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Marinobacter adhaerens sp. nov., prominent in aggregate formation
with the diatom Thalassiosira weissflogii
Eva C. Kaeppel 1 , Astrid Gärdes 1 , Shalin Seebah 1 , Hans-Peter Grossart 2 , Matthias S.
Ullrich 1
1 Jacobs University Bremen, Campus Ring 1, 28759 Bremen, Germany
2 Leibniz Institute of Freshwater Ecology and Inland Fisheries - Neuglobsow, Alte Fischerhütte 2, 16775
Stechlin, Germany
Correspondence:
Matthias S. Ullrich
m.ullrich@jacobs-university.de
______________________________________________________________________
The Gram-negative, motile, and rod-shaped bacterial strain, HP15 T , was isolated from
particles sampled in surface waters of the German Wadden Sea. It was identified
among 82 other marine isolates due to its high potential to induce production of
transparent exopolymeric particles and aggregate formation while interacting with the
diatom, Thalassiosira weissflogii. HP15 T grew optimally at a range of 34-38 °C, a pH of
7-8.5, and was able to tolerate salt concentrations between 0.5-20 % (w/v) NaCl. HP15 T
was chemotaxonomically characterized by possessing ubiquinone-9 as the major
respiratory lipoquinone as well as C 16:0 , C 18:1 !9c, C 16:1 !7c/iso, and C 15:0 2-OH as
predominant fatty acids. The G+C content of its DNA was 56.9 mol%. The closest
relative by means of 16S rRNA sequence analysis was Marinobacter flavimaris with a
similarity level of 99 %. The whole-genome relatedness of HP15 T to M. flavimaris, M.
salsuginis, M. lipolyticus, and M. algicola was determined to be lower than 70 % by
DNA-DNA hybridization. On the basis of phenotypic and chemotaxonomic properties as
well as phylogenetic analyses, strain HP15 T (=DSM 23420 T = CIP 110141 T ) is proposed
to represent the novel species, Marinobacter adhaerens sp. nov.
______________________________________________________________________
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain HP15 T is
AY241552.
A transmission electron micrograph of HP15 T and the comparison of cellular fatty acid composition between
strain HP15 T and type strains of seven other Marinobacter species are available as supplementary
material in IJSEM Online.
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Fig. 1. Transmission electron micrograph of
strain HP15 T cultivated in marine broth for 24
hours.
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Table 1. Phenotypic and genotypic differentiation between HP15 T and the closest
related Marinobacter type strains.
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Fig. 2. Maximum likelihood phylogenetic tree based on 16S rRNA sequences of
HP15 T , all type strains of the genus Marinobacter and the type strains of Halospina
denitrificans HGD 1-3 T (DQ072719) and Salicola marasensis 7Sm5 T (DQ019934) as
out groups. The tree was inferred from 1,531 alignment positions using the RAxML
algorithm (Stamatakis, 2006). Support values from 1,000 bootstrap replicates were
displayed above branches if larger than 50 %. Bar, 0.01 nucleotide substitutions per
site.
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)
$5C525B357$
Antunes, A., Franca, L., Rainey, F. A., Huber, R., Nobre, M. F., Edwards, K. J. & Da
Costa, M. S. (2007). Marinobacter salsuginis sp. nov., isolated from the brine-seawater interface
of the Shaban Deep, Red Sea. Int J Syst Evol Microbiol 57, 1035-1040.
Gauthier, M. J., Lafay, B., Christen, R., Fernandez, L., Acquaviva, M., Bonin, P. &
Bertrand, J. C. (1992). Marinobacter hydrocarbonoclasticus gen. nov., sp. nov., a new,
extremely halotolerant, hydrocarbon-degrading marine bacterium. Int J Syst Evol Microbiol 42,
568-576.
Green, D. H., Bowman, J. P., Smith, E. A., Gutierrez, T. & Bolch, C. J. S. (2006).
Marinobacter algicola sp. nov., isolated from laboratory cultures of paralytic shellfish toxinproducing
dinoflagellates. Int J Syst Evol Microbiol 56, 523-527.
Huu, N. B., Denner, E. B. M., Ha Dang, T. C., Wanner, G. & Stan-Lotter, H. (1999).
Marinobacter aquaeolei sp. nov., a halophilic bacterium isolated from a Vietnamese oilproducing
well. Int J Syst Evol Microbiol 49, 367-375.

Martin, S., Marquez, M. C., Sanchez-Porro, C., Mellado, E., Arahal, D. R. & Ventosa, A.
(2003). Marinobacter lipolyticus sp. nov., a novel moderate halophile with lipolytic activity. Int
J Syst Evol Microbiol 53, 1383-1387.
Montes, M. J., Bozal, N. & Mercade, E. (2008). Marinobacter guineae sp. nov., a novel
moderately halophilic bacterium from an Antarctic environment. Int J Syst Evol Microbiol 58,
1346.
Romanenko, L. A., Schumann, P., Rohde, M., Zhukova, N. V., Mikhailov, V. V. &
Stackebrandt, E. (2005). Marinobacter bryozoorum sp. nov. and Marinobacter sediminum sp.
nov., novel bacteria from the marine environment. Int J Syst Evol Microbiol 55, 143-148.
Yoon, J. H., Yeo, S. H., Kim, I. G. & Oh, T. K. (2004). Marinobacter flavimaris sp. nov. and
Marinobacter daepoensis sp. nov., slightly halophilic organisms isolated from sea water of the
Yellow Sea in Korea. Int J Syst Evol Microbiol 54, 1799-1803.
$

Martin, S., Marquez, M. C., Sanchez-Porro, C., Mellado, E., Arahal, D. R. & Ventosa, A.
(2003). Marinobacter lipolyticus sp. nov., a novel moderate halophile with lipolytic activity. Int
J Syst Evol Microbiol 53, 1383-1387.
Montes, M. J., Bozal, N. & Mercade, E. (2008). Marinobacter guineae sp. nov., a novel
moderately halophilic bacterium from an Antarctic environment. Int J Syst Evol Microbiol 58,
1346.
Romanenko, L. A., Schumann, P., Rohde, M., Zhukova, N. V., Mikhailov, V. V. &
Stackebrandt, E. (2005). Marinobacter bryozoorum sp. nov. and Marinobacter sediminum sp.
nov., novel bacteria from the marine environment. Int J Syst Evol Microbiol 55, 143-148.
Yoon, J. H., Yeo, S. H., Kim, I. G. & Oh, T. K. (2004). Marinobacter flavimaris sp. nov. and
Marinobacter daepoensis sp. nov., slightly halophilic organisms isolated from sea water of the
Yellow Sea in Korea. Int J Syst Evol Microbiol 54, 1799-1803.
$

Complete genome sequence of Marinobacter adhaerens type strain
(HP15), a diatom-interacting marine microorganism
Astrid Gärdes 1 , Eva Kaeppel 1 , Aamir Shehzad 1 , Shalin Seebah 1 , Hanno Teeling 2 , Pablo
Yarza 3 , Frank Oliver Glöckner 2 , Hans-Peter Grossart 4 and Matthias S. Ullrich 1 *
1 Jacobs University Bremen, School of Engineering and Science, Bremen, Germany
2 Max Planck Institute for Marine Microbiology, Microbial Genomics and Bioinformatics
Group, Bremen, Germany
3 Institut Mediterrani d'Estudis Avançats, Marine Microbiology Group, Esporles, Spain
4 IGB-Neuglobsow, Dept. Limnology of Stratified Lakes, Stechlin, Germany
* Corresponding author: Matthias S. Ullrich (m.ullrich@jacobs-university.de)
Keywords: marine heterotrophic bacteria, diatoms, attachment, marine aggregate
formation
______________________________________________________________________
Marinobacter adhaerens HP15 is the type strain of a newly identified marine species,
which is phylogenetically related to M. flavimaris, M. algicola, and M. aquaeolei. It is of
special interest for research on marine aggregate formation because it showed specific
attachment to diatom cells. In vitro it led to exopolymer formation and aggregation of
these algal cells to form marine snow particles. M. adhaerens HP15 is a free-living,
motile, rod-shaped, Gram-negative Gammaproteobacterium, which was originally
isolated from marine particles sampled in the German Wadden Sea. M. adhaerens
HP15 grows heterotrophically on various media, is easy to access genetically, and
serves as a model organism to investigate the cellular and molecular interactions with
the diatom Thalassiosira weissflogii. Here we describe the complete and annotated
genome sequence of M. adhaerens HP15 as well as some details on flagellaassociated
genes. M. adhaerens HP15 possesses three replicons; the chromosome
comprises 4,422,725 bp and codes for 4,180 protein-coding genes, 51 tRNAs and three
rRNA operons, while the two circular plasmids are ~187 kb and ~42 kb in size and
contain 178 and 52 protein-coding genes, respectively.
______________________________________________________________________
Introduction
Strain HP15 (DSM 23420) is the type
strain of the newly established species
Marinobacter adhaerens sp. nov. and
represents one of 27 species currently
assigned to the genus Marinobacter [1].
Strain HP15 was first described by
Grossart et al. in 2004 [2] as a marine
particle-associated, Gram-negative,
gammaproteobacterium isolated from the
German Wadden Sea. The organism is
of interest because of its capability to
specifically attach in vitro to the surface
of the diatom Thalassiosira weissflogiiinducing
exopolymer and aggregate
formation and thus generating marine
snow particles [3]. Marine snow
formation is an important process of the
biological pump, by which atmospheric
carbon dioxide is taken up, recycled, and
partly exported to the sediments. This
sink of organic carbon plays a major role

for marine biogeochemical cycles [4].
Several studies reported on the
formation and properties of marine
aggregates [5–8]. Although it was shown
that heterotrophic bacteria control the
development and aggregation of marine
phytoplankton [3], specific functions of
individual bacterial species on diatom
aggregation have not been explored thus
far.
A better understanding of the molecular
basis of bacteria-diatom interactions that
lead to marine snow formation is
currently gained by establishing a
bilateral model system, for which M.
adhaerens sp. nov. HP15 serves as the
bacterial partner of the easy-to-culture
diatom, T. weissflogii [3]. Herein, we
present a set of features for M.
adhaerens sp. nov. HP15 (Table 1)
together with its annotated complete
genomic sequence, and a detailed
analysis of its flagella-associated genes.
Classification and features
M. adhaerens sp. nov. strain HP15 is a
motile, Gram-negative, non-sporeforming
rod (Figure 1). Based on its
16S rRNA sequence, strain HP15 was
affiliated to the Marinobacter genus of
Gammaproteobacteria. Two other
Marinobacter species were identified
based on their interactions with
eukaryotes - M. algicola isolated from
dinoflagellate cultures [9] and M.
bryozoorum derived from Bryozoa [10].
Strain HP15’s 16S rRNA gene is most
closely related to those of the type
strains of M. flavimaris (99%), M.
salsuginis (98%) and M. algicola (96%).
These four type strains form a discrete
cluster in the phylogenetic tree
(Figure 2). In contrast, DNA-DNA
hybridization experiments revealed that
the genome of M. adhaerens sp. nov.
HP15 showed about 64% binding to that
of M. flavimaris [1], which is below the
generally accepted species
differentiation limit of 70% [11].
Figure 1. Transmission electron micrograph of
M. adhaerens sp. nov. strain HP15.
Chemotaxonomy
Strain HP15 can grow in artificial sea
water with a nitrogen-to-phosphorus ratio
of 15:1 supplemented with glucose as
the sole carbon source. In presence of
diatom cells but without glucose, HP15
utilized diatom-produced carbohydrates
as sole source of carbon. Furthermore,
M. adhaerens sp. nov. HP15 differed
from M. flavimaris and other
Marinobacter species in a number of
chemotaxonomic properties, such as
utilization of glycerol, fructose, lactic
acid, gluconate, alanine, and glutamate
[1]. Additionally, strain HP15 showed a
unique fatty acid composition pattern.
Genome sequencing and annotation
Genome project history
M. adhaerens HP15 was selected for
sequencing because of its phylogenetic

position, its particular feature as a
diatom-interacting marine organism [3],
and its feasible genetic accessibility to
act as a model organism. The respective
genome project is deposited in the
Genome OnLine Database [17] and the
complete genome sequence in
GenBank. The main project information
is summarized in Table 2.
Growth conditions and DNA isolation
M. adhaerens sp. nov. HP15 was grown
in 100 ml Marine Broth medium [18] at
28°C. A total of 23 !g DNA was isolated
from the cell paste using a Qiagen
DNeasy Blood and Tissue Kit (Qiagen,
Hilden, Germany) according to the
manufacturer’s instructions.
Genome sequencing and assembly
The Marinobacter adhaerens sp. nov.
HP15 genome was sequenced at
AGOWA (AGOWA GmbH, Berlin,
Germany) using the 454 FLX Ti platform
of 454 Life Sciences (Branford, CT,
USA). The sequencing library was
prepared according to 454’s instructions
from genomic M. adhaerens sp. nov.
HP15 DNA with a final concentration of
153 ng/!l. Sequencing was carried out
on a quarter of a 454 picotiterplate,
yielding 258.645 reads with an average
length of 405 bp, totaling to almost
105 Mb. These reads were assembled
using the Newbler assembler version
2.0.00.22 (Roche), resulting in 253.285
fully and 4.763 partially assembled
reads, leaving 932 singletons, 226
Figure 2. Maximum likelihood phylogenetic tree based on 16S rRNA sequences of M. adhaerens type
strain (HP15) plus all type strains of the genus Marinobacter and the type species of the neighbor order
Pseudomonadales. Sequence selection and alignment improvements were carried out using the Living
Tree Project database [12] and the ARB software package [13]. The tree was inferred from 1,531
alignment positions using RAxML [14] with GTRGAMMA model. Support values from 1,000 bootstrap
replicates are displayed above branches if larger than 50%. The scale bar indicates substitutions per
site.

epeats and 371 outliers. The assembly
comprised 112 contigs, with 40
exceeding 500 bp. The latter comprised
more than 4.6 Mb, with an average
contig size of almost 116 kb and a
longest contig of more than 1.2 Mb.
Gaps between contigs were closed in a
conventional PCR-based gap closure
approach, resulting in a fully closed
circular chromosome of 4.421.911 bp,
and two plasmids of 187.465 bp and
42.349 bp, respectively. Together all
sequences provided 22.5x coverage of
the genome. The error rate of the
completed genome sequence is about 3
in 1,000 (99.7%).
Genome annotation
Potential protein-coding genes were
identified using GLIMMER v3.02 [19],
transfer RNA genes were identified using
tRNAScan-SE [20] and ribosomal RNA
genes were identified via BLAST
searches [21] against public nucleotide
databases. The annotation of the
genome sequence was performed with
the GenDB v2.2.1 system [22]. For each
predicted gene, similarity searches were
performed against public sequence
databases (nr, SwissProt, KEGG) and
protein family databases (Pfam, InterPro,
COG). Signal peptides were predicted
with SignalP v3.0 [23, 24] and
transmembrane helices with TMHMM
v2.0 [25]. Based on these observations,
annotations were derived in an
automated fashion using a fuzzy logicbased
approach [26]. Finally, the
predictions were manually checked with
respect to missing genes in intergenic
regions and putative sequencing errors,
and the annotations were manually
curated using the Artemis 11.3.2
program and refined for each putative
gene [27].
Genome properties
The genome of strain HP15 comprises
three circular replicons: the 4,422,725 bp
chromosome and two plasmids of
~187 kb and ~42 kb, respectively (Table
3A and Figure 3). The genome
possesses a 56.9% GC content
(Table 3B). Of the 4,482 predicted
genes, 4,422 were protein coding genes,
and 60 RNAs; 391 pseudogenes were
also identified. The majority of the
protein-coding genes (67.5%) were
assigned with a putative function, while
those remaining were annotated as
hypothetical proteins. The distribution of
genes into COGs functional categories is
presented in Table 4.
Table 1b. Classification and general features of M. adhaerens sp. nov. HP15 in
accordance to the MIGS recommendations [15]
MIGS ID Property Term Evidence
code
Domain Bacteria
Phylum Proteobacteria
Class Gammaproteobacteria TAS [16]
Current classification Order Alteromonadales TAS [16]
Family Alteromonadaceae TAS [16]
Genus Marinobacter TAS [1]
Species Marinobacter adhaerens TAS [1]
Type strain HP15 TAS [1]
Gram stain negative IDA

Table 1b. Classification and general features of M. adhaerens sp. nov. HP15 in
accordance to the MIGS recommendations [15]
MIGS ID Property Term Evidence
code
Cell shape rod-shaped IDA
Motility motile, single polar flagellum IDA
Sporulation non-sporulating NAS
Temperature range mesophilic IDA
Optimum temperature 34-38°C IDA
Salinity
0.4-10 g NaCl/l (optimum/growth IDA
within 1 day)
MIGS-22 Oxygen requirement strictly aerobic IDA
Carbon source
dextrin, Tween 40 and 80, pyruvic IDA
acid methyl ester, succinic acid
mono-methyl-ester, cis-aconitic
acid, "-hydroxybutyric acid, #-
hydroxybutyric acid, $-keto glutaric
acid, $-keto valeric acid, D,L-lactic
acid, bromosuccinic acid, L-
alaninamide, D-alanine, L-alanine,
L-glutamic acid, L-leucine and L-
proline
Energy source chemoorganoheterotrophic IDA
MIGS-6 Habitat sea water IDA
MIGS-15 Biotic relationship free-living and particle-associated TAS [2]
MIGS-13 Culture deposition no. DSM 23420 IDA
MIGS-14 Pathogenicity none NAS
Biosafety level 1 NAS
Isolation marine aggregates (0.1-1 mm) TAS [2]
MIGS-4 Geographic location German Wadden Sea TAS [2]
MIGS-4.1 Latitude 53°43’20’’N TAS [2]
MIGS-4.2 Longitude 07°43’20’’E TAS [2]
MIGS-4.3 Depth surface waters TAS [2]
MIGS-4.4 Altitude sea level TAS [2]
MIGS-5 Sample collection time 15 June 2000 TAS [2]
Evidence codes – IDA: inferred from Direct Assay (first time in publication); TAS: Traceable Author
Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e.,
not directly observed for the living, isolated sample, but based on a generally accepted property of
the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project
[17]. If evidence code is IDA, then the property was directly observed for a live isolate by one of the
authors or an expert mentioned in the acknowledgements.

Table 2: Genome sequencing project information for M. adhaerens sp.
nov. HP15
MIGS ID Property Term
MIGS-31 Finishing quality Finished
MIGS-28 Library used 454 pyrosequencing standard library
MIGS-29 Sequencing platforms 454 FLX Ti
MIGS-31.2 Sequencing coverage 22.5x pyrosequencing
MIGS-30 Assemblers Newbler version 2.0.00.22
MIGS-32 Gene calling method GLIMMER v3.02, tRNAScan-SE
Genbank ID
CP001978 (chromosome)
CP001979 (pHP-42)
CP001980 (pHP-187)
Genbank Date of Release September 18, 2010
GOLD ID
Gi06214
NCBI project ID 46089
Database: IMG
Project relevance
pending
Marine diatom-bacteria interactions
Table 3A. Genome composition for M. adhaerens HP15
Label
Size Topology RefSeq ID
(Mb)
Chromosome § 4.423 circular CP001978
Plasmid pHP-187 0.187 circular CP001980
Plasmid pHP-42* 0.042 circular CP001979
§ Number of protein-coding genes: 4,180; Number of protein-coding
genes: 178;
* Number of protein-coding genes: 52

Table 3B. Genome statistics for M. adhaerens HP15
Attribute
Genome (total)
Value % of total a
Genome size (bp) 4,651,725
DNA Coding region (bp) 4,178,502 89.8
DNA G+C content (bp) 2,644,970 56.9
Number of replicons 3
Extrachromosomal elements 2
Total genes b 4,410
tRNA genes 51 1.16
5S rRNA genes 3 0.07
16S rRNA genes 3 0.07
23S rRNA genes 3 0.07
Protein-coding genes 4,355 98.66
Genes assigned to COGs 3,027 67.54
Genes with Pfam domains 2,918 65.1
1 Pfam domain 2,041 45.54
2 Pfam domains 598 13.34
3 Pfam domains 194 4.33
4 or more Pfam domains 85 1.9
Genes with signal peptides 765 17.07
Genes with transmembrane helices 1,043 23.27
1 transmembrane helix 341 7.61
2 transmembrane helices 154 3.44
3 transmembrane helices 72 1.61
4 or more transmembrane helices 476 10.62
Genes in paralogous clusters 570 12.72
Genes with 1 paralog 364 8.12
Genes with 2 paralogs 63 1.41
Genes with 3 paralogs 26 0.58
Genes with 4 or more paralogs 117 2.61
Pseudo/hypothetical genes 391 8.72
Conserved hypothetical genes 668 14.90
Genes for function prediction 3,363 75.03
a) The total is based on either the size of the genome in base pairs or the total number of
protein coding genes in the annotated genome.
b) Also includes 54 pseudogenes and 5 other genes.

Figure 3. Graphical circular maps of the genome and the two plasmids of HP15. From outside
to the center: Genes on forward strand (color by COG categories), Genes on reverse strand
(color by COG categories), RNA genes (tRNAs green, rRNAs red, other RNAs black), GC
content, GC skew.

Table 4. Number of genes associated with the 21 general COG functional
categories
Code Value % of total a Description
J 162 3.7 Translation
A 0 0 RNA processing and modification
K 161 3.6 Transcription
L 132 3 Replication, recombination and repair
B 0 0 Chromatin structure and dynamics
D 32 0.7 Cell cycle control, mitosis and meiosis
Y 0 0 Nuclear structure
V 0 0 Defense mechanisms
T 199 4.5 Signal transduction mechanisms
M 151 3.4 Cell wall/membrane biogenesis
N 166 3.8 Cell motility
Z 0 0 Cytoskeleton
W 0 0 Extracellular structures
U 0 0 Intracellular trafficking and secretion
O
127 2.9 Posttranslational modification, protein
turnover, chaperones
C 192 4.3 Energy production and conversion
G 82 1.9 Carbohydrate transport and metabolism
E 254 5.7 Amino acid transport and metabolism
F 51 1.1 Nucleotide transport and metabolism
H 97 2.2 Coenzyme transport and metabolism
I 141 3.2 Lipid transport and metabolism
P 138 3.1 Inorganic ion transport and metabolism
Q
76 1.7 Secondary metabolites biosynthesis,
transport and catabolism
R 330 7.5 General function prediction only
S 251 5.7 Function unknown
multiple 285 6.4
COGs
3,027 68.6 Total
- 1,383 31.4 Not in COGs
a) The total is based on the total number of protein coding genes in the annotated genome
b) Also includes 54 pseudogenes and 5 other genes.

Flagella-associated gene clusters of
M. adhaerens HP15
Because M. adhaerens HP15 was
experimentally shown to adhere to
diatom cells, gene clusters coding for
secretion, assembly, and mechanistic
function of the polar flagellum were
analyzed in detail (Figure 4). Besides
several other chemotactic mechanisms
and various cell surface interactions,
bacterial flagella and other cell
appendages had previously been shown
to be instrumental for chemotactic
movement towards and adhesion to
biotic surfaces [28, 29]. The amino acid
sequences of proteins encoded by the
three identified gene clusters showed
significant similarities to orthologous and
experimentally well-described gene
products of P. aeruginosa PAO1 and
various other bacterial species as
determined by BLASTP algorithm
comparison using the Blosum 62
substitution matrix [21]. Not surprisingly,
hook and motor switch complex
components were most conserved.
However, gene products involved in
flagellar filament formation encoded by
Cluster II also showed 53 to 78%
similarity to the respective PAO1
proteins. Mutagenesis of flagella-
Figure 4. Schematic presentation of the three flagella-associated gene clusters of M.
adhaerens HP15 coding for the basal body, the filament, and the hook and motor
switch complex. Identities to the respective orthologs in the genome of P. aeruginosa
PAO1 are indicated by gray-scale code. Numbers of CDS are shown below gene
names.

associated genes of M. adhaerens HP15
will be carried out in the near future to
study the role of flagella in bacteriadiatom
interactions and to further our
understanding of the cell-to-cell
communication between those
organisms.
Acknowledgements
We thank Yannic Ramaye for help with
TEM operation and Christian Quast for
computer support. The work was
financially supported by the Max Planck
Society, the Helmholtz Foundation, and
Jacobs University Bremen.
References
[1] Kaeppel EC, Gärdes A, Seebah S,
Grossart HP, Ullrich MS. Marinobacter
adhaerens sp. nov., prominent in aggregate
formation with the diatom Thalassiosira
weissflogii. Int J Syst Evol Microbiol 2010
(Submitted).
[2] Grossart HP, Schlingloff A, Bernhard M,
Simon M, Brinkhoff T. Antagonistic activity of
bacteria isolated from organic aggregates of
the German Wadden Sea. FEMS Microbiol
Ecol 2004; 47:387-396. doi:10.1016/S0168-
6496(03)00305-2
[3] Gärdes A, Iversen M, Grossart H,
Passow U, Ullrich MS. Diatom-associated
bacteria are required for Thalassiosira
weissflogii aggregation. J Int Soc Mic Ecol
2010, in press.
[4] Fowler SW, Knauer GA. Role of large
particles in the transport of elements and
organic compounds through the oceanic
water column. Prog Oceanogr 1986; 16:147-
194. doi:10.1016/0079-6611(86)90032-7
[5] Alldredge AL, Silver MW. Characteristics,
dynamics and significance of marine snow.
Progr Oceanogr 1988; 20:41-82.
doi:10.1016/0079-6611(88)90053-5
[6] Passow U. Transparent exopolymer
particles (TEP) in aquatic environments.
Progr Oceanogr 2002; 55:287-333.
doi:10.1016/S0079-6611(02)00138-6
[7] Simon M, Grossart HP, Schweitzer B,
Ploug H. Microbial ecology of organic
aggregates in aquatic ecosystems. Aquat
Microb Ecol 2002; 28:175-211. IDS number:
574DB
[8] Verdugo P, Alldredge AL, Azam F,
Kirchman DL, Passow U, Santschi PH. The
oceanic gel phase: a bridge in the DOM-
POM continuum, p. 67-85. Symposium on
New Approaches in Marine Organic
Biogeochemistry held in Honor of the Life
and Science of John I Hedges. 2003;
Elsevier Science Bv.
doi:10.1016/j.marchem.2004.06.017
[9] Green DH, Bowman JP, Smith EA,
Gutierrez T, Bolch CJS. Marinobacter
algicola sp. nov., isolated from laboratory
cultures of paralytic shellfish toxin-producing
dinoflagellates. Int J Syst Evol Microbiol
2006; 56:523-527.
doi:10.1099/ijs.0.63447-0
[10] Romanenko LA, Schumann P, Rohde
M, Zhukova NV, Mikhailov VV, Stackebrandt
E. Marinobacter bryozoorum sp. nov. and
Marinobacter sediminum sp. nov., novel
bacteria from the marine environment. Int J
Syst Evol Microbiol 2005; 55:143-148.
doi:10.1099/ijs.0.63258-0
[11] Wayne LG, Brenner DJ, Colwell RR,
Grimont PAD, Kandler O, Krichevsky MI,
Moore LH, Moore WEC, Murray RGE,
Stackebrandt E, Starr MP, Truper HG.
Report of the ad hoc committee on
reconciliation of approaches to bacterial
systematics. Int J Syst Evol Microbiol 1987;
37:463. IDS number: K3838
[12] Yarza P, Richter M, Peplies J, Euzéby
J, Amann R, Schleifer K-H, Ludwig W,
Glöckner FO, Rosselló-Móra R. The All-
Species Living Tree Project: a 16S rRNAbased
phylogenetic tree of all sequenced
type strains. /System. Appl. Microbiol/. 2008;

31:241-250.
doi:10.1016/j.syapm.2008.07.001
[13] Ludwig W, Strunk O, Westram R,
Richter L, Meier H, Yadhukumar A, Buchner
T, Lai S, Steppi G, Jobb W, Forster I,
Brettske S, Gerber AW, Ginhart O, Gross S,
Grumann S, Hermann A, Vilbig M, Lenke T,
Ludwig A. ARB: a software environment for
sequence data. /Nucleic Acids Res/ 2004;
32:1363-1371.
doi: 10.1093/nar/gkh293
[14] Stamatakis A. RAxML-VI-HPC:
maximum likelihood-based phylogenetic
analyses with thousands of taxa and mixed
models. Bioinfo 2006; 22:2688.
doi:10.1093/bioinformatics/btl446
[15] Field D, Garrity G, Gray T, Morrison N,
Selengut J, Sterk P, Tatusova T, Thomson
N, Allen MJ, Angiuoli SV. Towards a richer
description of our complete collection of
genomes and metagenomes: the “Minimum
Information about a Genome Sequence”
(MIGS) specification. Nat Biotechnol 2008;
26:541-547.
doi:10.1038/nbt1360
[16] Euzéby JP. List of Bacterial Names with
Standing in Nomenclature: a folder available
on the Internet. Int J Syst Bacteriol 1997;
47:590-592. doi:10.1099/00207713-47-2-590
[17] Lilios K, Mavromatis K, Tavernarakis N,
Kyrpides NC. The Genome On Line
Database (GOLD) in 2007: Status of
genomic and metagenomic projects and their
associated metadata. Nuc Acids Res 2008;
36:475-479. doi:10.1093/nar/gkp848
[18] Zobell, C. E. 1941. Studies on marine
bacteria. I. The cultural requirements of
heterotrophic aerobes. J Mar Res 1941;
4:42-75.
[19] Delcher AL, Bratke KA, Powers EC,
Salzberg SL. Identifying bacterial genes and
endosymbiont DNA with Glimmer. Bioinfo
2007; 23:673-679.
doi:10.1093/bioinformatics/btm009
[20] Lowe TM, Eddy SR. tRNAscan-SE: a
program for improved detection of transfer
RNA genes in genomic sequence. Nuc Acids
Res 1997; 25:955-964. PMID: 9023104
PMCID: PMC146525
[21] Altschul SF, Gish W, Miller W, Myers
EW, Lipman DJ. Basic local alignment
search tool. J Mol Biol 1990; 215:403-410.
doi:10.1016/S0022-2836(05)80360-2
[22] Meyer F, Goesmann A, McHardy AC,
Bartels D, Bekel T, Clausen J, Kalinowski J,
Linke B, Rupp O, Giegerich R, Puhler A.
GenDB--an open source genome annotation
system for prokaryote genomes. Nucl Acids
Res 2003; 31:2187-2195. doi:
10.1093/nar/gkg312
[23] Emanuelsson O, Brunak S, Von Heijne
G, Nielsen H. Locating proteins in the cell
using TargetP, SignalP and related tools.
Nat Protoc 2007; 2:953-971.
doi:10.1038/nprot.2007.131
[24] Nielsen H, Brunak S, Von Heijne G.
Machine learning approaches for the
prediction of signal peptides and other
protein sorting signals. Prot Eng 1999; 12:3-
9. PMID: 10065704
[25] Krogh A, Larsson B, Von Heijne G,
Sonnhammer EL. Predicting transmembrane
protein topology with a hidden Markov
model: application to complete genomes. J
Mol Biol S 2001; 305:567-580.
doi:10.1006/jmbi.2000.4315
[26] Quast C. MicHanThi - design and
implementation of a system for the prediction
of gene functions in genome annotation
projects. Master Thesis 2006 (Available on
request).
[27] Rutherford K, Parkhill J, Crook J,
Horsnell T, Rice P, Rajandream MA, Barrell
B. Artemis: sequence visualization and
annotation. Bioinformatics 2000; 16:944-945.
PMID: 11120685

[28] O'Toole GA, Kolter R. Flagellar and
twitching motility are necessary for
Pseudomonas aeruginosa biofilm
development. Mol Microbiol 1998; 30:295-
304. doi: 10.1046/j.1365-2958.1998.01062.x
[29] Pallen MJ, Matzke NJ. From The Origin
of Species to the origin of bacterial flagella.
Nat Rev Micro 2006; 4:784-790.
doi:10.1038/nrmicro1493

[28] O'Toole GA, Kolter R. Flagellar and
twitching motility are necessary for
Pseudomonas aeruginosa biofilm
development. Mol Microbiol 1998; 30:295-
304. doi: 10.1046/j.1365-2958.1998.01062.x
[29] Pallen MJ, Matzke NJ. From The Origin
of Species to the origin of bacterial flagella.
Nat Rev Micro 2006; 4:784-790.
doi:10.1038/nrmicro1493

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Swimming diameter in mm
18
16
14
12
10
8
6
4
2
0
0 2 4 6 8 10
Time in days
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