VAAM-Jahrestagung 2012 18.–21. März in Tübingen

71CEP027Characterisation of the Esx secretion system of Staphylococcus aureusH. Kneuper*, T. PalmerUniversity of Dundee, Division of Molecular Microbiology, Dundee,United KingdomThe Sec-independent Esx (or Type VII) protein secretion pathway ispredominantly found in members of the Actinobacteria and Firmicutes.Initially characterised for its role in virulence of Mycobacteriumtuberculosis [1], Type VII secretion systems have since been associatedwith a number of diverse processes including conjugative DNA transfer[2] and iron aquisition [3]. In Staphylococcus aureus, a Type VII secretionsystem is present that contributes to virulence by enhancing murineabscess formation and establishing persistent infections [4, 5].To further assess the role of individual Esx components in proteinsecretion, unmarked chromosomal deletions of single esx genes as well asthe whole esx operon were created in S. aureus strains RN6390 and COL.As the Esx system was found to be involved in various processes notdirectly related to virulence in Actinobacteria and is furthermore present ina wide range of non-pathogenic Firmicutes bacteria, we also tried toidentify alternative pathways affected by deletions of core Esxcomponents.[1] Abdallah et al. (2007), Nature Rev. Microbiol. 5, 883-891[2] Coros et al. (2008), Mol. Microbiol. 69(4), 794-808[3] Siegrist et al. (2009), PNAS 106(44), 18792-18797[4] Burts et al. (2005), PNAS 102(4), 1169-1174[5] Burts et al. (2008), Mol. Microbiol. 69(3), 736-746CEP028Identification of a Tat signal peptide-processing proteaseN. Nigusie Woldeyohannis* 1 , S. Richter 2 , B. Hou 1 , T. Brüser 11 Leibniz University Hannover, Institute of Microbiology, Hannover, Germany2 Leibniz-institut für Pflanzenbiochemie, Halle, GermanyProteins can be translocated by the twin-arginine translocation (Tat)pathway in a folded conformation. The N-terminal signal peptide of suchTat-substrates is unfolded in solution, even when the remainder of theprotein is fully folded. After transport, the signal peptide is usually cleavedoff by a signal peptidase. Precursor proteins can Tat-independently interactwith membranes via their signal peptide. It has been suggested that thismembrane interaction is important for functional transport, as signalpeptides can adopt secondary structures at the membrane surface thatcould trigger the recognition by the Tat system. In our studies on the Tatsystem of Escherichia coli, we noted the generation of a distinct partiallyprocessed Tat substrate in the cytoplasm, whereas in the periplasm therewas only correctly processed HiPIP detectable. In vitro studies revealedthat a component of the cytoplasmic membrane catalyzed this specifictransport-independent proteolytic turnover. We were able to identify theresponsible enzyme as well as the exact cleavage site in the signal peptideand could characterize the requirements for the processing. Interestingly,the cleavage had almost no influence on the translocation efficiency.Together, our data indicate that membrane-interacting Tat substratesencounter proteases that do not abolish transport, at least if the Tatsubstrate is correctly folded. This observation is discussed in terms ofpossible roles for a membrane interaction prior to Tat transport.CEP029TatA and TatE are membrane-permeabilizing components ofthe Tat system in Escherichia coliD. Mehner*, C. Rathmann, T. BrüserLeibniz University Hannover, Institute of Microbiology, Hannover, GermanyThe twin-arginine translocation (Tat) pathway transports folded proteinsacross the cytoplasmic membrane of most prokaryotes and the thylakoidmembrane of plant plastids. A basic prerequisite for translocation is astable membrane potential. In Escherichia coli, the functional Tattranslocaseconsists of multiple copies of the proteins TatA, TatB andTatC. There exists a second paralog of TatA, TatE that can functionallysubstitute TatA. While TatB and TatC form stable complexes in thecytoplasmic membrane that are believed to mediate the specificrecognition of Tat substrates, TatA forms separate complexes that onlytransiently interact with TatBC complexes during translocation. It isbelieved that TatA complexes somehow facilitate the protein passage. Wenoted that recombinant TatA strongly affects growth. This effect could betraced back to the N-terminus that forms a trans-membrane domain.Further analyses indicated that this N-terminus alone has the capacity topermeabilize the membrane, which is very unusual for a natural transmembranedomain and thus strongly suggests a direct function of the TatAN-terminus in the facilitation of the membrane passage. The data supportthe view that TatA as well as TatE are pore-forming or membraneweakeningconstituents of the Tat system.CEP030Mode of action of theta-defensins against Staphylococcus aureusM. Wilmes* 1 , A. Ouellette 2 , M. Selsted 2 , H.-G. Sahl 11 Universität Bonn, Institut für Med. Mikrobiologie, Immunologie undParasitologie (IMMIP), Bonn, Germany2 University of Southern California, Pathology & Laboratory Medicine,Los Angeles, United StatesMulticellular organisms defend themselves against infectiousmicroorganisms by producing a wide array of antimicrobial peptidesreferred to as host defense peptides (HDPs). These evolutionary ancientpeptides are important effector molecules of innate immunity and display -in addition to their immunomodulatory functions - potent directantimicrobial activity against a broad range of pathogens. Generally, HDPsare short (12 to 50 amino acids), positively charged and able to adopt anamphipathic structure.Among HDPs defensins are an important peptide family characterized bydisulfide-stabilized -sheets as a major structural component. Their modeof action was long thought to result from electrostatic interaction betweenthe cationic peptides and negatively charged microbial membranes,followed by pore-formation or unspecific membrane permeabilization.Recently, it has been demonstrated that defensin activities can be muchmore targeted and that fungal (Schneider et al., 2010), invertebrate(Schmitt et al., 2010) and human defensins (Sass et al., 2010; De Leeuw etal., 2010) bind to and sequester the bacterial cell wall building block lipidII, thereby specifically inhibiting cell wall biosynthesis in staphylococci.Interestingly, the antistaphylococcal mode of action of the cyclic rhesusmacaque theta-defensins (RTD-1 and RTD-2) differ from this since RTDsdo not affect cell wall biosynthesis. Moreover, the peptides do notcompromise the membrane integrity. S. aureus cells treated with RTDsshow membranous structures, protrusions of cytoplasmic contents and cellwalls peeling off the cell. These morphological changes indicate prematureactivation of peptidoglycan lytic enzymes involved in cell separation asmechanism of killing.CEP031Analyses of the alkaline shock protein 23 (Asp23) ofStaphylococcus aureusM. Müller 1 , S. Reiß 1 , R. Schlüter 1 , W. Reiß 1 , U. Mäder 2 , J. Marles-Wright 3 , R. Lewis 3 , S. Engelmann 1 , M. Hecker 1 , J. Pané-Farré* 11 Ernst-Moritz-Arndt-Universität, Institut für Mikrobiologie, Greifswald, Germany2 Ernst-Moritz-Arndt-Universität, Functional Genomics Group, Greifswald,Germany3 University of Newcastle upon Tyne, Newcastle Structural Biology,Newcastle, United KingdomWith a copy number of about 20,000 molecules per cell, Asp23 is one ofthe most abundant proteins in S. aureus. Asp23 has been characterized as aprotein with an apparent molecular mass of 23 kDa that, following analkaline shock, accumulates in the soluble protein fraction. Moreover, itwas shown that the transcription of the asp23 gene is exclusively regulatedby the alternative sigma factor SigB. The function of Asp23, however, hasremained elusive. Sequence analysis identified Asp23 as a Pfam DUF322family member, precluding functional predictions based on its sequence.Using fluorescence microscopy we found that Asp23 co-localizes with thestaphylococcal cell membrane. Interestingly, Asp23 appeared to beexcluded from sites of active cell division. Since Asp23 has norecognizable transmembrane spanning domains, we initiated a search forproteins that link Asp23 to the cell membrane. To gain evidence for thefunction of Asp23, a deletion mutant was constructed and comparativeanalyses of the wild type and mutant proteome and transcriptome werecarried out. These analyses identified a rather small number ofdifferentially regulated transcripts and proteins. Furthermore, usingtransmission electron microscopy of negatively stained Asp23 protein weshowed that it forms large spiral complexes in vitro, the formation ofwhich appears to be dependent on the presence of magnesium ions.In summary, we identified Asp23 as a membrane associated protein in S.aureus that forms large, ordered complexes in vitro. Identification of theAsp23 function is the subject of ongoing research.BIOspektrum | Tagungsband 2012