptide).
Transmembrane helices were identified by the TMHMM v2.0 program (http://www.cbs.dtu.dk/
services/TMHMM/). This program predicts transmembrane segments in proteins, using a Hidden
Markov Model (Krogh et al., 2001).
Denomination of the proteins
Proteins with similarity to a B. subtilis protein were named accordingly. Proteins with no homolog in B.
subtilis received the gene ID of the sequencing project.
Dual channel imaging, quantification and colour coding
The 2-D gel images were analyzed using the DECODON Delta2D software (Decodon GmbH
Greifswald). Artificial coloring and warping of the gel images were done as described by Bernhardt et
al. (Bernhardt et al., 1999). Spot quantities were also determined by the Delta2D software. The
numbers given represent the relative portion (% volume) of an individual spot of the total protein
present on the gel. For comparison of the protein patterns during the different starvation conditions the
“union fusion” approach of Delta2D for the generation of a proteome map was applied. For this
purpose the gel images of the phosphate, glucose and nitrogen starvation were warped to the gel image
of the control (exponential growth phase) and a fusion gel (union fusion) was created by combining the
images. The spots were color coded according to their expression profile. Only spots induced more
than 2.5-fold by the starvation conditions compared to the exponential phase were considered. Each
spot subset, i.e., all spots induced in response to one starvation condition or a certain combination of
starvation conditions, received a defined colour.
Results and discussion
Prediction of signal peptides
The recently finished genome sequence of B. licheniformis allows the prediction of all
proteins containing signals for secretion through one of the so-far-known protein secretion
systems. From the B. licheniformis genome data there are 296 proteins predicted to have an
N-terminal signal peptide for export from the cytoplasm. Most of these signal peptides would
direct proteins to the Sec secretion machinery, but 19 have a twin-arginine motif (4 RR, 15
KR) that may direct the corresponding precursor proteins into the Tat pathway, and 4 have the
potential to direct proteins into a pseudopilin export pathway (Supplemental table Va;
proteins with one or more membrane spanning domains in addition to the signal peptide were
excluded). Of the 296 identified signal peptides, 220 have recognition sites for cleavage by a
type I signal peptidase (e.g., SipS, SipT or SipV), 72 have recognition sites for cleavage by a
lipoprotein-specific signal peptidase II (LspA), and 4 have recognition sites for a pseudopilin
signal peptidase (ComC).
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The extracellular proteome of B. licheniformis and the comparison of the predicted to
the real secretome
The highest level of protein secretion in B. licheniformis was observed in the stationary
growth phase (Figs. 1, 2). Many of the degradative extracellular enzymes were secreted at low
levels during the exponential growth phase and were induced in the stationary phase. The
induction of such enzymes in B. subtilis in the stationary growth phase is dependent on the
DegSU two component system (Msadek et al., 1995; Ogura et al., 2001). As in B. subtilis,
cells of B. licheniformis secrete much higher levels of protein when grown in a complex
medium compared to cells grown in minimal medium (this is not apparent from our gels,
because the same protein amount was loaded onto each gel).
Figure 1. Dual channel images of the extracellular proteome of B. licheniformis. Cells were grown in LB and
the images were created with the Delta 2D software (Decodon GmbH Greifswald). Proteins were prepared during
exponential growth (OD 2), in the transition phase (OD 4) and in the stationary growth phase (OD 6). Proteins
were separated in a pH gradient 3–10 and stained with colloidal CBB. Spots labeled in italics are presumably
intracellular proteins; fr: fragment.
From about 200 protein spots visible on the gels, altogether 143 proteins could be identified
(Supplemental tables Vb and Vc). Some of the proteins, e.g., Vpr and YfnI, occur as multiple
spots. Only 89 of the identified proteins were expected to be secreted because they either have
a predicted N-terminal signal peptide sequence (79 proteins) or are known to be secreted by
Sec independent pathways (10 proteins, Supplemental table Vb). These extracellular proteins
of B. licheniformis include carbohydrate degrading enzymes, several proteases and
peptidases, enzymes involved in nucleic acid degradation, phosphodiesterases and
phosphatases, enzymes involved in cell wall turnover, transport related proteins, flagellum-
and phage-related proteins, proteins involved in sporulation and membrane bioenergetics and
some proteins of yet unknown function.
Of the 79 identified proteins containing an N-terminal signal peptide, 18 are involved in
transport processes, among them several ABC transporter binding proteins. These proteins
would be expected to be lipid-anchored in the membrane. It is most likely that they are
released from the membrane by “proteolytic shaving” (Antelmann et al., 2001).
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The extracellular proteome of Bacillus licheniformis grown in different media
and under different nutrient starvation conditions
Figure 2. The extracellular proteome of B. licheniformis. Cells were grown in minimal medium under different
starvation conditions. (A) Exponential growth. (B) Stationary phase phosphate starvation. (C) Stationary phase
glucose starvation. (D) Stationary phase nitrogen starvation. Proteins were separated in a pH gradient 3–10 and
stained with colloidal CBB. The graphs show the quantification of the ten most prominent spots in the gels given as
% volume (representing the relative portion of an individual spot of the total protein present on the gel).
Quantification was done with the Delta 2D software. Spots labelled in italics are presumably intracellular
proteins; fr: fragment.
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There were also some cell wall-related proteins present in the extracellular medium. These
cell wall proteins were present to a higher extent in the extracellular proteome of
exponentially growing B. licheniformis cells (e.g., YvcE, YwtD; Supplemental table Vb). The
same was described for B. subtilis (Antelmann et al., 2002). In B. subtilis such proteins are
retained in the cell wall because they contain specific wall-binding domains additionally to
the signal peptide (Tjalsma et al., 2000). They are probably released into the medium by the
action of extracellular proteases as was shown by Antelmann et al. (Antelmann et al., 2002),
since in multiple protease-deficient strains the cell wall proteins were stabilised.
In the signal peptides of four secreted proteins found in this study, a potential twin-arginine
signal peptide was identified (containing the canonical twin-arginine motif R-R-XH- H,
where H is a hydrophobic amino acid, Supplemental table Vb) (Tjalsma et al., 2000;
Jongbloed et al., 2000). One of these proteins is PhoD, one of the two proteins which have
been actually shown to be translocated Tat dependently in B. subtilis (Jongbloed et al., 2000).
Additional 15 proteins were predicted from the sequence to contain a K-R-X-H-H motif that
may be functional in directing proteins to the Tat machinery (Tjalsma et al., 2004).
The ten proteins known to be secreted by other pathways included seven flagellum-related
proteins and three phage-related proteins, which typically lack signal peptides. The flagellum
proteins are probably transported via a specific flagellum pathway. Hirose et al. (Hirose et al.,
2000) found that Hag is not transported via the Sec pathway, because SecA depletion in a
mutant strain did not affect Hag secretion. This was confirmed by studies of Jongbloed et al.
(Jongbloed et al., 2002), who inhibited SecA activity by sodium azide and found that the
secretion of the flagellum related proteins FliD and Hag was not affected. The proteins with
phage-related functions may be secreted via prophage-encoded holins that can form
membrane pores (Krogh et al., 1998).
In addition, 55 proteins were identified in the extracellular proteome of B. licheniformis,
which were not expected to be secreted because they have no predicted signal for secretion
through one of the known systems, and which, taking into account their function, in most
cases would be expected to have a cytoplasmic localisation (Supplemental table Vc). The
secretion of proteins without known export signals has been also described in other studies of
the extracellular proteome (Antelmann et al., 2001; Hirose et al., 2000). It is not yet known
whether these proteins are secreted via an unknown secretion mechanism, but more likely
they have been released into the medium by cell lysis. Remarkably, the two type I signal
peptidases SipS and SipT, which are membrane proteins with an N-terminal membrane
anchor, could both be detected in the extracellular proteome of B. licheniformis cells grown in
LB medium (Figure 1). The molecular mechanism underlying this unprecedented observation
is presently not clear.
Proteins secreted under all starvation conditions
In response to phosphate, carbon and nitrogen starvation B. licheniformis cells secreted
different proteins into the extracellular medium. There were, however, also proteins that were
secreted regardless of the growth conditions. Among these proteins was flagellin (Hag),
which formed a prominent spot under all conditions tested. Furthermore, most of the
proteases were secreted in response to all conditions, although not to the same level. The main
protease secreted by B. licheniformis was Vpr. In B. subtilis the majority of the extracellular
proteases are induced in the stationary growth phase (Antelmann et al., 2001). This induction
is mainly dependent on the two component system DegSU and the regulator Hpr (Mäder et
al., 2002; Antelmann et al., 2004).
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The extracellular proteome of Bacillus licheniformis grown in different media
and under different nutrient starvation conditions
Phosphate starvation
Surprisingly the main protein induced by phosphate starvation in B. licheniformis was the
phytase (13.9% relative volume, Figure 2b, Tab. 1). The phytase hydrolyses phytate, the salt
of phytic acid (myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate) (Tye et al., 2002).
Phytate is the major storage form of phosphorus in plants and therefore present in soil, where
it accounts for up to 50% of the organic phosphorus. B. licheniformis is able to grow on
phytate as a sole phosphate source, and in this case phytase is already secreted during
exponential growth (data not shown). In B. subtilis the phytase is expressed at a low level and
the protein was not found in the extracellular proteome of phosphate-starving cells
(Antelmann et al., 2004; Antelmann et al., 2000).
Phosphate starvation also led to the specific secretion of the phosphatases PhoB (there is no
homolog of the phoA gene encoded in the B. licheniformis genome) and PhoD. Secretion of
phosphatases was also noticed in B. subtilis cells subjected to phosphate starvation, where
they are among the main extracellular proteins. In B. licheniformis, however, these
phosphatases belonged to the minor proteins secreted in response to phosphate starvation. In
phosphate-starved B. licheniformis cells, YhdW, a protein that shows similarity to GlpQ, a
glycerophosphodiester phosphodiesterase, was secreted to a high amount (GlpQ itself was
only found in the proteome of cells grown in LB). Furthermore, there was a strong secretion
of some proteins involved in the metabolism of nucleic acids (YfkN, YhcR, NucB and
BLi03719). Induction of nucleic acid degrading enzymes during phosphate starvation has also
been described for Corynebacterium glutamicum (Ishige et al., 2003). To be able to
effectively take up the limiting amount of phosphate, B. licheniformis cells secreted PstS, the
phosphate binding protein of an ABC transporter involved in the high-affinity phosphate
uptake. In B. subtilis, PstS, probably attached to the membrane via a lipid anchor, is also an
abundant protein in the secretome (Antelmann et al., 2000).
Growth of B. subtilis under phosphate-starvation conditions results in the specific induction of
genes belonging to the Pho regulon (Eymann et al., 1996; Prágai and Harwood, 2002). This
regulon comprises several genes, which allow the cells to adapt to the limiting concentration
of phosphate. The expression of these genes is under control of at least three two-component
signal transduction systems (Hulett, 1996; Birkey et al., 1998). The main regulatory system is
PhoPR consisting of the sensor kinase PhoR and the transcriptional activator PhoP
(Antelmann et al., 2000; Liu and Hulett, 1998). Although there is not yet any experimental
evidence, it can be suggested that the regulation in B. licheniformis is similar, because the
genes for the two component system PhoPR are present in the genome.
Glucose starvation
We also investigated the extracellular protein pattern of B. licheniformis cells grown in a
glucose limited medium (Figure 2c, Supplemental table Vb). The main protein secreted
during glucose starvation was flagellin (Hag), which represents 10.3% (relative volume) of
the total protein on the gel. To our surprise, we identified only two proteins involved in the
degradation of carbohydrates in the extracellular proteome of glucose-starving cells (YvfO, an
arabinogalactan endo-1,4-β-galactosidase, and YvpA, a pectate lyase), although many genes
coding for such proteins have been identified in the genome. There was, however, a strong
secretion of some proteases (Vpr, Mpr and Bpr) and of the oligopeptide ABC transporter
binding proteins AppA and OppA. Other proteases (Epr, Ggt, BLi01109, BLi01747) were
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secreted only at a low level. Beside this, some proteins of still unknown function (e.g., YusA,
BLi02210, BLi01431) were strongly secreted.
Figure 3. Colour-coded extracellular proteome map of B. licheniformis. Cells were grown in MM under
phosphate, glucose and nitrogen starvation conditions. Proteome images of the three starvation conditions were
fused with an exponential growth phase proteome image to create a proteome map, which contains all protein
spots from the four individual images. Colour coding was done in such a way that all proteins belonging to a spot
subset, i.e., all proteins induced in response to one starvation condition or a certain combination of starvation
conditions received a defined, subset specific colour (colour scheme see upper left corner, P: phosphate
starvation, G: glucose starvation, N: nitrogen starvation). Only proteins induced more than 2.5 fold compared to
the exponential growth phase were included in the colour coding. Quantification, gel fusion and colour coding was
done with the Delta 2D software.
Perhaps B. licheniformis expresses some of the exoenzyme genes and secretes the
corresponding hydrolyzing enzymes only when the carbon sources are present in the medium.
Similar results were presented by Hirose et al. (Hirose et al., 2000) and Chu et al. (Chu et al.,
2000) who found new proteins exported into the medium when they cultured Bacillus cells on
different carbon sources, like cellobiose and xylan. Antelmann et al. (Antelmann et al., 2004)
reported the secretion of SacB and Phy, enzymes not seen in earlier experiments, when B.
subtilis was grown in a medium containing maltodextrin. Our results with cells grown in
complex medium also point to such a mechanism, because these cells secrete a considerable
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The extracellular proteome of Bacillus licheniformis grown in different media
and under different nutrient starvation conditions
number of carbohydrate degrading exoenzymes (Supplemental table Vb, Figure 1), although
not at a high level.
Such a substrate induction mechanism is a known regulatory phenomenon. It has been
described for instance for the usage of lichenan and levan by B. subtilis (Tobisch et al., 1997;
Steinmetz et al., 1989). The genes for the usage of these carbohydrates are induced by the
corresponding degradation products. Some other catabolic genes, like amyE and xynA,
however, are not regulated by substrate induction but only by carbon catabolite repression
(Stülke and Hillen, 2000). Carbon catabolite repression is the global regulatory mechanism to
coordinate the carbon metabolism in Bacillus. In the presence of the preferred sugar, glucose,
the regulator CcpA represses the catabolic genes whose expression is required for utilising
alternative carbon sources. Most of the genes encoding extracellular carbohydrate degrading
enzymes are under CcpA control in B. subtilis (Henkin et al., 1991; Martin-Verstraete et al.,
1999; Yoshida et al., 2001).
Nitrogen starvation
The flagellin protein Hag was also the main protein secreted from nitrogen-starving cells
(24.7% relative volume, Figure 2d). In addition, nitrogen starvation led to the secretion of
some proteases and peptidases (Supplemental table Vb). The main protease, Vpr, accumulated
during nitrogen starvation to the highest relative volume in all three conditions (14.5%
compared to 7.1% in glucose starvation and 4.5% in phosphate starvation). This was also the
case for some other proteases/ peptidases (e.g., Ggt, BLi01747, data not shown), although
these proteins are not secreted at high levels. The aminopeptidase YwaD was secreted
exclusively during nitrogen starvation conditions. In addition, the substrate binding proteins
of several peptide transporters were found in the extracellular protein fraction of nitrogenstarving
cells (DppE, OpuAC, BLi02811).
The nitrogen metabolism of B. subtilis is controlled by different regulatory proteins in
response to nutrient availability (Fisher, 1999). The regulatory proteins TnrA and GlnR
control many genes that are expressed at high levels in nitrogen starving cells. TnrA induces
the expression of genes involved in the usage of alternative nitrogen sources in nitrogenstarving
cells. GlnR, on the other hand, represses such genes in cells growing with excess
nitrogen. Although there are no data about the regulation of the nitrogen metabolism in B.
licheniformis available, the regulation might be similar as in B. subtilis, because the genes
tnrA and glnR are both present in the B. licheniformis genome.
Comparison of the extracellular proteome under the different starvation conditions
In Figure 3, an image fused from the images of the extracellular proteomes of the exponential
growth phase and from all three starvation conditions is presented. To gain an overview over
the differences in the proteins patterns, all proteins induced in response to the different
starvation conditions more than 2.5 fold compared to the exponential growth phase were
colour coded. Colour coding was done in such a way that all proteins belonging to a spot
subset, i.e., all proteins induced by one starvation condition or a certain combination of
starvation conditions, received a defined, subset specific colour. Seven protein subsets could
be defined: (1) proteins induced only by phosphate starvation (red colour), (2) proteins
induced only by glucose starvation (yellow), (3) proteins induced only by nitrogen starvation
(blue), (4) proteins induced by phosphate as well as by glucose starvation (orange), (5)
proteins induced by phosphate as well as by nitrogen starvation (green), (6) proteins induced
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by glucose as well as by nitrogen starvation (magenta), (7) proteins induced by all three
conditions (cyan). This colour coding revealed the differences in the pattern of the
extracellular proteins in response to phosphate, glucose and nitrogen starvation.
Concluding remarks
Prediction of N-terminal signal peptide sequences revealed 296 proteins in B. licheniformis,
most of which are presumably Sec-dependently exported to the extracellular medium. This
number is (almost) identical to the estimated number of 297 signal peptide-containing
proteins of B. subtilis 168 (Tjalsma et al., 2004), which would be consistent with the fact that
B. subtilis 168 and B. licheniformis DSM13 have about 4100 and 4200 protein-encoding
genes, respectively. However, there appear to be some interesting differences with respect to
the numbers of predicted secretory proteins and lipoproteins. While 179 predicted signal
peptides of B. subtilis have a recognition site for a type I signal peptidase, and 114 for a type
II signal peptidase, 220 predicted signal peptides of B. licheniformis have a recognition site
for a type I signal peptidase and 72 for a type II signal peptidase. Thus, the B. licheniformis
genome seems to encode a relatively higher number of secretory proteins, and a relatively
lower number of lipoproteins, than the B. subtilis genome. The most effective technique to
verify the localisation of proteins in certain cellular compartments is proteomics. Using this
technique 89 proteins were identified in the extracellular proteome of B. licheniformis that
either contain an N-terminal signal peptide or are known to be exported in a Sec-independent
manner. These proteins are involved in different physiological processes like cell wall
turnover, mobility, transport of low molecular weight substrates and utilisation of alternative
nutrient sources. The highest level of protein secretion was reached in the stationary growth
phase in complex medium, when cells secreted a high number of degradative enzymes to
make alternative nutrient sources accessible. Growth in defined medium under different
nutrient starvation conditions led to the secretion of specific proteins, presumably involved in
counteracting the starvation.
Acknowledgements
We thank Anne Krause for excellent technical assistance. We are grateful to the Decodon
GmbH Greifswald for cooperation and the pre-release access to new software tools. This
work received financial support from the Bundesministerium für Bildung und Forschung
(0312707, 031U214B), the Henkel KGaA, the Bildungsministerium of the country
Mecklenburg-Vorpommern, the Fonds of Chemical Industry of Germany and the EU (LSHC-
CT- 2004–503468/005257).
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“I will choose free will”
-Neil E. Peart-
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Chapter 9
General summary and discussion
163

Chapter 9
General summary and discussion
The human body functions as a host to many bacteria. Normally, we have no problem
carrying all these bacteria, and some are even beneficial to us. One can think of the bacteria
that are present in our colon that help us by synthesizing vitamins (Vitamine K, biotin), or
bacteria that help to break down food components that we cannot digest, such as starch and
various other fibers. Other bacteria are dangerous pathogens, causing diseases that can be
lethal to human beings. Several of these primary pathogens cause well known diseases, like
Clostridium tetani (tetanus), Corynebacterium diphtheria (diphtheria), Treponema pallidum
(syphilis), Vibrio cholerae (cholera), Mycobacterium leprae (leprosy) and Mycobacterium
tuberculosis (tuberculosis).
Staphylococcus aureus and Staphylococcus epidermdis are two of those bacteria that are
living as commensals in the moist places and the skin of the human body. For healthy
humans, S. aureus is not considered as a serious threat and ~20% of the population carries this
organism in the nose. However, when the host defenses are breached and S. aureus is able to
spread throughout the human body, this organism can turn into a dangerous pathogen. The
infective properties of S. aureus are caused by the arsenal of virulence factors that are
expressed by this organism. As with all pathogens, these virulence factors are often
proteinaceous compounds that are necessary for the bacterium to invade, colonize and spread
throughout the (human) host and compounds that contribute to the symptoms of disease. All
these virulence factors are synthesized in the cytoplasm of the cell and translocated across the
bacterial membrane to an extracytoplasmic location, such as the cell surface or the host
milieu. Well-characterized virulence factors include the many exotoxins of S. aureus.
Most virulence factors are synthesized with an N-terminal signal peptide. This signal peptide
is recognized by cytoplasmic chaperones that lead the protein to translocation pathways that
are present in the cell. At the trans-side of the membrane, the translocated virulence factors
are processed into their mature form and folded into the correct three-dimensional
conformation with or without the help of other proteins. While several proteins are released
into the environment (e.g. exotoxins), other proteins are retained at the surface of the cell. In
Gram-positive bacteria, at least four extracytoplasmic cellular locations are distinguished: the
membrane, the membrane-cell wall interface, the cell wall and the cell surface. All these
locations can contain important virulence factors.
Soon after the introduction of penicillin to eradicate the threat of S. aureus infections,
resistant strains emerged and today the multidrug resistant S. aureus strains are imposing both
therapeutic and financial challenges to our health care. The last resort antibiotic is
vancomycin, but in the last 10-15 years S. aureus strains that are less susceptible or even fully
resistant to vancomycin have been isolated. The need to search for alternatives to antibiotics
such as vaccines and target-directed drugs is therefore increasing. These alternative drugs or
vaccines should be directed against a specific protein or process which decreases the chance
of survival of the pathogenic bacteria in the human host without interfering with the host’s
health.
This thesis describes investigations on the secretome of S. aureus. By definition, the
secretome includes all proteins that are translocated across the bacterial membrane to
extracytoplasmic locations plus all the proteins that make up the protein translocation
machinery.
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General summary and discussion
Prediction of the secretome of Gram-positive bacteria
In Chapter 2, the secretome of S. aureus is investigated with the help of bioinformatics. The
genomes of thirteen sequenced and annotated S. aureus strains were used to search for
components of known translocation pathways, such as the general Sec pathway, the Twinarginine
translocon, the Com pathway, ABC-transporters, holins and the Ess pathway. The
proteins that make up these pathways are all present in S. aureus, including proteins that are
involved in the processing (e.g. signal peptidases, sortases) and folding (e.g. PrsA, DsbA)
after translocation of these proteins. In addition, the deduced protein sequences were searched
for the presence of N-terminal signal peptides. The search patterns developed for Bacillus
subtilis were used to identify S. aureus signal peptides and the available proteomic data for
extracellular proteins of S. aureus were used to further refine the respecitive staphylococcal
consensus motifs. Five different signal peptides were classified according to the translocation
pathway that is used and to the signal peptidase that processes the protein. In total, 145-168
proteins, depending on the S. aureus strain investigated, were found that carry an N-terminal
signal peptide for membrane translocation. This number is relatively high compared to other
Gram-positive bacteria, such as Bacillus licheniformis (Chapter 10), B. subtilis, and S.
epidermidis. In fact, B. subtilis has been claimed to be a secretion factory and various
biotechnological industries have been using this organism for the production of commercially
interesting enzymes. The high numbers of secreted proteins in S. aureus are partly related to
the many exotoxins that are produced. From the proteins that have (predicted) signal peptides
two groups were defined. The core exoproteome consists mainly of housekeeping proteins
that seem to be necessary to maintain the position of S. aureus in its ecological niche in the
(human) host. This is especially true for the proteins that are retained at the cell wall by
sortase A. It is known that many of these proteins have adhesive properties and are involved
in colonization of the host tissue. Some of these proteins (e.g. protein A) are also involved in
protection of the staphylococcal cell against the immune system of the host. The proteins of
the core exoproteome are interesting candidates for future vaccine development as is indicated
in Chapter 3. The second group of proteins is defined as the variable exoproteome, which
mainly consists of the exotoxins that are produced by S. aureus. These proteins can be
regarded as special S. aureus “gadgets” and the proteins with a yet unknown function should
be regarded as potentially relevant virulence factors. Whether these proteins of unknown
function are all important for virulence remains to be assessed. This is especially true for the
lipoproteome, where many lipid-modified proteins (lipoproteins) have unknown functions. In
recent years it has become clear that lipoproteins play very important roles in the process of
infection.
Using a proteomics approach, the exoproteomes of 25 clinical isolates that have been
collected at the University Medical Center Groningen were analysed (Chapter 3). It is
surprising that from the 69 core proteins only seven proteins were identified in all
exoproteomes of these isolates. This difference can partly be explained by the retention of
certain proteins in the cell wall, such as the covalently attached proteins (e.g. protein A,
clumping factors, fibronectin and fibrinogen binding proteins, SasG) and proteins with other
cell wall binding properties (e.g. LytN, SA0620, Atl). Other proteins are expressed only under
specific growth conditions, such as iron-limiting conditions, oxygen stress, and other stress
conditions, and these will therefore escape detection under the tested laboratory conditions.
Nevertheless, these proteins are potential candidates for vaccine development. It will
therefore be necessary to determine the exoproteomes of clinical S. aureus strains that are
grown under infection mimicking conditions.
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Systematic analysis of translocation machineries of S. aureus
To investigate the importance of translocation pathways in the secretion of proteins, a set of
isogenic S. aureus mutants were created and the exoproteomes of these mutants were
analyzed. A summary of all mutants that were analyzed has been presented in Chapter 4, and
a few of selected mutants were analyzed in more detail (Chapters 5, 6 and 7). To select
proteins that are important in proteins translocation, the thirteen sequenced and annotated S.
aureus genomes that are publicly available were scanned for these proteins. This in silico
analysis of S. aureus genomes revealed that the components for the Sec pathway, Twinarginine
pathway, Com pathway, ABC transporters for the translocation of bacteriocins,
holins and Ess pathway are present in all strains. Differences were only detected for the Ess
pathway in the S. aureus MRSA252 strain, where one of the substrates (EsxB) is not present
and one other component (EsaC) is absent as well. This suggests either that EsaC is
dispensable and that EsxB is dependent on the presence of EsaC, or that the Ess pathway may
be not functional in this particular S. aureus strain. In this case, EsaC could still be required
for the proper function of this pathway.
Most proteins are translocated across the membrane via the general Sec pathway.
Translocation via this pathway occurs in three distinguishable stages: chaperoning of the
newly synthesized protein to the translocon, translocation, and modification and processing of
the translocated proteins. In this thesis, several components that are involved in the latter two
stages have been analyzed in more detail. In general, a translocon consists of a translocation
motor and a channel. The translocation motor in the Sec pathway of S. aureus is the SecA
protein that drives the translocation through repeated cycles of ATP binding and hydrolysis.
The core channel is composed of the SecYEG proteins. Both SecA and SecY are known to be
essential for growth and viability, and it was therefore decided not to attempt making the
respective mutant strains. As has been found for other Gram-positive pathogens, the genome
of S. aureus contains an additional set of secA and secY genes, encoding the SecA2 and
SecY2 proteins. Usually, this accessory translocon is used only for a few selective proteins.
Since the substrate and genes encoding glycosylation proteins are present in the same operon
as the secA2 and secY2 genes it is conceivable that this accessory translocon only transports
glycosylated proteins as was shown for Streptococcus gordonnii. In Chapter 5, the analyses
of the isogenic secG and secY2 mutants were investigated. While the deletion of secY2 had no
detectable effect on protein secretion, the importance of SecG became clearly evident upon
analysis of the exoproteom of a secG mutant by 2-D PAGE. The extracellular accumulation
of nine abundant exoproteins and seven cell wall-bound proteins was significantly affected in
the secG mutant. Among these proteins are some known virulence factors, such as protein A,
immunodominant antigen A, lipase and α-hemolysin. Interestingly, deletion of secY2
exacerbated the secretion defects of secG mutants, affecting the extracellular accumulation of
one additional exoprotein and one cell wall protein. Furthermore, the secG secY2 double
mutant displayed a synthetic growth defect. These findings suggest that SecY2 can interact
with the main Sec channel of S. aureus. This was in fact already predicted on the basis of
genome analyes, because all sequenced S. aureus strains have only a single set of the secE
and secG genes in S. aureus (Chapter 2). Another interesting finding was that the second
IgG-binding protein Sbi was almost completely absent from the cell wall of the secG mutant
strain. Despite these interesting phenotypes of the secG mutant, it is questionable whether
SecG might be a good target for novel antibiotics, since this protein is homologous to SecG of
other bacteria and the Sec61β unit of the mammalian Sec61p complex. Moreover, infection
experiments in a mouse model did not reveal any attenuation of the secG mutant strain,
166

General summary and discussion
suggesting that this component of the secretion machinery is dispensable for host subversion
by S. aureus.
Chapter 6 of this thesis deals with the modification and processing of certain translocated
proteins by the class A sortase (SrtA) of S. aureus and S. epidermidis. SrtA attaches proteins
with an LPxTG motif to the cell wall, by cleaving this motif between the conserved Thr and
Gly residues and simultaneously attaching the Thr residue to peptidoglycan. In this study, the
extracellular proteins of sortase A mutants from S. aureus and S. epidermidis were analyzed.
Deletion of the srtA genes of these bacteria resulted in the dislocation of several LPxTG
proteins, such as ClfA, SasG, SdrC, SdrD, and protein A of S. aureus, and the Aap protein of
S. epidermidis 1457, from the cell wall to the growth medium. Nevertheless, substantial
amounts of these proteins remained cell wall-bound through non-covalent interactions
facilitated by cell wall-binding domains. The protein dislocation phenotypes of srtA mutations
in S. aureus and S. epidermidis were fully reversed by ectopic expression of srtA genes of
either species. Interestingly, the class C sortase (SrtC) from S. epidermidis 12228 was capable
of reversing the dislocation of ClfA, SasG and Aap to significant extents, showing that the
substrate specificities of SrtA and SrtC overlap at least partially. By contrast, SrtC was unable
to restore the covalent cell wall attachment of protein A and the SdrC and SdrD proteins in
the S. aureus srtA mutant. Interestingly, biofilm formation was affected in srtA mutants of S.
aureus, but not in the S. epidermidis srtA mutant. This difference can be correlated to the
expression levels of particular covalently cell wall-bound proteins involved in proteindependent
biofilm formation, such as S. aureus SasG and its S. epidermidis homologue Aap.
Remarkably, SrtA activity was a limiting determinant for protein-dependent biofilm
formation in S. aureus and S. epidermidis, whereas SrtC expression interfered with biofilm
formation in S. epidermidis 1457. Taken together, these findings imply that sortases can have
modulating roles in staphylococcal biofilm formation.
While the large mechano-sensitive membrane channel MscL can form pores that are large
enough to allow the passage of small proteins across the membrane, no evidence was obtained
that an S. aureus mscL mutant would be blocked in the release of certain proteins. In chapter
7, an interesting phenotype of the mscL mutant strain is however described. This relates to the
production of the extremely stable and broad-spectrum lantibiotic sublancin 168 by Bacillus
subtilis strain 168. Known sublancin 168 susceptible organisms include important pathogens,
such as S. aureus. Nevertheless, since its discovery, the mode of action of sublancin 168 has
remained elusive. The studies in chapter 7 were, therefore, aimed at the identification of
cellular determinants for bacterial susceptibility towards sublancin 168. Growth inhibition and
competition assays on plates and in liquid cultures revealed that sublancin 168-mediated
growth inhibition of susceptible B. subtilis and S. aureus cells is affected by the NaCl
concentration in the growth medium. Added NaCl did not influence the production, activity or
stability of sublancin 168 but, instead, lowered the susceptibility of sensitive cells towards
this lantibiotic. Importantly, the susceptibility of B. subtilis and S. aureus cells towards
sublancin 168 was shown to depend on the presence of MscL. These findings demonstrate
that MscL is a critical and specific determinant in bacterial sublancin 168 susceptibility that
may either serve as a direct target for this lantibiotic, or as a gate of entry to the cytoplasm
both in B. subtilis and S. aureus.
The combination of bioinformatics and proteomics to investigate the secretome of S. aureus
has been proven to be a powerful approach for dissecting the functions of secretome
components. That this approach is useful not only for the analyses of pathogens, but also for
industrial workhorses like Bacillus licheniformis is shown in chapter 8. From the genome
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Chapter 9
sequence of B. licheniformis DSM 13, 296 proteins were predicted to contain an N-terminal
signal peptide for secretion via the Sec system. Using 2-D PAGE, the extracellular proteome
of B. licheniformis grown in different media was studied. From the approximately 200 spots
visible on the gels, 89 were identified that either contain an N-terminal signal sequence or are
known to be secreted by other mechanisms than the Sec pathway. The extracellular proteome
of B. licheniformis was shown to include proteins from different functional classes, like
enzymes for the degradation of macromolecules, proteins involved in cell wall turnover,
flagellum- and phage-related proteins and some proteins of yet unknown function. Protein
secretion was shown to be highest during the stationary growth phase. Furthermore, cells
grown in a complex medium were found to secrete considerably higher protein amounts than
cells grown in a minimal medium. Limitation of phosphate, carbon and nitrogen sources
resulted in the secretion of specific proteins that may be involved in counteracting the
potentially negative effects of the respective starvation.
In conclusion, comparative secretomics approaches are applicable to the functional dissection
of secretomes of bacterial pathogens, such as S. aureus, and biotechnologically relevant cell
factories, such as B. subtilis and B. licheniformis.
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“Good music is good music and everything else can go to hell”
-David J. Matthews-
170

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and I like to put that to good use”
-James J. Brown-
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Bacteriën
Bacteriën behoren tot de oudste levensvormen op aarde en ze worden beschouwd als de
voorgangers van al het andere leven op aarde. De oudste bacteriën zijn zo’n 4 miljard jaar
geleden ontstaan, terwijl de meest recente “moderne” bacteriesoorten zo’n 3 miljard jaar
geleden zijn ontstaan. Rond deze tijd waren bacteriën en archaea, die beide waarschijnlijk
ontstaan zijn uit een gezamenlijke voorouder, de dominante levensvormen op aarde. Bacteriën
en archaea zijn eencellig en bevatten in deze ene cel alle informatie, die ze nodig hebben om
te overleven onder de meest verschillende omstandigheden. Zo kunnen we deze organismen
vinden op plaatsen waar geen enkel ander organisme zou kunnen overleven, zoals op de
bodem van de oceaan (hoge druk) en nabij vulkanen in de oceaan (zeer giftig en hoge
temperatuur) tot onder het ijs van antarctica (extreem lage temperatuur). De eerste eukaryote
cellen (cellen waaruit alle dieren en planten bestaan) zijn waarschijnlijk ontstaan door de
opname van bacteriën door de voorouders van de eukaryote cellen. Deze endosymbionten
hebben zich vervolgens ontwikkeld tot organellen zoals mitochondriën (de “energiecentrales”
van eukaryote cellen) en chloroplasten (de organellen die nodig zijn voor fotosynthese in
groene algen en planten). De Nederlander Antoni van Leeuwenhoek was in 1676 de eerste die
met behulp van een miscroscoop bacteriën kon waarnemen.
Bacteriën zijn afgesloten compartimenten (cellen), die bestaan uit een waterige oplossing
(cytoplasma), waarin het DNA, eiwitten en alle andere componenten aanwezig zijn, die nodig
zijn voor de bacterie om te kunnen groeien en zich te vermenigvuldigen, ofwel om te leven.
Het cytoplasma is omgeven door een membraan, die het cytoplasma volledig scheidt van de
buitenwereld. Om deze cytoplasmamembraan zit meestal een stevige celwand en, bij
sommige bacteriesoorten een tweede membraan, die de bacterie weerbaar maken tegen
fysieke stress. Er zijn vele verschillende vormen van bacteriën bekend, maar de meest
voorkomende vormen zijn de cocci (bolletjes) en bacilli (staafjes). Verder wordt er vaak nog
onderscheid gemaakt tussen Gram-positieve bacteriën (bacteriën met slechts één membraan
en een dikke celwand) en de Gram-negatieve bacteriën (bacteriën met een dubbele membraan
waartussen een marginale celwand ligt).
Bacteriën hebben een grote invloed op het welbevinden van de mens. Aangenomen wordt dat
er tussen de 500 en 1000 verschillende soorten bacteriën in het menselijke darmkanaal leven
en ongeveer een even groot aantal op de huid. Onder normale omstandigheden vormen deze
bacteriën geen bedreiging voor ons en een aantal soorten zijn zelfs heel nuttig. Denk hierbij
bijvoorbeeld aan bacteriën in ons darmkanaal die vitamines maken (Vitamine K, biotine) of
helpen zetmeel en andere vezels af te breken (wat de mens uit zichzelf niet goed kan). Ook
gebruiken we al eeuwen bacteriën bij het bereiden van verschillende voedingsmiddelen (vaak
Lactobacillus en Lactococcus), zoals kaas, yoghurt en worst. Daarnaast zijn er helaas ook een
groot aantal bacteriën, die schadelijk kunnen zijn. Ze kunnen ziektes veroorzaken die in
sommige gevallen uiteindelijk tot de dood van hun gastheer kunnen leiden. Enkele
voorbeelden hiervan zijn de bacteriën die tetanus (Clostridium tetani), difterie
(Corynebacterium diphtheriae), syfilis (Treponema pallidum), cholera (Vibrio cholerae),
lepra (Mycobacterium leprae) of tuberculose (Mycobacterium tuberculosis) veroorzaken.
Sommige voor de mens gevaarlijke bacteriën (pathogenen) maken deel uit van de normale
microbiële flora (microbiota) van de mens. Zo zitten Staphylococcus epidermidis en
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Staphylococcus aureus bijvoorbeeld op de huid en in de neus waar deze organismen normaal
gesproken geen problemen veroorzaken, terwijl ze toch ook vele onaangename of zelfs
levensbedreigende infecties kunnen veroorzaken. In dit proefschrift is onderzocht over welke
ziekmakende factoren bacteriën zoals S. aureus kunnen beschikken. Daarnaast is de
verworven kennis ook gebruikt om belangrijke eigenschappen van bacteriën zoals Bacillus
subtilis en Bacillus licheniformis te bestuderen. Dit laatste was interessant omdat deze twee
bacilli veel gebruikt worden voor de productie van nuttige en waardevolle eiwitten in de
biotechnologische industrie.
DNA en Eiwitten
Al het leven op aarde is mogelijk door het bestaan van DNA. Alle informatie die nodig is om
te (over)leven is opgeslagen in dit molecuul. DNA is opgebouwd uit vier verschillende
bouwstenen, die we nucleotiden noemen. Deze vier zijn Adenine (A), Guanine (G), Thymine
(T) and Cytosine (C) en de combinatie van deze vier nucleotiden vormt de juiste informatie.
Genen zijn opgebouwd uit deze vier bouwstenen en elk gen heeft een unieke combinatie van
deze bouwstenen. Chromosomen zijn DNA strengen die vele genen bevatten. De meeste
bacteriën hebben maar één chromosoom. DNA kan naast het chromosoom op andere
manieren meegedragen worden, bijvoorbeeld in de vorm van plasmiden. Dit zijn meestal
ronde DNA moleculen waar extra informatie op kan zitten, die voordelig voor een bacterie
zou kunnen zijn. In een heel enkel geval kunnen deze plasmiden (of gedeeltes daarvan) ook
worden ovegebracht naar een andere bacteriesoort. De genen die zijn opgeslagen in deze
chromosomen en plasmiden coderen voor eiwitten. Deze eiwitten zijn vaak enzymen, die een
bepaalde chemische reactie versnellen. Elk eiwit heeft zo zijn eigen specifieke functie. Zo zijn
er enzymen die DNA kunnen kopiëren of kunnen afbreken, enzymen die schadelijke stoffen
kunnen omzetten in ongevaarlijke stoffen, en enzymen die kleine, maar vaak cruciale
veranderingen aan andere eiwitten aanbrengen. Sommige eiwitten zijn alleen nodig voor het
organisme om bepaalde processen in de cel te kunnen uitvoeren, terwijl andere eiwitten ter
verdediging zijn of juist worden gebruikt om de omliggende cellen, zowel prokaryoot als
eukaryoot, aan te vallen. Voor een goede werking van eiwitten is de juiste vorm (conformatie)
nodig en daarvoor moeten ze op de juiste manier worden opgevouwen. Eiwitten worden in het
cytoplasma gesynthetiseerd om vervolgens naar de juiste plek te worden gebracht om hun
functie te kunnen vervullen. Zo kunnen eiwitten naar verschillende locaties worden geleid
waar hun functie nodig is. Eiwitten die buiten de cel hun functie moeten vervullen worden
met een signaalpeptide gesynthetiseerd. Dit signaalpeptide zorgt ervoor dat het eiwit naar een
bepaald transportsysteem wordt geleid en vervolgens door het membraan naar het
celoppervlak of buiten de cel wordt gebracht. Tijdens of kort na de passage van het membraan
wordt het signaal peptide afgeknipt en het eiwit in de juiste vorm gevouwen. Sommige
eiwitten zijn juist nodig aan het opervlak van de cel en deze eiwitten kunnen op verschillende
manieren aan de cel gehecht worden. Ook hiervoor zijn er bepaalde signalen in het eiwit
aanwezig, die ervoor zorgen dat ze aan het oppervlak van de cel blijven vastzitten en daar hun
functie kunnen vervullen. Wanneer er helemaal geen signalen zijn om aan het oppervlak te
hechten, zullen de eiwitten vrij in de omgeving van de bacterie terecht komen.
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Inleiding – Hoofdstuk 1
S. aureus is een bacterie, die als commensaal bij de mens voorkomt. Deze bacterie komt men
vaak tegen op de slijmvliezen in de neus en op andere vochtige plekken in het lichaam. Bij
30-40% van de populatie kan men S. aureus aantreffen. Een deel van deze groep draagt S.
aureus continu bij zich, terwijl het andere deel deze bacterie ook weer spontaan kwijtraakt.
De meeste mensen die S. aureus bij zich dragen zullen hier nooit last van hebben. Echter,
wanneer S. aureus in de bloedbaan terecht komt, dan kan dit leiden tot ernstige infecties.
Infecties worden onder andere opgelopen in het ziekenhuis wanneer invasieve medische
hulpmiddelen zoals katheters en implantaten gebruikt worden. Samen met S. epidermidis is S.
aureus berucht om het vermogen zich op oppervlakken van katheters en implantaten te
hechten en op de plaatsen in het lichaam waar deze hulpmiddelen aangebracht zijn infecties te
veroorzaken. Een van de grote problemen bij het bestrijden van S. aureus is het vermogen van
deze bacterie om resistent te worden tegen antibiotica. Al vrij snel na de eerste inzet van
penicilline als antibioticum tegen S. aureus infecties, werden penicilline-resistente S. aureus
stammen geïsoleerd. Sinds een aantal jaren wordt vancomycine gebruikt als laatste redmiddel,
maar vancomycine-resistente stammen zijn nu ook al gevonden. Een goed voorbeeld hiervan
zijn de S. aureus JH1 en JH9 stammen waarvan de genomen gesequenced zijn. De
vancomycine-sensitieve S. aureus stam JH1 werd geïsoleerd uit een patient die behandeld
werd met vancomycine. Een aantal dagen later werd de vancomycine-intermediair sensitieve
S. aureus stam JH9 uit dezelfde patiënt geïsoleerd. JH9 stamt af van de JH1 stam, waaruit
duidelijk blijkt hoe snel S. aureus ongevoelig kan worden voor antibiotica. Het vermogen van
S. aureus om bijna alle weefsels en organen van de mens te infecteren is gebaseerd op het feit
dat deze bacterie een heel arsenaal aan virulentiefactoren kan produceren. De meeste van deze
virulentiefactoren zijn eiwitten die in het cytoplasma van de cel gemaakt worden, om
vervolgens over het membraan te worden getransporteerd naar het oppervlak of het externe
milieu van de bacterie.
Elke bacterie heeft transportsystemen die bedoeld zijn om eiwitten over het membraan te
transporteren. In dit proeschrift wordt ingegaan op de verschillende eiwittransportsystemen
die aanwezig zijn in S. aureus en S. epidermidis. Door te onderzoeken welke
transportsystemen aanwezig kunnen zijn en wat voor eiwitten via deze systemen naar buiten
worden getransporteerd kan inzicht verkregen worden in het belang van deze systemen voor
de virulentie van S. aureus en S. epidermidis. Door de individuele componenten van elk
systeem te bestuderen, kan tevens inzicht worden verkregen in het belang van deze
componenten voor het transport van specifieke eiwitten. De verkregen kennis kan in de
toekomst wellicht gebruikt worden voor de ontwikkeling van nieuwe diagnostische of
therapeutische middelen om Staphylococcus infecties te voorkomen of te behandelen.
Hoofdstuk 2
In Hoofdstuk 2 wordt gebruik gemaakt van de genoomsequenties van verschillende S. aureus
isolaten om te onderzoeken welke (potentiële) eiwittransportsystemen aanwezig zijn en om
een voorspelling te doen hoeveel eiwitten er worden gesecreteerd via de aanwezige
transportsystemen. Alle eiwitten die worden getransporteerd over het membraan, worden
gesynthetiseerd met een signaalpeptide. Met behulp van programma’s die zoeken naar
verschillende signaalpeptidemotieven werd een voorspelling gedaan hoeveel eiwitten er
mogelijk over het membraan getransporteerd kunnen worden. In de veertien S. aureus
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genomen die inmiddels zijn gesequenced werden zes verschillende eiwittransportsystemen
gevonden, waarvan het meest gebruikte transport systeem het Sec-systeem is. Dit systeem
transporteert ongevouwen eiwitten via het SecYEG kanaal over het membraan. Het Tat
transportsysteem kan gevouwen eiwitten transporteren, maar er zijn veel minder eiwitten
gevonden die mogelijk via dit systeem worden getransporteerd. Daarnaast zijn er nog een
aantal specifieke transportsystemen (Ess, Com, holins en ABC transporters) die maar enkele
eiwitten transporteren. In totaal zijn ~150 eiwitten gevonden, die via deze systemen over het
membraan worden getransporteerd en vervolgens vastgehouden worden in de celwand of
losgelaten in het externe milieu van de bacterie. Dit betreft daarom allemaal potentiële
virulentiefactoren. Uit een vergelijking van de datasets die voor de verschillende S. aureus
stammen verkregen zijn blijkt dat 117 eiwitten in alle stammen voorkomen. Deze eiwitten
vormen samen het zogenaamde kern-exoproteoom. Veel van de eiwitten die covalent aan de
celwand worden gekoppeld vallen onder deze categorie. De eiwitten uit het kern-exoproteoom
lijken belangrijke “huishoud”-functies te vervullen. Het betreft bijvoorbeeld proteases,
fibrinogeen- en IgG-bindende eiwitten die S. aureus nodig heeft om zich in de gastheer te
handhaven en het immuunsysteem te omzeilen. De eiwitten die niet in alle stammen aanwezig
zijn vormen samen het zogenaamde variabele exoproteoom. Deze groep van eiwitten omvat
voornamelijk de toxines en andere eiwitten die nodig zijn om nieuwe plekken in het menselijk
lichaam te veroveren. Deze eiwitten zijn vaak de veroorzakers van ziekteverschijnselen.
Hoofdstuk 3
Ten tijde van het schrijven van dit proefschrift waren de genomen van dertien humane S.
aureus isolaten gesequenced. Doordat de genomen van deze isolaten in grote mate
overeenkomen zou men verwachten dat dit ook het geval is voor de virulentiefactoren die
door de verschillende S. aureus stammen geproduceerd worden. Dat dit niet altijd het geval is,
wordt zichtbaar gemaakt in Hoofdstuk 3. Hier zijn de exoproteomen van 25 klinische isolaten
uit het Universitair Medisch Centrum Groningen onderzocht. Deze isolaten zijn geïsoleerd uit
verschillende patiënten met verschillende soorten infecties. In deze analyses werden 63
verschillende extracellulaire eiwitten geïdentificeerd, waarvan er slechts zeven door alle
isolaten worden gesecreteerd. Hieruit blijkt dat er een enorme heterogeniteit bestaat in de
gesecreteerde eiwitten van deze ene soort bacteriën. Deze heterogeniteit wordt veroorzaakt
door verschillen in de genomen van de verschillende S. aureus isolaten en door variaties in de
expressie van de gesecreteerde eiwitten. De verworven kennis over het constante
exoproteoom is van groot belang voor de ontwikkeling van een geschikt vaccin ter
voorkoming van (liefst alle) S. aureus infecties. Dit onderzoek dient bij voorkeur in de
toekomst uitgebreid te worden tot de eiwitten die bij alle S. aureus stammen aan het
celoppervlak gehecht zijn.
Hoofdstuk 4
De virulentiefactoren van S. aureus worden in het cytoplasma gesynthetiseerd en vervolgens
over het membraan getransloceerd. Daarna worden deze eiwitten ofwel met een
retentiesignaal in het membraan of de celwand vastgehouden ofwel, als retentiesignalen
ontbreken, gesecreteerd in het externe milieu. Bij het proces van eiwittranslocatie en retentie
zijn verschillende eiwitten specifiek betrokken. In Hoofdstuk 4 worden de effecten van het
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verwijderen van deze componenten van eiwittransportystemen op de secretie van eiwitten
beschreven. Het betreft hierbij componenten van de Sec-, Com-, Tat- en holin-systemen. Deze
componenten worden verwijderd door de desbetreffende genen van het chromosoom van S.
aureus te deleteren hetgeen leidt tot mutante S. aureus stammen met defecten in de
machinerie voor eiwittransport. De verwijderde componenten die bij het Sec-systeem
betrokken zijn vervullen verschillende rollen tijdens de verschillende stadia van het
eiwittransportproces. SecG en SecY2 zijn betrokken bij de vorming van transportkanalen in
het membraan waardoor eiwitten het membraan kunnen passeren, Lgt en LspA zijn betrokken
bij het modificeren en processen van eiwitten met een vetzuurmodificatie (lipoproteinen) voor
hun retentie aan de buitenkant van het membraan, DsbA en PrsA hebben een functie bij het
goed vouwen van getransporteerde eiwitten en SrtA en SrtB verankeren bepaalde eiwitten
covalent aan de celwand. Uit de analyses van de exoproteomen van de geconstrueerde
mutanten blijkt, dat het merendeel weinig invloed heeft op de secretie van eiwitten. De deletie
van secG heeft een sterke invloed op eiwitsecretie en dit wordt in meer detail beschreven in
Hoofdstuk 5. Ook de lgt mutant vertoont een sterk veranderd exoproteoom hetgeen te maken
heeft met het feit dat een aantal lipoproteinen niet meer goed aan het membraan hechten en
daardoor in het exoproteoom van de mutante cellen terecht komen.
Hoofdstuk 5
De meeste eiwitten (en virulentiefactoren) worden via het Sec-systeem gesecreteerd. De kern
van het Sec-systeem bestaat uit de translocatiemotor SecA en het kanaal dat door de SecY,
SecE en SecG eiwitten wordt gevormd. Het is bekend dat de genen voor SecY en SecA
essentieel zijn voor de bacterie en daardoor niet kunnen worden uitgeschakeld. S. aureus en
een aantal andere Gram-positieve pathogenen, waaronder S. epidermidis, Bacillus anthracis
en Listeria monocytogenes, hebben nog een tweede set secA en secY genen. De SecA2 en
SecY2 eiwitten lijken een zeer beperkt aantal specifieke eiwitten te transporteren. Voor S.
aureus is in de vakliteratuur gesuggereerd dat zelfs maar één eiwit SecA2/SecY2-afhankelijk
wordt gesecreteerd. In Hoofdstuk 5 wordt beschreven wat de gevolgen zijn van de deletie
van de niet-essentiële secG en secY2 genen op het transport van eiwitten naar het externe
milieu van S. aureus en naar de celwand van dit organisme. De extracellulaire accumulatie
van acht gesecreteerde eiwitten en zeven celwand eiwitten blijkt aanzienlijk te zijn veranderd
in de secG mutant. Een aantal van deze eiwitten zijn bekende en belangrijke
virulentiefactoren, zoals Spa, IsaA en Hla. Een eiwit, waarvan het transport naar de celwand
zeer sterk is verminderd is het antilichaam-bindende eiwit Sbi. De secY2 mutant vertoonde
geen veranderingen in het exoproteoom. Echter, de secretie van twee extra eiwitten was
veranderd in een dubbelmutant waarin de secY2 mutatie gecombineerd was met de secG
mutatie. Daarnaast vertoonde de secG secY2 dubbelmutant een duidelijk groeidefect wat erop
duidt dat SecY2 interacties aan kan gaan met het reguliere Sec kanaal.
Hoofdstuk 6
Een aantal eiwitten in S. aureus vervullen hun functie aan het oppervlak van de cel. Deze
eiwitten zijn bijvoorbeeld nodig voor de hechting van bacteriecellen aan weefsels van de
gastheer of aan elkaar. Ook kunnen dergelijke geëxponeerde eiwitten op een dusdanige
manier antilichamen binden, dat de bacteriecel niet meer door het menselijke immuunsysteem
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als lichaamsvreemd herkend wordt. De enzymen die voor de covalente koppeling van eiwitten
aan de celwand zorgen heten sortases. Ze zijn ingedeeld in vier verschillende klassen: sortase
A-D. S. aureus heeft twee sortases: SrtA dat meerdere verschillende eiwitten met een LPxTG
of LPxAG motief aan de celwand bindt, en SrtB dat slechts één substraat met een NPQTN
motief aan de celwand bindt. Afhankelijk van de stam zijn er in S. epidermidis één of twee
sortases te vinden: SrtA dat dezelfde functie heeft als SrtA in S. aureus en SrtC. Op basis van
genomische informatie werd aangenomen dat SrtC ook eiwitten met een LPxTG motief
herkent, maar de exacte activiteit van dit eiwit was nog niet onderzocht. Hoofdstuk 6
beschrijft onderzoek naar de functies van SrtA en SrtC. Allereerst werden de effecten van
srtA mutaties op de binding van LPxTG-eiwitten aan de celwand in S. aureus en S.
epidermidis onderzocht. Het blijkt dat een aantal eiwitten zoals ClfA, SasG, SdrC, SdrD en
Spa in de S. aureus srtA mutant voor een groot deel in het externe milieu terechtkomen.
Echter, een substantieel deel van deze eiwitten blijft ook aan de celwand gebonden via nietcovalente
interacties. Expressie vanaf een plasmide van S. aureus of S. epidermidis SrtA
resulteerde in herstel van het fenotype van de niet-mutante (wild-type) cellen wat betreft de
locatie van eerder genoemde eiwitten. Ook expressie vanaf een plasmide van SrtC van S.
epidermidis zorgde ervoor dat een aantal eiwitten, te weten ClfA, SasG en Aap, weer covalent
aan de celwand werden gebonden, net als in wild-type cellen. Dit betekent dat SrtA en SrtC
een gedeeltelijk overlappende substraatspecificiteit hebben. Daarnaast werd de betrokkenheid
van sortases bij de vorming van een biofilm onderzocht. Het vermogen van de S. aureus srtA
mutant om een biofilm te vormen was beduidend minder dan dat van wildtype cellen. Deletie
van srtA in S. epidermidis had daarentegen geen gevolg voor de vorming van een biofilm. Dit
verschil kan gerelateerd worden aan de expressieniveaus van een beperkt aantal specifieke
eiwitten, die betrokken zijn bij biofilm vorming, zoals Spa en SasG in S. aureus en Aap in S.
epidermidis. Een interessante waarneming was dat de activiteit van SrtA bepalend bleek te
zijn voor de vorming van biofilms door S. aureus en S. epidermidis. De activiteit van SrtC
had daarentegen een remmende werking op de vorming van een biofilm door S. epidermidis.
Hoofdstuk 7
Het eiwit sublancin 168, dat van nature door de B. subtilis stam 168 geproduceerd wordt,
vertoont antibacteriële activiteiten tegen een groot aantal (pathogene) Gram-positieve
bacteriën, zoals S. aureus, Streptococcus pyogenes en Bacillus cereus. Hoofdstuk 7 beschrijft
een studie naar de activiteit van sublancin 168 onder verschillende groeicondities. Verhoging
van de zoutconcentratie in het groeimedium zorgde voor een verlaging van de gevoeligheid
van cellen voor sublancin 168, maar had geen effect op de productie, activiteit of stabiliteit
van dit antimicrobiële eiwit. Tevens bleek dat het eiwit MscL, dat mechanosensitieve kanalen
in de cytoplasmamembraan vormt, een bepalende rol heeft in de gevoeligheid van de cellen
voor sublancin 168. De resultaten van dit onderzoek suggereren dat MscL ofwel het
aangrijpingspunt voor sublancin 168 is, ofwel een porie in het membraan die sublancin 168
toegang geeft tot een vooralsnog onbekend aangrijpingspunt binnen in de cel.
Hoofdstuk 8
Één van de bacteriën die in de biotechnologische industrie gebruikt worden voor de productie
van gesecreteerde eiwitten is Bacillus licheniformis. Al lang wordt van deze bacterie gebruik
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gemaakt, maar er was nog nooit onderzocht welke eiwitten er nu precies worden gesecreteerd.
Met het beschikbaar komen van de genoomsequentie van B. licheniformis DSM 13 werd het
mogelijk om te voorspellen welke eiwitten in het medium terecht zouden komen. Hoofdstuk
8 beschrijft onderzoek naar de transportsystemen die aanwezig zijn in B. licheniformis. Met
behulp van de genoomsequentie werden voorspellingen gedaan over de eiwitten die een
signaal peptide hebben en via welke transportsystemen deze eiwitten naar buiten worden
getransporteerd. In totaal werden 298 eiwitten met signaalpeptiden voorspeld, waarvan de
meeste naar het Sec-systeem worden gestuurd voor secretie. Met behulp van 2-D
gelelectroforese werd het exoproteoom van B. licheniformis bestudeerd. Dit heeft geleid tot
identificatie van 89 daadwerkelijk gesecreteerde eiwitten, die gesynthetiseerd worden met een
duidelijk herkenbaar signaalpeptide. Tot deze groep van eiwitten behoren enzymen, die nodig
zijn voor de afbraak van macromoleculen, enzymen die nodig zijn voor verandering en
afbraak van de celwand, bacteriofaag- en flagel-gerelateerde eiwitten en meerdere eiwitten
waarvan de functie nog niet bekend is. Onder verschillende groeicondities worden
verschillende eiwitten in het exoproteoom gevonden. Zo worden er meer verschillende en
grotere hoeveelheden extracellulaire eiwitten gevonden wanneer de bacterie in een rijk en
complex medium wordt gekweekt dan wanneer de bacterie in een minimaal medium wordt
gekweekt.
Hoofdstuk 9
In Hoofdstuk 9 worden de onderwerpen die in dit proefschrift zijn beschreven besproken en
in een breder perspectief geplaatst.
196

Appendix I
Dankwoord
197

Appendix I
Dankwoord
En nu je eindelijk het hele proefschrift hebt doorgelezen (yeah, right!!!), het meest gelezen
“Dankwoord”. Laat ik eerst maar beginnen met het bedanken van mijn ouders, zodat ze met trots
bovenaan staan; en terecht wat mij betreft.
Pap en mam, ik wil jullie hier in de eerste plaats heel erg bedanken voor de steun in de afgelopen vijf
jaar. Het ging in de eerste twee jaar fantastisch goed en jullie hebben gedurende mijn AIO-tijd altijd
interesse getoond naar de vorderingen op en naast het werk en volgens mij zijn jullie een van de
weinige ouders die (doordeweeks) op het lab hebben rondgekeken. Ook (of misschien wel juist) tijdens
en na de persoonlijke tegenslag met het verlies van Regina in het laatste jaar, heb ik ontzettend veel
steun aan jullie gehad. Daarnaast wil ik jullie bedanken dat jullie me altijd vrij hebben gelaten in mijn
keuzes (soms met een klein beetje bijsturen).
Pap, ondanks dat je dit niet meer mee mocht maken, weet ik zeker dat je nu apetrots zou zijn geweest.
Je bent in een aantal opzichten een groot voorbeeld geweest, waar ik tegenop kon kijken. Je interesse
voor muziek heb je aan mij doorgegeven, en muziek is altijd een goede uitlaatklep voor mij geweest.
Dat heeft me zeker door de vele AIO-dipjes geholpen. Bedankt voor de “unconditional love” and het
feit dat je zo blij met mijn Eleni was. We vinden het erg jammer dat jij en Regina er volgend jaar niet
bij kunnen zijn op onze trouwerij in Griekenland. Ik mis je.
Mam, voor mij ben je altijd de sterkste vrouw geweest die ik ken. Ik ben altijd een mama’s kindje
geweest en ik weet dat ik altijd naar jou kon gaan om over problemen te praten (ook al deed ik dat erg
weinig). In 2007 en 2008 werd (te) veel van jou en ons gevraagd, maar ondanks dit heb jij je staande
weten te houden. Hierdoor is mijn respect voor jou, die al groot was, nog groter geworden. Ook hier
komen we doorheen! Bedankt voor al die bezoekjes in Groningen samen met Pap, Elvira en Lisa,
Tamara en Patrick, of Regina en Feiko. Samen met Pap heb je de basis gelegd voor mijn interesse in
muziek. Ook dank voor de “muzikale opvoeding” thuis en in de auto naar Brabant, waar ik met veel
plezier aan terug denk.
Mijn drie zusjes Elvira, Tamara en Regina wil ik bedanken voor de jaren thuis op de Skarren in
Bolsward en de jaren daarna in Groningen/Sneek/Leeuwarden. Ik ben altijd trots op alledrie geweest en
ik hoop dat jullie nu ook trots op mij kunnen zijn met het behalen van de Doctors titel.
Lieve Elvira (Zus 1), vroeger waren we al twee handen op één buik, maar daar is volgens mij helemaal
niks mis mee. Juist daarom weet ik dat je er altijd voor me zult zijn wanneer dat nodig is en daar heb ik
de laatste 2 jaar heel veel aan gehad. Ook de komst van jullie dochter Lisa en het mogen oppassen op
haar heeft mij door het laatste jaar geholpen. Elvira en Jan, heel veel plezier met jullie kleine aliens en
stuur ze maar langs bij oom Ma’k en tante ‘Nena als ze vervelend beginnen te worden. Ik zal het even
langs wippen voor een lekker hapje eten of gewoon een praatje bij jullie in het restaurant missen.
Lieve Tamara (Zus 2), heel erg bedankt voor het meehelpen opknappen van het huisje op de
Goudsbloemstraat. Samen hebben we er toch iets heel moois van gemaakt (al heb ik de trap nog een
keer moeten verven) en ik heb er de afgelopen jaren met veel plezier gewoond. Ook jouw steun in de
laatste 2 jaar is heel belangrijk voor mij geweest. Veel plezier samen met Patrick in jullie nieuwe
huisje!
Lieve Regina (Zus 3), de avonden met Feiko bij mij of bij jullie waren voor mij altijd dikke pret.
Lekker lullen over niks tijdens een spelletje doen of een filmpje kijken. Bedankt dat ik bij jou helemaal
en totaal mezelf kon zijn. Je hebt de betekenis van “Carpe Diem” wel een beetje op een rare manier
duidelijk gemaakt. Ik mis je.
Hoe vaak ik er ook om gezeurd heb, een broertje heb ik nooit gekregen. Gelukkig was er die
buurjongen van nummer 8 die een aantal jaar ouder is en gemakkelijk ingezet kon worden als “grote
broer”. Marco, hartelijk dank voor de vele uurtjes ontspanning in de vorm van een filmpje of een potje
Mario Kart of Burnout. Het heeft misschien wel wat schrijftijd gekost, maar het heeft ook veel uurtjes
plezier (bij winst) en mateloze irritatie (bij verlies) opgeleverd. Ook de vakanties waren van
198

Dankwoord
onschatbare waarde (Montpellier, Italië en 2x USA) en ik heb daar veel mooie foto’s en een
fantastische tattoo van een “Injun” aan overgehouden. De potjes MarioKart moeten we nu dan maar
online doen of de keren dat ik in Nederland ben, en als je goed oefent kun je misschien net zo goed
worden als ik (………).
Jan Maarten, promoter, full-time optimist en vertrouwenspersoon. De eerste keer dat we elkaar
ontmoetten was tijdens een kennismakingsgesprek bij jou in je kamer toen je nog bij Farmaceutische
Biologie werkte. Ik wilde toen als vrijwilliger aan de slag om mijn labvaardigheden een beetje op peil
te houden. Wat me toen al opviel, was dat je altijd open staat voor dingen. Toen je vertelde dat je een
eigen groep ging beginnen, wilde ik graag meegaan. Je hebt me gelukkig niet heel lang hoeven laten
wachten, want je belde al vrij snel dat ik bij je kon beginnen. Door mij zo snel mogelijk heel veel
dingen te laten doen, heb je meer uit me gehaald dan ik zelf voor mogelijk hield. Het presenteren, waar
ik altijd een hekel aan heb gehad, werd minder vervelend. De vele congressen waar je ons naar
toestuurde, waren altijd nuttig, heel erg gezellig en natuurlijk vol met mooie uitstapjes (met als
hoogtepunten Kreta en Australië). Ook heel erg bedankt voor het feit je altijd wilde luisteren en klaar
stond op de momenten wanneer dat nodig was (half twee ’s nachts) en voor de gesprekken die we
hebben gehad in de tijden dat ik het moeilijk had. Je altijd positieve instelling heeft mij veel geholpen
wanneer het even wat minder ging. Ook ben ik heel blij dat ik je heb kunnen overtuigen dat Eleni een
goede AIO zou zijn en prima bij ons zou kunnen werken. Succes met de groep en wie weet, komen we
elkaar in de toekomst weer tegen.
I would like to thank the reading committee, Tarek Msadek, Wim Quax and Arnold Driessen for
reading this thesis and for their critical comments. A special thanks goes out to Tarek for his
enthusiasm at my first presentation at the BACELL meeting at the Pasteur Institute in 2005 in Paris.
Also many thanks for the possibility to stay for a month and play with worms in your lab. Too bad we
never got to play some music together, but maybe we can arrange something for the party!!
Also many thanks to the people who have helped one way or another to make this thesis as it is now.
Anne, Birgit, Haike, Susanne, Theresa, Vanessa, and Michael, thank you very much for the pictures of
the 2D gels and the nice discussions we had on meetings or via mail. Anne en Harold, hartelijk dank
voor het begin met de predicties. Dit hoofdstuk heeft een mooie basis gelegd voor de rest van het
proefschrift. Dörte also thanks for your contribution to Chapter 6 and the identification of the many
protein samples I have sent to you. Ietse, bedankt voor de mooie EM-plaatjes van de Staphylococcus
aureus stammen. To the people from Tarek’s group, and especially Olivier: thank you for your warm
welcome in Paris and introducing me to the worms. Many thanks to the BACELL-HEALTH,
StaphDynamics, and TIPharma AntiStaph communities for giving me the chance to present my work
and the many helpful discussions.
Beste paranimfen, Thijs en Monika; de afgelopen 5 jaar (en met name de laatste twee jaar) heb ik
ontzettend veel steun aan jullie gehad.
Thijs, als collega, goede vriend en metalhead heb ik je beter leren kennen. Op het lab en tijdens de
werkbesprekingen had je vaak wel antwoord op mijn vragen en/of respons op wat ik te vertellen had. Je
bent als vriend een enorme steun geweest na het overlijden van Regina en de zware tijden die daarop
volgden. Altijd klaar om ’s avonds even langs te komen als het niet ging en dan maar even te praten
en/of met de PS2 of Gamecube de gedachten op iets anders te zetten. En dan zijn er natuurlijk de vele
concerten waar we met Esther, Astrid, Ralph en anderen naartoe zijn geweest (Iron Maiden (2x), Judas
Priest, Dragonforce, Fields of Rock en Apocalyptica). Ik hoop dat we er nog een aantal leuke concerten
aan kunnen toevoegen. We hebben veel plezier gehad om jouw cabaret in elkaar te zetten, dus doe je
best met die van mij! Veel succes met je nieuwe baan bij DSM.
Monika, (Zus 4), de vele uurtjes die we tijdens en na het werk hebben benut om je Nederlands te
verbeteren, onze frustraties te kunnen uiten, zijn erg waardevol geweest. Frustraties van het werk of
prive heb ik aan jou kunnen vertellen. Ik was wel blij met iemand op het lab die ook wel van
199

Appendix I
regelmatig schoonmaken hield. Het tripje naar Polen wat je samen met Gosia hebt georganiseerd, was
zeker een van leukste vakanties die ik heb gehad. Vooral de wandelingen door de Poolse bossen naar
de bergtoppen met de soundtrack van Lord of the Rings op mijn iPod zijn herinneringen die me altijd
bij zullen blijven. Ik kijk ernaar uit wanneer je jouw proefschrift af hebt, zodat ik eindelijk je cabaret in
elkaar kan zetten (materiaal genoeg!!).
En dan er natuurlijk al die andere collega’s die op een of andere manier invloed hebben gehad op het
tot stand komen van dit proefschrift. René, less talking, more eating!! Verbazingwekkend, dat het toch
goed gegaan is met een Sneker en een Bolswarder op één lab. Samen met Thijs waren we “The Three
Musketeers” (of “The Three Stooges”) die het samen met Jan Maarten en Jean-Yves allemaal zijn
begonnen. Ik vond het leuk om op vrijdag ochtend te gaan zwemmen voordat we weer aan het werk
moesten. Veel succes met je onderzoek in Amsterdam.
En ik kan natuurlijk niet al die andere collega’s vergeten waar ik samen mee heb gewerkt en samen een
aantal leuke dingen hebben gedaan; Jean-Yves, Gosia, Girbe, Jessica, May, Dennis, Rense, Matty,
Willy, Sjouke, Emma, Jetta, Jolanda, Magda, Arthur, Lakshmi, Henrik, Vahid, Carmine, Danai,
Marcus, Hermie, Vivianne, Eleni en alle studenten die bij ons hebben rond gelopen, allemaal hartelijk
dank voor jullie hulp, gezelligheid, alle borrels die we samen hebben gedaan en natuurlijk het labuitje
naar Franeker en het godvergeten Bolsward (hoe verzin je het!?!?!). Ook de UMCG volleybal
toernooien waarin we twee keer hebben gewonnen waren voor mij leuke sportieve uitjes.
Natuurlijk kan ik de studenten die mee hebben geholpen in het onderzoek niet vergeten. Marloes,
Magda, Jessica, Vincent, Eleni en Diana, jullie ook hartelijk dank voor jullie inzet en voor de
mogelijkheid voor mij om onderwijs te geven. Het kan geen toeval zijn dat drie van mijn studenten
uiteindelijk ook bij Jan Maarten gaan promoveren (zei hij met een knipoog).
Vier jaar bezig zijn met een proefschrift kan niet zonder enige vorm van ontspanning. Gelukkig waren
er genoeg mensen om te klaverjassen. Samen met Rik, Rene en Thijs zijn we begonnen met
klaverjassen en zijn uiteindelijk verder gegaan met pokeren. Het klaverjassen hebben we daarna weer
opgepikt met Thijs, Esther en Annechien of met Thijs, Esther en Chris. Bedankt voor de vele avonden
met goed eten, bier, chips en een hoop onzinnig geklets. Mattijs, Ykelien, Almer, en alle anderen ook
hartelijk dank voor het ontzettend mooie jaar bij FarmBio, met de vele borrels, filmpjes en Kolonisten
avonden.
Because limited exercise is being performed during board- and cardgames, there was need for some
physical training to get rid of all the calories obtained from the chips and beers. Many thanks to Tao
and Yoshitaka, who convinced me to join them for playing badminton every Tuesday. Within the
department, several other people were interested in playing badminton, with the one condition that after
playing we would go out for dinner. Tao and May “ ”, Yoshitaka “ ”,Ank, Rudi, and
Erwin “bedankt” for these very nice moments of physical exhaustion and the very nice dinners
afterwards.
Dennis en Grietje (en kinderen), Folkert en Klaske, Ebbo en Ingrid (en kinderen), hartelijk dank voor
de bezoekjes en de etentjes die we hebben georganiseerd. Ik hoop dat we hiermee nog door kunnen
gaan. Many thanks to the Greek community in Groningen (Basilis and Xrysa, Dimitris, Evi, Evelina
and Danai) for your warm welcome and introduction to the Greek people and their food.
Ανέστη, Βέτα και Θράσο σας ευχαριστώ για το θερµό καλοσώρισµα στην οικογένεια Τσοµπανίδη.
Τώρα που τελείωσα το διδακτορικό µου θα έχω επιτέλους τον χρόνο να µάθω ελληνικά ώστε να
µπορούµε να µιλάµε άµεσα.
Danai, you thought you were going to share a house with Eleni, but you were getting another guest as
well. Thank you for your understanding that you accepted me as a housemate and slowly took Eleni to
be my housemate.
200

Dankwoord
And last but not least: Eleni, zouzounaki mou, “euxaristo para para polu” for your help as a student, for
being there at the right time, for your love and understanding and accepting me the way I am, for you
making me laugh again in difficult times, for sharing our passion for music (I can’t wait to go to
Jamaica to visit Bob) and the concerts we went to and will go to, for showing me the nice places in the
Netherlands where, without you, I probably would not have gone, for showing me the nice parts of
Greece, for going out with me for dinner every 24 th of the month, for introducing me to your family,
and for being part of my family. I don’t know where it will go from here, but I want you to know that I
will always love you. Sagapo!
Mark
201

202

Appendix II
Publication list
203

Appendix II Publication list
Publication list
1. Huchon, D., Madsen, O., Sibbald, M.J., Ament, K., Stanhope, M.J., Catzeflis, F., de
Jong, W.W., Douzery, E.J. (2002) Rodent phylogeny and a timescale for the evolution of
Glires: evidence from an extensive taxon sampling using three nuclear genes. Mol. Biol.
Evol. 19, 1053-1065.
2. Bokma, E., Rozeboom, H.J., Sibbald, M., Dijkstra, B.W., Beintema, J.J. (2002)
Expression and characterization of active site mutants of hevamine, a chitinase from the
rubber tree Hevea brasiliensis. Eur. J. Biochem. 269, 893-901.
3. Voigt, B., Schweder, T., Sibbald, M.J.J.B., Albrecht, D., Ehrenreich, A., Feesche, J.,
Maurer, K.-H., Gottschalk, G., van Dijl, J.M., Hecker, M. (2005) The extracellular
proteome of Bacillus licheniformis grown in different media and under different nutrient
starvation conditions. Proteomics. 6, 268-281.
4. Sibbald, M.J.J.B., Ziebandt, A.-K., Engelmann, S., Hecker, M., de Jong, A., Harmsen,
H.J.M., Raangs, G.C., Arends, J., Dubois, J.-Y.F., van Dijl, J.M. (2006) Mapping the
pathways to staphylococcal pathogenesis by comparative secretomics. Microbiol. Mol.
Biol. Rev. 70, 755-788.
5. van Dijl, J.M., Buist, G., Sibbald, M.J.J.B., Zweers, J.C., Dubois, J.-Y.F., and Tjalsma,
H. (2006) In's and out's of the Bacillus subtilis membrane proteome. In: Bacillus: Cellular
and Molecular Biology (P. Graumann ed.), Horizon Scientific Press, Hethersett, Norwich,
UK.
6. Sibbald, M.J.J.B., van Dijl, J.M. (2008) Secretome mapping in Gram-positive pathogens.
In: Bacterial secreted proteins: secretory mechanisms and role in pathogenesis (K.
Wooldridge ed.). Horizon Scientific Press, Hethersett, Norwich, UK.
7. Ziebandt, A.-K., Degner, M., Sibbald, M.J.J.B., Arends, J.P., Chlebowicz, M.A., Kusch,
H., Albrecht, D., Pantuček, R., Doškar, J., Ziebuhr, W., Bröker, B.M., Hecker, M., van
Dijl, J.M., Engelmann1, S. Proteogenomics uncovers extreme heterogeneity of the
Staphylococcus aureus exoproteome. Proteomics. In revision.
8. Kouwen, T.R.H.M., Trip, E.N., Denham, E.L., Sibbald, M.J.J.B., Dubois, J.-Y.F., van
Dijl, J.M. (2009) The large mechanosensitive channel (MscL) determines bacterial
susceptibility to the bacteriocin sublancin 168. Antimicrob. Agents Chemother. 53, 4702-
4711.
9. Sibbald, M.J.J.B. # , Winter, T. # , van der Kooi-Pol, M.M., Bosma, T., Schäfer, T., Ohlsen,
K., Hecker, M., Antelmann, H., Engelmann, S., van Dijl, J.M. SecG of Staphylococcus
aureus plays a crucial role in the translocation of virulence factors. In revision.
10. Sibbald, M.J.J.B., Winter, T., ten Brinke M., Buist, G., Koedijk, D.G.A.M., van der
Kooi-Pol, M.M., Msadek, T., Poupel, O., Rühmling, V., Stokroos, I., Tsompanidou, E.,
Antelmann, H., Hecker, M., Engelmann, S., van Dijl, J.M. Characterization of
Staphylococcus aureus secretion mutants. To be submitted.
11. Sibbald, M.J.J.B. # , Yang, X.M. # , Tsompanidou, E., Hecker, M., Becher, D., Buist, G.,
van Dijl, J.M. Partially overlapping substrate specificities of sortases A and C of
staphylococci. Submitted.
# both authors contributed equally to this work
204

Appendices III-V
Supplemental tables
205

Appendix III: Supplemental tables to Chapter 2
a. Identified proteins in extracellular proteomes of various S. aureus strains with a
known signal peptide
b. Identified proteins in the extracellular proteomes of various S. aureus strains without
a known signal peptide
c. The core exoproteome of S. aureus; proteins with predicted Sec type signal peptides
present in all sequenced strains
d. The variant exoproteome of S. aureus; proteins with predicted Sec type signal
peptides present in at least one sequenced strain
e. The core lipoproteome of S. aureus; proteins with predicted lipoprotein signal
peptides present in all sequenced strains
f. The variant lipoproteome of S. aureus; proteins with predicted lipoprotein signal
peptides present in at least one sequenced strain
g. Composition of the variant exoproteome of sequenced S. aureus strains
h. Composition of the variant lipoproteome of sequenced S. aureus strains
i. Specific S. epidermidis proteins with a predicted signal peptide
j. Proteins with predicted Sec type signal peptides present in S. aureus RF122
Appendix IV: Supplemental tables to Chapter 3
a. Characteristics of the clinical S. aureus isolates used in this study
b. Identified proteins on 2-D gels of the S. aureus isolates
c. Extracellular proteins in different S. aureus isolates
Appendix V: Supplemental tables to Chapter 9
a. Proteins of B. licheniformis DSM 13 with a predicted signal peptide
b. Extracellular proteins of B. licheniformis which either contain an N-terminal signal
peptide or which are known to be secreted by other pathways
c. Proteins detected in the extracellular proteome lacking known export signals
206

Appendix III Supplemental Table IIIa
Supplemental table IIIa. Identified proteins in extracellular proteomes of various S. aureus
strains a with a known signal peptide
PID Protein Function -3 -2 -1 +1 Localization Motif
15925728 b SA0022 hypothetical protein S N A A Extracellular LPKTG
15925799 Plc 1-phosphatidylinositol
phosphodiesterase precurosr
A H A S Extracellular
15925800 SA0092 hypothetical protein T A G C Extracellular Lipo
15925815 b Spa immunoglobulin G binding A N A A Membrane / LPETG
protein A precursor
Cell Wall /
Extracellular
15925838 SasD hypothetical protein A H A D Extracellular LPAAG
15925848 SA0139 hypothetical protein S L A I Extracellular
15925933 Coa staphylocoagulase precursor A D A I Extracellular
15925978 LytM peptidoglycan hydrolase A D A A Extracellular
15925983 SA0270 hypothetical protein A Q A Y Extracellular
15926008 SA0295 hypothetical protein A F A K Extracellular
15926022 Geh glycerol ester hydrolase A Q A S Extracellular
15926073 SA0359 hypothetical protein L T A C Extracellular Lipo
15926099 Set6 exotoxin 6 V Q A K Extracellular
15926104 Set11 exotoxin 11 V H A K Extracellular
15926111 Set15 exotoxin 15 V K A S Extracellular
15926112 SA0394 hypothetical protein A E A S Extracellular
15926142 SA0423 hypothetical protein A N A A Extracellular LysM
15926239 b SdrC Ser-Asp rich fibrinogen- A K A A Membrane / LPETG
binding, bone sialoproteinbinding
protein
Cell Wall
15926240 SdrD Ser-Asp rich fibrinogen- A K A A Membrane / LPETG
binding, bone sialoproteinbinding
protein
Cell Wall
15926241 SdrE Ser-Asp rich fibrinogenbinding,
bone sialoproteinbinding
protein
A K A A Extracellular LPETG
15926291 SA0570 hypothetical protein A E A A Extracellular
15926342 b SA0620 hypothetical protein A Q A S Extracellular
15926373 SA0651 hypothetical protein A L A K Extracellular
15926385 SA0663 hypothetical protein L G A C Extracellular Lipo
15926417 SA0695 hypothetical protein I S A C Extracellular Lipo
15926464 b ClfA fibrinogen-binding protein A A D A S Membrane /
Cell Wall
LPQTG
15926548 GlpQ glycerophosphoryl diester
phosphodiesterase
A G A E Extracellular
15926570 SA0841 hypothetical protein V S A A Extracellular
15926634 SspB cysteine protease precursor A K A D Extracellular
15926635 SspA serine protease, V8 protease,
glutamyl endopeptidase
A N A L Extracellular
15926639 Atl autolysin, N-acetylmuramyl-Lalanine
amidase and endo-β-Nacetylglucosaminidase
V Q A A Extracellular GW
15926648 SA0914 hypothetical protein A D A T Extracellular
15926713 IsdB iron-regulated heme-iron
binding protein
A Q A A Extracellular LPQTG
15926714 IsdA cell surface protein V N A A Extracellular LPKTG
15926715 IsdC hypothetical protein A N A A Extracellular NPQTN
15926739 SA1001 hypothetical protein A K A F Extracellular
15926746 SA1007 α-hemolysin precursor A N A A Extracellular
15926796 SA1056 hypothetical protein V A G C Extracellular Lipo
207

Appendix III Supplemental Table IIIa
PID Protein Function -3 -2 -1 +1 Localization Motif
15926830 b LytN hypothetical protein A Y A D Membrane /
Cell Wall
15926969 c SA1221 thioredoxin reductase L G A C Extracellular Lipo
15927068 SA1318 hypothetical protein L S G C Extracellular Lipo
15927308 SA1552 hypothetical protein A Q A A Extracellular LPKTG
15927383 SplF serine protease SplF A K A E Extracellular 2 TM
15927384 SplD serine protease SplD A K A E Membrane /
Cell Wall
2 TM
15927385 SplC serine protease SplC A N A E Extracellular
15927386 SplB serine protease SplB A K A E Extracellular
15927387 SplA serine protease SplA A K A E Membrane /
Cell Wall
15927389 SA1633 probable β-lactamase A K A E Extracellular
15927393 LukD leukotoxin, LukD V D A A Extracellular
15927394 LukE leukotoxin, LukE S R A N Extracellular
15927483 SA1725 staphopain, cysteine proteinase A N A E Extracellular
15927512 Map truncated map-w protein A S A A Extracellular
15927517 SA1755 hypothetical protein A K A F Extracellular
15927520 Sak staphylokinase precursor V S A S Membrane /
Cell Wall
15927579 Hlb truncated β-hemolysin A K A E Extracellular
15927580 SA1812 hypothetical protein S Y A K Extracellular
15927581 SA1813 hypothetical protein T Q A N Extracellular
15927586 SA1818 hypothetical protein A K A E Extracellular
15927607 SA1839 hypothetical protein V E A K Extracellular
15927670 SceD hypothetical protein A H A S Extracellular
15927741 b FmtB FmtB protein A S A D Membrane /
Cell Wall
LPDTG
15927757 SA1979 hypothetical protein V A A C Extracellular Lipo
15927785 SA2006 hypothetical protein A S A D Extracellular
15927879 SsaA hypothetical protein A H A S Extracellular
15927884 SA2097 hypothetical protein A D A A Extracellular
15927890 SA2103 hypothetical protein V A A K Extracellular
15927988 SA2198 hypothetical protein L T A C Membrane /
Cell Wall
Lipo
15927996 Sbi IgG-binding protein SBI A K A S Extracellular
15927997 HlgA γ-hemolysin chain II precursor S K A E Extracellular
15927998 HlgC γ-hemolysin component C A K A A Extracellular
15927999 HlgB γ-hemolysin component B A N A E Extracellular
15928076 SA2285 hypothetical protein A E A A Extracellular LPKTG
15928148 IsaA immunodominant antigen A A H A A Extracellular
15928216 ClfB clumping factor B A Q A S Extracellular LPETG
15928223 Aur zinc metalloproteinase A L A I Membrane /
aureolysin
Cell Wall
15928230 SA2437 N-acetylmuramoyl-L-alanine
amidase
A Y A D Extracellular
15928232 b SasF hypothetical protein A Q A A Membrane /
Cell Wall
LPKAG
15928254 IcaB intercellular adhesion protein A N A D Membrane /
B
Cell Wall
15928257 Lip triacylglycerol lipase precursor A Q A A Extracellular
49482650 d Set16 exotoxin 16 V Q A K Extracellular
49482656 d Set11 exotoxin 11 V Q A A Extracellular
49482662 d Set26 exotoxin 26 V K A S Extracellular
49482663 d SAR0436 putative exported protein A E A S Extracellular
49483672 SAR1494 hypothetical protein L S G C Extracellular Lipo
208

Appendix III Supplemental Table IIIa
PID Protein Function -3 -2 -1 +1 Localization Motif
49484059 d SAR1905 serine protease A K A E Extracellular
48494887 d Cna collagen adhesin precursor A F A A Extracellular LPKTG
49484898 d SAR2788 putative exported protein A E A S Extracellular
57652419 e Pls methicillin-resistant surface
protein
A E A A Extracellular LPDTG
57651309 e SACOL0468 exotoxin 3, putative V Q A K Extracellular
57651319 e SACOL0478 exotoxin 3, putative V K A S Extracellular
57651320 e SACOL0479 surface protein, putative A E A S Extracellular
57650159 e Sek staphylococcal enterotoxin A S A Q Extracellular
57650160 e Sei staphylococcal enterotoxin
type I
A Y A D Extracellular
57651597 e Seb staphylococcal enterotoxin B V L A E Extracellular
57651598 e SACOL0908 hypothetical protein A K A S Extracellular
57650600 e SACOL1865 serine protease SplE, putative A K A E Extracellular
57650605 e SACOL1870 hypothetical protein A K A E Extracellular
57650692 e Hlb β-hemolysin/phospholipase C A K A E Extracellular
57651004 e SACOL2505 cell wall surface anchor family
protein
A E A A Extracellular LPKTG
24636603 f Etd exfoliative toxin D S H A E Extracellular
24636604 f probable glutamylendopeptidase
V S A S Extracellular
37196678 f Ser enterotoxin R V S A K Extracellular
a
The annotation of proteins is based on that of S. aureus N315, except for those proteins that are not encoded by
the N315 genome
b
Identified in membrane/cell wall fraction by Nandakumar et al. (2005) and/or Gatlin et al. (2006)
c
Note that SA1221 is probably not a thioredoxin reductase, but a phosphate binding protein
d
Proteins encoded by S. aureus MRSA252 and absent from N315
e
Proteins encoded by S. aureus COL and absent from N315
f
Note that the corresponding gene is not present in the sequenced S. aureus genomes
209

Appendix III Supplemental Table IIIb
Supplemental table IIIb. Identified proteins in the extracellular proteomes of various S.
aureus strains without a known signal peptide
PID Protein Function
15925714 SerS seryl-tRNA synthetase
15925746 a MecR1 methicillin resistance protein
15925747 a MecI methicillin resistance regulatory protein
15925748 XylR hypothetical protein
15925892 SA0182 hypothetical protein, similar to indole-3-pyruvate decarboxylas
15925929 PflB formate acetyltransferase
15925944 a LctE L-lactate dehydrogenase
15925982 SA0269 hypothetical protein
15925985 SA0272 hypothetical protein
15926071 SA0357 hypothetical protein
15926081 AhpF alkyl hydroperoxide reductase subunit F
15926082 AhpC alkyl hydroperoxide reductase subunit C
15926088 SA0372 hypothetical protein
15926091 GuaB inositol-monophosphate dehydrogenase
15926092 GuaA GMP synthase
15926167 MetS methionyl-tRNA synthetase
15926175 a SpoVG stage V sporulation protein G homologue
15926178 RplY 50S ribosomal protein L25
15926188 a FtsH cell-division protein
15926190 CysK cysteine synthase (o-acetylserine sulfhydrylase) homologue
15926194 LysS lysyl-tRNA synthetase
15926202 ClpC HSP100/Clp ATPase
15926205 GltX glutamyl-tRNA synthetase
15926216 RplA 50S ribosomal protein L1
15926225 Fus translational elongation factor G
15926226 TufA translational elongation factor TU
15926229 SA0509 chaperone protein HchA
15926232 IlvE branched-chain amino acid aminotransferase
15926236 SA0516 hypothetical protein
15926244 SA0524 hypothetical protein
15926266 Pta phosphotransacetylase
15926283 Adh1 alcohol dehydrogenase
15926294 SarA staphylococcal accessory regulator A
15926363 SA0641 transcriptional regulator
15926396 YfnI hypothetical protein with 5 transmembrane segments and a potential SPase I
cleavage site
15926420 a PepT aminotripeptidase
15926429 SA0707 hypothetical protein
15926441 TrxB thioredoxine reductase
15926445 ClpP peptidase
15926449 Gap glyceraldehyde-3-phosphate dehydrogenase
15926450 Pgk phosphoglycerate kinase
15926451 Tpi triosephosphate isomerase
15926452 Pgm 2, 3-diphosphoglycerate-independentphosphoglycerate mutase
15926453 Eno enolase
15926469 a CspC cold-shock protein C
15926497 a SA0769 ABC transporter ATP-binding protein homologue
15926503 SA0775 hypothetical protein
15926543 SA0806 hypothetical protein
15926546 RocD ornithine-oxo-acid transaminase
15926551 Pgi glucose-6-phosphate isomerase A
15926554 a SpsB type-1 signal peptidase 1B
15926559 Cdr coenzyme A disulfide reductase
210

Appendix III Supplemental Table IIIb
PID Protein Function
15926572 FabH 3-oxoacyl-(acyl-carrier protein) synthase homologue
15926589 SA0859 hypothetical protein
15926603 SA0873 hypothetical protein
15926632 MenB naphthoate synthase
15926642 SA0908 hypothetical protein; predicted to have an uncleaved signal peptide
15926657 PurM phosphoribosylformylglycinamidine cyclo-ligase PurM
15926669 PtsH phophocarrier protein Hpr phosphohistidin-containing protein
15926670 PtsI phosphoenolpyruvate-protein phosphatase
15926679 PdhB pyruvate dehydrogenase E1 component beta subunit
15926680 PdhC dihydrolipoamide S-acetyltransferase component of pyruvate dehydrogenase
complex E2
15926681 PdhD dihydrolipoamide dehydrogenase component of pyruvate dehydrogenase E3
15926699 PycA pyruvate carboxylase
15926716 IsdD hypothetical protein
15926723 PheT Phe-tRNA synthetase β chain
15926735 SA0998 hypothetical protein
15926759 SA1019 hypothetical protein
15926764 a PbpA penicillin-binding protein 1
15926776 IleS Ile-tRNA synthetase
15926779 a LspA lipoprotein signal peptidase
15926817 a Smc chromosome segregation SMC protein
15926828 SucD succinyl-CoA synthetase, β subunit
15926829 SucC succinyl-CoA synthetase, α subunit
15926838 CodY transcriptional repressor CodY
15926839 RpsB 30S ribosomal protein S2
15926840 Tsf elongation factor TS
15926857 PnpA polyribonucleotide nucleotidyltransferase
15926883 GlpK glycerol kinase
15926884 GlpD aerobic glycerol-3-phosphate dehydrogenase
15926892 GlnA glutamine-ammonia ligase
15926901 a SA1157 hypothetical protein, similar to ABC transporter integral membrane protein
15926915 KatA catalase
15926919 SA1173 hypothetical protein
15926923 Tkt transketolase
15926930 CitB aconitate hydratase
15926936 a AlsT amino acid carrier protein (sodium/alanine symporter)
15926982 b CspA major cold shock protein CspA
15926992 OdhB dihydrolipoamide succinyltransferase
15926993 OdhA 2-oxoglutarate dehydrogenase E1
15927003 SA1255 PTS system, glucose-specific enzyme II, A component
15927005 SA1257 peptide methionine sulfoxide reductase
15927015 SA1267 hypothetical protein, similar to streptococcal adhesin emb
15927031 Pbp2 penicillin-binding protein 2
15927035 AsnC asparaginyl-tRNA synthetase
15927054 SA1305 DNA-binding protein II
15927057 SA1308 30S ribosomal protein S1
15927062 EbpS elastin binding protein
15927086 SA1336 glucose-6-phosphate 1-dehydrogenase
15927092 Gnd phosphogluconate dehydrogenase
15927107 a AccC acetyl-CoA carboxylase AccC, biotin carboxylase subunit
15927109 SA1359 translation elongation factor EF-P
15927115 SA1365 glycine dehydrogenase subunit 2
15927132 Pbp3 penicillin-binding protein 3
15927133 SodA superoxide dismutase SodA
15927145 GlyS glycyl-tRNA synthetase
15927160 DnaK DnaK protein
211

Appendix III Supplemental Table IIIb
PID Protein Function
15927161 GrpE GrpE protein
15927190 GreA transcription elongation factor
15927206 SA1453 hypothetical protein
15927229 SA1475 hypothetical protein
15927242 ValS valine t-RNA Ligase
15927254 Tig trigger factor (prolyl isomerase)
15927257 RplT 50S ribosomal protein L20
15927261 ThrS threonyl-tRNA synthetase
15927265 GapB glyceraldehyde 3-phosphate dehydrogenase 2
15927273 CitZ citrate synthase II
15927286 Ald alanine dehydrogenase
15927287 SA1532 hypothetical protein
15927288 AckA hypothetical protein
15927309 Fhs formyltetrahydrofolate synthetase
15927327 SA1571 D-alanine aminotransferase
15927328 SA1572 hypothetical protein
15927365 PckA phosphoenolpyruvate carboxykinase
15927403 a Sem enterotoxin SEM
15927409 TRAP signal transduction protein TRAP
15927412 SA1656 Hit-like protein involved in cell-cycle regulation
15927425 FumC fumarate hydratase
15927428 SA1671 hypothetical protein
15927495 SA1737 hypothetical protein
15927503 SA1743 hypothetical protein
15927524 Sep enterotoxin P
15927539 SA1774 hypothetical protein
15927584 Sel enterotoxin L
15927604 GroEL GroEL protein
15927605 GroES GroES protein
15927638 SA1868 hypothetical protein
15927681 AtpF ATP synthase subunit B
15927687 GlyA serine hydroxymethyl transferase
15927699 FbaA fructose-bisphosphate aldolase
15927712 DeoD purine nucleoside phosphorylase
15927753 SAS074 hypothetical protein
15927762 Asp23 alkaline shock protein 23, Asp23
15927782 a HysA hyaluronate lyase precursor
15927798 RplM 50S ribosomal protein L13
15927803 RplQ 50S ribosomal protein L17
15927804 RpoA DNA-directed RNA polymerase α-subunit
15927825 RplV 50S ribosomal protein L22
15927911 HutU urocanate hydratase
15927994 SA2204 phosphoglycerate mutase, Pgm homolog
15928070 SA2279 hypothetical protein
15928081 FnbB fibronectin binding protein B
15928082 FnbA fibronectin binding protein A
15928103 Ddh D-specific D-2-hydroxyacid dehydrogenase
15928107 SrtA sortase A
15928125 MvaS 3-hydroxy-3-methylglutaryl CoA synthase
15928133 RocA 1-pyrroline-5-carboxylate dehydrogenase
15928160 SA2367 hypothetical protein
15928185 PanB 3-methyl-2-oxobutanoate hydroxymethyltransferase
15928188 a SA2395 L-lactate dehydrogenase
15928192 SA2399 fructose-bisphosphate aldolase
15928221 ArcA arginine deiminase
15928284 SA2490 hypothetical protein
212

Appendix III Supplemental Table IIIb
PID Protein Function
16119211 BlaR1 bla regulator protein BlaR1
15924938 c Sep enterotoxin P
15925528 c PtsG PTS system, glucose-specific II ABC component
18920604 d 18920604 phi ETA orf 18-like protein
24636605 d Edin-B epidermal cell differentation inhibitor B
49484190 e Sea enterotoxin type A precursor
57650441 f SACOL1528 hypothetical protein
57651702 f PdhA pyruvate dehydrogenase complex E1 component, α subunit
a
Identified in membrane/cell wall fraction by Nandakumar et al. (2005) and/or Gatlin et al. (2007)
b
Contains a pseudopilin SPase recognition and cleavage site
c
Proteins encoded by S. aureus Mu50 and absent from N315
d
Note that the corresponding gene is not present in the sequenced S. aureus genomes
e
Proteins encoded by S. aureus MRSA252 and absent from N315
f Proteins encoded by S. aureus COL and absent from N315
213

Appendix III Supplemental Table IIIc
Supplemental table IIIc. The core exoproteome of S. aureus; proteins with predicted Sec
type signal peptides present in all sequenced strains
PID Protein -3 -2 -1 +1 Function
15925728 a,b SA0022 S N A A hypothetical protein
15925815 a Spa A N A A Immunoglobulin G binding protein A precursor
15925838 c,d SasD A H A D hypothetical protein
15925848 e SA0139 S L A I hypothetical protein
15925933 Coa A D A I staphylocoagulase precursor
15925978 f LytM A D A A peptidoglycan hydrolase
15925983 SA0270 A Q A Y hypothetical protein, similar to precursor SsaA
15926008 e SA0295 A F A K hypothetical protein
15926022 e Geh A Q A S glycerol ester hydrolase
15926106 g Set13 V H A K exotoxin 13
15926107 h Set 14 G H A K exotoxin 14
15926113 SA0395 A D A K hypothetical protein
15926142 e,i Aaa A N A A hypothetical protein
15926239 a,e SdrC A K A A Ser-Asp rich fibrinogen-binding
15926291 e SA0570 A E A A hypothetical protein
15926319 e,j Pbp4 A Q A T penicillin binding protein 4
15926342 e,i SA0620 A Q A S secretory antigen SsaA homologue
15926373 e SA0651 A L A K hypothetical protein
15926432 e,i SA0710 A H A Q hypothetical protein
15926464 a ClfA A D A S fibrinogen-binding protein A
15926466 Ssp A K A A extracellular ECM and plasma binding protein
15926467 SA0745 A N A L hypothetical protein
15926468 Nuc A N A S staphylococcal nuclease
15926548 e,j GlpQ A G A E glycerophosphoryl diester phosphodiesterase
15926570 SA0841 V S A A hypothetical protein
15926634 SspB A K A D cysteine protease precursor
15926635 e SspA A N A L serine protease
15926639 e,i Atl V Q A A autolysin
15926648 e SA0914 A D A T hypothetical protein
15926713 a IsdB A Q A A hypothetical protein
15926714 a IsdA V N A A cell surface protein
15926715 e,k IsdC A N A A hypothetical protein
15926738 SA1000 S H A Q hypothetical protein
15926741 Efb A D A S hypothetical protein
15926742 SA1004 A D A S hypothetical protein
15926749 SA1009 A K A Y hypothetical protein
15926750 SA1010 A K A Y hypothetical protein
15926751 SA1011 A K A Y hypothetical protein
15926830 i LytN A Y A D LytN protein
15927055 e GpsA V L A E glycerol-3-phosphate dehydrogenase
15927385 SplC A N A E serine protease SplC
15927456 e SA1698 S L A D hypothetical protein
15927483 e SspB2 A N A E staphopain, cysteine proteinase
15927498 e SAS056 A F A Y hypothetical protein
15927580 l SA1812 S Y A K hypothetical protein
15927581 SA1813 T Q A N hypothetical protein
15927607 e SA1839† V E A K hypothetical protein
15927670 e SA1898 A H A S hypothetical protein
15927785 SA2006 A S A D hypothetical protein
15927879 e SsaA A H A S hypothetical protein
15927884 e SA2097 A D A A hypothetical protein
15927890 e SA2103 V A A K hypothetical protein
15927996 Sbi A K A S IgG-binding protein Sbi
214

Appendix III Supplemental Table IIIb
PID Protein -3 -2 -1 +1 Function
15927997 HlgA S K A E γ-hemolysin chain II precursor
15927998 HlgC A K A A γ-hemolysin component C
15927999 HlgB A N A E γ-hemolysin component B
15928114 SA2323 A Y A H hypothetical protein
15928123 e SA2332 S H A A hypothetical protein
15928145 e SA2353 A Q A A hypothetical protein
15928148 e IsaA A H A A immunodominant antigen A
15928216 a ClfB A Q A S clumping factor B
15928223 e,m Aur A L A I zinc metalloproteinase aureolysin
15928224 e,n IsaB A Q A A immunodominant antigen B
15928225 SA2432 I Y A A hypothetical protein
15928230 e SA2437 A Y A D hypothetical protein
15928232 c,e SasF A Q A A conserved hypothetical protein
15928254 IcaB A N A D intercellular adhesion protein B
15928257 e Lip A Q A A triacylglycerol lipase precursor
Signal peptide predictions were performed with SignalP-NN and SignalP-HMM version 2.0 (Nielsen et al., 1997;
http://www.cbs.dtu.dk/services/SignalP-2.0/), PrediSi (Hiller et al, 2004; http://www.predisi.de/), Phobius (Kall et
al., 2004; http://phobius.cgb.ki.se/) and LipoP version 1.0 (Juncker et al., 2003; (http://www.cbs.dtu.dk /services
/LipoP/). These programs are designed to identify Sec-type signal peptides, amino-terminal membrane anchors
(Phobius), or lipoprotein signal peptides in Gram-negative bacteria (LipoP). The TMHMM-program version 2.0
(Cserzö et al., 1997; http://www.cbs.dtu.dk/services/TMHMM/) was used to identify transmembrane segments in
proteins.
a
Proteins with an LPxTG-motif
b
Protein homologue of B. subtilis is found in extracellular proteome(Tjalsma et al., 2004)
c
Proteins with an LPxAG-motif
d
Only the S. aureus NCTC 8325 protein is truncated at the C-terminus, thereby missing the LPxTG-motif
e
Proteins that are also present in S. epidermidis
f
Only the S. aureus Newman protein is truncated at the N-terminus, thereby missing the signal peptide
g
The S. aureus COL, NCTC8325, Newman, USA300 and USA300_TCHC1516 have a Gly on the -3 position,
which is not included in the recognition and cleavage pattern
h
The homologue in S. aureus RF122 contains a recognition and cleavage site, but the proteins in all other strains
contain a Gly on the -3 position, which is not included in the recognition and cleavage pattern
i
Proteins with a LysM or GW domain motif
j
Protein homologues of B. subtilis are classified as Sec-attached membrane protein (Tjalsma and van Dijl, 2005)
k
Proteins with an NPQTN-motif
l
Only the S. aureus COL protein is truncated at the N-terminus, thereby missing the signal peptide. All other S.
aureus strains conform the proposed pattern
m
Protein homologue of B. subtilis is classified as secretory protein (Tjalsma and van Dijl, 2005)
n
Only for the S. aureus MRSA252 protein; protein homologues in other strains are predicted to have two
transmembrane domains and were excluded from the list
215

Appendix III Supplemental Table IIId
Supplemental table IIId. The variant exoproteome of S. aureus; proteins with predicted
Sec type signal peptides present in at least one sequenced strain
PID Protein -3 -2 -1 +1 Function
15925799 Plc A H A S 1-phosphatidylinositol phosphodiesterase
precursor
15926099 a Set6 V Q A K exotoxin 6
15926100 a Set7 V H A E exotoxin 7
15926101 a Set8 V K A E exotoxin 8
15926102 a Set9 A N A T exotoxin 9
15926103 a Set10 V N A S exotoxin 10
15926104 a Set11 V H A K exotoxin 11
15926105 a Set12 V N A K exotoxin 12
15926111 a Set15 V K A S exotoxin 15
15926112 SA0394 A E A S hypothetical protein
15926240 b SdrD A K A A Ser-Asp rich fibrinogen-binding, bone
sialoprotein-binding protein; low Smax score
15926241 b SdrE A K A A Ser-Asp rich fibrinogen-binding, bone
sialoprotein-binding protein; low Smax score
15926465 SA0743 A S A V hypothetical protein
15926739 SA1001 A K A F hypothetical protein
15926746 Hly A N A A α-hemolysin precursor
15927016 c EbhB A H A A hypothetical protein
15927120 SA1370 I D A S hypothetical protein
15927308 b Fhs A Q A A hypothetical protein
15927384 d SplD A K A E serine protease SplD
15927386 SplB A K A E serine protease SplB
15927387 SplA A K A E serine protease SplA
15927389 SA1633 A K A E probable β-lactamase
15927393 LukD V D A A leukotoxin, LukD
15927394 e LukE S R A N leukotoxin, LukE
15927398 SEG V N A Q extracellular enterotoxin type G precursor
15927399 SEN V N A E enterotoxin SeN
15927402 SEI T Y A Q extracellular enterotoxin type I precursor
15927404 SEO A Y A N enterotoxin SeO
15927512 Map A S A A truncated Map-W protein
15927513 Hlb A K A E truncated β-hemolysin
15927516 SA1754 A Q A S hypothetical protein
15927517 SA1755 A K A F hypothetical protein
15927520 Sak V S A S staphylokinase precursor
15927522 SA1760 A K A I hypothetical protein
15927579 c,f SA1811 A K A E truncated β-hemolysin
15927585 SEC3 V L A E enterotoxin type C3
15927586 SA1818 A K A E hypothetical protein
15927587 TSST-1 A K A S toxic shock syndrome toxin-1
15927741 b,g FmtB A S A A FmtB protein
15928076 b,c,h SA2285 A E A A hypothetical protein
15928174 b,i SA2381 A N A E hypothetical protein
15928182 SA2389 V L A D hypothetical protein
16119203 j SAP003 A Y A N hypothetical protein
16119219 k SAP019 A E A A hypothetical protein
15923851 SAV0861 S D A I hypothetical protein
14141830 j SAVP008 A N A E hypothetical protein
21281780 SEH A K A E enterotoxin H
21282111 Set16 V Q A K hypothetical protein
21282116 Set21 V K A A hypothetical protein
21282123 Set26 V K A I hypothetical protein
216

Appendix III Supplemental Table IIIb
PID Protein -3 -2 -1 +1 Function
21283107 LukF V D A A Panton-Valentine leukocidin chain F precursor
21283108 LukS S K A D Panton-Valentine leukocidin chain S precursor
21283486 MW1757 A K A E hypothetical protein
21283490 EpiP A S A S epidermin leader peptide processing serine
protease EpiP
21283666 SEG2 A Y A D staphylococcal enterotoxin G
21283667 SEK A S A Q staphylococcal enterotoxin K
49482651 SAR0423 V H A E exotoxin
49482652 SAR0424 A N A E exotoxin
49482653 SAR0425 A N A E exotoxin
49482925 c SAR0721 T F A E multicopper oxidase protein
49484047 SAR1886 A Y A F putative exported protein
49484058 SplE A K A E serine protease
49484059 c SAR1905 A K A E serine protease
49484887 b Cna A L A A collagen adhesin precursor
49484898 SAR2788 A E A S putative exported protein
57651309 SACOL0468 V Q A K exotoxin 3, putative
57651319 SACOL0478 V K A S exotoxin 3, putative
57651320 SACOL0479 A E A S putative surface protein
57651597 SEB V L A E staphylococcal enterotoxin B
57652419 b Pls A E A A methicillin-resistant surface protein
87159841 pUSA010004 A Q A Q hypothetical protein
a
Although these proteins share homology with exotoxins from other S. aureus strains these proteins are highly
variable (Holtfreter et al., 2004)
b
Proteins with an LPxTG motif
c
Proteins that are also present in S. epidermidis
d
Only for the S. aureus COL protein; protein homologues in other strains are predicted to have two
transmembrane domains and were excluded from the list
e
Only the S. aureus NCTC 8325 protein is truncated in the N-terminus and therefore missing the signal peptide
f
Truncated in all S. aureus strains, except COL, RF122 and S. epidermidis, thereby missing the signal peptide
g
S. aureus NCTC8325 misses the LPxTG retention signal; annotated as two proteins in S. aureus USA300
h
The S. aureus Mu50, N315 and USA300 proteins are truncated in their C-termini, therefore missing the LPxTG
motif
i
Only the S. aureus MW2 protein is truncated in the N-terminus, therefore missing the signal peptide
j Protein encoding gene lies on a plasmid
k Protein encoding gene lies on a plasmid, except for S. aureus MRSA252
217

Appendix III Supplemental Table IIIe
Supplemental table IIIe. The core lipoproteome of S. aureus; proteins with predicted
lipoprotein signal peptides present in all sequenced strains
PID Gene -3 -2 -1 +1 +2 Function
15925800 SA0092 T A G C G hypothetical protein
15925819 SirA L A G C S lipoprotein
15925912 Slp L S G C G RGD-containing lipoprotein
15925918 SA0207 V T A C G hypothetical protein, similar to maltose/
maltodextrin-binding protein
15925928 a,b SA0217 L S S C A hypothetical protein, similar to periplasmic-ironbinding
protein BitC
15925940 c,d SA0229 L S G C G hypothetical protein, similar to nickel ABC
transporter nickel-binding protein
15926044 SA0331 I A A C G conserved hypothetical protein
15926073 SA0359 L T A C G conserved hypothetical protein
15926079 a SA0363 L T G C A hypothetical protein
15926141 a,e SA0422 L A A C G hypothetical protein, similar to lactococcal
lipoprotein
15926287 a,f SA0566 L S G C G hypothetical protein, similar to iron-binding
protein
15926308 a SA0587 V A A C G lipoprotein, Streptococcal adhesin PsaA
homologue
15926354 a SA0632 L T G C G conserved hypothetical protein
15926385 a,g SA0663 L G A C G hypothetical protein
15926413 a,h SA0691 L A A C G lipoprotein, similar to ferrichrome ABC
transporter
15926417 a SA0695 I S A C G hypothetical protein
15926461 SA0739 L G A C G conserved hypothetical protein
15926499 a,I SA0771 L A A C G conserved hypothetical protein
15926579 a SA0849 L S G C A hypothetical protein, similar to peptide binding
protein OppA
15926625 f SA0891 V A G C G hypothetical protein, similar to ferrichrome ABC
transporter
15926678 a SA0943 L A G C T conserved hypothetical protein
15926717 j IsdE L T S C Q hypothetical protein
15926796 a SA1056 V A G C S hypothetical protein
15926969 j SA1221 L G A C G thioredoxin reductase
15927111 a SA1361 L A G C G hypothetical protein
15927372 j SA1616 L S S C G hypothetical protein
15927373 k SA1617 L S A C S hypothetical protein
15927375 a,l SA1619 L T A C G hypothetical protein
15927415 a PrsA L G A C G peptidyl-prolyl cis/trans isomerase homolog
15927477 a SA1719 L A A C G conserved hypothetical protein
15927757 a,m SA1979 V A A C G hypothetical protein, similar toferrichrome ABC
transporter (binding protein)
15927859 a ModA L A G C S probable molybdate-binding protein
15927864 i SA2079 L A A C G hypothetical protein, similar to ferrichrome ABC
transporter FhuD precursor
15927948 a SA2158 L A A C G hypothetical protein, similar to TpgX protein
15927961 a,j SA2171 L I V C I hypothetical protein
15927984 a DsbA L A A C G DsbA; hypothetical protein, similar to Zn-binding
lipoprotein AdcA
15927987 c SA2197 L T A C G conserved hypothetical protein
15927988 c,f SA2198 I S G C G hypothetical protein
15927992 a,i SA2202 L A A C G hypothetical protein, similar to ABC transporter,
periplasmic amino acid-binding protein
218

Appendix III Supplemental Table IIIb
PID Gene -3 -2 -1 +1 +2 Function
15928025 a OpuCC L S G C S glycine betaine/carnitine/choline ABC transporter
OpuC
15928037 a SA2247 L S A C G conserved hypothetical protein
15928046 a Opp-1A L T G C G oligopeptide transporter putative substrate
binding domain
15928066 a,m SA2275 I G A C G hypothetical protein
15928267 j SA2473 L Y S C S hypothetical protein
a
Proteins that are also present in S. epidermidis
b
Excluded for S. aureus JH1, JH9, Mu3, Mu50 and N315 because of the motif proposed by Sutcliffe and
Harrington (2002)
c
Excluded for S. epidermidis because of the motif proposed by Sutcliffe and Harrington (2002)
d
The S. aureus JH1, JH9 and NCTC 8325 proteins are truncated at the N-terminus and thereby missing the signal
peptide
e
Contains the lipoprotein release motif (Tjalsma and van Dijl, 2005) with one amino acid changes, except for S.
aureus COL, NCTC 8325, Newman, USA300 and S. epidermidis
f
The S. aureus NCTC 8325 and USA300 proteins are truncated at the N-terminus and thereby missing the signal
peptide
g
Contains the lipoprotein release motif (Tjalsma and van Dijl, 2005) with one amino acid change for all
staphylococcal strains
h
Contains the exact lipoprotein release motif (Tjalsma and van Dijl, 2005), except in S. epidermidis
i
Contains the lipoprotein release motif (Tjalsma and van Dijl, 2005) with one amino acid change, except in S.
epidermidis
j
Excluded for all S. aureus strains because of the motif proposed by Sutcliffe and Harrington (2002)
k
Excluded for all S. aureus strains because of the motif proposed by Sutcliffe and Harrington (2002), except for S.
aureus MRSA252
l
Annotated as two proteins in the S. aureus USA300 strain
m
Contains the lipoprotein release motif (Tjalsma and van Dijl, 2005) with one amino acid change, only for S.
epidermidis
219

Appendix III Supplemental Table IIIf
Supplemental table IIIf. The variant lipoproteome of S. aureus; proteins with predicted
lipoprotein signal peptides present in at least one sequenced strain
PID Gene -3 -2 -1 +1 +2 Function
15925801 a SA0093 F A G C G hypothetical protein
15925802 SA0094 V A G C G hypothetical protein
15925803 SA0095 T A G C G hypothetical protein
15925804 SA0096 T A G C G hypothetical protein
15925847 b SA0138 A A A C G hypothetical protein, similar to
alkylphosphonate ABC tranporter
15925877 a SA0167 I T G C D hypothetical protein, similar to membrane
lipoprotein SrpL
15926004 SA0291 L A G C S hypothetical protein
15926114 c Lpl1 I A G C G hypothetical protein
15926115 a Lpl2 I I G C D hypothetical protein
15926116 Lpl3 I A G C G hypothetical protein
15926118 Lpl4 I I G C G hypothetical protein
15926119 Lpl5 V A G C G hypothetical protein
15926120 a Lpl6 I I G C D hypothetical protein
15926121 a Lpl7 I I G C N hypothetical protein
15926122 a Lpl8 A T S C G hypothetical protein
15926123 Lpl9 I G G C G hypothetical protein
15926465 a SA0743 G A L C V hypothetical protein
15926580 d SA0850 L S A C G hypothetical protein, similar to
oligopeptide ABC transporter
oligopeptide-binding protein
15927067 SA1317 L S G C S hypothetical protein
15927068 b SA1318 L S G C S hypothetical protein
15927069 SA1319 L S G C S hypothetical protein
15927071 e SA1321 L G G C S hypothetical protein
15927396 c SA1640 L V A C G conserved hypothetical protein
15928064 a SA2273 I G G C I hypothetical protein
16119210 b,f BlaZ L S A C N β-lactamase precursor
14141829 a SAVP006 L V S C N hypothetical protein
15923793 SAV0803 L T A C S hypothetical protein
15924991 b SAV2001 L S A C G hypothetical protein
21281801 MW0072 T A G C G Staphylococcus tandem lipoprotein
21282126 Lpl10 I A G C G hypothetical protein
21282127 a Lpl11 V T S C G hypothetical protein
21282129 a Lpl13 I I G C D hypothetical protein
21283103 MW1374 L S G C S conserved hypothetical protein
21283167 MW1438 L T A C G hypothetical protein
21283173 g MW1444 L G G C S hypothetical protein
21284135 MW2406 I G A C G hypothetical protein
21284306 MW2577 V S G C S hypothetical protein
49482670 SAR0445 I G G C G putative lipoprotein
49483474 SAR1288 L S A C G putative lipoprotein
49483672 SAR1494 L S G C S hypothetical protein
49484287 a SAR2149 L I V C G hypothetical protein
49484977 b,h SAS0074 I G G C G putative lipoprotein
57650161 SACOL0888 L G A C G pathogenicity island, putative lipoprotein
57650444 SACOL1531 L S G C S hypothetical protein
57650485 SACOL1574 L S A C G hypothetical protein
57650996 a SACOL2497 I G G C V staphylococcus tandem lipoprotein
57651323 a SACOL0482 I M G C D staphylococcus tandem lipoprotein
57651324 SACOL0483 M A G C E staphylococcus tandem lipoprotein
57651327 i SACOL0486 I G G C G staphylococcus tandem lipoprotein
220

Appendix III Supplemental Table IIIb
PID Gene -3 -2 -1 +1 +2 Function
57652445 SACOL0081 T A G C G hypothetical protein
150392787 SaurJH1_0313 L S A C G hypothetical protein
150393510 SaurJH1_1042 L G A C G hypothetical protein
87159856 pUSA03_0017 L A G C G transfer complex protein TraH
87160691 a SAUSA300_0411 I G G C D staphylococcus tandem lipoprotein
87160733 b,j SAUSA300_0079 L S A C S putative lipoprotein
87161538 SAUSA300_0073 L G A C G peptide ABC transporter, peptide-binding
protein
151220617 a NWMN_0405 I G G C D truncated staphylococcal tandem
lipoprotein
a
Excluded for all S. aureus strains because of the motif proposed by Sutcliffe and Harrington (2002)
b
Proteins that are also present in S. epidermidis
c
Excluded for S. aureus COL, Mu3, Mu50, N315, NCTC 8325, Newman and USA300 because of the motif
proposed by Sutcliffe and Harrington (2002)
d
Contains the lipoprotein release motif (Tjalsma and van Dijl, 2005) with one amino acid change
e
The S. aureus COL, Mu3, Mu50, N315, NCTC 8325, Newman and USA300 proteins are truncated at the Nterminus,
thereby missing the signal peptide
f
The S. aureus JH1, JH9, MSSA476 and N315 proteins are encoded by genes that lie on a plasmid
g
The S. aureus COL, NCTC 8325 and USA300 proteins are truncated at the N-terminus, thereby missing the
signal peptide
h
Contains the lipoprotein release motif for S. epidermidis (Tjalsma and van Dijl, 2005) with one amino acid
change
i
The S. aureus NCTC 8325 protein is truncated at the N-terminus, thereby missing the signal peptide
j
The S. epidermidis ATCC 12228 protein is truncated at the N-terminus, thereby missing the signal peptide
221

Appendix III Supplemental Table IIIg
Supplemental table IIIg. Composition of the variant exoproteome of sequenced S. aureus strains
PID Protein Function COL JH1 JH9 MRSA 252 MSSA 476 Mu3 Mu50
15925799 Plc 1-phosphatidylinositol phosphodiesterase precursor Y Y Y Y N Y Y
15926099 Set6 exotoxin 6 N Y Y N N Y Y
15926100 Set7 exotoxin 7 Y Y Y N Y Y Y
15926101 Set8 exotoxin 8 N Y Y N Y Y Y
15926102 Set9 exotoxin 9 Y Y Y N Y Y N
15926103 Set10 exotoxin 10 N Y Y Y Y Y Y
15926104 Set11 exotoxin 11 N Y Y Y Y Y Y
15926105 Set12 exotoxin 12 N Y Y N Y Y Y
15926111 Set15 exotoxin 15 N Y Y N N Y Y
15926112 SA0394 hypothetical protein N Y Y Y Y Y Y
15926465 SA0743 hypothetical protein Y Y Y N Y Y Y
15926739 SA1001 hypothetical protein Y Y Y N Y Y Y
15926746 HlY α-hemolysin precursor Y Y Y N Y Y Y
15927016 EbhB hypothetical protein Y Y Y Y N Y Y
15927120 SA1370 hypothetical protein N Y Y Y Y Y Y
15927308 Fhs hypothetical protein Y Y Y N Y Y Y
15927384 SplD serine protease SplD Y N N N N Y Y
15927386 SplB serine protease SplB Y Y Y N Y Y Y
15927387 SplA serine protease SplA Y Y Y N Y Y Y
15927389 SA1633 probable β-lactamase N Y Y N N Y Y
15927393 LukD leukotoxin, LukD Y Y Y N Y Y Y
15927394 LukE leukotoxin, LukE Y Y Y N Y Y Y
15927398 SEG extracellular enterotoxin type G precursor N Y Y Y N Y Y
15927399 SEN enterotoxin SEN N Y Y Y N Y Y
15927402 SEI extracellular enterotoxin type I precursor N Y Y Y N Y Y
15927404 SEO enterotoxin SEO N Y Y Y N Y Y
15927512 Map truncated map-w protein Y N Y Y N Y Y
15927513 Hlb β-hemolysin/ phospholipase C Y Y Y N N Y Y
15927516 SA1754 hypothetical protein N Y Y Y Y Y Y
15927517 SA1755 hypothetical protein N Y Y Y N N N
15927520 Sak staphylokinase precursor N Y Y Y Y Y Y
15927522 SA1760 hypothetical protein N Y Y Y Y Y Y
15927579 SA1811 phospholipase C Y Y Y N N Y Y
15927585 SEC3 enterotoxin typeC3 Y N N Y N Y Y
15927586 SA1818 hypothetical protein Y Y Y N N Y Y
222

Appendix III Supplemental Table IIIg
PID Protein Function COL JH1 JH9 MRSA 252 MSSA 476 Mu3 Mu50
15927587 TSST-1 toxic shock syndrome toxin-1 N N N N N Y Y
15927741 FmtB FmtB protein Y Y Y N N Y Y
15928076 SA228 hypothetical protein Y Y Y N Y Y Y
15928174 SA2381 hypothetical protein N Y Y N N Y Y
15928182 SA2389 hypothetical protein N Y Y N N Y Y
16119203 SAP003 hypothetical protein N Y Y N N N N
16119219 SAP019 hypothetical protein N N N Y Y N N
15923851 SAV0861 hypothetical protein N N N N N Y Y
14141830 SAVP008 hypothetical protein N N N N N N Y
21281780 SEH enterotoxin H N N N N Y N N
21282111 Set16 hypothetical protein N N N Y Y N N
21282116 Set21 hypothetical protein N N N N Y N N
21282123 Set26 hypothetical protein N N N Y Y N N
21283107 LukF Panton-Valentine leukocidin chain F precursor N N N N N N N
21283108 LukS Panton-Valentine leukocidin chain S precursor N N N N N N N
21283486 MW1757 hypothetical protein Y N N N Y N N
21283490 EpiP epidermin leader peptide processing serine protease EpiP Y N N N Y N N
21283666 SEG2 staphylococcal enterotoxin G Y N N N Y N N
21283667 SEK staphylococcal enterotoxin K Y N N N Y N N
21284341 Cna collagen adhesin precursor N N N Y Y N N
49482651 SAR0423 exotoxin N N N Y N N N
49482652 SAR0424 exotoxin N N N Y N N N
49482653 SAR0425 exotoxin N N N Y N N N
49482925 SAR0721 multicopper oxidase protein N N N Y N N N
49483328 SAR1139 exotoxin N N N Y N N N
49484047 SAR1886 putative exported protein N N N Y N N N
49484058 SplE serine protease Y N N Y N N N
49484059 SAR1905 serine protease N N N Y N N N
49484898 SAR2788 putative exported protein N N N Y N N N
57651309 SACOL0468 exotoxin 3, putative Y N N N N N N
57651319 SACOL0478 exotoxin 3, putative Y N N N N N N
57651320 SACOL0479 hypothetical protein Y N N N N N N
57651597 SEB staphylococcal enterotoxin B Y N N N N N N
57652419 Pls methicillin-resistant surface protein Y N N N N N N
87159841 pUSA010004 hypothetical protein N N N N N N N
223

Appendix III Supplemental Table IIIg
Supplemental Table IIIg. Continued
PID Protein Function MW2 N315 NCTC 8325 Newman USA300 USA300 TCHC1516
15925799 Plc 1-phosphatidylinositol phosphodiesterase precursor Y Y Y Y Y Y
15926099 Set6 exotoxin 6 N Y N N N N
15926100 Set7 exotoxin 7 Y Y Y Y Y Y
15926101 Set8 exotoxin 8 Y Y Y Y Y Y
15926103 Set10 exotoxin 10 Y Y Y Y Y Y
15926104 Set11 exotoxin 11 Y Y Y Y Y Y
15926105 Set12 exotoxin 12 Y Y Y Y Y Y
15926106 Set13 exotoxin 13 Y Y N N N N
15926111 Set15 exotoxin 15 N Y N N N N
15926465 SA0743 hypothetical protein Y Y Y Y Y Y
15926739 SA1001 hypothetical protein Y Y Y Y Y Y
15926746 HlY α-hemolysin precursor Y Y Y Y Y Y
15927016 EbhB hypothetical protein Y Y Y Y Y Y
15927120 SA1370 hypothetical protein Y Y Y Y N Y
15927308 Fhs hypothetical protein Y Y Y Y Y Y
15927384 SplD serine protease SplD N Y Y Y Y Y
15927386 SplB serine protease SplB Y Y Y Y Y Y
15927387 SplA serine protease SplA Y Y Y Y Y Y
15927389 SA1633 probable β-lactamase N Y N N N N
15927393 LukD leukotoxin, LukD Y Y Y Y Y Y
15927394 LukE leukotoxin, LukE Y Y Y Y Y Y
15927398 SEG extracellular enterotoxin type G precursor N Y N N N N
15927399 SEN enterotoxin SEN N Y N N N N
15927402 SEI extracellular enterotoxin type I precursor N Y N N N N
15927404 SEO enterotoxin SEO N Y N N N N
15927512 Map truncated map-w protein Y Y Y Y N Y
15927513 Hlb β-hemolysin/ phospholipase C Y Y Y Y Y Y
15927516 SA1754 hypothetical protein Y Y Y Y Y Y
15927517 SA1755 hypothetical protein N Y Y Y Y Y
15927520 Sak staphylokinase precursor Y Y Y Y Y Y
15927522 SA1760 hypothetical protein Y Y Y Y Y Y
15927579 SA1811 phospholipase C Y Y Y Y Y Y
15927585 SEC3 enterotoxin typeC3 Y Y N N N N
15927586 SA1818 hypothetical protein Y Y N N Y Y
15927587 TSST-1 toxic shock syndrome toxin-1 N Y N N N N
224

Appendix III Supplemental Table IIIg
PID Protein Function MW2 N315 NCTC 8325 Newman USA300 USA300 TCHC1516
15927741 FmtB FmtB protein Y Y Y Y Y N
15928076 SA228 hypothetical protein Y Y Y Y Y N
15928174 SA2381 hypothetical protein Y Y N N N N
15928182 SA2389 hypothetical protein N Y N N N N
16119203 SAP003 hypothetical protein N Y N N N N
16119219 SAP019 hypothetical protein N Y N N N N
15923851 SAV0861 hypothetical protein N N N N N N
14141830 SAVP008 hypothetical protein N N N N N N
21281780 SEH enterotoxin H Y N N N N N
21282111 Set16 hypothetical protein Y N N N N N
21282116 Set21 hypothetical protein Y N Y Y Y Y
21282123 Set26 hypothetical protein Y N N N N N
21283107 LukF Panton-Valentine leukocidin chain F precursor Y N N N Y Y
21283108 LukS Panton-Valentine leukocidin chain S precursor Y N N N Y Y
21283486 MW1757 hypothetical protein Y N Y Y Y Y
21283490 EpiP epidermin leader peptide processing serine protease
EpiP
Y N Y Y Y Y
21282666 SEG2 staphylococcal enterotoxin G Y N N N Y Y
21282667 SEK staphylococcal enterotoxin K Y N N N Y Y
21284341 Cna collagen adhesin precursor Y N N N N N
49482651 SAR0423 exotoxin N N N N N N
49482652 SAR0424 exotoxin N N N N N N
49482653 SAR0425 exotoxin N N N N N N
49482925 SAR0721 multicopper oxidase protein N N N N N N
49484047 SAR1886 putative exported protein N N N N N N
49484058 SplE serine protease N N Y Y Y Y
49484059 SAR1905 serine protease N N N N N N
49484898 SAR2788 putative exported protein N N N N N N
57651309 SACOL0468 exotoxin 3, putative N N Y Y Y Y
57651319 SACOL0478 exotoxin 3, putative N N Y Y Y Y
57651597 SEB staphylococcal enterotoxin B N N N N N N
57652419 Pls methicillin-resistant surface protein N N N N N N
87159841 pUSA010004 hypothetical protein N N N N Y N
225

Appendix III Supplemental Table IIIh
Supplemental table IIIh. Composition of the variant lipoproteome of sequenced S. aureus strains
PID Protein Function COL JH1 JH9 MRSA252 MSSA476 Mu3 Mu50
15925801 SA0093 hypothetical protein N Y Y N N Y Y
15925802 SA0094 hypothetical protein N Y Y N N Y Y
15925803 SA0095 hypothetical protein N Y Y N N Y Y
15925804 SA0096 hypothetical protein Y Y Y N N Y Y
15925847 SA0138 hypothetical protein, similar to alkylphosphonate ABC
tranporter
Y Y Y Y Y Y Y
15925877 SA0167 hypothetical protein N Y Y Y Y Y Y
15926004 SA0291 hypothetical protein Y Y Y N Y Y Y
15926114 Lpl1 hypothetical protein Y Y Y Y N Y Y
19526115 Lpl2 hypothetical protein N Y Y N N Y Y
15926116 Lpl3 hypothetical protein Y Y Y N N Y Y
15926118 Lpl4 hypothetical protein N Y Y N N Y Y
15926119 Lpl5 hypothetical protein N Y Y N N Y Y
15926120 Lpl6 hypothetical protein N Y Y N Y Y Y
15926122 Lpl8 hypothetical protein Y Y Y Y N Y Y
15926123 Lpl9 hypothetical protein Y Y Y Y Y Y Y
15926465 SA0743 hypothetical protein, similar to staphylocoagulase precursor Y Y Y N Y Y Y
15926580 SA0850 hypothetical protein, similar to oligopeptide ABC transporter
oligopeptide-binding protein
Y Y Y N Y Y Y
15927067 SA1317 hypothetical protein N Y Y N N Y Y
15927068 SA1318 hypothetical protein N Y Y N Y Y Y
15927069 SA1319 hypothetical protein N Y Y Y Y Y Y
15927071 SA1321 hypothetical protein Y Y Y N N Y Y
15927396 SA1640 conserved hypothetical protein N Y Y N N Y Y
15928064 SA2273 hypothetical protein Y Y Y N N Y Y
16119210 BlaZ β-lactamase precursor N Y Y Y Y N N
14141829 SAVP006 hypothetical protein N N N N N N Y
15923793 SAV0803 hypothetical protein Y N N N Y Y Y
15924491 SAV2001 hypothetical protein N N N Y N Y Y
21281801 MW0072 hypothetical protein Y N N N Y N N
21282126 Lpl10 hypothetical protein N N N Y Y N N
21282127 Lpl11 hypothetical protein N N N Y Y N N
21282129 Lpl13 hypothetical protein N N N N Y N N
226

Appendix III Supplemental Table IIIh
PID Protein Function COL JH1 JH9 MRSA252 MSSA476 Mu3 Mu50
21283103 MW1374 conserved hypothetical protein N N N N Y N N
21283167 MW1438 hypothetical protein N N N Y Y N N
21283173 MW1444 hypothetical protein Y N N N Y N N
21284135 MW2406 hypothetical protein N N N N Y N N
21284306 MW2577 hypothetical protein N N N N Y N N
49482670 SAR0445 putative lipoprotein N N N Y N N N
49483474 SAR1288 putative lipoprotein N N N Y N N N
49483672 SAR1494 hypothetical protein Y N N Y N N N
49484287 SAR2149 putative exported protein N N N Y N N N
49484977 SAS0074 putative lipoprotein N N N N Y N N
57650161 SACOL0888 pathogenicity island, putative lipoprotein Y N N N N N N
57650444 SACOL1531 hypothetical protein Y N N N N N N
57650485 SACOL1574 hypothetical protein SA1574 Y N N Y N N N
57650996 SACOL2497 staphylococcus tandem lipoprotein Y N N N N N N
57651323 SACOL0482 Staphylococcus tandem lipoprotein Y N N N N N N
57651324 SACOL0483 staphylococcus tandem lipoprotein Y N N N N N N
57651327 SACOL0486 staphylococcus tandem lipoprotein Y N N N N N N
57652445 SACOL0081 hypothetical protein SA0081 Y N N N N N N
150392787 SaurJH1_0313 hypothetical protein N Y Y N N N N
150393510 SaurJH1_1042 hypothetical protein N Y Y N N N N
87159856 pUSA03_0017 transfer complex protein TraH N N N N N N N
87160691 SAUSA300_0411 staphylococcus tandem lipoprotein N N N N N N N
87160733 SAUSA300_0079 putative lipoprotein N N N N N N N
87161538 SAUSA300_0073 peptide ABC transporter, peptide-binding protein N N N N N N N
151220617 NWMN_0405 truncated staphylococcal tandem lipoprotein N N N N N N N
227

Appendix III Supplemental Table IIIh
Supplemental Table IIIh. Continued
PID Protein Function MW2 N315 NCTC 8325 Newman USA300 USA300 TCHC1516
15925801 SA0093 hypothetical protein N Y N N N N
15925802 SA0094 hypothetical protein N Y N N N N
15925803 SA0095 hypothetical protein N Y Y Y Y Y
15925804 SA0096 hypothetical protein N Y Y Y Y Y
15925847 SA0138 hypothetical protein, similar to
alkylphosphonate ABC tranporter
Y Y Y N Y Y
15925877 SA0167 hypothetical protein Y Y Y Y Y Y
15926004 SA0291 hypothetical protein Y Y Y Y Y Y
15926114 Lpl1 hypothetical protein N Y Y Y Y Y
19526115 Lpl2 hypothetical protein N Y N N N N
15926116 Lpl3 hypothetical protein N Y N N N N
15926118 Lpl4 hypothetical protein N Y N N N N
15926119 Lpl5 hypothetical protein N Y N N N N
15926120 Lpl6 hypothetical protein Y Y N N N N
15926122 Lpl8 hypothetical protein N Y Y Y Y Y
15926123 Lpl9 hypothetical protein N Y N Y Y Y
15926465 SA0743 hypothetical protein, similar to
staphylocoagulase precursor
Y Y Y Y Y Y
15926580 SA0850 hypothetical protein, similar to
oligopeptide ABC transporter
oligopeptide-binding protein
Y Y Y Y Y Y
15927067 SA1317 hypothetical protein N Y N N N N
15927068 SA1318 hypothetical protein Y Y N N N N
15927069 SA1319 hypothetical protein Y Y N N N N
15927071 SA1321 hypothetical protein N Y Y Y Y Y
15927396 SA1640 conserved hypothetical protein N Y N N N N
15928064 SA2273 hypothetical protein N Y N N N N
16119210 BlaZ β-lactamase precursor N Y N N N Y
14141829 SAVP006 hypothetical protein N N N N N N
15923793 SAV0803 hypothetical protein Y N Y Y Y Y
15924491 SAV2001 hypothetical protein N N N Y N N
21281801 MW0072 hypothetical protein Y N Y Y Y Y
228

Appendix III Supplemental Table IIIh
PID Protein Function MW2 N315 NCTC 8325 Newman USA300 USA300 TCHC1516
21282126 Lpl10 hypothetical protein Y N N N N N
21282127 Lpl11 hypothetical protein Y N N Y Y Y
21282129 Lpl13 hypothetical protein Y N N N N N
21283103 MW1374 conserved hypothetical protein Y N N N N N
21283167 MW1438 hypothetical protein Y N N N Y Y
21283173 MW1444 hypothetical protein Y N Y Y Y Y
21284135 MW2406 hypothetical protein Y N N N N N
21284306 MW2577 hypothetical protein Y N N N N N
49482670 SAR0445 putative lipoprotein N N N N N N
49483474 SAR1288 putative lipoprotein N N N N Y Y
49483672 SAR1494 hypothetical protein N N Y Y Y Y
49484287 SAR2149 putative exported protein N N N N N N
49484977 SAS0074 putative lipoprotein N N N N N N
57650161 SACOL0888 pathogenicity island, putative lipoprotein N N N N N N
57650444 SACOL1531 hypothetical protein N N Y Y Y Y
57650485 SACOL1574 hypothetical protein SA1574 N N N N N N
57650996 SACOL2497 staphylococcus tandem lipoprotein N N Y Y Y Y
57651323 SACOL0482 Staphylococcus tandem lipoprotein N N N Y Y Y
57651324 SACOL0483 staphylococcus tandem lipoprotein N N N Y Y Y
57651327 SACOL0486 staphylococcus tandem lipoprotein N N Y Y Y Y
57652445 SACOL0081 hypothetical protein SA0081 N N N N N N
150392787 SaurJH1_0313 hypothetical protein N N N N N N
150393510 SaurJH1_1042 hypothetical protein N N N N N N
87159856 pUSA03_0017 transfer complex protein TraH N N N N Y N
87160691 SAUSA300_0411 staphylococcus tandem lipoprotein N N N Y Y Y
87160733 SAUSA300_0079 putative lipoprotein N N N N Y Y
87161538 SAUSA300_0073 peptide ABC transporter, peptide-binding
protein
N N N N Y Y
151220617 NWMN_0405 truncated staphylococcal tandem
lipoprotein
N N N Y N N
229

Appendix III Supplemental Table IIIi
Supplemental table IIIi. Specific S. epidermidis proteins with a predicted signal peptide
Sec type signal peptide
PID Protein -3 -2 -1 +1 Function
27467163 SE0245 V H A A triacylglycerol lipase precursor
27467176 SE0258 A K A Q immunodominant antigen B
27467249 a,b SE0331 A K A E Ser-Asp rich fibrinogen-binding, bone sialoprotein-binding protein
27467501 c SE0583 S F A N hypothetical protein
27467545 c SE0627 T L A D poly D-alanine transfer protein
27467746 a SE0828 A H A E lipoprotein VsaC
27467938 c SE1020 I K A Q hypothetical protein
27468418 a SE1500 S Y A Q hypothetical protein
27468493 SE1575 A Q A H immunodominant antigen B
27468546 a SE1628 V Y A D hypothetical protein
27468838 SE1920 V Y A Q hypothetical protein
27468875 SE1957 A H A T copper export proteins
27469060 c SE2142 T L A F 2-dehydropantoate 2-reductase
27469070 a SE2152 T H A A hypothetical protein
27469115 c SE2197 S Y A S alkaline phosphatase III precursor
27469119 SE2201 A D A Z phage-related protein
27469291 SE2373 A Q A S 1,4-β-N-acetylmuramidase
27469316 SE2398 S S A S hypothetical protein
32470521 P601 A S A S hypothetical protein
32470527 P607 I N A D hypothetical protein
32470549 a P517 A K A E hypothetical protein
32470583 P202 T F A L hypothetical protein
Lipoprotein signal peptide
PID Protein -3 -2 -1 +1 +2 Function
27466952 SE0034 L S A C S hypothetical protein
27467000 SE0082 L T A C G hypothetical protein
27467062 SE0144 V S G C G hypothetical protein
27467063 SE0145 V S G C G hypothetical protein
27467067 d SE0149 L A G C D hypothetical protein
27467309 d SE0391 L T T C S hypothetical protein
27468024 SE1106 V T A C S ABC transporter
27468425 d SE1507 L Y G C G hypothetical protein
27469012 d SE2094 L I I C S hypothetical protein
27469069 d SE2151 L A G C G hypothetical protein
27469130 SE2212 V S G C S hypothetical protein
27469141 d SE2223 L G S C S hypothetical protein
27469317 SE2399 L S A C G hypothetical protein
32470551 SE_p519 L A G C S hypothetical protein
a Proteins with an LPxTG-motif
b This protein has a lower SignalP score than our threshold score for all S. aureus strains
c All S. aureus strains have one or more residues in the cleavage site, which are not included in the search pattern
d Excluded for S. epidermidis because of the motif proposed by Sutcliffe and Harrington (2002)
230

Appendix III Supplemental Table IIIj
Supplemental table IIIj. Proteins with predicted Sec type signal peptides present in S.
aureus RF122
PID Protein -3 -2 -1 +1 Function
82749800 a,b SAB0023 A R A E 5' nucleotidase
82749803 c SAB0026 A H A S enterotoxin protein
82749815 Plc S L A I 1-phosphatidylinositol phosphodiesterase
82749847 a,d SasD A D A I surface protein
82749858 a SAB0085 A D A A hypothetical protein
82749938 a Coa A Q A S staphylocoagulase precursor
82749981 a LytM A K A S peptidoglycan hydrolase
82750007 a,e SAB0244 A I A K hypothetical protein
82750020 a Geh V L A E glycerol ester hydrolase
82750121 TSST-1 V Q A K toxic shock syndrome toxin-1
82750124 SEC3 V H A E staphylococcal enterotoxin C-bovine
82750136 Set6 V K A E superantigen-like protein
82750137 Set7 V N A S superantigen-like protein
82750138 Set8 V Q A K superantigen-like protein
82750139 Set10 V N A K superantigen-like protein 5
82750140 Set11 V H A K superantigen-like protein 7
82750141 Set12 S H A K superantigen-like protein
82750142 a Set13 V K A D superantigen-like protein
82750143 a Set14 A E A S superantigen-like protein
82750145 Set26 A D A K superantigen-like protein
82750146 SAB0387 A E A A hypothetical protein
82750147 a SAB0388c A Q A A hypothetical protein
82750172 a,f Aaa A Q A S autolysin
82750320 a SAB0566 A L A K hypothetical protein
82750345 a Pbp4 A N A E penicillin binding protein 4
82750367 a SAB0614c A S A V secretory antigen SsaA-like protein
82750398 a SAB0645 A N A S hypothetical protein
82750460 a,f SAB0708 A N A L hypothetical protein
82750491 c SAB0739 A N A S hypothetical protein
82750496 a,b,g ClfA A D A S truncated clumping factor
82750497 SAB0745 A K A I secreted von Willebrand factor-binding protein precursor
82750498 a Ssp T N A E extracellular matrix and plasma binding protein precursor
82750499 a SAB0747 V D A A hypothetical protein
82750500 a Nuc A G A E staphylococcal thermonuclease precursor
82750530 SAB0780 V S A A phage-associated holin
82750532 LukS A K A D leukocidin chain lukM precursor
82750533 LukF A N A L Panton-Valentine leukocidin LukF-PV chain precursor
82750575 a GlpQ V Q A A glycerophosphoryl diester phosphodiesterase
82750593 a SAB0846 A D A T hypothetical protein
82750659 a SspB S H A Q cysteine protease precursor
82750660 a SspA A K A F glutamyl endopeptidase serine protease
82750663 a,f Atl A D A S autolysin
82750672 a SAB0928c A D A S hypothetical protein
82750736 a,b IsdB A N A A iron-regulated cell wall-anchored protein
82750737 a,b IsdA A K A Y cell surface transferrin-binding protein
82750738 a,h IsbC A K A Y iron-regulated cell surface protein
82750761 a SAB1018 A K A Y hypothetical protein
82750762 SAB1019c A H A A formyl peptide receptor-like 1 inhibitory protein
82750764 a Efb V L A E fibrinogen-binding protein
82750765 a SAB1022 I D A S fibrinogen-binding protein precursor
82750770 Hly A Q A A α-hemolysin precursor
82750773 a SAB1030c A N A E superantigen-like protein
82750774 a SAB1031c A K A D superantigen-like protein
82750775 a SAB1032c A S A S superantigen-like protein
231

Appendix III Supplemental Table IIIj
PID Protein -3 -2 -1 +1 Function
82751035 EbhB V D A A truncated cell surface fibronectin-binding protein
82751071 a GpsA S R A N glycerol-3-phosphate dehydrogenase
82751144 SAB1412c V N A E hypothetical protein
82751320 i Fhs S Y A Q surface-anchored iron-regulated surface protein
82751393 j SplE D K A E serine proteinase
82751394 a SplC A Y A N serine proteinase
82751395 k SplB A K A E serine proteinase
82751397 SAB1675 S L A D hypothetical protein
82751401 EpiP A N A E serine protease precursor
82751408 LukD A F A Y leukotoxin D subunit
82751409 LukE A S A A leukotoxin E subunit
82751419 SEN A K A E enterotoxin N
82751421 SEI S Y A K enterotoxin I
82751422 SEO T Q A N enterotoxin O
82751535 a SAB1814 V E A K hypothetical protein
82751566 a SspB2 A H A S staphopain cysteine proteinase
82751581 a SAB1860c A S A D hypothetical protein
82751594 Map A H A S cell surface protein
82751595 SAB1874 A D A A β-hemolysin
82751596 a SAB1875c V A A K leukocidin F subunit
82751597 a SAB1876c A K A S leukocidin S subunit
82751634 a SAB1916c S K A E membrane anchored Ser-Asp rich fibrinogen-binding protein
82751697 a SAB1980c A K A A hypothetical protein
82751757 b FmtB A N A E truncated methicillin resistance-related surface protein
82751802 a SAB2085 A Y A H hypothetical protein
82751888 a SsaA S H A A secretory antigen precursor
82751892 a SAB2176 V K A K exported secretory antigen precursor
82751903 a SAB2187c A Q A A transcriptional regulator
82752013 a Sbi A H A A immunoglobulin G-binding protein
82752015 a HlgA A L A I γ-hemolysin component A precursor
82752016 a HlgC I Y A A γ-hemolysin component C
82752017 a HlgB A Y A D γ-hemolysin component B
82752118 a SAB2409c A N A D hypothetical protein
82752126 a SAB2418 A Q A T secretory antigen SsaA-like protein
82752129 c SAB2421c A N A A hypothetical protein
82752147 a SAB2439c A H A Q secretory antigen precursor
82752151 a IsaA S N A A immunodominant antigen A
82752211 a,b ClfB A Q A A clumping factor B
82752218 a Aur V N A A zinc metalloproteinase aureolysin
82752220 a SAB2514 A S A A hypothetical protein
82752225 a SAB2519 A Q A S N-acetylmuramoyl-L-alanine amidase
82752227 a,c SasF A Q A A surface anchored protein
82752246 a IcaB A H A D intercellular adhesion protein B
82752249 a Lip A N A A triacylglycerol lipase precursor
a
Proteins belonging to the S. aureus core proteome
b
Proteins with an LPxTG-motif
c
Proteins that are only present in S. aureus RF122
d
Proteins with an LPxAG-motif
e
The S. aureus RF122 protein has an Ile on the -2 position, which is not included in the recognition and
cleavage pattern; all other S. aureus strains are conform the proposed pattern
f
Proteins with a LysM or GW domain motif
g
Only the S. aureus RF122 protein is truncated at the N-terminus, thereby missing part of the signal peptide
h
The S. aureus RF122 protein is truncated at the C-terminus, therefor missing the LPxTG motif
i
The S. aureus RF122 protein has an Asp on the -3 position , which is not included in the
j
The S. aureus RF122 protein has an Asp on the -3 position , which is not included in the recognition and
cleavage pattern; the MRSA252 protein is conform the proposed pattern
k
Only the S. aureus RF122 protein is predicted to have two membrane domains
232

Appendix III Supplemental Table IIIj
Supplemental table IVa. Characteristics of the clinical S. aureus isolates used in this study
Patient Age Isolate Related Acquired
isolates
b Date of Origin Infection / Antibiotic resistance others MLST CC agrisolation
Colonization
Type
1 2 A B398 C 04.07.2003 umbilicus phlegmone ciprofloxacin, erythromycin, MSSA, pvl- 398 398 1
penicillin
positive
2 35 B B398 C 17.06.2003 pleural pneumonia ciprofloxacin, erythromycin, caMRSA, 398 398 1
fluid
oxacillin, penicillin
pvl-positive
3 76 C G228 a H 23.01.2004 nose colonization ciprofloxacin, erythromycin,
gentamicin, oxacillin, penicillin
MRSA 228 5 2
D G228 a H 23.12.2004 nose colonization amoxicillin-clavulanate,
ciprofloxacin, erythromycin,
gentamicin, oxacillin, penicillin
MRSA 228 5 2
E E8 a H 13.11.2003 feet chronic wound ciprofloxacin, co-trimoxazole, MSSA 8 8 1
infection
penicillin
4 62 F E8 a H 16.12.2003 blood postoperative wound ciprofloxacin, co-trimoxazole, MSSA 8 8 1
infection, sepsis erythromycin, penicillin
G G228 a H 17.12.2003 blood postoperative wound amoxicillin-clavulanate,
MRSA 228 5 2
infection, sepsis ciprofloxacin, erythromycin,
gentamicin, oxacillin, penicillin
5 74 H G228 a H 06.01.2004 blood postoperative wound amoxicillin-clavulanate,
MRSA 228 5 2
infection, sepsis ciprofloxacin, co-trimoxazole,
erythromycin, gentamicin, oxacillin,
penicillin,
I G228 a H 12.01.2005 perineum colonization ciprofloxacin, erythromycin,
gentamicin, oxacillin, penicillin
MRSA 228 5 2
6 62 J G228 a H 12.01.2004 perineum colonization amoxicillin-clavulanate,
ciprofloxacin, erythromycin,
gentamicin, oxacillin, penicillin,
rifampin
MRSA 228 5 2
K G228 a H 28.09.2004 feet chronic wound amoxicillin-clavulanate,
MRSA 228 5 2
infection
ciprofloxacin, erythromycin,
gentamicin, oxacillin, penicillin
7 32 L L80 a C 13.04.2002 buttock abscess oxacillin, penicillin, tetracycline caMRSA,
pvl-positive
80 3
8 2 M L80 a C 13.04.2004 finger panaritium oxacillin, penicillin, tetracycline caMRSA,
pvl-positive
80 3
9 57 N C 06.01.2004 blood pneumonia, sepsis no resistance MSSA 15 15 2
10 65 O C 09.02.2004 blood pneumonia, sepsis ciprofloxacin, erythromycin,
penicillin
233
MSSA 5 5 2

Appendix IV Supplemental Table IVa
Patient Age Isolate Related Acquired
isolates
b Date of Origin Infection / Antibiotic resistance others MLST CC agrisolation
Colonization
Type
11 72 P C 29.09.2004 blood sepsis Penicillin MSSA 7 1
12 47 Q C 15.01.2004 blood sepsis, arthritis Penicillin MSSA 30 30 3
13 47 R C 16.07.2005 blood sepsis penicillin, tetracyclin MSSA 15 15 2
14 22 S C 03.07.2005 blood sepsis Erythromycin MSSA 8 8 1
15 54 T C 06.02.2001 blood sepsis, rheumatoid Penicillin MSSA 870 8 1
arthritis
16 72 U C 19.03.2005 blood sepsis, meningitis,
probably
endocarditis
Penicillin MSSA 5 5 2
17 13 V C 12.06.2005 blood septic arthritis Penicillin MSSA 903 5 2
18 4 W C 25.09.2005 blood septic arthritis Ciprofloxacin, penicillin MSSA 869 2
19 34 X X8 H 15.09.2005 throat colonization, ciprofloxacin, erythromycine, MSSA 8 8 1
cholangitis
Y X8 H 15.09.2005 nose colonization,
cholangitis
a strains involved in hospital outbreaks
b Community (C); Hospital (H)
234
penicillin, tetracycline
ciprofloxacin, erythromycine,
oxacillin, penicillin, tetracycline
MRSA 8 8 1

Appendix IV Supplemental Table IVb
Supplemental table IVb. Identified proteins on 2D gels of the S. aureus isolates
precursor mature
Protein Accession # MW (kD) pI MW (kD) pI SP localization A B C D E F G H I J K L M N O P Q R S T U V W X Y
AckA 15927288 44,60 5,70 cytosolic + +
AhpC 13700295 20,96 4,88 cytosolic + + + + + + + + + + + + + + + + + + + +
AhpF 13700294 54,70 6,70 cytosolic +
Ald 13701504 40,05 5,58 cytosolic +
Aly 13702602 69,19 5,96 66,3 5,8 + extracellular + + + + + + + + + + + + + + + + + + + + + + + + +
Asp23 13701982 19,18 5,13 unknown + + + + + + + +
AtlE 13700854 136,67 9,60 133,7 9,6 + extracellular + + + + + + + + + + + + + + + +
Aur 13702595 56,34 5,14 33,4 4,8 + extracellular + + + + + + + + + + + + + + + + +
Bbp 49482792 123,30 4,30 117,6 4,2 + cellwall + +
BlaR1 16119211 69,00 9,50 membrane + + + + +
CitB 13701147 98,91 4,83 cytosolic + + + + + + + + + + + + + + + + +
ClfB 15925620 93,60 3,92 88,7 3,8 + cellwall + + + + + + +
ClpC 13700415 90,98 5,51 cytosolic + +
ClpP 13700659 21,50 5,13 cytosolic +
Cna 49484887 133,00 5,90 169,7 5,8 + cellwall +
Coa 13700145 74,50 8,25 129,7 5,8 + extracelular + + + + + + + +
CspA 13701199 7,32 4,52 cytosolic + + + + + + + +
CysK 13700403 33,00 5,20 cytosolic +
DeoD 13701932 25,89 4,85 unknown + + + + + + + + + + + +
Ddh 15928103 39,30 5,40 cytosolic +
Dnak 13701378 66,32 4,65 cytosolic + + + + + + + + + +
Ear 57650605 20,40 7,70 16 5,2 + unknown +
EbpS 13701280 53,15 5,97 membrane + + + + + + + + + + + + + + + + + +
Edin-B 24636605 27,50 9,38 23,64 9,3 + extracellular + +
EF-G 13700438 76,56 4,80 cytosolic + + + + + + + + + + + + +
EF-TS 13701057 32,50 5,15 cytosolic + + + + + + +
EF-TU 13700439 43,08 4,74 cytosolic + + + + + + + + + + + + + + +
Eno 13700667 47,09 4,55 cytosolic + + + + + + + + + + + + + + + + + + +
Etd 24636603 30,80 8,90 27,9 8 + extracellular + +
FabH 13700787 33,90 4,90 unknown + +
FbaA 13701919 30,82 5,01 cytosolic + + + + + + + + + + + + + + + + +
Fhs 13701527 59,83 5,69 cytosolic + + + + + + + + + + + + + + + +
Fnb 15928082 113,60 4,60 109,7 4,5 + cellwall + +
FnbA 57651010 109,10 4,56 107,8 5,6 + cellwall + + + + +
235

Appendix IV Supplemental Table IVb
precursor mature
Protein Accession # MW (kD) pI MW (kD) pI SP localization A B C D E F G H I J K L M N O P Q R S T U V W X Y
FnbB 57651008 101,00 4,60 99,7 4,6 + cellwall + + +
Gap 13700663 36,26 4,89 cytosolic + + + + + + + + + + + + + + + + + + + +
Geh 13700235 76,50 8,99 72,4 8,9 + extracellular + + + + + + + + + + + + + + + + + +
GlnA 13701109 50,80 5,00 cytosolic + + + +
GlpQ 13700763 35,29 8,67 32,2 8 + extracellular/wall + + + + + + + + + + + + + + + + + + +
GltX 13700418 56,25 5,21 cytosolic + + +
GlyA 13701907 45,14 5,75 cytosolic + + + +
Gnd 13701310 51,80 4,80 cytosolic + + +
GreA 13701408 17,70 4,50 cytosolic +
GroEL 13701823 57,54 4,56 cytosolic + + + + + + + + +
GroES 13701824 10,40 4,60 cytosolic + +
GrpE 13701379 24,00 4,10 cytosolic +
GuaA 13700305 58,17 5,03 cytosolic + + + + +
GuaB 13700304 52,82 5,61 cytosolic + + + + + + + + + +
Hla 13700962 35,95 8,70 33 7,9 + extracellular + + + + + + + + + + + + + + + + + + + + +
Hlb 13701798 33,04 8,56 33,7 7,3 + extracellular + +
HlgA 13702368 32,00 9,50 31,9 9,3 + extracellular + +
HlgB 13702370 36,69 9,35 33,9 9,3 + extracellular + + + + + + + + + + + + + + + + +
HlgC 13702369 35,56 9,29 32,5 9,1 + extracellular + + + + + + + + + + + + + +
Hpr 13700884 9,49 4,50 cytosolic + +
IleS 13700992 105,00 5,20 cytosolic + + + + + + + +
IsaA 13702519 24,19 5,90 21,5 5,3 + extracellular + + + + + + + + + + + + + + + + + + + + + + + + +
IsdA 15926714 38,70 9,60 33,7 9,6 + cellwall + +
IsdB 49483291 73,00 9,00 68,6 8,8 + cellwall + +
IsdC 15926715 24,90 8,90 21,8 8,5 + unknown + + + +
IsdD 15926716 41,50 8,80 37,9 8,3 + unknown +
KatA 13701132 58,58 5,27 cytosolic + + + + + + + + +
Lip 13702629 76,62 6,58 21,5 5,3 + extracellular + + + + + + + + + + + + + + + + + + + + + + +
LukD 13701612 36,88 9,21 34,2 9 + extracellular + + + + + +
LukE 13701613 34,80 9,48 31,9 9,3 + extracellular + + +
LytM 13700191 34,30 6,20 31,7 6 + extracellular + + + + + + + + + + + + + + + + + + + + + + + + +
MetS 13700380 74,80 4,95 cytosolic + +
MvaS 13702496 43,00 4,90 cytosolic +
Nuc 13700682 25,07 9,21 18,8 9,3 + extracellular + + + + + + + + + + + + + + + + + + + + + + + + +
OdhA 13701210 103,05 5,47 cytosolic + + + + +
PanB 13702556 29,20 5,80 unknown +
236

Appendix IV Supplemental Table IVb
precursor mature
Protein Accession # MW (kD) pI MW (kD) pI SP localization A B C D E F G H I J K L M N O P Q R S T U V W X Y
Pbp2 57650405 80,40 8,70 74,4 6,6 + extracellular + +
Pbp3 13701350 77,18 9,22 membrane + + + + + + + + + + + + + + + + +
PdhA 57651702 41,36 4,90 cytosolic + + + + + + +
PdhB 13700894 35,22 4,65 cytosolic + + + + + + + + + + + + + + + + + +
PdhC 13700895 46,30 4,90 cytosolic + + + + +
PdhD 13700896 49,40 4,95 cytosolic + + + + + + + + + + + + + + + + + + + + +
Pgi 13700766 49,76 4,82 cytosolic + + + + + + + + + + + + + + + + +
Pgk 13700664 42,58 5,17 cytosolic + + + + + + + + + + + + +
Pgm 13700666 56,42 4,74 cytosolic + +
PheT 13700939 88,88 4,66 cytosolic + +
Plc 13700011 37,06 7,71 34,2 6,4 + extracellular + + + + + + + + + + + + + + + + +
Pls 12644358 174,50 4,06 159,6 4 + cellwall + + + + + + +
PnpA 13701074 77,31 4,89 cytosolic + +
Pta 13700480 34,93 4,72 cytosolic + + +
PtsI 13700885 63,18 4,62 cytosolic + + + + +
PurM 13700872 37,00 4,50 unknown + + +
PycA 13700915 128,60 5,00 cytosolic + + + + + + +
RplA 15926216 24,70 9,00 cytosolic + + + + +
RplM 13702018 16,32 9,30 unknown + + + + +
RplY 15926178 23,80 4,40 cytosolic + + + + +
RpsA 13701275 43,30 4,40 cytosolic + + + + + + + + + + + + + + +
RpsB 15926839 29,10 5,40 cytosolic +
SA0022 13699940 83,45 9,24 80,5 9,2 + cellwall + + + + + +
SA0092 13700012 29,72 8,24 27,1 6,3 + extracellular + +
SA0129 15925838 26,50 9,60 23,6 9,3 + cellwall + +
SA0139 13700060 56,20 7,00 53,4 6 + unknown + + +
SA0182 13700104 60,49 5,10 unknown + +
SA0269 15925982 57,80 7,50 58 7,5 + unknown + +
SA0295 13700221 33,33 9,49 30,2 9,4 + unknown + +
SA0357 15926071 23,20 9,50 19,3 9,4 + unknown +
SA0270 13700196 33,07 5,85 30,5 5,6 + extracellular + + + + + + + + + + +
SA0272 15925985 114,70 6,20 membrane + + + + +
SA0423 13700355 35,81 9,67 33,4 9,6 + extracellular + + + + + + + + + + + + +
SACOL0468 57651309 25,60 8,50 22,4 6,3 + extracellular + +
SA0359 13700286 21,26 5,70 unknown + + + + + + +
SA0394 13700325 55,46 5,07 51,9 5 + unknown + +
237

Appendix IV Supplemental Table IVb
precursor mature
Protein Accession # MW (kD) pI MW (kD) pI SP localization A B C D E F G H I J K L M N O P Q R S T U V W X Y
SACOL0478 57651319 25,40 9,10 22,3 6 + extracellular + + + +
SACOL0479 57651320 56,40 4,80 52,9 4,7 + unknown + + + + + + + + + +
SA0516 15926236 17,10 8,30 cytosolic +
SA0570 13700505 18,60 9,17 15,9 9,2 + unknown + + + + + + + + + + + + + + + + + + + + + + +
SACOL0613 57651442 32,64 4,85 cytosolic +
SA0620 13700556 28,17 6,13 25,6 5,6 + extracellular + + + + + + + + + + + + + + + + + + + + + + + + +
SA0651 13700587 16,91 8,48 13,5 9 + unknown + + +
SA0663 13700599 16,04 9,15 unknown + +
SA0695 13700631 34,06 9,14 31,2 9,1 + extracellular + +
SA0707 13700643 22,20 5,15 cytosolic +
SA0775 13700717 48,49 5,44 cytosolic + +
SA0815 13700758 21,60 4,30 unknown +
SA0841 13700785 15,89 9,28 12,9 9,2 + unknown + + + + + + + + + +
SA0859 13700804 69,80 5,00 cytosolic + + +
SA0873 13700818 19,30 4,70 cytosolic +
SACOL0908 57651598 20,20 9,18 16,2 6,6 + unknown + +
SA0908 13700857 45,68 6,02 41,8 5,8 + unknown + + + + + + + + + + + + +
SA1056 15926796 35,80 7,60 unknown +
SA1173 13701136 39,50 9,40 unknown +
SA1255 13701220 17,95 4,52 cytosolic +
SA1257 13701222 20,60 6,37 cytosolic +
SA1336 13701304 56,90 5,30 cytosolic + + + +
SA0914 13700863 11,30 7,50 8,7 6,5 + unknown +
SA0998 13700951 21,40 4,91 cytosolic +
SA1001 13700955 15,20 9,12 12,3 8 + extracellular +
SA1475 13701447 30,99 9,04 unknown + + + + + + + + + + + + +
SACOL1528 57650441 34,60 6,40 32,7 5,7 unknown + +
SA1532 13701505 13,90 5,15 cytosolic + + +
SA1737 13701714 38,50 4,90 cytosolic + +
SA1743 15927503 22,00 5,20 unknown +
SA1755 13701736 17,06 9,45 14,1 9,1 + extracellular +
SA1774 15927539 42,20 5,30 unknown + + + +
SA1812 13701799 38,64 8,58 38,7 8,6 + extracellular + + + + + + + + + + + + + + + + + + +
SA1572 13701547 52,79 4,58 49,1 4,6 + extracellular + + + +
SA1633 13701608 20,86 8,48 16,5 5,3 + unknown + + + + + + + + +
SA1813 13701800 40,44 9,43 37,4 9,3 + extracellular + + + + + + + + +
238

Appendix IV Supplemental Table IVb
precursor mature
Protein Accession # MW (kD) pI MW (kD) pI SP localization A B C D E F G H I J K L M N O P Q R S T U V W X Y
SA1552 15927308 104,00 5,10 96 4,9 + cellwall +
SA1839 13701826 46,20 8,70 42,7 7,8 + cellwall + + + + + + + +
SACOL1870 57650605 20,30 6,80 20,4 6,8 + unknown + +
SA1979 15927757 36,60 9,40 29,3 9,4 + unknown +
SA2006 13702005 15,42 9,22 12,5 9,1 + unknown + + + + + + + +
SA2097 13702104 17,39 5,77 14,7 5,1 + extracellular + + + + + + + + + + + + + + + + + + + + + + + + +
SA2367 15928160 31,00 4,70 cytosolic + + +
SA2399 13702563 33,00 4,90 unknown + + + + +
SA2490 13702656 30,70 6,40 unknown +
SACOL2505 57651004 136,30 5,70 130,7 5,5 + cellwall + + + +
SAR0106 49482346 29,70 9,10 26,8 8,8 + unknown +
SAR0422 49482650 25,70 8,90 22,7 7,9 + extracellular +
SA2103 13702110 34,70 9,70 30,8 6,8 + unknown + +
SA2204 13702365 26,70 5,10 unknown + +
SA2279 13702441 66,10 6,50 cytosolic +
SA2285 15928076 48,90 8,60 43,4 6,4 + cellwall + + +
SAR0435 49482662 26,40 8,70 23,3 6,5 + extracellular + +
SAR0436 49482663 55,50 4,90 51,9 4,8 + extracellular + +
SAR1905 49484059 25,60 8,80 21,9 8,1 + extracellular +
SAR2788 49484898 27,70 9,70 25 9,7 + unknown +
SAS074 13701973 10,00 6,00 unknown +
SAS2383 49487275 149,90 5,70 144,4 5,5 + cellwall +
Sak 13701739 18,51 6,75 15,5 6,2 + extracellular + + + + + + + + + + + + + + + + + +
SasH 38259869 17,10 7,90 unknown + + +
Sbi 13702367 50,00 9,38 50,2 9,4 + unknown + + + + + + + + + + +
SceD 13701890 24,06 5,53 21,5 5,1 + extracellular + + + + + + + + + + + + +
Sed 758691 29,70 8,60 26,9 7,2 + extracellular + +
Set1 49482656 26,00 6,90 23 6,2 + extracellular + + + +
Set6 13700312 25,67 9,05 22,7 8,5 + extracellular + + + + + + +
Set11 15926104 26,20 9,20 23,2 9,1 + extracellular + + +
Set15 13700324 25,42 8,79 22,4 6,5 + extracellular + + + +
SodA 13701351 22,70 5,08 cytosolic + + + + + + + + + + + +
Sea 49484190 29,70 8,30 26,9 7,2 + extracellular + + + + +
Seb 57651597 31,40 8,90 28,4 8,3 + extracellular + + + +
Spa 13700027 48,84 5,60 45,3 5,2 + cellwall + + + + + + + + + + + + + + + + + + + + + +
SplA 13701606 25,40 9,60 21,7 8,8 + extracellular + +
239

Appendix IV Supplemental Table IVb
precursor mature
Protein Accession # MW (kD) pI MW (kD) pI SP localization A B C D E F G H I J K L M N O P Q R S T U V W X Y
SdrD 57651438 149,40 4,10 143,8 4,1 + cellwall + + +
SdrE 15926241 123,95 4,24 118,3 4,2 + cellwall + + + + + + + + + +
Sek 57650159 27,71 8,30 25,3 6,6 + extracellular + + +
Seq 15625528 28,17 8,33 25,1 6,6 + extracellular + + +
Sep 13701743 29,70 7,60 26,3 6,2 extracellular + + +
Ser 37196678 30,00 8,90 27,1 8,8 + extracellular + +
SerS 15925714 48,60 5,00 cytosolic + +
SplB 13701605 26,13 9,11 22,4 9 + extracellular + + + + + + + + + +
SplC 13701604 26,08 6,32 22,4 6,4 + extracellular + + + + + + + +
SplD 13701603 25,70 9,12 22 8,9 + extracellular + + +
SplE 57650600 25,70 9,80 22 9,2 + extracellular + + +
SplF 13701602 25,63 9,16 21,9 8,9 + extracellular + + + + + + + + + + +
SsaA 13702099 29,31 8,96 26,7 8,7 + extracellular + + + + + + + + + + + + + + + + +
SspA 13700850 36,95 5,00 33,4 4,8 + extracellular + + + + + + + + + + + + + + + + + + + + + + +
SspB 13700849 44,57 5,68 40,7 5,3 + extracellular + + + + + + + + + + + + + + + + +
Stp 13701702 44,18 9,64 41,5 9,6 + extracellular + + + + + + + + + + + + + +
SucD 13701046 31,52 5,47 cytosolic +
Tig 13701472 48,58 4,34 cytosolic + + + + + + + + + + +
Tkt 13701140 72,21 4,97 unknown + + + + + + + + + + + + + + + + + + +
Tpi 13700665 27,24 4,80 unknown + + + + +
Trap 13701628 19,54 6,13 cytosolic + + +
TrxB 13700655 33,60 5,20 cytosolic + + + + + + + + + +
ValS 13701460 102,00 4,80 cytosolic + + +
YfnI 13700610 74,35 9,04 71/49.3 8.9/ + membrane + + + + + + + + + + + + + + + + + + + + +
8.4
18920604 18920604 21,30 5,30 unknown +
24636604 24636604 25,40 7,00 22,8 6,6 + extracellular + +
90585739 90585739 20,30 9,10 16,3 6,8 + extracellular +
240

Appendix IV Supplemental Table IVb
Supplemental table IVc. Extracellular proteins in different S. aureus isolates
Name Function
Extracellular proteins identified in at least 80% of the strains
Aly hypothetical protein, similar to autolysin precursor
Aur zinc metalloproteinase aureolysin
Geh glycerol ester hydrolase
GlpQ glycerophosphoryl diester phosphodiesterase
Hla α-hemolysin precursor
HlgB γ-hemolysin component B
IsaA immunodominant antigen A
Lip triacylglycerol lipase precursor
LytM peptidoglycan hydrolase
Nuc staphylococcal nuclease
SA0570 hypothetical protein
SA0620 secretory antigen SsaA homologue
SA1812 hypothetical protein, similar to synergohymenotropic toxin precursor S. intermedius
SA2097 hypothetical protein, similar to secretory antigen precursor SsaA
SspA serine protease; V8 protease; glutamyl endopeptidase
SspB cysteine protease precursor
Extracellular proteins identified in less than 80% of the strains
Atl autolysin
Coa staphylocoagulase precursor
HlgC γ-hemolysin component C
LukD leukotoxin, LukD
Plc 1-phosphatidylinositol phosphodiesterase precurosr
SA0270 hypothetical protein, similar to secretory antigen precursor SsaA
SA0423 hypothetical protein, similar to autolysin (N-acetylmuramoyl-L-alanine amidase)
SACOL0479 surface protein, putative
SA0841 hypothetical protein, similar to cell surface protein Map-w
SA0908 conserved hypothetical protein
SA1633 probable β-lactamase
SA1813 hypothetical protein, similar to leukocidin chain lukM precursor
SA2006 hypothetical protein, similar to MHC class II analog
Sak staphylokinase precursor
Sbi IgG-binding protein SBI
SceD hypothetical protein, similar to SceD precursor
Sea enterotoxin type A precursor
Set1 exotoxin 1
Set6 exotoxin 6
SplB serine protease SplB
SplC serine protease SplC
SplF serine protease SplF
SsaA secretory antigen precursor SsaA homolog
Stp staphopain, cysteine proteinase
Extracellular proteins identified in less than 20% of the strains
Ear β-lactamase, putative
Edin-B epidermal cell differentiation inhibitor B
Etd exfoliative toxin D
Hlb truncated β-hemolysin
HlgA γ-hemolysin component A
IsdC hypothetical protein SA0978
IsdD isdD; hypothetical protein SA0979
LukE leukotoxin LukE
SA0092 hypothetical protein
SA0139 conserved hypothetical protein
SA0269 hypothetical protein SA0269
SA0295 hypothetical protein, similar to outer membrane protein precursor
241

Appendix IV Supplemental Table IVc
Name Function
Extracellular proteins identified in less than 20% of the strains
SA0357 hypothetical protein SA0357
SA0394 hypothetical protein
SA0651 hypothetical protein
SA0695 hypothetical protein
SA0914 hypothetical protein, similar to chitinase B
SA1001 hypothetical protein
SA1755 hypothetical protein
SA2103 hypothetical protein, similar to lyt divergon expression
SACOL0468 exotoxin 3, putative
SACOL0478 exotoxin 3, putative
SACOL0908 hypothetical protein
SACOL1870 hypothetical protein SACOL1870
SAR0106 putative lipoprotein
SAR0422 exotoxin
SAR0435 exotoxin
SAR0436 hypothetical protein SAR0436
SAR1905 serine protease
SAR2788 hypothetical protein SAR2788
SEB staphylococcal enterotoxin B
SED enterotoxin D precursor
SEK staphylococcal enterotoxin
SEP enterotoxin P
SEQ staphylococcal enterotoxin type I
SER enterotoxin R
Set11 exotoxin 11
Set15 exotoxin 15
SplA serine protease SplA
SplD serine protease SplD
SplE serine protease SplE, putative
24636604 probable glutamyl-endopeptidase
90585739 conserved hypothetical protein
242

Appendix V Supplemental Table Va
Supplemental table Va. Proteins of B. licheniformis DSM 13 with a predicted signal
peptide
ID number Name Cleavage Site Sequence Function
Secretory proteins
BLi00015 DacA 31 32 AKA AN D-alanyl-D-alanine carboxypeptidase
(penicillin-binding protein 5)
BLi00171 CwlD 27 28 FNN DD Germination-specific N-acetylmuramoyl-Lalanine
amidase (EC 3.5.1.28)
BLi00181 PbpX 42 43 GMR DH penicillin-binding protein
BLi00186 YbbC 23 24 AAA FP unknown
BLi00187 YbbD 26 27 REA EA unknown; similar to β-hexosaminidase
BLi00188 YbbE 23 24 AQT AI unknown; similar to β-lactamase
BLi00223 YflP 38 39 VPA EP unknown
BLi00238 YrkA1 25 26 EFA IV unknown; similar to hemolysin-like
BLi00255 33 34 LSE LT unknown
BLi00281 PhoD 46 47 VNA AP phosphodiesterase/alkaline phosphatase
(EC3.1.3.1)
BLi00302 YbdN 21 22 AFS AS unknown
BLi00321 YcdA 30 31 ASG EK unknown
BLi00338 32 33 AKA DS putative chitinase (EC 3.2.1.14)
BLi00339 26 27 ISA ET putative chitinase (EC 3.2.1.14)
BLi00340 Mpr 30 31 AQA AP glutamyl endopeptidase precursor (EC
3.4.21.19)(glutamate specific endopeptidase)
BLi00347 YvcE 30 31 ASA ET unknown; similar to cell wall-binding protein
BLi00411 24 25 IHA QE unknown
BLi00439 30 31 AGS AE putative sugar ABC transporter,
periplasmicsugar-binding protein
BLi00448 Phy 29 30 AEA SA 3-phytase (EC 3.1.3.8) / 6-phytase (EC 3.1.3.26)
BLi00478 25 26 AEE QT unknown
BLi00514 33 34 IAA AG putative transcriptional regulator, LytR family
BLi00628 YoaR 19 20 GHS DS unknown
BLi00654 30 31 INA SQ unknown
BLi00656 29 30 AAA NL α-amylase precursor (EC 3.2.1.1)
BLi00668 36 37 VKA SS hypothetical protein
BLi00669 27 28 SSA SD unknown
BLi00670 YdjM 27 28 ASA KT unknown
BLi00671 YdjN 20 21 AFA AV unknown
BLi00702 PurN 17 18 FEA IE phosphoribosylglycinamide formyltransferase
BLi00712 YerB 27 28 EQQ EK unknown
BLi00735 YdhT 24 25 SYA HT unknown; similar to mannan endo-1,4-βmannosidase
BLi00784 28 29 VYA AE unknown
BLi00824 YfkD 25 26 ADA AK unknown
BLi00827 YfjS 24 25 AEA IS unknown; similar to polysaccharide deacetylase
BLi00837 28 29 FNG NP unknown
BLi00840 YckD 26 27 AYG ET unknown
BLi00866 24 25 AST EE unknown
BLi00967 27 28 ASS QD unknown
BLi00976 25 26 AFS PE unknown
BLi00979 YhcP 29 30 VFS QE unknown
BLi00982 YhcR 33 34 THA SE unknown; similar to 5'-nucleotidase
BLi00983 YhcS 19 20 TFA YG unknown
BLi01008 LytE 25 26 ASA QT cell wall hydrolase (major autolysin)
BLi01039 YheN 40 41 SSA AT unknown; similar to endo-1,4-β-xylanase
BLi01079 YhaH 30 31 TSG KN unknown
BLi01109 29 30 ASA AQ subtilisin carlsberg precursor (EC 3.4.21.62)
243

Appendix V Supplemental Table Va
ID number Name Cleavage Site Sequence Function
BLi01123 Epr 26 27 IQA ES minor extracellular serine protease (EC 3.4.21.-)
BLi01138 23 24 VSA AE unknown
BLi01150 27 28 GSG NT unknown
BLi01154 YvgL 28 29 GGS AK unknown; similar to molybdate-binding protein
BLi01295 AbnA 32 33 SAA EP arabinan-endo 1,5-α-L-arabinase
BLi01299 32 33 VQA QE unknown
BLi01308 YoeB 22 23 SLA AK unknown
BLi01309 25 26 ASA KE putative cell wall-binding protein
BLi01364 Ggt 25 26 SKA EG γ-glutamyltranspeptidase
BLi01372 YesS 43 44 LFS AA unknown; similar to transcriptional regulator
(AraC/XylS family)
BLi01376 YesW 32 33 AEA DG unknown
BLi01404 Pel 24 25 IEA AD pectate lyase (EC 4.2.2.2)
BLi01455 27 28 AAA VW unknown
BLi01536 27 28 DVK KE hypothetical protein
BLi01539 25 26 IYA AK unknown
BLi01566 26 27 VDA TT putative phosphodiesterase
BLi01585 28 29 NNR EK unknown
BLi01590 YkvT 24 25 EHA QA unknown; similar to spore cortex-lytic enzyme
BLi01592 YkvV 27 28 ASA KQ unknown; similar to thioredoxin
BLi01595 15 16 AEA KV hypothetical protein
BLi01607 YkwD 26 27 ADA KE unknown
BLi01622 27 28 AKA GE unknown
BLi01644 MoaD 16 17 AGA QS molybdopterin converting factor (subunit 1)
BLi01697 YlaJ 26 27 ARN EA unknown
BLi01722 YlbL 35 36 GEA TE unknown
BLi01733 PbpB 41 42 VNG EV penicillin-binding protein 2B (cell-division
septum)
BLi01742 YlxW 28 29 ARE NK unknown; similar to proteins
BLi01743 YlxX 29 30 SLK AP unknown
BLi01747 27 28 VQA DT putative bacillopeptidase F
BLi01748 Bpr 30 31 SDA AA bacillopeptidase F (EC 3.4.21.-)
BLi01851 FliL 31 32 GSA SE flagellar protein required for flagellar formation
BLi01880 33 34 TRA AS putative endo-1,4-glucanase
BLi01882 33 34 ASG TS putative cellulase (EC 3.2.1.4)
BLi01883 31 32 ALA AS putative endo-1,4-β-mannosidase
BLi02014 YoaW 25 26 AEA AV unknown
BLi02027 37 38 IEK IP unknown
BLi02030 NucB 32 33 AEG AA sporulation-specific extracellular nuclease
BLi02033 YneA 28 29 AGK IE unknown
BLi02048 YneN 21 22 VWN FT unknown; similar to thioldisulfide interchange
protein
BLi02088 BglC 49 50 AAA AS endo-1,4-β-glucanase
BLi02095 YwoF 27 28 TGA KE unknown
BLi02100 26 27 VFG GN unknown
BLi02101 45 46 ANA AS unknown
BLi02120 DctB 28 29 VIG VD possible C4-dicarboxylate binding protein
BLi02165 33 34 EYA YM unknown
BLi02166 24 25 LNA GD unknown
BLi02178 YndF 27 28 SHE IE unknown; similar to spore germination protein
BLi02210 23 24 SYA AA unknown
BLi02213 YocH 25 26 ASA KE unknown; similar to cell wall-binding protein
BLi02237 YoqH 23 24 AYA QV unknown
BLi02255 YvgO 24 25 SEA KE unknown
BLi02264 YojL 26 27 VEA QT unknown; similar to cell wall-binding protein
244

Appendix V Supplemental Table Va
ID number Name Cleavage Site Sequence Function
BLi02271 YoaJ 25 26 ASA AY unknown; similar to extracellular endoglucanase
precursor
BLi02281 CtpA 36 37 VYS AS carboxy-terminal processing protease
BLi02310 YpmS 32 33 GGQ KE unknown
BLi02312 YpmQ 24 25 TSK ID unknown
BLi02321 YpjP 29 30 LMA DK unknown
BLi02340 YpcP 22 23 ATA VH unknown; similar to DNA polymerase I
BLi02367 PonA 62 63 VMV AD penicillin-binding proteins 1A/1B
BLi02372 AspB 22 23 AKA KE aspartate aminotransferase (EC 2.6.1.1)
BLi02373 YpmB 24 25 AGA NV unknown
BLi02387 YpjB 22 23 LKA KE unknown; similar to proteins
BLi02391 QcrA 39 40 RFA LD menaquinolcytochrome c oxidoreductase (ironsulfur
subunit)
BLi02420 GpsA 21 22 VLA DN NAD(P)H-dependent glycerol-3-phosphate
dehydrogenase
BLi02431 SleB 33 34 AFS EQ spore cortex-lytic enzyme
BLi02447 30 31 SEA SE unknown
BLi02450 22 23 SYG IY close homolog to LytR attenuator role for
lytABC and lytR expression
BLi02451 50 51 NSA AS putative peptidoglycan GlcNAc deacetylase
BLi02461 ResA 35 36 ESV AV essential protein similar to cytochrome c
biogenesis protein
BLi02465 DacB 27 28 AQA QP penicillin-binding protein 5* (D-alanyl-Dalaninecarboxypeptidase)
(EC 3.4.16.4)
BLi02476 YpuD 43 44 VSS EE unknown
BLi02479 32 33 VKV AE hypothetical protein
BLi02498 DacF 27 28 ESA KK penicillin-binding protein (putative D-alanyl-Dalanine
carboxypeptidase)
BLi02506 27 28 AEA LN putative PTS cellobiose-specific enzyme IIB
BLi02525 Lip 30 31 ASA AS extracellular lipase (EC 3.1.1.3)
BLi02527 31 32 AKG EE putative ABC transporter
BLi02543 23 24 AAA AG unknown
BLi02544 28 29 VSA DT unknown
BLi02564 YdhM 25 26 EYA HS unknown; similar to
cellobiosephosphotransferase system enzyme II
BLi02565 PhoB 34 35 AKK KE alkaline phosphatase III (EC 3.1.3.1)
BLi02590 YqiI 24 25 AFA AE unknown; similar to N-acetylmuramoyl-Lalanine
amidase
BLi02607 SpoIII
AH
31 32 EGE NV mutants block sporulation after engulfment
BLi02613 SpoIII
AB
22 23 EMA KP mutants block sporulation after engulfment
BLi02637 TasA 27 28 TWA AF translocation-dependent antimicrobial spore
component
BLi02639 YqxM 39 40 LSQ HT unknown
BLi02640 YqzG 23 24 AHA AA unknown
BLi02671 YqzC 32 33 GKA EA unknown; similar to proteins from B. subtilis
BLi02682 YqfZ 39 40 AAE AP unknown
BLi02744 YqxA 28 29 ANN GM unknown
BLi02820 31 32 SDA SP putative phosphatase
BLi02825 YndA 27 28 AAG AG unknown
BLi02826 YvaG 25 26 AIA AS unknown; similar to 3-oxoacyl- acylcarrierprotein
reductase
BLi02827 SacC 22 23 FSA AA levanase (EC 3.2.1.65)
BLi02833 24 25 ALT FE unknown
BLi02844 32 33 AAS EK hypothetical protein
245

Appendix V Supplemental Table Va
ID number Name Cleavage Site Sequence Function
BLi02850 32 33 AAE DS putative cell wall-associated protease
precursor(EC 3.4.21.-)
BLi02859 YrrR 26 27 RLA EI unknown; similar to penicillin-binding protein
BLi02865 YrrL 43 44 VKS AL unknown; similar to folate metabolism
BLi02884 YrvJ1 28 29 ASA AI unknown; similar to N-acetylmuramoyl-Lalanine
amidase
BLi02902 BofC 29 30 ALA EK forespore regulator of the sigma-K checkpoint
BLi02914 NadB 36 37 ASV KD L-aspartate oxidase
BLi02979 27 28 GKA EF hypothetical protein
BLi03010 32 33 TEA SE unknown
BLi03024 AraN 31 32 DQA DG L-arabinose transport (sugar-binding protein)
BLi03029 27 28 ESG KA close homolog to AbnA arabinan-endo 1,5-α-Larabinase
BLi03053 PelB 30 31 ASA AN pectate lyase (EC 4.2.2.10)
BLi03092 SppA 27 28 LLA VF putative signal peptide peptidase required for
efficient processing of pre-proteins (EC 3.4.21.-)
BLi03095 25 26 VCG YY unknown
BLi03138 YtzB 17 18 AAA VV unknown
BLi03164 YteS 21 22 SCG KD unknown
BLi03168 YtcQ 30 31 DQA SS unknown; similar to lipoprotein
BLi03201 YtlA 22 23 SCG GQ unknown
BLi03262 27 28 EAA SQ unknown
BLi03304 29 30 AFA AS putative sugar hydrolase
BLi03331 PbpD 28 29 REA QN penicillin-binding protein 4
BLi03343 29 30 TRE QT unknown
BLi03371 20 21 VSK AG putative lipase precursor
BLi03389 YuiC 27 28 VEA QD unknown; similar to proteins
BLi03405 29 30 GDA EF hypothetical protein
BLi03421 YutC 23 24 ALN DT unknown; similar to proteins
BLi03423 YunA 21 22 ALA KE unknown
BLi03433 24 25 SQA AD unknown
BLi03441 YurI 25 26 AEA FQ unknown; similar to ribonuclease
BLi03490 GerA
C
23 24 DSR QI germination response to L-alanine
BLi03538 BdbD 35 36 TQN AS thiol-disulfide oxidoreductase
BLi03540 18 19 AFS AG putative ABC transporter sugar binding protein
BLi03544 34 35 SQA DE putative sugar hydrolase
BLi03547 MntA 27 28 SSS EE manganese ABC transporter (membrane protein)
BLi03670 25 26 SFA KD unknown
BLi03706 SacB 29 30 TFA KE levansucrase (EC 2.4.1.10)
BLi03707 YveB 32 33 EKK GE unknown; similar to levanase
BLi03719 30 31 AEG PQ putative ribonuclease (EC 3.1.27.-)
BLi03739 38 39 LHS SH unknown
BLi03741 YvpA 29 30 ALA AE unknown; similar to pectate lyase
BLi03749 YvnB 28 29 SSA SG unknown
BLi03765 YvjB 36 37 ASA AE unknown; similar to carboxy-terminal
processing protease
BLi03767 25 26 LKS NH putative cell wall-binding protein
BLi03796 YvhJ 48 49 ISA AD unknown; similar to transcriptional regulator
BLi03808 LytC 24 25 VFA AN N-acetylmuramoyl-L-alanine amidase
(EC3.5.1.28)
BLi03809 LytB 25 26 AQA AD modifier protein of major autolysin LytC
BLi03811 LytR 31 32 SYA YY attenuator role for lytABC and lytR expression
BLi03821 LytD 28 29 ALA AY N-acetylglucosaminidase (major autolysin)
(EC3.2.1.96)
BLi03833 YwtF 44 45 ANA SK unknown; similar to transcriptional regulator
246

Appendix V Supplemental Table Va
ID number Name Cleavage Site Sequence Function
BLi03835 YwtD 33 34 VRA DT unknown; similar to murein hydrolase
BLi03836 YwtC 25 26 FKY SD unknown
BLi03837 YwtB 46 47 GSA KT unknown; similar to capsular polyglutamate
biosynthesis
BLi03892 SpoII
Q
48 49 ASN ND required for completion of engulfment
BLi03919 Ywm
D
17 18 AFA AE unknown
BLi03920 Ywm
C
23 24 AFA AE unknown
BLi03923 Ywm
B
29 30 EAA GN unknown
BLi03942 SpoII
R
31 32 ETA QS required for processing of pro-σ-E
BLi03981 24 25 AGA AK unknown
BLi04019 Vpr 28 29 VQA TS minor extracellular serine protease (EC 3.4.21.-)
BLi04029 25 26 ALL KE unknown
BLi04074 YwaD 30 31 AQA AP unknown; similar to aminopeptidase
BLi04089 LicB 23 24 EKS AE PTS lichenan-specific enzyme IIB component
BLi04102 YweA 30 31 EEA SA unknown; similar to proteins from B. subtilis
BLi04124 24 25 AQA KE unknown
BLi04129 27 28 AEA AS putative pectate lyase (EC 2.1.3.3)
BLi04148 YxeA 24 25 IHN EV unknown
BLi04156 29 30 AGA QE unknown; similar to glycerophosphoryl diester
phosphodiesterase
BLi04157 YhjA 27 28 AEA KT unknown
BLi04166 22 23 ASA EA carbamate kinase (EC 2.7.2.2)
BLi04182 28 29 ANS QD putative sugar ABC transporter sugar
bindingprotein
BLi04185 33 34 AHA AN unknown
BLi04206 25 26 RPA KT hypothetical protein
BLi04220 YxiA 28 29 ASA QT unknown; similar to arabinan endo-1,5-α-Larabinosidase
BLi04232 YdaJ 28 29 IKA ED unknown
BLi04236 YdaN 23 24 AAA KD putative cellulose synthase
BLi04254 GlpQ 28 29 AEA AS glycerophosphoryl diester phosphodiesterase
(EC 3.1.4.46)
BLi04272 24 25 LQK AT unknown
BLi04276 YvfO 26 27 AEA AR unknown; similar to arabinogalactan endo-1,4-βgalactosidase
BLi04294 27 28 AYA QS hypothetical
BLi04306 33 34 VSR DT unknown
BLi04308 41 42 SFA WV unknown
BLi04333 YycH 27 28 IWG FQ unknown
Lipoproteins
BLi00173 GerD 19 20 VTA CA germination response to L-alanine and to the
combination of glucose, fructose, L-asparagine,
and KCl
BLi00260 24 25 VAG CS putative sugar ABC transporter, periplasmic-
binding protein
BLi00261 19 20 LGA CS unknown
BLi00280 PenP 26 27 LAG CG β-lactamase precursor (EC 3.5.2.6)
(penicillinase)
BLi00301 19 20 LAG CG putative serine protease
BLi00384 YckB 25 26 TAA CS unknown; similar to amino acid ABC transporter
(binding protein)
247

Appendix V Supplemental Table Va
ID number Name Cleavage Site Sequence Function
BLi00397 21 22 LSG CG putative spermidine/putrescine-binding
periplasmic protein 2 precursor
BLi00420 YckK 19 20 MAA CG unknown; similar to glutamine ABC transporter
(glutamine-binding protein)
BLi00442 19 20 VTA CS putative sugar ABC transporter, periplasmicbinding
protein
BLi00466 YclQ 19 20 VAA CG unknown; similar to ferrichrome ABC
transporter (binding protein)
BLi00550 YdcC 20 21 LSA CG unknown
BLi00659 YvdG 22 23 LAA CS unknown; similar to maltose/maltodextrinbinding
protein
BLi00708 YybP 18 19 AGG CG unknown
BLi00717 YerH 18 19 LSA CA unknown
BLi00894 22 23 LMG CS putative oligopeptide ABC transporter (binding
protein) (initiation of sporulation, competence
development)
BLi00942 SsuA 17 18 LAG CS aliphatic sulfonate ABC transporter (binding
lipoprotein)
BLi00974 YhcJ 19 20 IAG CA unknown; similar to ABC transporter (binding
lipoprotein)
BLi00978 YhcN 21 22 TAG CG unknown
BLi01011 16 17 LGA CT putative oxidoreductase
BLi01072 PrsA 19 20 LSA CS protein secretion (post-translocation molecular
chaperone)
BLi01111 YhfQ 19 20 MTA CS unknown; similar to iron(III) dicitrate-binding
protein
BLi01140 MsmE 20 21 LAG CS multiple sugar-binding protein
BLi01199 Ipi 15 16 VSG CG intracellular proteinase inhibitor
BLi01218 Med 17 18 LSG CG positive regulator of comK
BLi01226 AppA 23 24 LTA CN oligopeptide ABC transporter (oligopeptidebinding
protein)
BLi01232 OppA 20 21 LSA CG oligopeptide ABC transporter (binding protein)
(initiation of sporulation, competence
development)
BLi01241 22 23 LGG CG unknown
BLi01368 YesO 21 22 LFG CS unknown; similar to sugar-binding protein
BLi01384 LplA 23 24 LIG CS lipoprotein
BLi01396 DppE 19 20 LFG CT dipeptide ABC transporter (dipeptide-binding
protein) (sporulation)
BLi01417 21 22 LAG CG unknown
BLi01431 19 20 LAA CS unknown
BLi01505 PbpC 20 21 AGA CS penicillin-binding protein 3
BLi01570 18 19 LTA CN unknown
BLi01673 YkyA 19 20 LTG CL unknown
BLi01680 Slp 19 20 TSG CS small peptidoglycan-associated lipoprotein
BLi01936 18 19 AAA CL unknown
BLi02013 20 21 LSG CN unknown
BLi02263 YojM 19 20 AAA CT unknown; similar to superoxide dismutase
BLi02284 YodJ 23 24 GTG CT unknown; similar to D-alanyl-D-alanine
carboxypeptidase
BLi02311 YpmR 18 19 LSA CT unknown
BLi02417 YphF 19 20 LSG CL unknown
BLi02557 RocC 22 23 AAA CL amino acid permease
BLi02558 22 23 LAA CG unknown
BLi02577 YqiX 19 20 LTA CG unknown; similar to amino acid ABC transporter
(binding protein)
248

Appendix V Supplemental Table Va
ID number Name Cleavage Site Sequence Function
BLi02591 YqiH 17 18 LAG CG unknown; similar to lipoprotein
BLi02626 OpuAC 20 21 LAA CG glycine betaine ABC transporter (glycine
betaine-binding protein)
BLi02656 YqgU 21 22 AAG CT unknown
BLi02658 FhuD 23 24 LTA CG ferrichrome ABC transporter (ferrichromebinding
protein)
BLi02676 PstS 22 23 AAA CG phosphate ABC transporter (binding protein)
BLi02763 YqeF 17 18 LSG CG unknown
BLi02811 26 27 LAG CT putative oligopeptide transporter putative
substrate binding domain
BLi02821 19 20 LFA CT putative lipase/esterase
BLi02910 CoxA 20 21 LSA CG spore cortex protein
BLi02986 GerM 22 23 LSG CG germination (cortex hydrolysis) and sporulation
(stage II, multiple polar septa)
BLi03178 22 23 LAA CG unknown
BLi03208 YtkA 18 19 LSA CS unknown
BLi03213 YcdH 20 21 TAG CS unknown; similar to ABC transporter (binding
protein)
BLi03455 YusA 19 20 LAA CG unknown
BLi03475 YfiY 20 21 LAA CG unknown; similar to iron(III) dicitrate transport
permease
BLi03476 YusW 18 19 MTG CG unknown
BLi03506 YvrC 20 21 LSG CG unknown; similar to iron-binding protein
BLi03623 19 20 AAG CE unknown
BLi03649 OpuCC 22 23 ISG CA glycine betaine/carnitine/choline ABC
transporter(osmoprotectant-binding protein)
BLi03657 19 20 LAA CG putative iron(III) transporter binding protein
BLi03770 CccB 18 19 LAA CG cytochrome c551
BLi03810 17 18 LSA CG unknown
BLi03845 RbsB 18 19 LSA CS ribose ABC transporter (ribose-binding protein)
BLi03906 FeuA 19 20 AAG CG iron-uptake system (binding protein)
BLi04191 22 23 TTG CG unknown
BLi04262 YxeB 20 21 VSA CG unknown; similar to ABC transporter (binding
protein)
BLi04280 YvfK 22 23 LTA CG unknown; similar to maltose/maltodextrinbinding
protein
Pseudopilins
BLi02642 ComGG 6 7 KG FIYPA pseudopilin
BLi02644 ComGE 7 8 KG FTTVE pseudopilin
BLi02645 ComGD 5 6 KG FTLLE pseudopilin
BLi02646 ComGC 5 6 KG FTLIE pseudopilin
Signal peptide prediction was done with the SignalP 2.0 software for proteins that contain a signal sequence for
one of the type I signal peptidases (http://www.cbs.dtu.dk/services/SignalP-2.0/). Proteins with more than one
membrane spanning domain were excluded from the list. The cleavage site is the position of the amino acids in the
protein between which the signal peptidase is predicted to cleave. Note that signal peptides with a proline at the
+1 position are probably not cleaved by signal peptidase (Tjalsma et al., 2000). Lipoproteins were predicted by
using the search pattern described by Sutcliffe and Harrington (2002) with the PATTINPROT software
(http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA /npsa_server.html). The pseudopilins were also
predicted by using the PATTINPROT software and by BLAST searches with ComG proteins of B. subtilis.
249

Appendix V Supplemental Table Vb
Supplemental table Vb: Extracellular proteins of B. licheniformis which either contain an N-terminal signal peptide or which are known to be secreted
by other pathways
Protein Function pI Mr ID number md signal peptide sequence P G N LB ex LB st
Metabolism of carbohydrates
Pel pectate lyase (EC 4.2.2.2) 5.85 48.7 BLi01404 MKRFFSVIILGALLLLGTSAPIEA AD x
SacB levansucrase (EC 2.4.1.10) 8.73 53.7 BLi03706 MNIKNIAKKASALTVAAALLAGGAPQTFA KE x
SacC levanase (EC 3.2.1.65) 7.18 75.5 BLi02827 MKKRMIQMGIIGAMMFPEAFSA AA x x
YheN similar to endo-1,4-β-xylanase 9.94 34.6 BLi01039 MRQVSKKTAPSVAYLLTKAACFFVLCLILLYV
WDLSQSSA AT
x
YvfO similar to arabinogalactan endo-1,4β-galactosidase
5.87 46.2 BLi04276 MKNVLAVFVVLIFVLGAFGTSGPAEA AR x x
YvpA similar to pectate lyase 9.00 23.7 BLi03741 MMKRLAGTVILSGLLVCGFGQALPEKALA AE x
YxiA similar to arabinan endo-1,5-α-Larabinosidase
6.63 53.1 BLi04220 MNMRKCFIQVLALLFIIAACFAPNQASA QT x
BLi00338 putative chitinase (EC 3.2.1.14) 4.97 65.8 BLi00338 MLINKSKKFFVFSFIFVMMLSLSFVNGEVAKA
DS
x
BLi03029 a close homolog to AbnA arabinanendo
1,5-α-L-arabinase
8.62 35.6 BLi03029 MKNVLRKMSLAALIFGLLLSFSMPESGKA AF x a)
BLi04129 putative pectate lyase (EC 2.1.3.3) 9.32 37.4 BLi04129 MKKLISIIFIFVLGVVGSLTAAVSAEA AS x x
Metabolism of proteins and peptides
Bpr bacillopeptidase F (EC 3.4.21.-) 5.04 155.0 BLi01748 MKRKLRKKAFSTILSGLLIGSLFMPAVSDAA
AK
x x x x
Epr minor extracellular serine protease
(EC 3.4.21.-)
10.2 63.0 BLi01123 MKKLWKIAVSAAMFVGFFANSPRIQA ES x x x
Ggt γ-glutamyltranspeptidase (EC
2.3.2.2)
4.81 64.0 BLi01364 1 MRRLAFLVVAFCLAVGCFFSPVSKA EG x x x x
Mpr glutamyl endopeptidase precursor
(EC 3.4.21.19)
9.78 33.7 BLi00340 MVSKKSVKRGLITGLIGISIYSLGMHPAQA AP x x x
Vpr extracellular serine protease (EC
3.4.21.-)
8.87 85.6 BLi04019 MRKSIVRYFVMAFILLFALSTFLTGVQA TS x x x x
YwaD similar to aminopeptidase 9.09 48.2 BLi04074 MKRKMMMFGLALSIIAGGVVADGTGNAAQA
AP
x x x
BLi00301 putative serine protease 6.65 45.0 BLi00301 MKSKWSAMVVIAGLLLLAG CGA x
250

Appendix V Supplemental Table Vb
Protein Function pI Mr ID number md signal peptide sequence P G N LB ex LB st
BLi01109 subtilisin carlsberg precursor (EC
3.4.21.62)
8.63 38.9 BLi01109 MMRKKSFWLGMLTALMLVFTMAFS DS x x x
BLi01747 putative bacillopeptidase F 9.83 54.2 BLi01747 MKKKPLFRTFMCAALIGSLLAPVAVQA DT x x x
Metabolism of nucleotides and nucleic acids
NucB sporulation-specific extracellular
nuclease (EC 3.-.-.-)
9.05 15.3 BLi02030 MIKKWAVHLLFSALVLLGLSGGAA YS x
YfkN similar to 2',3'-cyclic-nucleotide 2'- 5.11 161.7 BLi00814 2 MVGIQKRRFSRKNILRILLTSVMILSLLMPNTQT x x x
phosphodiesterase (EC 3.1.4.16)
YA EE
YhcR similar to 5'-nucleotidase 4.68 130.6 BLi00982 MVNVVKSRFMAGLTITFMMIASFLTPFADVTH
A SE
x x x x
YurI similar to ribonuclease 5.14 30.5 BLi03441 MNRKCVIPFILMLSAMCAPAQNAEA FQ x x
BLi03719 putative ribonuclease (EC 3.1.27.-) 9.58 16.6 BLi03719 MKKILSTLALGFVLALGFLAGNLFTSSA EG x x x x
Metabolism of lipids
GlpQ glycerophosphoryl diester
phosphodiesterase (EC 3.1.4.46)
YhdW similar to glycerophosphodiester
phosphodiesterase (EC 3.1.4.46)
9.17 33.8 BLi04254 MKRLVRSIFLITAAIAAFGFGFSGHAEA AS x x
9.43 31.5 BLi04156 MSALFKKLMLSSLIGVSIGSALFAPNAGA QE x x
Metabolism of phosphate
PhoB alkaline phosphatase III (EC 3.1.3.1) 9.51 50.3 BLi02565 MGFLRNRIVGITLAGAVALGSAGTGSA AM x
PhoD phosphodiesterase/alkaline
7.25 65.8 BLi00281 MKKLSEESLKDNTFDRRRFIQGAGKIAGLSLGL x
phosphatase (EC 3.1.3.1)
AIAQSMGAMEVNA AP
Phy 3-phytase (EC 3.1.3.8) / 6-phytase
(EC 3.1.3.26)
4.66 42.0 BLi00448 MNFYKTLALSTLAASLLSPSWSILPRAEA SA x x
Metabolism of the cell wall
LytD N-acetylglucosaminidase (major
autolysin) (EC 3.2.1.96)
9.60 97.0 BLi03821 MKNIRKTVIFAAIILLVHTAVPA IP x
PbpB penicillin-binding protein 2B (celldivision
septum)
9.70 78.8 BLi01733 MPKKNKFMNRGAAILSICFALFFFVIVGRFA FI x
YodJ similar to D-alanyl-D-alanine
carboxypeptidase (EC 3.4.16.4)
6.03 30.9 BLi02284 MNGKYKYVTIASLLSAAVLLGTG CTM x
YvcE similar to cell wall-binding protein 9.66 49.1 BLi00347 MKKKVYTFGLASILGTASLFTPFMNNTASA ET x
251

Appendix V Supplemental Table Vb
Protein Function pI Mr ID number md signal peptide sequence P G N LB ex LB st
YrvJ1 similar to N-acetylmuramoyl-Lalanine
amidase
? ? BLi02884 MKKRAVLILSMMMLAAQAAFYTSSNTASA AI x x x x x
YwtD similar to murein hydrolase 9.61 45.6 BLi03835 1 MIKKAANKKLVLFCGIAVLWMSLFLTNHNDVR
A DT
x
BLi01309 putative cell wall-binding protein 9.13 25.9 BLi01309 MKKTIMSLAAAAAMSATAFGATASA KE x
BLi03767 putative cell wall-binding protein 9.52 46.3 BLi03767 MKRKLMTLGLTAVLGSSAVLIPLKSNHALA YE x x
Transport/binding proteins and lipoproteins
AppA oligopeptide ABC transporter
(oligopeptide-binding protein)
4.77 62.7 BLi01226 MNKRKTGFSILSLLLILSIFLTA CNS x x x
DppE dipeptide ABC transporter
(dipeptide-binding protein)
(sporulation)
4.85 61.2 BLi01396 MKRLTSVLASAFAVILLFG CTA x
FeuA iron-uptake system (binding protein) 6.46 34.9 BLi03906 MRKISIFLFILLLALGAAG CGN x x
MntA manganese ABC transporter
(membrane protein)
5.21 34.3 BLi03547 MKWKQTLAIAAALILILAAGCSSKSSS EE x
OppA oligopeptide ABC transporter
(binding protein)
5.52 60.8 BLi01232 MKKRLSFISLMLIFTLVLSA CGF x x x x
OpuAC glycine betaine ABC transporter
(glycine betaine-binding protein)
6.00 32.0 BLi02626 MWKKIAGIGTAAVLTLGLAA CGS x
PstS phosphate ABC transporter (binding
protein)
4.56 32.6 BLi02676 MKPFKKITLMFIMSVLVVFAAA CGS x
YcdH similar to ABC transporter (binding
protein)
5.02 38.4 BLi03213 MKKTFGIASAFILAAGLTAG CSS x
YclQ similar to ferrichrome ABC
transporter (binding protein)
5.41 34.7 BLi00466 MKKLSLLIMALITVLVVAA CGN x x x x
YesO similar to sugar-binding protein 4.89 48.7 BLi01368 MLMRRFVFVSLCILLTLGLFG CSS x
YfiY similar to iron(III) dicitrate transport
permease
5.56 36.7 BLi03475 MKRWSIVGFIALLAISILAA CGG x
YflE similar to anion-binding protein 5.67 74.3 BLi00793 5 MKRFIKERGLAFFLIAAILLWLKTYA AY x x x x
YfnI similar to anion-binding protein 5.95 72.6 BLi04159 5 MKKIFSHKLSFFVLAVVFVWAKTYASYFLEFN
LGVKGSTQHMLLFINPLSFTIAALGLALFAKGR
RSA IW
x x x x
252

Appendix V Supplemental Table Vb
Protein Function pI Mr ID number md signal peptide sequence P G N LB ex LB st
YhcJ similar to ABC transporter (binding
lipoprotein)
4.67 29.4 BLi00974 MKKFACVVIFLLLAAVIAG CAA x
YqgS similar to putative molybdate binding
protein
5.65 73.2 BLi02659 5 MRKSFFSKISFLLIATLLMWLKTYVVYK TS x
BLi02527 putative ABC transporter 6.49 57.0 BLi02527 MAYIAKRMIIPIIFLFILASCSAGGAGSAKG EE x x x
BLi02811 putative oligopeptide transporter
(putative substrate binding domain)
5.83 61.1 BLi02811 MMSGRISLKIKIIFILMLAFSILLAG CTT x x
BLi03657 putative iron(III) transporter binding
protein
Membrane bioenergetics
QcrA menaquinolcytochrome c
oxidoreductase (iron-sulfur subunit)
5.81 34.9 BLi03657 MKRFKWFALFAALILLLAA CGN x x
7.67 19.4 BLi02391 MKMSEKRHRVSRRQFLNYTLTGVGGFMAAG
MLMPMVRFA LD
Mobility and chemotaxis
FlgE flagellar hook protein 4.63 28.0 BLi01849 x x
FlgK flagellar hook-associated protein 1
(HAP1)
4.44 54.3 BLi03785 x x x
FlhO flagellar basal-body rod protein 4.74 30.7 BLi03875 x
FlhP flagellar hook-basal body protein 6.55 30.3 BLi03874 x
FliD flagellar hook-associated protein 2
(HAP2)
5.19 54.2 BLi03778 x x
FliK flagellar hook-length control 4.74 51.2 BLi01847
FliL flagellar protein required for flagellar
formation
5.06 16.0 BLi01851 MNKKLLGIMMTIILAIAVLGTAAFFVIKGSA SE x
Hag flagellin protein 5.28 33.2 BLi03780 x x x x x
Sporulation
TasA translocation-dependent
antimicrobial spore component
5.27 28.8 BLi02637 MGTKKKLGLGVASAALGLALVGGGTWA AF x x x x x
RNA synthesis and regulation
YwtF similar to transcriptional regulator 9.64 35.8 BLi03833 1 MLRSQRTKKKRLRKWVKYSLFFIALILTATA
AA
253
x
x x

Appendix V Supplemental Table Vb
Protein Function pI Mr ID number md signal peptide sequence P G N LB ex LB st
Phage-related functions
XkdG PBSX prophage 5.14 34.3 BLi01331 x x x
XkdK PBSX prophage 4.54 48.5 BLi01337 x x x x
XkdM PBSX prophage 4.70 16.4 BLi01338 x x x x
Unknown
YbdN 5.35 30.7 BLi00302 MKKSLFLFVFSLFLMAIPAFS AS x x x x
YdaJ 5.04 41.3 BLi04232 MKAPVRYIWIGMILCFLSVSLAVGCIKA ED x
YkwD 10.16 26.6 BLi01607 MKKAFLLSAAAAATLFTFSGVQHA DA x
YpjP 5.38 23.4 BLi02321 MKMWMRKALVALFTIATFGLVSPPAALMA DK x
YqgU 5.24 41.4 BLi02656 MAGLRVSLLIIAALMAVAAAG CTP x x
YusA 6.45 30.5 BLi03455 MKKGLLTALFIIFAGVLAA CGS x x x x
YusW 4.46 16.6 BLi03476 MNQFRMAVIALVLILMTG CGS x x
YwoF 4.57 51.5 BLi02095 MRKWYFILSACILVSVIIAFA YD x x x x
BLi00654 6.10 25.9 BLi00654 MKKAMAILGFLVLTASLLFIINKGTSQINA SQ x x x
Unknown
BLi00784 9.06 60.6 BLi00784 MKIQKRVQALLATSAMFAGLMLSDAVYA AE x
BLi01431 4.56 18.6 BLi01431 MKKFLVLFLSFGLALALAA CSS x x x
BLi02210 4.63 34.9 BLi02210 MLMSAFVLVLAACQQADPKGSYA AA x x x
BLi02558 5.12 34.9 BLi02558 MKKWKSLSWMVLLLTLVMGLAA CGS x x x x
BLi03010 9.50 34.9 BLi03010 MSKIKWIITLTICTALAFSLFIFFNKA NF x x
BLi03178 5.89 22.9 BLi03178 MKKLLKWTSALGLALSLALLAA CGN x
BLi03260 4.68 37.4 BLi03260 2 MKKRLMSLLVCILVLVPAAGAFA AP x
BLi03670 4.06 17.6 BLi03670 MKKLLFMVILSVLTLVFGSSSVSFA KD x
BLi04124 6.80 48.5 BLi04124 MKRIYIFLLCFAVLLPVGGKTAQA KE x x x
BLi04294 9.46 37.5 BLi04294 MVFKKPKVFIAAVILALSSFAGTAAYA QS x x x x x
BLi04308 10.4 14.1 BLi04308 MKNHLYEKKKRKPLTRTIKATLAVLTMSIALV
GGATVPSFA WV
x x
Signal peptides were predicted as described in the section on signal peptide prediction. The signal peptidase cleavage site is indicated with a gap in the amino acid sequence and the twinarginine
motif in the signal peptides is highlighted in bold letters.
md: number of additional predicted membrane spanning domains, P: phosphate starvation, G: glucose starvation, N: nitrogen starvation, LB ex : cells grown in LB, exponential growth phase,
LB st : cells grown in LB, stationary growth phase.
a this protein was found only in the extracellular proteome after 72 h cultivation (gel not shown).
254

Appendix V Supplemental Table Vb
Supplemental table Vc. Proteins detected in the extracellular proteome lacking known
export signals
Protein Function P G N LB
Cell envelope related functions
AtpD ATP synthase (subunit β) (EC 3.6.1.34) x
MntB manganese ABC transporter (ATP-binding protein) x
YkuA similar to penicillin-binding protein x
Protein secretion
SipS signal peptidase I (EC 3.4.21.89) x
SipT signal peptidase I x
Sporulation
SpoVG required for spore cortex synthesis x
Carbohydrate metabolism
CitB aconitate hydratase (aconitase) (EC 4.2.1.3) x x x x
Eno enolase (EC 4.2.1.11) x x x
GapA glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) x x
Icd isocitrate dehydrogenase (EC 1.1.1.42) x x
IolS myo-inositol catabolism x
Mdh malate dehydrogenase (EC 1.1.1.37) x
PdhA pyruvate dehydrogenase (E1 α subunit) (EC 1.2.4.1) x x
PdhB pyruvate dehydrogenase (E1 β subunit) (EC 1.2.4.1) x
PdhC pyruvate dehydrogenase (dihydrolipoamide acetyltransferase E2
subunit) (EC 2.3.1.12)
x
PdhD pyruvate dehydrogenase / 2-oxoglutarate dehydrogenase
(dihydrolipoamide dehydrogenase E3 subunit) (EC 1.8.1.4)
x x x x
Pgk phosphoglycerate kinase (EC 2.7.2.3) x x x x
PtsH histidine-containing phosphocarrier protein of the PTS (HPr protein) x
YjeA similar to chitooligosaccharide deacetylase x
YwjH similar to transaldolase (pentose phosphate) (EC 2.2.1.2) x
Amino acid metabolism
GlnA glutamine synthetase (EC 6.3.1.2) x x x
GlyA serine hydroxymethyltransferase (EC 2.1.2.1) x
IlvC ketol-acid reductoisomerase (EC 1.1.1.86) x x x
MetE cobalamin-independent methionine synthase (EC 2.1.1.14) x
YjbG similar to oligoendopeptidase (EC 3.4.24.-) x
BLi04164 ornithine carbamoyltransferase, catabolic (EC 2.1.3.3) x
BLi04275 close homolog to Ald L-alanine dehydrogenase x
Lipid metabolism
Bcd leucine dehydrogenase S81735 (EC 1.4.1.9) x
Nucleotide and nucleic acid metabolism
GuaB inosine-monophosphate dehydrogenase (EC 1.1.1.205) x
Ndk nucleoside diphosphate kinase (EC 2.7.4.6) x
DNA packaging and segregation
Hbs non-specific DNA-binding protein Hbsu x x
RNA synthesis and modification
TenA transcriptional regulator of extracellular enzyme genes x
YugI similar to polyribonucleotide nucleotidyltransferase x
Translation
FusA elongation factor G x
Frr ribosome recycling factor x
RplJ ribosomal protein L10 (BL5) x
RplL ribosomal protein L12 (BL9) x
RpsF ribosomal protein S6 (BS9) x
255

Appendix V Supplemental Table Vc
Protein Function P G N LB
Protein modification and folding
GroES class I heat-shock protein (chaperonin) x
PpiB peptidyl-prolyl isomerase (EC 5.2.1.8) x x
Tig trigger factor (prolyl isomerase)(EC 5.2.1.8) x
Detoxification and adaptation to atypical conditions
AhpC alkyl hydroperoxide reductase (small subunit) (EC 1.6.4.-) x
ClpP ATP-dependent Clp protease proteolytic subunit (class III heat-shock
protein) (EC 3.4.21.92)
x
SodA superoxide dismutase (EC 1.15.1.1) x x x x
PspA phage shock protein A homolog x
YceD similar to tellurium resistance protein x
YvtA similar to HtrA-like serine protease x
Unknown
YdhD Unknown x
YkrZ Unknown x x x
YkuV Unknown x
YqbG Unknown x
YxjG Unknown x
BLi00303 Unknown x x x x
BLi03379 Unknown x
P: phosphate starvation, G: glucose starvation, N: nitrogen starvation, LB: Luria Broth.
256