Comparative Genomics of Gardnerella vaginalis Strains
Reveals Substantial Differences in Metabolic and
Carl J. Yeoman1, Suleyman Yildirim1, Susan M. Thomas1, A. Scott Durkin2, Manolito Torralba2, Granger
Sutton2, Christian J. Buhay3, Yan Ding3, Shannon P. Dugan-Rocha3, Donna M. Muzny3, Xiang Qin3,
Richard A. Gibbs3, Steven R. Leigh4, Rebecca Stumpf4, Bryan A. White1,5, Sarah K. Highlander3,6, Karen E.
Nelson2, Brenda A. Wilson1,7*
1Institute for Genomic Biology, University of Illinois, Urbana, Illinois, United States of America, 2J. Craig Venter Institute, Rockville, Maryland, United States of America,
3Human Genome Sequencing Center, Baylor College of Medicine, Houston, Texas, United States of America, 4Department of Anthropology, University of Illinois, Urbana,
Illinois, United States of America, 5Department of Animal Sciences, University of Illinois, Urbana, Illinois, United States of America, 6Department of Molecular Virology and
Microbiology, Baylor College of Medicine, Houston, Texas, United States of America, 7Department of Microbiology, University of Illinois, Urbana, Illinois, United States of
Background: Gardnerella vaginalis is described as a common vaginal bacterial species whose presence correlates strongly
with bacterial vaginosis (BV). Here we report the genome sequencing and comparative analyses of three strains of G.
vaginalis. Strains 317 (ATCC 14019) and 594 (ATCC 14018) were isolated from the vaginal tracts of women with symptomatic
BV, while Strain 409-05 was isolated from a healthy, asymptomatic individual with a Nugent score of 9.
Principal Findings: Substantial genomic rearrangement and heterogeneity were observed that appeared to have resulted
from both mobile elements and substantial lateral gene transfer. These genomic differences translated to differences in
metabolic potential. All strains are equipped with significant virulence potential, including genes encoding the previously
described vaginolysin, pili for cytoadhesion, EPS biosynthetic genes for biofilm formation, and antimicrobial resistance
systems, We also observed systems promoting multi-drug and lantibiotic extrusion. All G. vaginalis strains possess a large
number of genes that may enhance their ability to compete with and exclude other vaginal colonists. These include up to
six toxin-antitoxin systems and up to nine additional antitoxins lacking cognate toxins, several of which are clustered within
each genome. All strains encode bacteriocidal toxins, including two lysozyme-like toxins produced uniquely by strain 409-
05. Interestingly, the BV isolates encode numerous proteins not found in strain 409-05 that likely increase their pathogenic
potential. These include enzymes enabling mucin degradation, a trait previously described to strongly correlate with BV,
although commonly attributed to non-G. vaginalis species.
Conclusions: Collectively, our results indicate that all three strains are able to thrive in vaginal environments, and therein
the BV isolates are capable of occupying a niche that is unique from 409-05. Each strain has significant virulence potential,
although genomic and metabolic differences, such as the ability to degrade mucin, indicate that the detection of G.
vaginalis in the vaginal tract provides only partial information on the physiological potential of the organism.
Citation: Yeoman CJ, Yildirim S, Thomas SM, Durkin AS, Torralba M, et al. (2010) Comparative Genomics of Gardnerella vaginalis Strains Reveals Substantial
Differences in Metabolic and Virulence Potential. PLoS ONE 5(8): e12411. doi:10.1371/journal.pone.0012411
Editor: Wenjun Li, Duke University Medical Center, United States of America
Received June 5, 2010; Accepted July 22, 2010; Published August 26, 2010
Copyright: ? 2010 Yeoman et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: We would like to acknowledge and thank the National Institutes of Health for funding this project to The J. Craig Venter Institute (N01 AI 30071; U54-
AI084844) and Baylor College of Medicine (U54-HG003273; U54-HG004973). The authors also wish to acknowledge the support of the Institute for Genomic
Biology and the Research Board of the University of Illinois at Urbana-Champaign. The funders had no role in study design, data collection and analysis, decision
to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Gardnerella vaginalis is a facultatively anaerobic bacterium of the
Bifidobacteriaceae family. G. vaginalis is often described as a Gram-
variable organism but has a Gram-positive wall type . Although
G. vaginalis commonly occurs in the vaginal microbiota of healthy
individuals [2–4], it has been identified as one of the frequent and
predominant colonists of the vaginal tract in women diagnosed
with bacterial vaginosis (BV) [2,5,6]. The presence of high
numbers of G. vaginalis also correlates with both infertility and
pre-term labor [5,7]. Moreover, BV may increase the risk of
sexually transmitted diseases including HIV . Although the
etiology of BV with respect to G. vaginalis remains poorly
understood, G. vaginalis has been identified in 95% of clinically
diagnosed cases. Recent research indicates that G. vaginalis may be
more virulent than other organisms commonly associated with the
PLoS ONE | www.plosone.org1August 2010 | Volume 5 | Issue 8 | e12411
disease . In addition to BV, G. vaginalis has also been linked to
vertebral osteomyelitis , retinal vasculitis  and acute hip
arthritis . Consequently, G. vaginalis is of significant interest to
both clinicians and researchers.
Several strains of G. vaginalis have been targeted for genome
sequencing as part of the National Institutes of Health (NIH)-
funded Human Microbiome Project (HMP). For two of these
strains, 317 (ATCC 14019) and 409-05, genome sequencing has
been completed, while a draft sequence is available for a third, 594
(ATCC 14018). Strains 317 and 594 were isolated from vaginal
secretions of women suffering from BV . Strain 409-05 was
isolated from a healthy, asymptomatic individual with a Nugent
score of 9 , which is indicative of a BV state . Further
investigation of the microbiome from this individual revealed an
enriched population of G. vaginalis (25% of the microbiome) and a
reduction in the lactobacilli (20%); lactobacilli typically account for
70–90% of the microbiome in a healthy individual . Here we
report the comparative genomic analyses of the two finished
genomes, making comparisons, where possible, to the draft
genome of strain 594. We define and contrast their potential
virulence features, and provide much needed genomic insights into
the metabolic potentials and implicated lifestyle of G. vaginalis.
These analyses show potentially significant differences among
closely-related strains along many important dimensions, raising
critical questions about bacterial pathogenesis and evolution.
Results and Discussion
The genus Gardnerella comprises a single species, G. vaginalis.
Phylogenetic reconstruction of the 16S rDNA shows G. vaginalis
forms a distinct clade within the Bifidobacteraceae family most closely
related to Bifidobacterium coryneforme and B. minimum (Fig. 1). The
16S rDNA of the two G. vaginalis strains isolated from BV patients
appear to be very similar, differing by just a single nucleotide. On
the other hand strain 409-05 appears to be deeper rooting and
shares only 98% 16S rDNA identity, close to the maximum 16S
rDNA-dissimilarity commonly tolerated for strains of a single
species. Given the 16S rDNA difference between strain 409-05
and the other two strains, comparison of their genomes therefore
provides a useful indicator of the potential genetic variability
within G. vaginalis species.
General genome features
The general features of the three G. vaginalis strains are shown in
Table 1. The genome of G. vaginalis strain 409-05 (1,617,545 bp) is
smaller than that of strain 317 (1,667,350 bp), both being
consistent with previous estimates by Lim et al. . Their
genomes are smaller than other sequenced members of the
Bifidobacteriaceae family, which range from 1.9–2.8 Mb. All three of
the G. vaginalis strains appear to have a single chromosomal
genomic architecture, with no evidence of episomal elements. The
two closed genome sequences (from strains 409-05 and 317) have a
standard GC skew, suggesting replication occurs through a typical
bidirectional theta mechanism (Fig. 2). All three genomes have a
low %GC content (41–42%) although there appeared to be some
variation throughout each and in particular the genome of strain
317 (Fig. 2). Strain 317 has 63 more protein encoding genes than
strain 409-05, which is solely accounted for by the larger genome.
Their comparison provides early evidence of a large pangenome
for G. vaginalis, with the strains sharing a core genome of just 846
orthologues and 939 strain-variable genes (949 and 681 genes,
respectively, if just considering the two complete genome
sequences; Fig. 3). Consistent with the 16S rDNA phylogenetic
similarity, the two G. vaginalis strains isolated from BV patients
share more orthologues (n=1120; Fig. 3), and just eight of those
orthologues had less than 97.5% amino acid sequence identity
(Fig. S1 and Tables 2–3). In contrast almost half (46% and 47%,
respectively) of the orthologues shared by strain 409-05 and either
strain 317 and/or 594 have less than 80% amino acid sequence
identity (Fig. S1).
All strains have two ribosomal RNA (rRNA) operons, consistent
with a previous analysis of strains 317, 594 and several other tested
G. vaginalis strains. Strains 409-05 and 317 share the same number
of transfer RNAs (tRNAs; n=45), while 34 are currently evident
in the strain 594 genome. None of the tRNAs found in the three
strains were unique in terms of amino acids transferred or
recognized anti-codons (Table S1).
Whole genome comparisons of strains 409-05 and 317, the two
Gardnerella strains with closed genomes show substantial genomic
rearrangement, including a large ca. 500 kb inversion encompass-
ing 413 genes, including the region encoding the chromosomal
replication initiation protein, DnaA, the rRNA operons, as well as
the replication origin, which is typically proximal to dnaA (Fig. 4).
Evidence presented below suggests both mobile DNA elements
and lateral gene acquisition through inducible competence may
have played a role in the rearrangement and heterogeneity
observed in these genomes.
Competence and indications of lateral gene transfer
All three strains were found to encode the competence-
promoting proteins ComEA, ComEC and CinA (Table S2).
Orthologues of ComEA, ComEC and CinA have been identified
in many Gram-positive (and Gram-negative) bacteria found to be
naturally competent, including Bacillus subtilis , Streptococcus
pneumoniae  Thermoanaerobacter spp.  and Thermus thermophilus
. ComEA and ComEC, have been shown to be involved in the
transport of single-stranded DNA across the membrane, where it is
then bound by CinA-localized RecA (also present in all three
genomes) and integrated into the genome . In characterized
systems knockouts of any one of these genes results in the dramatic
reduction or inhibition of transformability [15,17].
To gain further insight into the potential contributions of lateral
gene transfer (LGT) in shaping the G. vaginalis genomes, we
initially determined the propensity of the genes encoded by each of
the G. vaginalis strains to match the respective codon usage mode of
that genome. As genes that differ from this ‘native’ codon usage
are potential candidates for LGT . Analyses revealed that a
significant number of genes (n=832, 892 and 889 in strains 409-
05, 317 and 594, respectively) did not match the modal codon
usage of their respective genome (p,0.1). Ninety, 93 and 75 of
these, respectively, were genes that typically have a high basal level
of expression and were expected to differ from the mode due to
their optimized codon frequencies. Constructing an axis from the
mode of the overall genome to the mode of the highly expressed
genes, as previously described , we found, in total, just 597,
598 and 570 genes, respectively, that based upon their codon
adaptation were native to the G. vaginalis genomes (p.0.1).
Indicating the remaining 53–56% (661, 723 and 715 genes,
respectively), were potentially foreign to these genomes.
Work by Davis and Olsen , analyzed 923 genomes available
in the SEED database  using their Native codon usage analysis
software; we placed the three G. vaginalis strains within this data set
(Jim Davis, personal communication) and found each strain to be
within the 98thpercentile for the fraction of genes not matching
the native codon axis (Fig. S2) along with Neisseria spp, which are
The Genomics of G. vaginalis
PLoS ONE | www.plosone.org2August 2010 | Volume 5 | Issue 8 | e12411
Figure 1. Bifidobacteriaceae phylogeny. Maximum likelihood 16S rDNA based phylogentic reconstruction of the Bifidobacteriaceae. Bootstrap
values less than 100 are shown at each node. Larger taxonomic clusters have been collapsed for clarity.
The Genomics of G. vaginalis
PLoS ONE | www.plosone.org3 August 2010 | Volume 5 | Issue 8 | e12411
known to be naturally competent . These findings support the
hypothesis that LGT has contributed to the genetic composition of
each of the G. vaginalis genomes. Consistent with this hypothesis
290, 253 and 280 genes, respectively, did not have orthologues in
the completed genomes of Bifidobacterium adolescentis ATCC15703,
B. dentium BD1, B. longum NCC2705 or in the draft genomes of B.
breve DSM 20213 and B. catenulatum DSM16992 suggesting these
genes were either lost in these Bifidobacterial strains, had diverged
significantly or had been acquired by G. vaginalis following their
phylogenetic split in the Bifidobacteriaceae (refer to Fig. 1 and listed
in Table S3). BlastP alignments of each of these genes against the
NCBI non-redundant database revealed the best non-Gardnerella
hits (e-value ,0.1; BBH) to be mostly to human associated isolates
particularly from the genera Lactobacillus (47 genes), Ruminococcus
(12 genes), Rothia (9 genes), Coprococcus (8 genes), and Oribacterium (6
genes) and included common vaginal isolates Atopobium vaginae (57
genes), Lactobacillus iners (27 genes), L. jensenii (3 genes), Mobiluncas
mulieris (12 genes) Peptostreptococcus anaerobius (4 genes), Actinomyces
urogenitalis (3 genes), Anaerococcus lactolyticus (2 genes) and Streptococcus
mitis (2 genes). Several genes also aligned to more distantly-related
members of the Bifidobacteriaceae, including those from the genera
Scardovia (10 genes) and Parascardovia (15 genes) supporting the
former hypothesis that some of these genes had been lost in more
The majority of these genes (67%) were unable to be assigned
an annotated function but additionally included many of the genes
identified as being potentially important to virulence (discussed
below) including vaginolysin (BBH: Streptococcus intermedius), iso-
chorismatase (BBH: Atopobium vaginae), the Rib-family protein
(BBH: Lactobacillus iners), the GA module protein (BBH: Lactobacillus
jensenii), several antibiotic resistance proteins including those
potentially important to methicillin (BBH: Mobiluncus mulieris) and
lantibiotic (BBH: Clostridiales genomosp.) resistances, as well as many
genes potentially important to biofilm formation, adhesion to the
epithelium and nine TA system toxin or antitoxin components. In
addition to natural competence, other potential mechanisms for
LGT exist and in G. vaginalis may include transposon or phage-
mediated gene shuttling through mobile elements.
Accounting for the large 500-kb inversion observed between
strains 409-05 and 317, the first loss of gene synteny between these
strainscorresponds to the insertionof an IS3509a-family transposon
in strain 317. The IS3509a-family compound transposon comprises
21 genes (HMPREF0421_20010–HMPREF0421_20030), flanked
by an annotated IS3509a-family transposase and a recom-
binase. Genes annotated to encode a transcriptional regulator
(HMPREF0421_20023), a toxin-antitoxin (TA) system toxin
(HMPREF0421_20022) and two permeases map within the
transposon; the remaining genes were unable to be assigned a
function. The permeases appear to be specific for the efflux of
multiple drugs (HMPREF0421_20015) and the uptake of colicin
(HMPREF0421_20011), respectively. Orthologues of nine of the
twenty one IS3509a-family transposon genes, including the
transposase and recombinase (open reading frames (ORFs) 433
and 1192, respectively), were identified in strain 594, however
none of the twenty one genes were found in the genome of strain
409-05, suggesting the IS3509a-family transposons acquisition
was a recent event on an evolutionary timescale. Strain 409-05
contains just a single transposase belonging to IS150 family
(HMPREF0424_509; Table S4). Each strain possessed several
bacteriophage-associated genes though most of those present in
either strain 317 or 594 did not have orthologues in strain 409-05
and vice versa.
Toxin-Antitoxin system mediated competitive exclusion
Despite the paucity of transposases, a notable feature of each G.
vaginalis genome is an abundance of TA systems. TA systems
typically comprise two genes; one that encodes a stable toxin that
would be harmful to the host cell if it was not for the expression of
a more labile cognate antitoxin. They are self-promoting as the
loss of either the antitoxin component or both causes the cell to
lose viability, and are often found on mobile elements . Strain
409-05 encodes six TA system toxins, while strains 317 and 594
each encode four. The toxins represent an assortment of TA
system families (Table 2). While these are likely remnants from
prior infection of mobile elements, their retention may provide G.
vaginalis with a competitive advantage over other vaginal colonists
through the production of an assortment of toxins.
Recently, it has been shown TA system toxins can be delivered
to co-inhabitants via a type VI secretion system and effectively
retard their growth . For strain 409-05, all of the toxin genes
are contiguous with those encoding annotated cognate antitoxin
components, while three of the toxins in strains 317 and 594 show
no evidence of a contiguous antitoxin component. Each strain
encodes many more antitoxins than toxins. Strain 409-05 encodes
15 antitoxin components, while strains 317 and 594 each encode
eight. The unpaired antitoxin genes may be retained to enable
resistance to toxins produced by other bacterial strains that are co-
inhabitants of the vaginal biome. Consistent with the hypothesis
that these TA systems are being retained for competitive purposes,
the components are strongly conserved between strains and six
Table 1. General genome features.
Strain 409-05 317 5941
genome sequence (bp)
Genome statusClosedClosed Draft (5x)
% G+ +C content 42 4141
Coding density (%) 8686 82
Average gene length (bp)1,0911,0771,016
Assigned function 9181103935
Conserved hypothetical 141208132
Conserved domain88 1339
1Sequence, and consequently statistics, are incomplete.
2ABC transporters were defined to consist of, at least, a permease and either a
cognate ATP-binding protein or a substrate binding protein.
The Genomics of G. vaginalis
PLoS ONE | www.plosone.org4August 2010 | Volume 5 | Issue 8 | e12411
(five in the BV-isolates) of the TA system genes are clustered within
the G. vaginalis genomes. Collectively, these findings suggest that G.
vaginalis may competitively exclude a wide-range of vaginal co-
inhabitants and is resistant to a larger number of bacterial toxins
than either of the strains produce.
In addition to the TA systems, each strain possesses additional
genes likely to promote competitive exclusion. All three strains
encode two CHAP domain proteins and an ABC-transport system
involved in the secretion of antimicrobials (Table 2). A recent
study of the CHAP domain found it to have a strong lytic ability
. Strain 409-05 uniquely encodes two glycoside hydrolase
(GH) family 25 proteins, a family that exclusively comprises
lysozyme, and two Abi-proteins. Recent analysis of Abi-proteins
has shown they confer resistance to a broad-range of related
bacteriocins . These findings are in agreement with a recent
publication that found G. vaginalis strains produced substances that
were antagonistic to bacterial isolates common to the vaginal
Based on the closed genome sequences, the predicted metabolic
capabilities of strains 409-05 and 317 were modeled and
compared (Fig. 5). This analyses focuses on those with predicted
enzymatic activity and ignores other proteins, including those
involved in substrate transport, which are abundant in all strains
(Table 1). Approximately one third of the genes identified in
strains 409-05 (n=450) and 317 (n=487) (currently strain 594 has
465) were determined to encode enzymes. The majority of these
enzymatic functions were shared by all three genomes (n=429). In
terms of lifestyle, all strains appear capable of catabolizing
glycogen and glucose, the most abundant carbohydrate sources
in the vagina , along with other less abundant carbohydrate
sources such as fructose and starch. All strains were equipped with
numerous (n=13–14) proteases and peptidases that may be
involved in proteolysis for nitrogen (excluding signal peptidases,
pre-pilin peptidases, penicillin-binding protein transpeptidases and
those involved in cellular homeostasis, e.g. FtsH). Each strain is
predicted to be capable of using amino acids and ammonia as
nitrogen sources, but no strain appeared capable of using urea
(Table S5). Major metabolic pathways including glycolysis and the
pentose-phosphate pathway are present, however in contrast to
most Bifidobacteria, both G. vaginalis strains appear to have lost most
of the TCA cycle, retaining only succinate dehydrogenase (EC
18.104.22.168) and malate dehydrogenase (EC 22.214.171.124; Table S7).
A number of differences were observed between the strains
(Fig. 5 and Table S6) in various metabolic pathways including
Figure 2. Genome atlases. Genome atlases of the two G. vaginalis strains with completed genome sequences: 409-05 (left) and 317 (right). From
outside to inside the circles illustrate ORFs of the ‘+’ (1) and ‘2’ (2) strands, GC Skew (3) and % AT (4).
The Genomics of G. vaginalis
PLoS ONE | www.plosone.org5 August 2010 | Volume 5 | Issue 8 | e12411
carbohydrate, amino acid, nucleic acid and vitamin metabolisms.
As genes present in strains 409-05, 317 or both, but not in 594
may be present in the missing genome sequence of strain 594,
those differences will not be discussed in text. Of particular interest
were the differences observed in either or both of the two strains
isolated from patients with symptomatic BV, but not seen in strain
409-05. These included pathways enabling the catabolism of both
galactose and arabinose (Table S7), and enzymes involved in
A particularly significant difference among the strains was the
encoded ability of the two BV-isolates to degrade the N- and O-
glycan portions of glycoproteins (Table 4). Glycoproteins are the
primary component of mucins that are secreted to form the
protective barriers of host mucosal epithelial layers. In contrast,
strain 409-05, isolated from an asymptomatic individual, does not
have this capacity. The presence of genes in the two BV-isolates
involved in the catabolic degradation of N-acetylglucosamine (the
major sugar in glycans) further emphasizes this difference and
suggests that these strains may have a greater propensity to invade
the mucosa and that this may be important to the causation of
symptomatic BV. Previous measures of mucolytic enzymes in the
vaginal cavity show strong correlations with BV [29,30]. The
ability to degrade mucins has been proposed to be a necessary step
in the colonization of vaginal epithelial cells and in the
displacement of the normally dominant Lactobacilli . Further,
there is evidence that the degradation of vaginal mucins impairs
specific-immunoglobulin A immune responses . Given this,
along with the importance of glycoproteins to the integrity, growth
and function of vaginal epithelial cells, disruption of the mucin-
layer may predispose hosts to further complications, such as those
currently associated with BV, including an increased risk of
infection with HIV and other sexually transmitted diseases . All
three strains encode glycoproteases (EC 126.96.36.199) suggesting each
can exploit glycoproteins as a nitrogen source, though the BV-
isolates are able to exploit the carbohydrate component. None of
the strains were annotated as encoding enzymes enabling the
utilization of fucose or mannose.
Several common features of virulence are evident in the
genomes of one or more of the G. vaginalis strains including mucin
degradation (described above), cytotoxicity, hemolysis, adhesion to
the epithelium, biofilm production, iron scavenging, and antimi-
crobial resistance (Tables 3 and 4).
Effector protein translocation.
effector protein translocation were identified in all three strains
(Table S8). These genes appear to be randomly distributed
throughout each genome suggesting such a system may exist.
However, as secretion systems are less well elucidated in Gram-
counterparts, the presence or absence of a secretion system is
The ability of G. vaginalis to adhere to
vaginal epithelial cells has been demonstrated . Epithelial
adhesion is typically mediated by pili . Genes encoding both
type I and II pili are present in each strain (Table 3). The presence
of a type IV prepilin peptidase suggests that they may also encode
a type IV Flp pilin/pseudopilin. Although there is no Flp1 pilin
gene apparent in the G. vaginalis genomes, strain 409-05 uniquely
encodes TadE. The tadE gene typically occurs as part of a larger
type IV pilin-related gene cluster, and the protein resembles the
domain architecture of some Flp1 pilins. Like the Flp1 pilins,
TadE is post-translationally processed by prepilin peptidases, but
are considered pseudopilins . In Aggregatibacter actinomy-
cetemcomitans (formerly designated Actinobacillus actinomycetemcomi-
tans), TadE has been shown to be important to epithelial adhesion
. The same study found preprocessed TadE to be important
for biofilm formation.
G. vaginalis can form a biofilm . Our
genomic analysis suggests this is, at-least in part, due to
predominantly type II, but also types I and IV glycosyl-
transferases (GTs). Strain 409-05 uniquely encodes eight GTs,
while the two BV-isolates (317 and 594) encode nine GTs that are
likely to be important for the biosynthesis of exopolysaccharide
(EPS) for biofilm formation (Table 3). Five of the GTs appear to be
orthologous among all strains, while three 409-05 GTs showed
little or no sequence similarity to genes of the other two strains. All
GTs present in strain 317 were identified in the available genome
sequence of strain 594 and had 100% amino acid identity. Biofilm
formation also typically involves additional factors, in addition to
those facilitating EPS formation. In Gram-positive bacteria this
commonly includes proteins carrying an LPxTG motif that are
attached to the cell surface by a sortase enzyme. Each of strain
409-05, 317 and 594 are predicted to encode 6, 4 and 4 sortase
enzymes, respectively, and 15, 13 and 12 proteins, respectively,
carrying an LPxTG motif (Table S9). These include a Rib protein
(HMPREF0424_1196, HMPREF0421_21226) and a protein with
two G-related albumin-binding (GA) modules (HMPREF0424_
0399, HMPREF0421_20447 and ORF 683), both which have
further virulence potential (as discussed below), along with two of
the proteins predicted to be involved in the biogenesis of fimbria/
pili (HMPREF0424_1026 and HMPREF_0424_1164), each of
which may additionally contribute to biofilm formation.
vaginalis was first described in 1955  and has since been
attributed to a single protein that is excreted from G. vaginalis cells
during exponential growth . The lytic activity of the toxin is
specific for human erythrocytes, neutrophils and endothelial cells
[34,35]. The 59-kDa hemolysin was first isolated and charac-
A few genes implicated in
to their Gram-negative
Figure 3. Orthologue distribution. Venn diagram showing the
number of orthologues shared between the three strains of G. vaginalis.
The Genomics of G. vaginalis
PLoS ONE | www.plosone.org6 August 2010 | Volume 5 | Issue 8 | e12411
terized by Cauci et al. . The thiol-activated cytolysin of G.
vaginalis strain 594 was subsequently cloned, sequenced (NCBI
accession ACD39461) and characterized by Gelber et al.  and
found it to be a cholesterol-dependent cytolysin (CDC) family
toxin, which the authors designated vaginolysin. Vaginolysin
(HMPREF0424_0103, HMPREF0421_20066 and ORF 992 in
strains 409-05, 317 and 594, respectively) is encoded by all three
G. vaginalis strains, is highly conserved between strains 409-05 and
317 (94% aa ID) and is identical in strains 594 and 317. The
predicted molecular masses (56.7 kDa for 409-05 and 60 kDa for
317 and 594) along with predicted extracellular localizations of
vaginolysin in each strain is consistent with previous observations.
Vaginolysin is also strongly conserved in G. vaginalis strain T11
(95% amino acid identity; NCBI accession number ACD63042
), from which the encoding genes have been sequenced.
Another frequent vaginal inhabitant, Lactobacillus iners, also
possesses a vaginolysin homolog, annotated as perfringolysin O
in the L. iners strain DSM 13335 (NCBI accession number
ZP_05744302, 94% amino acid identity, e-value 26102148). In
addition to vaginolysin, we found evidence for the existence of a
second hemolytic/cytolytic toxin in each strain (HMPREF00424_
0679, HMPREF0421_20593 and 655, for strains 409-05, 317 and
Table 2. TA system components and other competitive exclusion genes.
409-05 (a)317 (b) 594 (c) Product
Orthology (% ID)
HMPREF0424_0078n/a n/aHicB-family TA system antitoxin-/-/-
HMPREF0424_0151 n/an/a Fic-family TA system toxin-/-/-
HMPREF0424_0152 HMPREF0421_21368158 Fic-family TA system antitoxin92/100/92
HMPREF0424_0173n/an/a TA-system toxin-/-/-
HMPREF0424_0174HMPREF0421_21334 1274 TA-system antitoxin98/100/98
HMPREF0424_0193n/an/a RelE-family TA system toxin-/-/-
HMPREF0424_0194n/a n/a RelE-family TA system antitoxin-/-/-
HMPREF0424_0434n/an/aTA system toxin -/-/-
HMPREF0424_0435n/a n/aTA system antitoxin -/-/-
HMPREF0424_0507HMPREF0421_213361275 RelE-family TA system toxin 96/100/96
HMPREF0424_0508 n/a n/aRelE-family TA system antitoxin-/-/-
HMPREF0424_0512 HMPREF0421_21058608 HipB-family TA system antitoxin98/100/98
HMPREF0424_0512HMPREF0421_21057 609 PHD-family TA system antitoxin99/100/99
HMPREF0424_0517HMPREF0421_21055610HigA-family TA system antitoxin 99/100/99
HMPREF0424_0518n/an/aRelB-family TA system antitoxin -/-/-
n/aHMPREF0421_21053 612RelE-family TA system toxin -/100/-
n/a HMPREF0421_21052613 RelE-family TA system toxin-/100/-
HMPREF0424_0564 HMPREF0421_21006 982HicB-family TA system antitoxin 93/100/93
HMPREF0424_0746n/a n/aRelB-family TA system toxin-/-/-
HMPREF0424_0747HMPREF0421_21051614 RelB-family TA system antitoxin 88/100/88
HMPREF0424_1061 n/a n/a RelB-family TA system antitoxin -/-/-
HMPREF0424_1165n/a n/a HipB-family TA system antitoxin-/-/-
HMPREF0424_1226 n/an/a TA system antitoxin -/-/-
n/aHMPREF0421_20168929 Fic-family TA system toxin-/100/-
n/aHMPREF0421_20084106Abortive infection protein -/100/-
Other genes with potential roles in competitive exclusion
HMPREF0424_0416n/a n/aAbi-like protein-/-/-
HMPREF0424_0398HMPREF0421_20542682CHAP domain protein49/100/49
HMPREF0424_1070 HMPREF0421_2092951 CHAP domain protein72/100/71
HMPREF0424_1002n/a n/aLysozyme, LysA-/-/-
HMPREF0424_1190n/an/aGH25 enzyme, LysB-/-/-
HMPREF0424_0641HMPREF0421_2069819SalY-family antimicrobial peptide ABC
transport system, ATP-binding protein
HMPREF0424_0642 HMPREF0421_20699 20SalY-family antimicrobial peptide ABC
transport system, permease
n/a - indicates protein was not identified within the genome.
The Genomics of G. vaginalis
PLoS ONE | www.plosone.org7August 2010 | Volume 5 | Issue 8 | e12411
594 respectively; Table 4). The collective evidence used to
annotate this protein suggested it was best annotated as an
rRNA methyltransferase (see Text S1), but the amino acid
sequence aligns strongly (e-values =2610236–4610242) to the
TIGRFAM TIGR00478, for which two characterized members
were found to be hemolytic (see Text S1).
In addition to hemolytic activity, each of
the G. vaginalis strains appears capable of acquiring iron through at
least two high-affinity iron transporters, consistent with previous
reports . These include an FTR1-family high-affinity iron
transporter and a Tpd-family pathogen-specific lactoferrin-
binding protein thought to function as a second high-affinity
iron transporter (Table 4). In a previous study by Jarosik et al. ,
strains 594 and 317 along with seven other G. vaginalis strains were
found to produce siderophores. In trying to determine the type of
siderophore produced and enzymatic machinery responsible, we
identified isochorismatase (E.C 188.8.131.52; Table 4), an enzyme
involved in the production of the siderophores vibriobactin,
enterochelin and bacillibactin (KEGG pathway KO1053). Other
enzymes important to siderophore biosynthesis proved more
elusive, though two proteins annotated as 3-oxoacyl-[acyl-carrier-
protein] reductases in both the G. vaginalis 594 and 317 genomes
(HMPREF0421_21015 and HMPREF0421_21034 in strain 317
and ORFs 1261 and 880 in strain 594) resemble the 2,3-dihydro-
NC_010943.1), an enzyme involved in the same siderophore
biosynthetic pathway. The genes HMPREF0421_21015 and 1261
also matched strongly to the Hidden Markov Model PRK08220,
that describes 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenases
(e-value =2610247). No orthologue of this enzyme, or other
potential candidate for a 2,3-dihydro-2,3-dihydroxybenzoate
dehydrogenase was identified for strain 409-05.
Strain 409-05 also encodes an oxygen-independent copropor-
phyrinogen III oxidase (HemN), while strain 317 and 594 encode
two. HemN is involved in the breakdown of porphyrins, such as in
hemoglobin or myoglobin, to release iron. Each strain encodes a
Ferric uptake regulator (FUR)-family global iron-binding tran-
scriptional repressor. As Fur-family repressors often regulate genes
involved in iron utilization , we attempted to glean more
information by identifying the DNA elements that bind Fur-family
regulators, namely Fur boxes (see methods). Several FUR-box
candidates were identified (Text S2 and Table S10), though most
occurred in agenic regions, although one Fur-box was identified
downstream of the isochorismatase in both strains 409-05 and 594,
but not in strain 317.
antimicrobial-specific resistance proteins that were identified in
each strain included those conferring resistance to bleomycin
and an unknown lantibiotic. Strains 317 and 594 also encoded
genes promoting resistance to methicillin and aminoglycosides
(Table 4). In Staphylococci, FemAB-family proteins (orthologous to
HMPREF0421_20446 and ORF 954) are involved in the
formation of pentaglycine interpeptide bridges, but have also
been found to be essential to methicillin resistance [39,40].
Although methicillin was not introduced until after strains 317
and 594 were isolated , the precursors of a resistance
mechanism were available. In contrast, aminoglycosides like
streptomycin and neomycin were used to treat vaginal infections
at the time strains 317 and 594 were isolated . These
Table 3. Genes potentially important to biofilm formation and epithelial adhesion.
409-05 (a)317 (b) 594 (c)Product
Orthology (% ID)
EPS production and Biofilm formation
HMPREF0424_0402 HMPREF0421_20545685 Family 1 glycosyltransferase90/100/88
HMPREF0424_1181n/an/aFamily 1 glycosyltransferase -/-/-
HMPREF0424_0821HMPREF0421_20842 565Family 2 glycosyltransferase81/100/81
HMPREF0424_1138HMPREF0421_20434 184 Family 2 glycosyltransferase92/100/92
HMPREF0424_1180n/a n/a Family 2 glycosyltransferase-/-/-
HMPREF0424_1189n/a n/aFamily 2 glycosyltransferase-/-/-
n/a HMPREF0421_20405 535Family 2 glycosyltransferase -/100/-
n/a HMPREF0421_20407537 Family 2 glycosyltransferase -/100/-
n/a HMPREF0421_20412 540Family 2 glycosyltransferase -/100/-
n/a HMPREF0421_20413541/1237 Family 2 glycosyltransferase-/100/-
HMPREF0424_1083HMPREF0421_20944262 Family 4 glycosyltransferase96/100/96
HMPREF0424_0590HMPREF0421_20996 224Glycosyltransferase (unclear family) 57/100/61
Fimbria/Pili biogenesis (Epithelial adhesion)
HMPREF0424_1026HMPREF0421_21115749 Type-I fimbrial major subunit precursor61/100/61
n/aHMPREF0421_20500 n/aType-I fimbrial major subunit precursor -/-/-
HMPREF0424_1164HMPREF0421_212041121 Type-II fimbrial major subunit precursor43/100/44
HMPREF0424_0378HMPREF0421_21089 774Type IV prepillin peptidase 52/100/52
HMPREF0424_0125n/a n/aTadE-like protein-/-/-
n/a - indicates protein was not identified within the available genome sequence.
The Genomics of G. vaginalis
PLoS ONE | www.plosone.org8August 2010 | Volume 5 | Issue 8 | e12411
strains each encode an aminoglycoside phosphotransferase
(HMPREF0421_20507 and ORF 1079), related to that of the
more modern nosocomial pathogen Stenotrophomonas maltophilia (e-
value 2610227) . The aminoglycoside phosphotransferase of
S. maltophilia, and a number of other organisms, has been shown
to significantly increase resistance to aminoglycosides and work
by inactivating the antibiotics through phosphorylation .
Each strain encoded three multi-drug extrusion transporters, and
strains 317 and 594 encode a heavy metal exporter that appears
to be specific for cadmium (HMPREF0421_21068 and ORF 602,
respectively). Collectively, these efflux transporters potentiate a
much broader antimicrobial tolerance. It is also likely, given the
heterogeneity observed among the G. vaginalis strains, that as a
species, a much broader antimicrobial resistance complement
exists. Like many Gram-positive pathogens, multiple antimi-
crobial resistances are fast becoming a major problem for the
healthcare sector [44,45]. The potential competence-promoting
nature of G. vaginalis strains (Table S2 and discussed above) also
suggests they may have the capacity to rapidly adapt to new
Immune response evasion and mitigation.
responses specific to G. vaginalis may be impaired by the ability
of the organism to evade detection by altering its surface antigens.
Both G. vaginalis strains 409-05 and 317, but not 594 encode a Rib-
protein. Rib proteins belong to the a-like protein (Alp)-family of
Figure 4. Genome Synteny. Overall synteny between G. vaginalis strain 317 (top) and strain 409-05 (bottom). Best Blastn alignments are indicated
by a red (same strand) or blue (opposite strand) line and indicate 40–100% ID (illustrated by the light to dark nature of the lines, respectively) over a
minimum of 125 contiguous bp. ORFs of the ‘+’ (above) and ‘2’ (below) strands are indicated surrounding the strain and contig information.
The Genomics of G. vaginalis
PLoS ONE | www.plosone.org9 August 2010 | Volume 5 | Issue 8 | e12411
pathogens . These proteins elicit protective immunity through
their inter-strain size variability. Size predictions based on the
primary amino acid sequences indicate the Rib protein encoded
by strain 409-05 is more than 2.5 times larger (predicted to be
338 kDa) than that encoded by strain 317 (predicted to be
128 kDa). Each strain may also be capable of further protecting
Figure 5. Metabolic potential. The metabolic pathways of G. vaginalis were mapped based upon genome information using iPath. Common
pathways are shown (green), along with those specific to strain 409-05 (blue), 317 (orange) and 594 (yellow). Those pathways common to the two BV-
isolates are shown in red, while those common to strain 409-05 and 317 are shown in purple. No enzymes were exclusively found in 409-05 and 594.
The Genomics of G. vaginalis
PLoS ONE | www.plosone.org10August 2010 | Volume 5 | Issue 8 | e12411
HMPREF0421_20840 and ORF 567, respectively), as has been
shown in other pathogenic bacteria .
peroxynitrite attack through
Other potential virulence factors.
were identified that have been linked to virulence or pathogenic
potential (Table 4). This includes a gene resembling a major
virulence factor of Listeria monocytogenes. Each strain encodes an
Several other genes
Table 4. Potential virulence genes.
409-05 (a)317 (b) 594 (c) Product
Orthology (% ID)
HMPREF0424_0103 HMPREF0421_20066 992Vaginolysin94/100/94
HMPREF0424_0679HMPREF0421_20593655 rRNA methyltransferase (possible TlyA-family
HMPREF0424_0074 HMPREF0421_20093 98MATE-family multidrug efflux permease71/100/71
163–165 Lantibiotic resistance ABC transporter
n/a HMPREF0421_20446954 Methicillin resistance protein-/100/-
HMPREF0424_0354HMPREF0421_20418 1243Multidrug resistance antiporter 83/100/83
HMPREF0424_1122HMPREF0421_20428 192Multidrug resistance ABC transporter 97/100/95
HMPREF0424_1123 HMPREF0421_20427191 Multidrug resistance ABC transporter94/100/94
HMPREF0424_0217HMPREF0421_20340283 Bleomycin hydrolase91/100/91
n/a HMPREF0421_205071079Aminoglycoside phosphotransferase -/100/-
HMPREF0424_0210 HMPREF0421_20333276 DedA-family protein92/100/92
HMPREF0424_0013 HMPREF0421_201601120 Ferritin92/100/87
HMPREF0424_0160 HMPREF0421_21358168FTR1-family iron permease 80/100/79
HMPREF0424_0161HMPREF0421_21357 169TPD-family pathogen-specific lactoferrin binding
n/a HMPREF0421_20888 n/aOxygen-independent coproporphyrinogen III oxidase-/-/-
HMPREF0424_0852 HMPREF0421_20889823Oxygen-independent coproporphyrinogen III oxidase94/100/88
HMPREF0424_1040 HMPREF0421_20881138 FUR-family transcriptional regulator 83/100/83
HMPREF0424_1242 HMPREF0421_203021223 Isochorismatase family protein97/100/97
n/a HMPREF0421_20186n/a Sialidase A precursor-/-/-
HMPREF0424_0937HMPREF0421_207401141 O-Sialoglycoprotein endopeptidase 95/100/95
HMPREF0424_0939HMPREF0421_20738 1139M22-family glycoprotease 66/100/66
Protection from or evasion of immune response
HMPREF0424_0003HMPREF0421_20840 567Peroxiredoxin 91/91/100
HMPREF0424_1196HMPREF0421_21226n/aRib-family surface protein 76/-/-
Other virulence-related genes
HMPREF0424_0545 HMPREF0421_20447427 GA module protein50/100/50
HMPREF0424_0888HMPREF0421_20630 972NLPA lipoprotein85/100/85
HMPREF0424_1075 HMPREF0421_20934n/aEndothelin-converting enzyme 91/-/-
HMPREF0424_1186n/an/aLicD protein -/-/-
HMPREF0424_1263HMPREF0421_20273114Raf-like phospholipid-binding protein77/100/77
n/an/a237Oxygen-insensitive NADPH nitroreductase, RdxA-/-/-
n/a - indicates protein was not identified within the available genome sequence.
The Genomics of G. vaginalis
PLoS ONE | www.plosone.org11August 2010 | Volume 5 | Issue 8 | e12411
invasion-associated hydrolase that has homology with the L.
monocytogenes p60 (e-value =6610217), an enzyme with murein
hydrolase activity that has been linked to fibroblast invasion,
hepatocyte and macrophage invasion . Other genes of
interest include those that encode a novel large (221 kDa)
extracellular protein with two GA modules (HMPREF0424_
0399, HMPREF0421_20447 and ORF 683)  and, in both
strains 409-05 and 317, a protein that aligns with the endothe-
HMPREF0421_20934; e-value =7610277) from endothelial
cells of the Norwegian rat (Rattus norvegicus) . In eukaryotic
systems ECE activates endothelin-1, a protein responsible for
constricting blood vessels and raising blood pressure . The
role of ECE within G. vaginalis is unclear, but it may help to
increase the availability of heme containing red blood cells.
Strain 409-05 encodes a LicD-family protein (HMPREF0424_
1186). In Streptococcus pneumoniae, the LicD protein has been
shown to have a role in virulence, specifically in cytoadhesion
and mutational inactivation of licD has been shown to decrease
transformation competence . In the same organism, LicD
has also been linked to phosphocholine metabolism . Along
with a Raf-like phospholipid binding protein (HMPREF0424_
1263, HMPREF0421_20473 and ORF114), which is present in
all genomes, LicD may further aid the ability of G. vaginalis to
adhere to epithelial cells.
Gardnerella vaginalis is one of several vaginal organisms whose
presence strongly correlates with BV. Biochemical and physi-
ological analyses demonstrate important virulence features such
as hemolytic activity, epithelial adhesion and biofilm formation
. Our genomic analyses support these findings and provide
detail of the genetic elements involved. We also identify other
features of the G. vaginalis pangenome, such as the ability to
degrade mucin, evade immune detection or resist a broad
spectrum of antimicrobials, which may be important to the role
of G. vaginalis in BV. Overall the two BV-isolates (strains 317
and 594) had a highly similar 16S rDNA sequence and genome
content. In contrast, substantial differences were observed
between the BV strains and 409-05. The BV isolates uniquely
encoded proteins enabling the degradation of mucin and had a
broader group of antibiotic resistance genes including an
aminoglycoside phosphotransferase and the precursors of
methicillin resistance. These genes were absent from strain
409-05, which was isolated from an asymptomatic subject with a
perturbed vaginal microbiome. Compared to the BV isolates,
strain 409-05 encoded more proteins predicted to be involved in
competitive exclusion. It will be interesting moving forward to
further elucidate the G. vaginalis pangenome and determine the
commonality of features like mucin degradation, and to
determine if such features are enriched in those strains isolated
from BV patients. It will also be interesting to determine the
capacity of strain 409-05, and others like it, to cause
symptomatic BV, or if these strains instead play a role in
maintaining a healthy vaginal microbiome through the com-
petitive exclusion of BV-causing isolates. We hope these
findings, along with the newly available genome sequences,
may enhance the efficacy of research into G. vaginalis physiology
and provide insights into the contribution of the organism to the
microbiome of the host, as well as lead to a more complete
understanding of its role in health and disease, in particular its
role in BV.
The isolation of all three Gardnerella vaginalis strains has
previously been reported, along with appropriate human subject
ethics considerations [4,12]. The work reported here does not
describe the isolation of these organisms from any human subject,
nor does it directly involve human participants.
G. vaginalis strain 409-05 was isolated from the vaginal swab of a
healthy female with a high Nugent score of 9 , while strains 317
and 594 were isolated from vaginal secretions of BV patients .
All strains are available from the ATCC-BEI Global Bioresource
SSU rDNA sequences from related species of the Bifidobacteriaceae
family were obtained from Greengenes, where available, or from
Genbank. Sequences were trimmed to remove overhangs and
aligned using ClustalW  without gap penalties to preserve
structural alignments. As Rubrobacter xylanophilus is believed to be one
of the deepest branching species of the Actinobacteria phyla , it
was selected for use as the outgroup. Maximum likelihood trees
wereconstructedwith 500bootstraps usingRaxML-III  andthe
resulting trees were drawn and analysed using phyloXML .
The closed genomes of G. vaginalis strains 409-05 and 317 were
sequenced as part of the NIH-sponsored Human Microbiome
Project (HMP) using 454 pyrosequencing technology and finished
by Sanger-sequencing of a 4 Kb library or PCR-based primer-
walking,respectively. Strain409-05was sequenced to 346coverage
at the J. Craig Venter Institute, while strain 317 was sequenced to
706coverage at the Baylor College of Medicine. Hybrid assemblies
of both Sangar and pyrosequencing data sets were performed using
the Celera v5.1  or Newbler v2.3 (454 Life Sciences, CT, USA)
assemblers, respectively. The assemblies were manually validated
using Consed v19.89 in conjunction with supporting sequence and
scaffold information. The draft sequence of strain 594, available in
145 contigs, was produced by the Stanford Genome Technology
Center and has 56coverage. Each sequence has been deposited in
GenBank  under theaccession
ACGF00000000 and ADNB00000000, respectively.
All genes discussed were manually reannotated using informa-
tion gleaned from the following resources: BlastP  searches of
both the NCBI non-redundant and SwissProt databases; modular
analysis using HMMER 3.0 , CDD  and InterPro scan
; protein localization predictions using Gpos-PLoc ; and
Protein molecular weight predictions, made using the ExPASy
proteomics server (http://ca.expasy.org/tools). Transfer RNAs
were determined using tRNAscan-SE . Orthologues shared
among the G. vaginalis strains were determined using BlastP
searches and defined as the best match with .40% identity over
.70% of the sequence of the largest protein, with exception of the
size-variable Rib protein. A less conservative ID threshold (.30%
identity) was used to determine orthologues to bifidobacterial
species. Enzyme commission (EC) numbers were assigned using
the Kyoto Encyclopedia of Genes and Genomes (KEGG)  and
metabolic pathways were mapped and analyzed using iPath 
(http://pathways.embl.de/). All annotations and analyses were
undertaken with careful consideration of the surrounding
The Genomics of G. vaginalis
PLoS ONE | www.plosone.org12August 2010 | Volume 5 | Issue 8 | e12411
literature and are available in SEED upon request to the authors.
Native codon usage analyses were undertaken as described ,
briefly, codon usage modes were determined as previously
described . An axis beginning at the modal codon usage of
the genome and extending through the point representing the
modal codon usage of genes within the genome that are
orthologous (e-value $161025, .70% coverage of the longest
gene and .20% sequence ID) to genes that have been shown to be
highly expressed in both Escherichia coli K-12 and Methanococcus
maripaludis S2 was then constructed in a 59-dimensional plot where
each dimension represents each of the synonymous codons,
excluding termination codons. Genes found to match any point
along this axis, as determined by the chi-square test (p.0.1; as
described in 9) are defined as native to the genome, those not
matching the mode are defined as foreign. Genome atlases were
created using the GeneWiz browser . The genome synteny
plot was created using ACT . For both genome atlas and
genome synteny analyses the genome sequences were reordered to
begin at the gene encoding DnaA.
the analyses applied to the toxins of G. vaginalis.
Found at: doi:10.1371/journal.pone.0012411.s001 (0.07 MB
Toxin characteristics. Further discussion surrounding
discussion surrounding the methodology and analysis of FUR-
regulatory elements and their proximal genes.
Found at: doi:10.1371/journal.pone.0012411.s002 (0.07 MB
Identification of Fur-regulated genes. More in-depth
determined orthologues sharing each degree of amino acid
sequence identity rounded to the nearest 5%.
Found at: doi:10.1371/journal.pone.0012411.s003 (0.07 MB
Orthologue protein sequence identity. Number of
relative to other bacteria. The number of genes matching the
modal codon usage relative to the number of genes within the
genome of Gardnerella vaginalis (red) is ploted along with 923 other
Found at: doi:10.1371/journal.pone.0012411.s004 (5.39 MB TIF)
Proportion of native genes within Gardnerella vaginalis
identified in each of the G. vaginalis genomes along with the
anticodon recognized and amino acid transferred.
Found at: doi:10.1371/journal.pone.0012411.s005 (0.10 MB
Transfer RNA distribution. The number of tRNAs
identified within the G. vaginalisgenomes whose annotated roles
have been shown to promote competence in other bacteria.
Found at: doi:10.1371/journal.pone.0012411.s006 (0.06 MB
Genes potentially involved in competence. Genes
These genes had no apparent orthologue in the completed
genomes of Bifidobacterium adolescentis ATCC15703, B. dentium BD1,
B. longum NCC2705 or in the draft genomes of B. breve DSM 20213
and B. catenulatum DSM16992. The best non-Gardnerella blast
G. vaginalis genes lacking orthologues in Bifidobacteria.
matches, locus tags/ORF number and annotated function are also
Found at: doi:10.1371/journal.pone.0012411.s007 (0.09 MB
identified within each G. vaginalis genome that are likely to have
been components of mobile elements.
Found at: doi:10.1371/journal.pone.0012411.s008 (0.06 MB
Genes characteristic of mobile elements. Genes
the G. vaginalis genomes that appear to encode functions important
for the utilization of various nitrogen sources.
Found at: doi:10.1371/journal.pone.0012411.s009 (0.08 MB
Nitrogen metabolism genes. Genes identified within
more of the G. vaginalis genomes that are predicted to encode
enzymes whose functions do not appear to be encoded for in the
Found at: doi:10.1371/journal.pone.0012411.s010 (0.09 MB
Strain variable enzymes. Genes identified from one or
or more of the G. vaginalis genomes that appear to encode functions
important to the utilization of various carbohydrates.
Found at: doi:10.1371/journal.pone.0012411.s011 (0.09 MB
Carbohydrate metabolism genes. Genes present in one
translocation. Genes identified within each G. vaginalis strain that
are potentially important to effector protein translocation.
Found at: doi:10.1371/journal.pone.0012411.s012 (0.06 MB
Genes potentially important to effector protein
motifs. All sortase encoding enzymes and proteins carrying
LPxTG motifs within the genomes of the G. vaginalis strains. Note
LPxTG motif-containing proteins are typically attached to the cell
surface by sortase enzymes.
Found at: doi:10.1371/journal.pone.0012411.s013 (0.08 MB
Sortase enzymes and proteins cantaining LPxTG
FUR-box sequences identified within each G. vaginalis strain along
with proximal downstream genes, which may be regulated by the
FUR-family transcriptional regulator.
Found at: doi:10.1371/journal.pone.0012411.s014 (0.08 MB
Potential FUR-regulatory sequences. All identified
The authors wish to thank Jim Davis, Nick Chia and Mengfei Ho for their
advice and technical support.
Conceived and designed the experiments: CJY RAG BAW SKH KEN
BAW. Performed the experiments: CJY SY SMT ASD MT GS CB YD
SDR DM XQ RAG BAW. Analyzed the data: CJY ASD MT GS CB YD
SDR DM XQ BAW. Contributed reagents/materials/analysis tools: CJY
SY SMT SRL RS BAW SKH KEN. Wrote the paper: CJY SY SMT ASD
SRL RS BAW SKH KEN BAW.
1. Harper JJ, Davis GHG (1982) Cell wall analysis of Gardnerella vaginalis
(Haemophilus vaginalis). Int J System Bacteriol 32: 48–50.
2. Fredricks DN, Fiedler TL, Marrazzo JM (2005) Molecular identification of
bacteria associated with bacterial vaginosis. N Engl J Med 353: 1899–1911.
The Genomics of G. vaginalis
PLoS ONE | www.plosone.org13 August 2010 | Volume 5 | Issue 8 | e12411
3. Hyman RW, Fukushima M, Diamond L, Kumm J, Giudice LC, et al. (2005)
Microbes on the human vaginal epithelium. Proc Nat Acad Sci USA 102:
4. Kim TK, Thomas SM, Ho M, Sharma S Reich, Frank CI, et al. (2009)
Heterogeneity of vaginal microbial communities within individuals. J Clin
Microbiol 47: 1181–1189.
5. Menard JP, Mazouni C, Salem-Cherif I, Fenollar F, Raoult D, et al. (2010) High
vaginal concentrations of Atopobium vaginae and Gardnerella vaginalis in women
undergoing preterm labor. Obstet Gynecol 115: 134–140.
6. Patterson JL, Stull-Lane A, Girerd PH, Jefferson KK (2010) Analysis of
adherence biofilm formation and cytotoxicity suggest a greater virulence
potential of Gardnerella vaginalis relative to other bacterial vaginosis-associated
anaerobes. Microbiology 156: 392–9.
7. Mania-Pramanik J, Kerkar SC, Salvi VS (2009) Bacterial vaginosis: a cause of
infertility? Int J STD & AIDs 20: 778–781.
8. Schmid G, Markowitz L, Joesoef R, Koumans E (2000) Bacterial vaginosis and
HIV infection. Sex Transm Infect 76: 3–4.
9. Graham S, Howes C, Dusmuir R, Sandoe J (2009) Vertebral osteomyelitis and
discitis due to Gardnerella vaginalis. J Med Microbiol 58: 1382–1384.
10. Neri P, Salvolini S, Giovannini A, Meriotti C (2009) Retinal vasculitis associated
with asymptomatic Gardnerella vaginalis infection: a new clinical entity. Ocul
Immunol Infamm 17: 36–40.
11. Sivadon-Tardy V, Roux AL, Piriou P, Herrmann JL, Rottman M (2009)
Gardnerella vaginalis acute hip arthritis in a renal transplant recipient. J Clin
Microbiol 47: 264–265.
12. Gardner HL, Dukes CD (1955) Haemophilus vaginalis vaginitis: A newly defined
specific infection previously classified ‘nonspecific’ vaginitis. Am J Ostet Gynecol
13. Nugent RP, Krohn MA, Hiller SL (1991) Reliability of diagnosing bacterial
vaginosis is improved by a standardized method of Gram stain interpretation.
J Clin Microbiol 29: 297–301.
14. Lim D, Trivedi H, Nath K (1994) Determination of Gardnerella vaginalis genome
size by Pulsed-field gel electrophoresis. DNA Res 1: 115–122.
15. Kramer N, Hahn J, Dubnau D (2007) Multiple interactions among the
competence proteins of Bacillus subtilis. Mol Microbiol 65: 454–464.
16. Peterson S, Cline RT, Tettelin H, Sharov V, Morrison DA (2000) Gene
expression analysis of the Streptococcus pneumoniae competence regulons by use of
DNA microarrays. J Bacteriol 182: 6192–6202.
17. Shaw AJ, Hogsett DA, Lynd LR. Natural competence in Thermoanaerobacter and
Thermoanaerobacter species. Appl Environ Microbiol, In press.
18. Friedrich A, Hartsch T, Averhoff B (2001) Natural transformation in mesophilic
and thermophilic bacteria: Identification and characterization of novel, closely
related competence genes in Acinetobacter sp. Strain BD413 and Thermus
thermophilus HB27. Appl Environ Microbiol 67: 3140–3148.
19. Chen I, Dubnau D (2004) DNA uptake during bacterial transformation. Nat
Rev Microbiol 2: 241–249.
20. Davis JJ, Olsen GJ. Characterizing the native codon usage of a genome: an
axis projection approach. Mol Biol Evol, In press.
21. Overbeek R, Begley T, Butler RM, Choudhuri JV, Chuang HY, et al. (2005)
The subsystems approach to genome annotation and its use in the project to
annotate 1000 genomes. Nucleic Acids Res 33: 5691–5702.
22. Snyder LAS, McGowan S, Rogers M, Duro E, O’Farrell EO, et al. (2007) The
repertoire of minimal mobile elements in the Neisseria species and evidence that
these are involved in horizontal gene transfer in other bacteria. Mol Biol Evol
23. Van Melderen L, De Bast MS (2009) Bacterial Toxin-Antitoxin systems: More
than selfish entities? Plos Genet 5: e1000437.
24. Hood RD, Singh P, Hsu F, Gu ¨vener T, Carl MA, et al. (2010) A type VI
secretion system of Pseudomonas aeruginosa targets a toxin to bacteria Cell. Host
Microbe 21: 25–37.
25. Horgan M, O’Flynn G, Garry J, Cooney J, Coffey A, et al. (2009) Phage lysine
LysK can be truncated to its CHAP domain and retain lytic activity against live
antibiotic-resistant Staphylococci. Appl Environ Microbiol 75: 872–874.
26. Kjos M, Snipen L, Salehian Z, Nes IF, Diep DB (2010) The Abi proteins and
their involvement in bacteriocin self-immunity. J Bacteriol, In-press.
27. Teixeira GS, Soares-Branda ˜o KLK, Branco KMGR, Sampaio JLM,
Nardi RMD, et al. Antagonism and synergism in Gardnerella vaginalis isolated
from women with bacterial vaginosis. J Med Microbiol, In press.
28. Rajan N, Cao Q, Anderson BE, Pruden DL, Sensibar J, et al. (1999) Roles of
glycoproteins and oligosaccharides found in human vaginal fluid in bacterial
adherence. Infec Immun 67: 5027–5032.
29. Cauci S, Driussi S, Monte R, Lanzafame P, Pitzus E, et al. (1998) Immunoglobin
A response against Gardnerella vaginalis hemolysin and sialidase activity in
bacterial vaginosis. Am J Obstet Gynecol 178: 511–515.
30. Wiggins R, Crowley T, Horner PJ, Soothill PW, Millar MR, et al. (2000) Use of
5-bromo-4-chloro-3-indolyl-a-D-N-acetylneuraminic acid in a novel spot test to
identify sialidase activity in vaginal swabs from women with bacterial vaginosis.
J Clin Microbiol 38: 3096–3097.
31. Roberton AM, Wiggins R, Horner PJ, Greenwood R, Crowley T, et al. (2005) A
novel bacterial mucinase glycosulfatase is associated with bacterial vaginosis.
J Clin Microbiol 43: 5504–5508.
32. Punsalang AP, Sawyer WD (1973) Role of pili in the virulence of Neisseria
gonorrhoeae. Infect Immun 8: 255–263.
33. Tomich M, Fine DH, Figurski DH (2006) The TadV protein of Actinobacillus
actinomycetemcomitans is a novel aspartic acid prepilin peptidase required for
maturation of the Flp1 pilin and TadE and TadF pseudopilins. J Bacteriol 188:
34. Rottini G, Dobrina A, Forgiarini O, Nardon E, Amirante GA, et al. (1990)
Identification and partial characterization of a cytolytic toxin produced by
Gardnerella vaginalis. Infect Immun 58: 3751–3758.
35. Gelber SE, Aguilar JL, Lewis KL, Ratner AJ (2008) Functional and phylogenetic
characterization of vaginolysin the human-specific cytolysin from Gardnerella
vaginalis. J Bacteriol 190: 3896–3903.
36. Zvirbliene A, Pleckaityte M, Lasicklene R, Kucinskaite-Kodze I, Zvirblis G
(2010) Production and characterization of monoclonal antibodies against
vaginolysin: Mapping of a region critical for its cytotoxic activity. Toxicon 56:
37. Jarosik GP, Land CB, Duhon P, Chandler R, Mercer T (1998) Acquisition of
Iron by Gardnerella vaginalis. Infect Immun 66: 5041–5047.
38. Escolar L, Perez-Martin J, de Lorenzo V (1999) Opening the iron box:
transcriptional metalloregulation by the Fur protein. J Bacteriol 181: 6223–6229.
39. Berger-Ba ¨chi B, Barberis-Maino L, Stra ¨ssle A, Kayser FH (1989) FemA, a host-
mediated factor essential for methicillin resistance in Staphylococcus aureus:
Molecular cloning and characterization. Mol Gen Genet 219: 263–269.
40. Strande ´n AM, Ehlert K, Labischinski H, Berger-Ba ¨chi B (1997) Cell wall
monoglycine cross-bridges and methicilin hypersusceptibility in a femAB null
mutant of methicillin-resistant Staphylococcus aureus. J Bacteriol 179: 9–16.
41. Foster TJ (1983) Plasmid-determined resistance to antimicrobial drugs and toxic
metal ions in bacteria. Microbiol Rev 47: 361–409.
42. Weinberg W (1955) Topical neomycin in cervical and vaginal infections with
special reference to Bacillus proteus infections. Sth Afr Med J 29: 14–16.
43. Okazaki A, Avison MB (2007) Aph(39)-IIc, an aminoglycoside resistance
determinant from Stenotrophomonas maltophilia. Antimicrob Agents Chemother
44. Roe VA (2008) Antibiotic resistance: a guide for effective prescribing in woman’s
health. J Midwifery Womens Health 53: 216–226.
45. Wilcox MH (2009) Future gazing in the management of multiply drug-resistant
Gram-positive infection. J Infect 59: S75–80.
46. Lindahl G, Stalhammer-Carlemalm M, Areschoug T (2005) Surface proteins of
Streptococcus agalactiae and related proteins in other bacterial pathogens. Clin
Microbiol Rev 18: 102–127.
47. Piacenza L, Peluffo G, Alvarez MN, Kelly JM, Wilkinson SR, et al. (2008)
Peroxiredoxins play a major role in protecting Trypanosoma cruzi against
macrophage- and endogenously-derived peroxinitrite. Biochem J 1: 359–368.
48. Sashinami H, Hu D, Li S, Mitsui T, Hakamada K, et al. (2010) Virulence factor
p60 of Listeria monocytogenes modulates innate immunity by inducing tumor
necrosis factor a. FEMS Immun Med Microbiol, In press.
49. Hess J, Gentschev I, Szalay G, Ladel C, Bubert A, et al. (1995) Listeria
monocytogenes p60 supports host cell invasion by and in vivo survival of attenuated
Salmonella typhimurium. Infec Immun 63: 2047–2053.
50. Johansson MU, de Cha ˚teau M, Wikstro ¨m M, Forse ´n S, Drakenberg T, et al.
(1997) Solution structure of the albumin-binding GA module: a versatile
bacterial protein domain. J Mol Biol 266: 859–865.
51. Shimada K, Takahashi M, Tanzawa K (1994) Cloning and functional
expression of endothelin-converting enzyme from the rat endothelial cells.
J Biol Chem 15: 18275–18278.
52. Schmidt M, Kro ¨ger B, Jacob E, Seulberger H, Subkowski T, et al. (1994)
Molecular characterization of human and bovine endothelin converting enzyme
(ECE-1). FEBS Letters 356: 238–243.
53. Zhang JR, Idanpaan-Heikkila I, Fischer W, Tuomanen EL (1999) Pneumococ-
cal licD2 gene is involved in phosphocholine metabolism. Mol Microbiol 31:
54. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, et al. (2007)
Clustal W and Clustal X version 2.0. Bioinformatics 23: 2947–2948.
55. Gao B, Gupta RS (2005) Conserved indels in protein sequences that are
characteristic of the phylum Actinobacteria. Int J Sys Evol Microbiol 55:
56. Stamatakis A, Ludwig T, Meier H (2005) RaxML-III: a fast program for
maximum likelihood-based inference of large phylogenetic trees. Bioinformatics
57. Han MV, Zmasek CM (2009) phyloXML: XML for evolutionary biology and
comparative genomics. BMC Bioinformatics 10: 356.
58. Miller JR, Delcher AL, Koren S, Venter E, Walenz BP, et al. (2008) Aggressive
assembly of pyrosequencing reads with mates. Bioinformatics 24: 2818–2824.
59. Benson DA, Boguski MS, Lipman DJ, Ostell J (1997) GenBank. Nucleic Acids
Res 25: 1–6.
60. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local
alignment search tool. J Mol Biol 215: 403–410.
61. Eddy SR (1998) Profile hidden Markov models. Bioinformatics 14: 755–763.
62. Marchler-Bauer A, Anderson JB, Chitsaz F, Derbyshire ML, DeWeese-Scott C,
et al. (2009) CDD: specific functional annotation with the Conserved Domain
Database. Nuleic Acids Res 37: D205–210.
63. Hunter S, Apweiler R, Attwood TK, Bairoch A, Bateman A, et al. (2009)
InterPro: the integrative protein signature database. Nucleic Acids Res 37:
The Genomics of G. vaginalis
PLoS ONE | www.plosone.org 14August 2010 | Volume 5 | Issue 8 | e12411
64. Shen H, Chou K (2007) Gpos-PLoc: an ensemble classifier for predicting Download full-text
subcellular localization of Gram-positive bacterial proteins. Prot Engineer Des
Select 20: 39–46.
65. Lowe TM, Eddy SR (1997) tRNAscan-SE: a program for improved detection of
transfer RNA genes in genomic sequence. Nucleic Acids Res 25: 955–964.
66. Aoki-Kinoshita KF, Kanehisa M (2007) Gene annotation and pathway mapping
in KEGG. Methods Mol Biol 396: 71–91.
67. Letunic I, Yamada T, Kanehisa M, Bork P (2008) iPath: interactive exploration
of biochemical pathways and networks. Trends Biochem Sci 33: 101–103.
68. Davis JJ, Olsen GJ (2010) Modal codon usage: assessing the typical codon usage
of a genome. Mol Biol Evol 27: 800–810.
69. Hallin PF, Staerfeldt H, Rotenberg E, Binnewies TT, Benham CJ, et al. (2009)
GeneWiz browser: An interactive tool for visualizing sequenced chromosomes.
Stand Genomic Sci 1: 2.
70. Carver TJ, Rutherford KM, Berriman M, Rajandream MA, Barrell BG, et al.
(2005) ACT: the Artemis comparison tool. Bioinformatics 21: 3422–3423.
The Genomics of G. vaginalis
PLoS ONE | www.plosone.org15August 2010 | Volume 5 | Issue 8 | e12411