BACKGROUND
lactobacilli are an abundant and heterogeneous group of lactic acid bacteria which occupies a wide variety of carbohydrate-rich niches ranging from plant and dairy environments to host-associated habitats.
they reside in the oral cavity, gastrointestinal tract , and genitourinary tract  of vertebrates.
lately, their occurrence and activity in the human microbiota as well as their potential biotherapeutic effects have gained substantial interest  <cit> .
the healthy human vagina, for instance, is predominantly colonized by lactobacilli that have a profound impact on the health of women by protecting the host from aberrant urogenital conditions .
lactobacillus crispatus is an important urogenital species that is routinely found in the vaginas of healthy women .
it can account for more than 80% of all vaginal bacteria  <cit>  and is considered to be one of the most active species in a healthy vagina  <cit> .
l. crispatus also contributes to the maintenance of normal vaginal microbiota, while its absence has been associated with a range of vaginal abnormalities, especially bacterial vaginosis  .
strains of l. crispatus are even considered as biotherapeutic agents for reducing recurrent urinary tract infections  and bv in women  and have been shown to inhibit in vitro the growth, viability, and adhesion of uropathogens , suggesting a role for l. crispatus in protecting the vagina from invading pathogens.
specifically, l. crispatus was recently identified to reduce the adhesion of both commensal and pathogenic gardnerella vaginalis to hela cells  <cit> , indicating that competitive exclusion of this bv-associated species could be in key role in the health-promoting effects of l. crispatus.
besides the gut, l. crispatus has been detected in the git of animals.
the species is among the most profuse lactobacilli in the chicken crop  <cit>  and has, for example, been isolated from the stratified squamous epithelium of the non-secreting portion of the horse stomach  <cit>  and the feces of pigs  <cit> .
l. crispatus has also been recovered from human fecal samples  <cit> , but this result is best explained by its presence in oral cavity and rectum  <cit> .
intriguingly, the rectal reservoirs of l. crispatus have been associated with a lower prevalence of bv  <cit> , suggesting the role of rectal l. crispatus in the maintenance of the healthy vaginal flora  <cit> .
recently, the genome sequences of ten l. crispatus strains have become publicly available  <cit> .
the genomes are all about  <dig> – <dig>  mb in size, with a gc content of ~37%.
they possess a large number of trna molecules  and are predicted to encode  <dig> – <dig>  proteins, several of which are of potential importance to vaginal health.
for example, the potential to inhibit harmful microorganisms by direct inhibition through lactic acid, hydrogen peroxide, and bacteriocins or by displacing them through competitive adhesion is supported by the genome annotation data.
in addition, these genomes have verified the phylogenetic position of the species in the lactobacillus delbrueckii clade  <cit> .
out of the ten l. crispatus strains having had their genome defined, nine are vaginal isolates and were sequenced as a part of the human microbiome project  <cit> , including the strain ctv- <dig> that may have a role in the treatment and prevention of bv and ruti .
the remaining genome belongs to the chicken-isolated strain st <dig>  <cit> , known for its strong adherence not only to chicken epithelia but also to buccal and vaginal cells of human origin .
the strain st <dig> was recently also characterized to produce a lactobacillus epithelium adhesin  that displays specific binding to both crop epithelium and epithelial cells from human vagina  <cit> .
thus far, the genome sequences of different l. crispatus strains have been studied separately.
unfortunately, a single genome sequence may not reflect the entire genomic complement of a species or provide an understanding of the biological processes that are peculiar to the species.
instead, better knowledge of the genetic diversity of a bacterial species can be gained by comparative genomics .
for example, comparative genomic analyses have established considerable intraspecies genetic diversity within the l. delbrueckii clade , but have also unraveled specific mechanisms of the host-microbe interaction that are common for all strains of the given species , suggesting species-specific rather than strain-specific host interaction properties.
in the present study, we used comparative genomics to assess the overall genomic similarity of ten l. crispatus strains and defined their core and pan-genome.
this global view on the gene content of l. crispatus provided an accurate account of features associated with vaginal health and represents the first effort to describe the genomic potential of this central urogenital species.
specific focus was placed on the molecular mechanisms governing host-microbe and microbe-microbe interactions.
these mechanisms involve genes encoding or implicated in the production of antimicrobial peptides, adhesion-associated compounds, exopolysaccharide , and s-layer proteins forming a paracrystalline structure on the cell surface .
in addition, l. crispatus ortholog data was compared and contrasted with that of g. vaginalis, a frequent and predominant colonizer of the vagina of women with bv  <cit> , implicated also in the development of the disease  <cit> .
these analyses revealed collective molecular factors in l. crispatus antagonistic to g. vaginalis, such as a counterpart to a g. vaginalis major subunit pilin.
the detected factors provided an explanation for the previously reported ability of l. crispatus to reduce the adhesion of g. vaginalis to host cells  <cit>  and for the inverse association between l. crispatus and g. vaginalis colonization in the vagina  <cit> .
overall, this pan-genome study of l. crispatus broadens our knowledge of this central vaginal colonist and sheds light on the molecular mechanisms by which l. crispatus could prevent bv and protect the vagina from pathogens.
materials and methods
genome entries and strains
all available genome sequences of l. crispatus in public databases as of january  <dig> were included in this work .
in addition, all available genome sequences of g. vaginalis with annotated coding sequences  in their genome files as of may  <dig> were included in the g. vaginalis genome analyses .
to resolve the phylogenetic position of l. crispatus in respective to closely related lactobacilli, genomes of lactobacillus helveticus, lactobacillus acidophilus and bacillus subtilis were downloaded and analyzed together with the l. crispatus genomes.
the set of l. helveticus, l. acidophilus and b. subtilis genomes included in this phylogenetic analysis is listed in additional file  <dig>  the annotated genomes were retrieved in genbank format from genbank  <cit>  or patric  <cit> .
for the draft genomes, supercontigs were preferred, if available.table  <dig>
overview of
l. crispatus
strains, properties and main findings
*annotations are derived from patric.
bv: bacterial vaginosis; ruti: recurrent urinary tract infection; apec: avian pathogenic escherichia coli.
for adhesion assays, g. vaginalis strain  <dig> isolated from a woman with bv  <cit>  and a vaginal lactobacillus crispatus strain ex533959vc <dig> isolated in the scope of the project “the vaginal microbiome: disease, genetics and the environment” of the human microbiome project  <cit>  were used.
reference-based genome scaffolding
the draft genomes of l. crispatus , l. helveticus , and l. acidophilus  were subjected to reference-based genome scaffolding using progressive mauve genome alignment software with default settings  <cit> .
the genome sequences of the strains st <dig>  dpc  <dig>  and ncfm served as references for the l. crispatus, l. helveticus, and l. acidophilus draft genomes, respectively.
the contig order was confirmed through whole genome sequence comparisons that were generated using blastn  <cit> , and visualized using the artemis comparison tool   <cit> .
putative plasmid-derived contigs among l. crispatus genomes were separated from chromosome derived sequence fragments using cbar with default settings  <cit> .
potential plasmid-derived contigs  <dig>  kb or longer were then extracted and aligned to known plasmid sequences using patric’s blastn  <cit> .
contigs that aligned at ≥40% identity over ≥70% of their length were considered as plasmid-derived.
phylogenetic analyses
the organized scaffolds of the  <dig> strains of l. crispatus, l. helveticus, and l. acidophilus were aligned using mauve progressive aligner  <cit> .
fully conserved columns with single nucleotide polymorphism  were extracted with mauve genome alignment software  <cit> , and used for the construction of the phylogenetic tree using phyml with default settings  <cit> .
maximum-likelihood trees were visualized with itol  <cit> .
for correct rooting of the phylogenetic tree, a snp-based phylogenetic tree including the b. subtilis genome as an out-group was constructed using the same approach.
genome re-annotation
in order to ensure the identical quality standards for all the investigated genomes, a functional annotation update was performed for l. crispatus cdss.
additional annotation information for the cdss was obtained with blannotator  <cit> , best blast, rast  <cit> , the automatic annotation server   <cit> , cog functional classification system  <cit> , and by searching the predicted protein products against the pfam database release  <dig>   <cit> .
for blannotator and best blast approach, blastp was run with default parameter settings, and hits that aligned with more than 40% amino-acid identity and 80% coverage threshold were retained.
the rast  <cit>  and kaas  <cit>  and cog  <cit>  annotation was obtained using the services with default settings.
pfam searches were performed locally using the hmmer  <dig>  package  <cit> , relying on the pfam trusted cut-off for the score.
the eps gene clusters were identified by manual examination of the annotation information.
the presence of putative bacteriocin-encoding genes was determined with bagel <dig>  <cit>  with default settings.
to identify genes associated with clustered regularly interspaced short palindromic repeats , cdss were screened for the presence of crispr-associated  protein domains using the hmmscan program from the hmmer  <dig>  package  <cit> .
matches having scores exceeding the trusted cut-off values were considered significant.
cas protein domain models were obtained from the tigrfam database  <cit> .
integration of annotation information was done using in-house perl scripts producing tab-delimited cdss information files.
other bioinformatic analyses included identification of mobile genetic elements and crispr loci.
genomic regions potentially obtained by horizontal gene transfer  were predicted using islandpick, islandpath-dimob and sigi-hmm methods with the help of islandviewer meta-analysis tool with default settings  <cit> .
prophage-like gene-clusters were predicted with prohinder using default parameters  <cit> .
overlapping prophage-like genome regions were merged into single extended regions spanning a given genomic region and manually inspected.
putative crispr loci were identified with pilercr run with default settings  <cit>  and manually adjusted.
megablast   <cit>  was used for similarity searches between crispr-spacer sequences and virus  and plasmid  entries in the genbank database.
only matches showing 100% identity over the complete crispr-spacer were retained.
annotation of proteinaceous adhesion factors
l. crispatus cdss potentially involved in binding to the host were identified by searching the predicted protein sequences against adhesion associated pfams.
adhesion associated pfams were identified by searching the pfam database release  <dig>   <cit>  entries with various keywords related to adhesion, host tissue components, and bacterial surface components, and by manual examination of the literature.
the list of pfam domains is available in additional file  <dig>  in addition, non-adhesion related domains for the selected adhesion-related cdss were detected by searching the protein sequences against pfam release  <dig>  through the pfam website using gathering thresholds greater than or equal to the trusted cut-off.
ortholog prediction
ortholog groups among l. crispatus strains were identified using orthomcl  <cit> .
to estimate the development of the size of the core and pan-genome as a function of the number of sequenced l. crispatus strains, ortholog groups were determined iteratively for an increasing numbers of sequenced genomes.
at each sample size, the analysis was repeated  <dig> times with different random sets of l. crispatus genomes.
orthomcl was run with default settings, except for a percent match threshold of  <dig> and blastp set to print up to  <dig>  alignments.
the protein products of the original cdss were used for the analysis.
the same approach, but without the sampling procedure, was used to define the ortholog groups among g. vaginalis.
because of the draft quality of most of the g. vaginalis genomes, ortholog groups present in ≥ 30 g. vaginalis genomes were considered as core groups.
estimation of l. crispatuspan- and core genome sizes
the estimation of the l. crispatus core and pan-genome sizes was based on the orthomcl results and was performed according to previously described approaches  <cit> .
the core genome was extrapolated by fitting an exponential decaying function y = κ exp + Ω to the median number of core ortholog groups with a weighted least square regression.
in the equation, n is the number of sequenced strains and κ, τ, and Ω are free parameters optimized in the regression analysis.
the Ω describes the estimated core genome size.
the power law y = k nβ was fitted to pan-genome data with a weighted least square regression, where y is the median, n is the number of genomes, and k and β are free parameters.
regression analyses were done using the nls function of the statistical software r  <cit> .
identification of significant enrichment of genes in cog-categories
hypergeometric distribution was used to test the probability of the over-representation of core, strain-specific or variably conserved accessory genes in a given cluster of orthologous groups .
the obtained p-values were subjected to bonferroni adjustment to reduce the number of false positives introduced by multiple hypothesis testing.
only cog categories containing more than  <dig> cdss were included in the analysis.
statistical tests were performed using the statistical software r  <cit> .
identification of antagonistic factors against g. vaginalis
virulence-related g. vaginalis cdss were inferred from a recent comparative genomic analysis  <cit>  and by comparison to the pfam database  <cit> .
the pfam search was done using the hmmersearch program from the hmmer  <dig>  package.
hits were considered significant if their score was above a trusted cut-off value.
virulence-related pfam models were identified based on a literature review.
following the identification of g. vaginalis virulence factors, all the members of their ortholog groups were extracted, an alignment built using muscle with default settings  <cit> , and a hidden markov model  constructed using the hmmbuild command.
the constructed hmms were then searched against the predicted l. crispatus proteomes with the hmmersearch program from the hmmer  <dig>  package in order to identify counterparts.
hits with e-value greater than or equal to  <dig>  were accepted and manually inspected.
detection of enzymes and metabolic pathway reconstructions
using the automatic annotation server kaas  <cit> , l. crispatus cdss were assigned with ec numbers describing enzymatic activity.
each strain’s ability to ferment carbohydrates and synthetize bio-compounds was then tested by matching its ec complement against the sets of ecs of metabolic reactions providing the conversion of a given starting compound to a particular end product.
a route was accepted as intact if at least one match was found for each enzyme-catalyzed reaction.
metabolic routes between two given compounds were retrieved from the fmm server  <cit>  which connects different kegg reference reaction maps  <cit>  and reconstructs metabolic pathways between metabolites.
for the analysis, the amino acids were paired with amino acid synthesis starting materials and with each other; carbohydrates were paired with selected key intermediates of the central carbon metabolism; the selected central carbon metabolism intermediates were paired with pyruvate or pyruvate, acetate and ethanol; and pyruvate was paired with various end products.
the exact list of compound pairs screened is available in additional file  <dig>  to determine pathways encoded by the l. crispatus core genome, the above pathway reconstruction approach was repeated for the core genome-encoded ec complement.
finally, the mode of carbohydrate fermentation was studied based on metacyc pathways for homolactic and heterolactic fermentation  <cit> .
hydrogen peroxide generating enzymes were detected by screening for ec numbers of the enzymes having the compound h2o <dig>  as a product.
adhesion assays
bacteria were grown in supplemented brain heart infusion  containing 2%  gelatin ,  <dig> % yeast extract ,  <dig> % starch  and  <dig> % glucose , for 48 h at 37°c, in 10% co <dig>  bacterial suspensions were collected by centrifugation at  <dig>  g at 4°c for 10 min and washed once with sterile phosphate buffered saline .
bacteria were resuspended in pbs and the optical density at 600 nm  was determined.
correlations between od <dig> and colony forming units  were made prior to the experiments, and the bacterial suspensions were adjusted to 1 × 108 cfus/ml, as optimized before  <cit> .
for the adhesion assays, hela cells  were cultured in dmem supplemented with 10%  fetal bovine serum  and 1 iu penicillin-streptomycin/ml  at 37°c and in 5% co <dig>  cells were cultured in chamber slides  until they reached a density of 2 ×  <dig> cells per well , at 37°c in 5% co <dig>  before the adhesion assays, cells were washed twice with 200 μl of pbs to remove non-adherent cells and fixed with cold 4%  paraformaldehyde  in pbs for 10 min followed by washing three times with pbs.
fab fragments prepared by papain treatment of purified igg against lea protein of l. crispatus st <dig> and flagellum of escherichia coli strain mg <dig> Δfima-h were available from a previous study  <cit> .
fab fragments  in pbs supplemented with 5 mm phenylmethylsulfonyl fluoride  were mixed in independent experiments with g. vaginalis or l. crispatus cells, at room temperature, for 30 min, with rotational agitation at  <dig>  g. mixtures of fab fragments and bacteria or bacteria alone in pbs supplemented with 5 mm pmsf were incubated with pfa-fixed hela cells for 1 hour, at 37°c in 5% co <dig>  each well was carefully washed twice with 200 μl of sterile pbs to remove non-adherent bacteria.
bacterial quantification was done as previously described  <cit> .
briefly, after fixing with methanol, dapi  was added to the wells.
microscopic visualization was performed using an olympus bx <dig> epifluorescence microscope equipped with a ccd camera  and filters capable of detecting the dapi staining .
the number of adherent bacteria in  <dig> randomly chosen microscope fields was determined using image j software .
results were expressed as the bacteria per hela cells, according the mean ± standard deviation of the two independent experiments, with technical duplicates.
the data were analyzed using the student’s t-test with the statistical software package spss  <dig>  .
p-values of less than  <dig>  were considered significant.
RESULTS
general genomic features of l. crispatus
the genome sequences of ten l. crispatus strains were compared and analyzed .
these genomes contain  <dig>  cdss, of which  <dig>   had an assigned role in the original genome file.
after the annotation update,  <dig>  cdss  were functionally classified by at least one of the functional annotation tools.
for each cds, the results of the different protein classification analyses were collected together and analyzed as a group.
the obtained annotations are presented in additional file  <dig>  only one of the genomes  is in one contig whereas the rest are in 5– <dig> super-contigs.
putative plasmid-derived sequences, each with a length of  <dig>  bases or more were identified in three vaginal isolates , the rest having only chromosomal-associated super-contigs.
using conserved genomic synteny, the orientation and order of the chromosomal-associated super-contigs of each draft genome was determined.
analyses of the resulting architecture revealed that genomes were in general collinear  and shared on average ~90% of each other’s content, comparable to conservation ratios seen in lactobacillus johnsonii
<cit> , l. helveticus
<cit> , and l. plantarum
<cit> .
the genomes of the strains 214- <dig> and sj-3c-us were most conserved, with ~97% of their sequences conserved in at least one strain, whereas only roughly 82% and 84% of the genomes of strains st <dig> and fb077- <dig> could be aligned against some other l. crispatus genome .
these data indicate that each assembly presents a near complete chromosome, providing a solid foundation for inter-strain comparisons.figure  <dig>
whole-genome alignment of the
l. crispatus
genomes.
the contigs of the draft genomes were ordered with mauve using the st <dig> genome as a reference.
matching genome regions were identified with blastn and visualized using the artemis comparison tool .
vertical bands represent the blastn matches .
prophinder-predicted prophage-like genomic regions and islandviewer predicted gis are represented as blue boxes on the bottom and red boxes on the top strand of each genome, respectively.
the l. crispatuspan-genome
the microbial pan-genome is defined as the full complement of genes in a species  <cit> .
in total, this set of l. crispatus genomes comprised  <dig>  ortholog groups, including on average  <dig>  orthologs and  <dig>  co-orthologs per group.
this current pan-genome was defined using orthomcl and was almost twice the average number of cdss  and ortholog groups  present in a single l. crispatus strain .
the ortholog group accumulation curve describing the expansion of the pan-genome as a function of genomes added to the analysis fitted well a power law model and was far from saturated , indicating that the total gene pool accessible to the species has not yet been fully captured  <cit>  and suggesting yet-to-be discovered traits in l. crispatus, similar to that what has previously been reported for oenococcus oeni
<cit>  or lactobacillus paracasei
<cit> .
particularly, the regression model  <cit>  revealed an open pan-genome  that grows by at least ten ortholog groups per every additional genome until  <dig> isolates have had their genome defined.figure  <dig>
pan- and core genomes of
l. crispatus
.
development of the pan-  and core  genomes as a function of the number of sequenced l. crispatus strains.
the total number of genes found according to the pan- and core genome analysis is shown for increasing numbers of sequenced genomes.
the dashed lines represent least squares fits to the medians and the r
<dig> describes the suitability of the fit.
the box plots present median , 25th and 75th percentiles , with the data extremes shown by whiskers outside the box.
c) the distribution of core and accessory l. crispatus cdss within cog functional categories.
for each category, the top and bottom bars show the percentage of the assigned core and accessory cdss relative to the entire core and the accessory l. crispatus cdss, respectively.
the proportion of the strain-specific cdss is highlighted  in the accessory bars.
cogs significantly enriched  in core , shared accessory , or strain-specific  cdss are marked next to the cog identifiers.
only cog functional categories with more than  <dig> members are shown.
the cog categories are given in the inset at the bottom of the figure.
d) distribution of ortholog groups at different levels of conservation in each strain.
the orthomcl-defined ortholog groups were classified into different levels of conservation according to the number of strains they were detected in.
ortholog groups found in all the ten genomes represent the current core .
conservation levels are represented by different colors.
the l. crispatuscore genome
the core genome is defined as the orthologous genes present in every strain of a species  <cit> .
we identified the current l. crispatus core genome to be comprised of  <dig>  ortholog groups that were conserved across all the ten analyzed strains.
this common core captured ~57% of the ortholog groups of a given genome, which is slightly less than what orthologous grouping has revealed for another lactobacillus species  <cit> .
based on the examination of the cog functional categories and hypergeometric tests , the core was identified to be significantly enriched with genes belonging to cog categories j , t , and e  .
furthermore, ~10% of the ortholog groups in the core genome could not be assigned with a descriptive functional annotation , and thus may represent proteins with yet-to-be discovered housekeeping functions or other functions relevant to the basic aspects of the biology of the species.
we also predicted the core genome to contain genes encoding features likely to contribute to cell envelope biogenesis, antimicrobial activity, and host-microbe interaction, as illustrated in detail below.
to estimate the number of ortholog groups present in an infinite number of l. crispatus strains, the number of shared ortholog groups found on sequential addition of each new genome sequence was extrapolated by fitting an exponential decaying function to the medians of core genome sizes  <cit> .
as expected, the number of ortholog groups in the core genome initially decreased with the addition of each new genome sequence.
the extrapolation of the curve designated that the core genome plateaus at 1116 ±  <dig> ortholog groups for an infinite number of l. crispatus strains .
thus, the current l. crispatus core genome appears to be almost within the estimated error margin, indicating that the current core is nearly a perfect representation of the final core genome.
however, it should be noted that gaps and sequencing errors in draft assemblies might have affected our estimate  <cit> .
the l. crispatusvariome
we investigated the distribution of the l. crispatus pan-genome by assessing the number of strains sharing a particular ortholog group .
in total,  <dig>  ortholog groups were present in some, but not in all the ten l. crispatus strains, forming the current l. crispatus accessory genome, suggested to provide selective advantages for different strain of a species  <cit> .
the overall composition of the cogs in the core and accessory genomes was mainly similar , the most notable  over-representations of accessory genome-encoded genes being associated with cog categories l  and q .
enrichment in the l and q categories was driven by diversity in strain-specific transposon-associated classes and abc-type multidrug transporters, respectively.
included in the accessory genome were also  <dig>  ortholog groups found only in a single strain.
most of these ortholog groups belonged to the genomes of the strains fb077- <dig> and st <dig> , which also displayed the smallest  and largest  accessory gene pools, respectively.
fewest strain-specific groups were present in the genome of the strain mv-1a-us.
the mean number of the strain-specific ortholog groups found in the l. crispatus dataset was 131 ±  <dig>  which forms a slightly bigger portion of the genome than what comparative analyses have previously detected in another lactobacillus species  <cit>  and less than in some other lactic acid bacteria such as o. oeni
<cit> .
as expected, the strain-specific gene pool is poorly characterized, close to 40% lacking a functional annotation.
interestingly, transposase-related genes accounted for ~25% of all strain-specific genes with an informative functional annotation.
protein homology searches revealed that ~30% of all strain-specific genes had the highest similarity to genes found in other strains of the l. delbrueckii clade .
the species l. helveticus and lactobacillus kefiranofaciens were deduced to be the two most notable reservoirs of genetic variability, providing the best matching targets for about 10% and 5%  of the strain-specific ortholog groups in l. crispatus.
for example, up to 47% of strain-specific ortholog groups in strain sj-3c-us had the top match in l. helveticus.
in addition, more distant lactobacillus species appear to have interacted with l. crispatus.
specifically, the strain st <dig> seems to have received seven strain-specific ortholog groups from lactobacillus salivarius, which is only distantly related to l. crispatus st <dig> but known to exist in the same ecological niche  <cit> .
horizontal gene transfer
hgt is a major force in bacterial evolution and can contribute to the fitness, metabolic versatility, and niche-adaptation of bacteria  <cit> .
for example, genomic islands  harboring genes for carbohydrate utilization reflect to the lifestyle adaptation of lactobacillus plantarum
<cit> .
to determine the presence of gis and potentially horizontally acquired genes, the l. crispatus genomes were interrogated using islandviewer  <cit> .
this analysis identified between  <dig> and  <dig> gis in each genome .
some of these gis agreed with the observed interruptions in the genomic synteny whereas others were conserved , highlighting the imprecision of the prediction methods or indicating the presence of ancient gi acquisition events in l. crispatus.
the total span of gis was longest in l. crispatus 125-2-chn , shortest in the strain jv-v <dig> , and on average ~166 kb in a l. crispatus genome.
based on cog and prophage-cluster analysis, over  <dig> of the total of  <dig>  cdss in the gis encoded phage-related products or transposases, which is not surprising, given that many of the prophage-like genomic regions co-localize with the gis .
in addition to the mobile elements, the gis were found to be rich in metabolism and biosynthesis-related genes.
close to 20% of their gene content was predicted to be involved in sugar metabolism and amino acid biosynthesis, pointing a role for hgt in adaptation of l. crispatus to varying environments.
for example, hgt events may have contributed to acquisition of cellobiose and fructose-specific transport systems as well as genes implicated in sialic acid utilization to certain l. crispatus strains .
on the other hand, the more ancient gene acquisition events in l. crispatus provide an explanation for the observed presence of an additional copy of phosphoketolase genes missing in the closely related l. acidophilus and l. helveticus genomes included in the phylogenetic analysis.
similarly, the investigated l. acidophilus and l. helveticus strains also lacked a gi-associated mannosylglycerate hydrolase encoding genes present in some l. crispatus strains.
moreover, missing from l. acidophilus genomes were also a hydrogen peroxide producing glycolate oxidase  gene that was present in all the l. crispatus and most l. helveticus genomes, further supporting the role of hgt in environmental adaptation.
another hydrogen peroxide producing enzyme, puryvate oxidase, was in contrast predicted to be present in all except three l. crispatus, l. helveticus, and l. acidophilus genomes.
the l. crispatus gis comprised also several putative eps biosynthesis genes in strains st <dig>  125-2-chn, and fb049- <dig>  which is in accordance with the observation that eps gene clusters in lactobacilli often have abnormal gc content  <cit> .
finally,  <dig> strain-specific genes were associated with gis.
most of these were distributed somewhat randomly, but it was also possible to define eight long  gis with considerably many strain-specific genes and probably thus acquired rather recently by hgt.
in three of these gis , the majority of the cdss did not show significant similarities to proteins in the ncbi databases, suggesting a recent acquisition of yet-undiscovered traits.
phages
temperate phages are common in vaginal lactobacilli and can form a potential threat for lactobacillus populations maintaining a healthy vagina .
some studies have even suggested that bacteriophage attack is the causative agent triggering the breakdown of the protective vaginal microbiota during bv  <cit> .
in this study, a total of  <dig> prophage-like regions were identified comprising of  <dig>  cdss and accounting for more than a fifth of the ortholog groups in l. crispatus.
markedly, this fraction of prophage-like ortholog groups in l. crispatus is substantially higher than the 9% reported for l. paracasei
<cit> , indicating a large variation of prophage-related gene contents among different lactobacillus species.
interestingly, the prophage-like clusters were enriched in the nine vaginal isolates of l. crispatus, whereas there was none in the chicken isolate st <dig> , possibly reflecting exposure to phage in the human vagina.
specifically, the strains 125-2-chn, sj-3c-us, 214- <dig>  jv-v <dig>  fb077- <dig>  and fb049- <dig> each contained between one and three prophage-like regions composed mostly of cdss with phage-related or non-informative annotations and with no or limited homology with the genome sequence of other l. crispatus strains.
the remaining three vaginal isolates  carried six candidate prophages, each consisting mostly of orphan cdss with phage-like or non-informative annotations.
sequence analysis of l. crispatus st <dig> genome also revealed a prophage-like region, but this region was rejected, because associated with the strain’s own replication machinery.
overall, the results are in accordance with the high degree of lysogeny, namely 77%, observed for vaginal l. crispatus strains  <cit> .
this suggests that temperate phages are widespread in vaginal lactobacilli and that transduction is an important mechanism for genome evolution in these bacteria.
notably, the lack of common insertion sites between the isolates indicates that various sites of the l. crispatus genomes can serve as targets for phage integration .
crispr/cas-systems
crisprs are a family of dna repeats present in the genomes of many prokaryotes that are responsible for providing acquired immunity to exogenous dna from bacteriophages and plasmids.
this system consists of a set of cas genes and an array of direct repeats separated by intervening sequence spacers derived from the invading dna .
interestingly, distinct types of crispr/cas systems were identified for the vaginal l. crispatus isolates and the chicken isolated st <dig> .
all the vaginal isolates but the strain 125-2-chn were predicted to have several genes that could be classified to belong to the previously described type ii crispr/cas system  <cit> .
analysis of the genome of 125-2-chn also revealed traces of the type ii system, but the presence of universal cas <dig> and cas <dig> core genes and the cas9/csn <dig> signature genes could not be verified, because the region next to csn <dig> is disrupted by a sequencing gap.
nevertheless, the crispr arrays in each of the vaginal strains was composed of direct repeats with an identical consensus sequence of 36 bp and two to six spacer sequences each.
homology searches between the identified spacers and public virus and plasmid sequences did not reveal the putative targets of these systems, which is in line with the previous spacer annotation survey  <cit>  identifying a plasmid or virus target only for 30% of the spacer sequences in lactic acid bacteria.
the lack of identified targets of the l. crispatus spacers points to a pool of not yet sequenced vaginal phages and plasmids.
interestingly, many spacers were identical across several of the vaginal strains , suggesting that these strains may share a recent ancestor or have encountered similar invading genetic elements in their past.
it should be noted that these genome sequences are incomplete and that some spacers and repeats may have remained undetected.
in addition, a type i crispr/cas system  <cit>  was identified in the st <dig> genome comprising eight cas genes and three crispr-arrays composed of direct repeats of 28 bp, and  <dig>   <dig>  and  <dig> spacers.
the repeats were highly similar and resembled a repeat discovered in  <dig> vaginal samples by rho et al.
<cit> .
however, the shortest array was positioned within a 423 bp long putative lcris_ <dig> gene and thus is most likely a false prediction.
similarly to the vaginal isolates, the spacers of these systems did not match any known plasmid or virus sequence.table  <dig>
distribution of cas-proteins in
l. crispatus
the first number in the table cells describes the number of cdss part of the cas-protein associated ortholog group in a specific strain and the second number indicates the number of group members matching the cas-model.
variation in crispr/cas locus in
l. crispatus.
the arrows represent different genes and their orientation within a locus.
orthologous genes are positioned vertically.
the cas genes with conserved function are of the same color, whereas grey describes genes not matching a cas model.
outlines of genes orthologous to some cas gene, but not matching a cas model are color coded according to the ortholog groups.
dashed lines represent contig breaks.
diamonds represent direct repeats and boxes different spacer sequences.
identical spacers are represented by the same color.
the direct repeats of the top nine genomes are identical.
prompted by the observed differences between the prevalence of crispr/cas systems in the vaginal strains and the chicken isolate, the distribution of the cas genes in  <dig> publicly available lactobacillus genomes was tested .
markedly, the type ii crispr/cas system hits were more frequent in vaginal  than in non-vaginal lactobacilli , which suggests that the type ii system could be important in the vaginal environment.
the prevalence of the other types of crispr/cas systems was not significantly different at alpha level  <dig> .
metabolic pathway reconstruction
using the automatic annotation server kaas  <cit> , we were able to assign ec numbers to the members of  <dig>  ortholog groups.
surprisingly, the majority of the enzymes belonged to the core groups , which is somewhat different from the large intra-specific variation present within the metabolic contents of o. oeni
<cit>  or l. paracasei
<cit> .
in accordance with the high number of core genome-encoded enzymes, the in silico reconstruction of l. crispatus metabolic pathways suggests that the strains have a potential to utilize a rather same set of carbohydrates .
the data supports the presence of metabolic routes in each strain for the conversion of a variety of sugars into the key intermediates of the pentose phosphate , embden–meyerhof–parnas , and tagatose-6-phosphate  pathways.
pathways for the conversion of the d-xylulose 5-phosphate and d-fructose  <dig> -bisphosphate into several of their end products were also annotated for nine of the ten strains.
the aforementioned indicates the presence of both embden–meyerhof–parnas and pentose phosphate pathways in the nine strains, which is typical for a heterofermentative species and contradictory to the previous classification of l. crispatus as a homofermentative species  <cit> .
the exception is the strain ctv- <dig> that had only partial pathways for many end product conversions, most likely because of sequencing gaps in the corresponding genomic loci.
no routes were recorded for the conversion of tagatose-6-phosphate pathway intermediate into pyruvate in any of the strains.
interestingly, the data also shows evidence for the presence of strain-specific glycerone conversions in l. crispatus 125-2-chn.
regarding urogenital lifestyle, conserved pathways were annotated for the metabolism glucose and mannose, the former reported to be the major free monosaccharide and the latter a minor constituent of the vaginal fluid  <cit> .
although we did not detect complete routes for the metabolism of glycogen, seven vaginal strains were discovered to carry a gene coding for a type i pullulanase debranching enzyme , which could contribute to the degradation of glycogen.
moreover, l. crispatus core appears to encode a sialic acid utilization regulator  and an o-sialoglycoprotein endopeptidase that could contribute to the hydrolysis of o-sialoglycoproteins in the vaginal mucosa.
notably, the manual examination of the enzyme contents revealed that each strain may generate hydrogen peroxide from pyruvate, of which the former acts as an antimicrobial compound.
we also assessed the range of amino acids that l. crispatus has a potential to synthetize .
based on the in silico analyses of the biosynthetic capabilities, all strains can synthesize seven amino acids either de novo or as derivatives using the same pathways, which is three and four amino acids more than l helveticus dpc  <dig>  <cit>  or l. acidophilus ncfm  <cit> , respectively.
pathways for aspartate biosynthesis were also annotated in nine isolates, excluding the strain ctv- <dig> that did not share this property.
we again speculate that the lack of biosynthesis route for aspartate is rather due to the draft nature of the genome sequence of this strain than a genuine loss.
the other differences in amino-acid synthesis related to nuances in synthesis routes for cysteine, serine, and glycine, which seem to vary between isolates.
overall, the in silico analyses predicted a dependency on external supplies of amino acids for l. crispatus similar to that described for closely related lactobacilli  <cit>  and shows that the strains are rather similar in their biosynthetic power.
moreover, none of the detected conversions was deduced to be strain-specific, further highlighting the similarity.
proteinaceous adhesins
adhesion to host tissue has long been considered an important factor and a prerequisite for the long-term colonization of the human vagina, stimulation of the immune system, and antagonistic activity against harmful pathogens through competitive exclusion  <cit> .
we screened the l. crispatus proteomes for adhesion and host colonization related domains and identified  <dig> proteins governing the ability of l. crispatus to colonize and interact with the host.
these putative adhesins were associated with seven distinct types of adhesion-associated domains belonging to  <dig> ortholog groups of which seven are part of the l. crispatus core genome .
it should be noted, however, that members of the same ortholog group did not necessarily share adhesion domains.
in addition, six strain-specific adhesins were identified, all of which were predicted to be mucus-binding proteins.
interesting examples of the strain-specific adhesins include a sortase-anchored protein  with multiple mucus-binding domains, and lcris_ <dig> being the only member of its ortholog group  with adhesion-associated domains.
one notable core adhesin  was a putative fibronectin/fibrinogen-binding protein fbpa, which has recently been proposed to contribute to the fibronectin-binding properties of lactobacillus iners and to explain the stronger adhesion of l. iners to human fibronectin compared to other species of lactobacillus tested in the study  <cit> .
notably, our data does not support this hypothesis, since the presence of functional fbpa gene in the l. crispatus core genome should have resulted in equal adhesion abilities for the l. crispatus and l. iners strains tested in the study.
markedly, the recently characterized lea protein of l. crispatus st <dig>  <cit>  belonging to lact <dig> was not identified, indicating that this adhesin binds to crop epithelium and epithelial cells from human vagina with some novel domain.
in addition to the aforementioned putative adhesins, l. crispatus was predicted to harbor ~ <dig> putative s-layer protein-encoding genes that could potentially contribute to bacterial adhesion.
however, these predicted s-layer proteins were different from the s-layer proteins of other related lactobacilli reportedly implicated in bacterial adhesion  <cit> .table  <dig>
distribution of adhesion related proteins in
l. crispatus
the first number in the table cells describes the number of cdss part of the ortholog group in a specific strain and the second the number indicates the number of group members having the adhesion or colonization related pfam-domain.
cell wall exopolysaccharide
in the l. crispatus genomes, a highly variable genome region appears to be associated with eps biosynthesis.
this eps gene cluster was observed in eight l. crispatus strains and noted to comprise  <dig> eps biosynthesis genes, five of which were present within each operon .
the five conserved genes were predicted to encode a transcriptional regulator, a polymerization and chain length determination protein, a tyrosine-protein kinase, a protein-tyrosine phosphatase, and the priming glycosyltransferase.
the remaining genes coded for proteins with putative glycosyl transferase functions, indicating that the strains produce epss with different sugar monomers and glycosidic linkages.
markedly, eps gene clusters were not detected in the genomes of l. crispatus jv-v <dig> and 214- <dig> figure  <dig>
variation in eps gene cluster in
l. crispatus
.
the organization and conservation of the exopolysaccharide synthesis regions in l. crispatus.
orthologous genes are represented with the same color and stars indicate genes found in different loci.
dashed lines represent contig breaks in the mv-1a-us and sj-3c-us clusters.
antimicrobial potential in l. crispatus
lactobacillus species can maintain the vaginal ecosystem in a healthy condition by the production of antimicrobial substances such as lactic acid, hydrogen peroxide and bacteriocin-like substances  <cit> .
lactic acid is the main end product of the carbohydrate fermentation in lactobacilli and can contribute to the vaginal acidity and thereby inhibit the colonization and proliferation of harmful micro-organisms in the vagina  <cit> .
the l. crispatus strains studied here appeared to possess between three to four l-lactate dehydrogenases for the conversion of puryvate into lactic acid.
interestingly, one specific ldh locus found in five l. crispatus strains was flanked by a transposase enzyme gene that may affect its expression  <cit> .
we also discovered hydrogen peroxide producing enzymes  in each l. crispatus, which correlates well with the experimental data showing that hydrogen peroxide generation is common among vaginal l. crispatus
<cit> .
using bagel  <cit> , the bacteriocin content of l. crispatus was investigated .
this method was able classify several sets of putative bacteriocin gene clusters in each strain, including at least two regions encoding bacteriolysins .
in addition, regions implicated in the production of class ii bacteriocins were revealed in the vaginal isolates.
a pediocin-like bacteriocin that inhibits the growth of pathogenic listeria and clostridium species  <cit>  was present in five vaginal isolates and all nine encoded a two-component bacteriocin ls <dig> that inhibits the growth of isolates belonging to genera listeria, shigella, and yersinia
<cit> .
notably, the pediocin-like bacteriocin encoding genes were found in the vicinity of cdss encoding proteins harboring a domain for enterocin a immunity.table  <dig>
distribution of predicted bacteriocin related proteins in
l. crispatus
for the bacteriocin-like molecules with pfam-domain, the first number in the table cells describes the number of cdss part of the ortholog group in a specific strain, and the second number indicates the number of group members having the pfam-domain.
helveticin j refers to the antimicrobial molecule first identified in l. helveticus and bacteriocin helveticin j to one identified in l. acidophilus.
antagonistic activities against g. vaginalis
bv is the most common vaginal disorders, affecting up to a third of women  <cit> .
it has been associated with increased risk for preterm birth, urinary tract infections, and hiv infection, and represents a condition in which the normal protective lactobacilli community is replaced by an overgrowth of anaerobic bacteria  <cit> .
although the etiology of bv is not known, g. vaginalis is present in up to 95% of all bv cases  <cit> , indicating that it could have a role in bv.
in our efforts to decipher the genetic basis of the inhibitory actions of the species l. crispatus against g. vaginalis, we performed ortholog grouping of the available g. vaginalis data  and used comparative genomics to identify shared common molecular mechanisms between g. vaginalis and l. crispatus.
importantly, our analyses revealed several components by which l. crispatus could interfere with the attachment of g. vaginalis in the vagina.
firstly, fibronectin-binding could play a role in this process, given that proteins with fivar domains related to hyaluronate or fibronectin-binding were encoded in the core genomes of both g. vaginalis  and l. crispatus .
secondly, searching l. crispatus proteins against the g. vaginalis hmm database suggested another l. crispatus protein , which could play a role in preventing the cell adhesion of g. vaginalis to fibronectin.
intriguingly, this counterpart of the g. vaginalis fivar-proteins was distributed in nine l. crispatus strains, but had no known adhesion domains.
another interesting core orholog group of g. vaginalis was gvag <dig>  many members of this ortholog group contained a bacterial ig-like domain , which is distantly related to the interaction domains, namely fn <dig>  and big_ <dig> , associated with several l. crispatus core adhesins .
moreover, searches against the g. vaginalis hmms revealed two additional l. crispatus adhesins  that could act as counterparts of gvag <dig>  although having mucin-binding domains .
finally, of the three g. vaginalis pilus-encoding gene clusters that were identified based on the pilus-encoding genes listed by yeoman et al.
<cit> , the one associated with most isolates had borderline  counterparts in the l. crispatus core genome.
its major subunit pilin  appears to have two potential antagonists in the l. crispatus core genome encoding a  <dig> -kilodalton protein  and the lea protein .
in addition, the long cds  located next to the major subunit component in the cluster and showing similarity to known adhesins and surface antigens, could be inhibited by the members of the lact <dig> and lact <dig> based on the g. vaginalis hmm searches.
taken together, these findings indicate that l. crispatus could interfere with fibronectin-binding and pilus components of g. vaginalis.
of the other listed virulence-related factors in g. vaginalis
<cit> , the invasion-associated hydrolase , protein with two g-related albumin-binding modules , nlpa lipoprotein , and endothelin-converting enzyme  have potential antagonists encoded by the l. crispatus core based on the g. vaginalis hmm searches.
a noteworthy finding is that the g-related albumin-binding module protein  present in 17 g. vaginalis isolates shared similarity with  <dig> l. crispatus proteins, including all nine fivar-domain associated proteins of the lact <dig> .
adhesion inhibition assays to hela cells
our comparative analysis described several species-wide factors by which l. crispatus could compete with g. vaginalis in the vagina.
for example, the lea protein was identified as a prominent counterpart of one of the g. vaginalis core adhesins and was thereby predicted to participate in the adherence inhibition of this pathogen.
to validate the role of lea in the antagonism against g. vaginalis, the adhesion capacity of a vaginal l. crispatus isolate ex533959vc <dig> and bv-associated g. vaginalis  <dig> to hela cells was tested using the previously described approach  <cit>  with and without the pretreatment with fab fragments prepared against lea  <cit> .
markedly, the anti-lea fab fragments significantly reduced the adhesion level of both bacterial species to hela cells whereas the unrelated anti-flagellum fab fragments showed no inhibitory effect .
the reduction in adherence was most evident for the strain ex533959vc06; the anti-lea fab fragment pretreatment resulting in  <dig> %  and  <dig> %  reduction in adhesion to hela cells compared with the untreated or anti-flagellum fab fragment pretreated bacteria, respectively.
intriguingly, pretreating g. vaginalis  <dig> with the anti-lea fab fragments caused also a significant reduction in adherence compared with the untreated bacterial cells  or bacteria pretreated with the control anti-flagellum fab fragments .
these observations validated the predicted competitive character between lea and g. vaginalis, suggesting a role for lea in the previously identified ability of l. crispatus to exclude and displace g. vaginalis from hela cells  <cit> .
the results also provide an explanation to the inverse association between l. crispatus and g. vaginalis colonization in the vagina  <cit> .
based on our comparative genomic analyses, the lea protein achieves its inhibitory effect by competing with the same attachment sites as the pili of g. vaginalis.
of note, our adhesion assay provided a further support for the species-wide distribution of lea among l. crispatus, since the strain ex533959vc <dig> has not yet been sequenced.
furthermore, since lea has previously been studied only in the chicken isolate st <dig>  <cit> , our results serve as the first record of the functionality of lea in vaginal l. crispatus.figure  <dig>
inhibition of
l. crispatus
or
g. vaginalis
adhesion to hela cells by lea-specific fab fragments.
cells of l. crispatus ex533959vc <dig>  or g. vaginalis  <dig>  were pretreated with lea-specific igg fab fragments or unrelated anti-flagellum fab fragments or left untreated in pbs supplemented with 5 mm pmsf before the adhesion assays.
the number of adherent bacteria per epithelial cell in  <dig> randomly chosen microscopic fields was determined.
the assay was performed twice with duplicate samples and the results show mean values of adherent bacteria.
the asterisk indicates p <  <dig>  as calculated by student’s t test.
phylogenetic relations
phylogentic relations between the selected l. crispatus strains and strains of closely related species l. acidophilus and l. helveticus were examined based on a maximum-likelihood tree built from the snps of the core genome.
altogether  <dig>  conserved polymorphic sites were identified from the genome alignments and used for the construction of a phylogenetic tree.
the phylogenetic tree  clearly shows that strains of the same species cluster together and that each lactobacillus species has differentiated as a distinct entity.
the species l. crispatus and l. helveticus share the most recent common ancestor and form a sister group to species l. acidophilus, which is accordance with previously reported phylogenetic trees  <cit> .
among the l. crispatus cluster, the chicken isolated st <dig> branches off first from the vaginal isolates.figure  <dig>
phylogenetic tree.
phylogenetic relations of the selected l. crispatus , l. helveticus  and l. acidophilus  strains based on the snps of the core genome.
the b. subtilis genome was used as the out-group to root the tree, but is not shown in the figure.
in the inset, the branching pattern of the l. crispatus strains is highlighted.
CONCLUSIONS
the rapidly increasing number of complete microbial genomes offers previously unimaginable possibilities to understand the phenotypic and genomic diversity in a particular species  <cit> .
in this study, we have taken advantage of publicly available l. crispatus genomes and present the genetic landscape of this important urogenital lactic acid bacterium .
we assessed the overall genomic similarity of ten strains and defined the l. crispatus pan- and core genomes.
these analyses depicted high sequence identity and extensive synteny punctuated by several gis, and revealed a current pan-genome that is nearly two times larger than the number of ortholog groups present in an average l. crispatus strain.
about one third of all  <dig>  ortholog groups were assigned to all strains, constituting the current l. crispatus core genome and encoding the basic aspects of l. crispatus biology.
importantly, these core features comprised several cdss for the production of antimicrobial molecules and competitive exclusion of the bv associated species g. vaginalis, shedding light on the molecular mechanisms by which l. crispatus could maintain vaginal health.
the pan-genome analysis also revealed  <dig>  singleton ortholog groups associated with only one strain.
the enrichment of functions related to replication and repair among these genes indicates the influence of transposons in genome evolution in this species.
a third of the strain-specific ortholog groups had the highest similarity to genes found in the other strains of the l. delbrueckii clade, suggesting notable sequence influx from closely related lactobacilli.
our regression analysis indicates that the genetic diversity present within l. crispatus has not yet been comprehensively captured.
specifically, we estimate that over ten new ortholog groups will be discovered per every additional genome until almost 300 l. crispatus strains have had their genomes defined.
this estimation may be compromised by the uncertainty caused by the draft genomes that have up to  <dig> sequence gaps.
nevertheless, the data implies the presence of large repertoires of undiscovered l. crispatus genes to be sequenced in the future.
the phylogenetic tree based on core genome snps among the ten isolates revealed that the chicken isolated st <dig> branches off first from the l. crispatus cluster and that the l. acidophilus cluster is a sister taxon to l. helveticus and l. crispatus, as suggested earlier  <cit> .
from the perspective of vaginal health, the most interesting genomic diversity regions in l. crispatus include the loci related to eps biosynthesis, prophages and adaptive immunity, of which the latter two may play a role in bv.
firstly, the genetic differences in the composition of the eps gene region may participate in the l. crispatus adhesion, biofilm formation and competitive exclusion of pathogens.
the eps-deficient strains jv-v <dig> and 214- <dig> are particularly interesting, as the deprivation of eps has been reported to promote bacterial adhesion in other lactobacilli  <cit> .
secondly, the presence of prophage-like clusters in the vaginal l. crispatus genomes is in accordance with the previously observed  <cit>  high level of lysogeny in vaginal l. crispatus strains.
if truly inducible, the spontaneous release of the prophages could contribute to the development of bv  <cit> .
finally, a relationship was depicted between the life environment of the strains and their adaptive immunity systems, suggesting that different types crispr/cas systems could be beneficial in different environments.
this hypothesis is further supported by the analysis of the cas gene contents of  <dig> lactobacillus genomes that revealed higher rates of the type ii crispr/cas systems in vaginal than in non-vaginal lactobacilli.
in addition, the crispr-arrays of the vaginal l. crispatus strains carry evidence of encounters with common invaders, as several of the spacer sequences were identical between several strains.
the defined l. crispatus core genome helps to explain how this species can thrive in the vaginal environment and benefit vaginal health.
in the vaginal epithelium of reproductive age females, large quantities of glycogen are broken down and then metabolized into lactic acid, which is thought to result in acidification of the vagina  <cit> .
although l. crispatus lacks complete enzymatic machinery for glycogen degradation, the core genome encodes enzymatic pathways for the utilization of a range of carbohydrates available in the vaginal fluid, which could support the urogenital commensal lifestyle of l. crispatus.
encoded in the core are also several features potentially governing host-interactions and displaying an antagonistic activity against other micro-organisms.
interestingly, the bacteriocin-like molecules encoded by the l. crispatus genomes could inhibit biofilm integrated g. vaginalis cells, shown to be more resistant to hydrogen peroxide and lactic acid than the cells in planktonic state  <cit> .
specifically, as g. vaginalis is known to develop an adherent biofilm on the vaginal epithelium in bv  <cit>  this property could provide attractive means to restore the normal vaginal flora.
in addition to the antimicrobial properties, l. crispatus was detected to contain several proteins that could mediate the previously reported  <cit>  competitive exclusion of g. vaginalis from epithelial cells and explain the inverse association between l. crispatus and g. vaginalis colonization in the vagina  <cit>
.
most notably, these specific interference mechanisms might include blocking the attachment of g. vaginalis by disturbing the pilus-mediated adhesion of the pathogen.
this mechanism could involve lea, shown here to be universally present in all l. crispatus strains, and demonstrated using lea-specific fab fragments to inhibit the adhesion of g. vaginalis adhesion to hela cells.
although lea showed sequence similarity to a pilus component of g. vaginalis, further studies are still needed to decipher whether the counterpart of lea is indeed the pilin subunit or some other adhesion associated molecule of g. vaginalis.
in addition, we cannot rule out that surface molecules other than the ones recognized by the anti-lea fab fragments have participated in the contact between g. vaginalis and the host cell, since the fab fragments did not abolish the adhesion completely.
nevertheless, the lea protein appears to be a key mediator of the competitive exclusion of g. vaginalis.
in summary, we have presented a comparative analysis of ten l. crispatus genomes available within the public databases at the time of this study and provided a comprehensive look on the pan-genomic structure of this important urogenital species.
furthermore, our analyses revealed a list of core genes implicated in protecting the urogenital tract from g. vaginalis colonization, providing new insights into the treatment and prevention of bv.
electronic supplementary material
additional file 1:
overview of
g. vaginalis
strains and properties.
in the table, the genomic properties of the g. vaginalis strains used in this study are given.
hmp refers to the human microbiome project.
additional file 2:
list of
l. helveticus, l. acidophilus
and
b. subtilis
genomes included in the phylogenetic analysis.
the accession is given for each genome.
additional file 3:
list of pfam domains used in the annotation of putative
l. crispatus
adhesins.
the accession, id, and description are given for each pfam matching an adhesion or colonization related keyword.
ones in the remaining columns indicate that the pfam matched some l. crispatus cds, passed the manual curation process and was included in the final list of adhesion or colonization related domains.
additional file 4:
the start and end compounds used in the metabolism screens.
this table describes the compound pairs related to the de novo synthesis and interconversion of amino acids and carbohydrate metabolism.
additional file 5:
the
l. crispatus
data table.
results of the different bioinformatic analyses for each l. crispatus cds.
additional file 6:
reservoirs of genetic variability.
this table describes the distribution of best blast hits of strain-specific l. crispatus cdss.
additional file 7:
prevalence of different types of crispr/cas systems in vaginal and non-vaginal lactobacilli.
additional file 8:
variation in metabolism related enzymes in
l. crispatus.
the horizontal lines represent the orthologous groups with assigned ec numbers and the different strains are indicated at the bottom of the picture.
the presence of a given ortholog group in a specific strain is indicated with grey  or dark grey .
the absence of a given ortholog group is indicated with light grey.
the colored bar on the left describes the conservation level of the ortholog groups and follows that of the figure  <dig> .
the dendrogram was generated using ward linkage clustering of the presence/absence data.
additional file 9:
detected metabolic routes between different carbohydrate metabolism compounds and between different amino acid biosynthesis related compounds.
the presence of intact metabolic route from one compound to another is indicated for each l. crispatus genome.
pathways found in the core genome analysis are marked with yellow color.
additional file 10:
domain organization of
l. crispatus
adhesion and colonization factors.
a representative member of each orthomcl-group is presented graphically.
the larger colored blocks represent adhesion or colonization related pfam-domains and the thinner blocks other domains.
the names of each color-coded domain are given at the bottom of the picture.
additional file 11:
overview of
g. vaginalis
ortholog groups.
for each group, the associated proteins, pfam domains, matching proteins in l. crispatus, the total number of matching proteins in l. crispatus, and total number of l. crispatus genomes providing the matches are given in the table.
abbreviations
bvbacterial vaginosis
cascrispr-associated
cdscoding sequence
cfuscolony forming units
cogcluster of orthologous groups
crisprclustered regularly interspaced short palindromic repeat
epsexopolysaccharide
gigenomic island
gitgastrointestinal tract
gutgenitourinary tract
hgthorizontal gene transfer
hmmhidden markov model
lealactobacillus epithelium adhesin
odoptical density
pbsphosphate buffered saline
pfaparaformaldehyde
pmsfphenylmethylsulfonyl fluoride
rutirecurrent urinary tract infection
snpsingle nucleotide polymorphism.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
to designed experiments and the adhesion experiments, analyzed the genomic data and prepared the manuscript.
mk designed experiments, analyzed the genomic data and prepared the manuscript.
jc performed the adhesion assays, analyzed the adhesion data and participated in preparing the manuscript.
nc designed the adhesion experiments, analyzed the adhesion data and participated in preparing the manuscript.
se provided the fab fragments.
bww designed the adhesion experiments and participated in preparing the manuscript.
lp participated in preparing the manuscript.
lh participated in preparing the manuscript.
pa participated in preparing the manuscript.
all authors read, commented and approved the final manuscript.

 lactobacilli are an abundant and heterogeneous group of lactic acid bacteria which occupies a wide variety of carbohydrate-rich niches ranging from plant and dairy environments to host-associated habitats. they reside in the oral cavity, gastrointestinal tract, and genitourinary tract of vertebrates. lately, their occurrence and activity in the human microbiota as well as their potential biotherapeutic effects have gained substantial interest. the healthy human vagina, for instance, is predominantly colonized by lactobacilli that have a profound impact on the health of women by protecting the host from aberrant urogenital conditions. lactobacillus crispatus is an important urogenital species that is routinely found in the vaginas of healthy women. it can account for more than 80% of all vaginal bacteria and is considered to be one of the most active species in a healthy vagina. l. crispatus also contributes to the maintenance of normal vaginal microbiota, while its absence has been associated with a range of vaginal abnormalities, especially bacterial vaginosis. strains of l. crispatus are even considered as biotherapeutic agents for reducing recurrent urinary tract infections and bv in women and have been shown to inhibit in vitro the growth, viability, and adhesion of uropathogens, suggesting a role for l. crispatus in protecting the vagina from invading pathogens. specifically, l. crispatus was recently identified to reduce the adhesion of both commensal and pathogenic gardnerella vaginalis to hela cells, indicating that competitive exclusion of this bv-associated species could be in key role in the health-promoting effects of l. crispatus. besides the gut, l. crispatus has been detected in the git of animals. the species is among the most profuse lactobacilli in the chicken crop and has, for example, been isolated from the stratified squamous epithelium of the non-secreting portion of the horse stomach and the feces of pigs. l. crispatus has also been recovered from human fecal samples, but this result is best explained by its presence in oral cavity and rectum. intriguingly, the rectal reservoirs of l. crispatus have been associated with a lower prevalence of bv, suggesting the role of rectal l. crispatus in the maintenance of the healthy vaginal flora. recently, the genome sequences of ten l. crispatus strains have become publicly available. the genomes are all about –  mb in size, with a gc content of ~37%. they possess a large number of trna molecules and are predicted to encode – proteins, several of which are of potential importance to vaginal health. for example, the potential to inhibit harmful microorganisms by direct inhibition through lactic acid, hydrogen peroxide, and bacteriocins or by displacing them through competitive adhesion is supported by the genome annotation data. in addition, these genomes have verified the phylogenetic position of the species in the lactobacillus delbrueckii clade. out of the ten l. crispatus strains having had their genome defined, nine are vaginal isolates and were sequenced as a part of the human microbiome project, including the strain ctv- that may have a role in the treatment and prevention of bv and ruti. the remaining genome belongs to the chicken-isolated strain st, known for its strong adherence not only to chicken epithelia but also to buccal and vaginal cells of human origin. the strain st was recently also characterized to produce a lactobacillus epithelium adhesin that displays specific binding to both crop epithelium and epithelial cells from human vagina. thus far, the genome sequences of different l. crispatus strains have been studied separately. unfortunately, a single genome sequence may not reflect the entire genomic complement of a species or provide an understanding of the biological processes that are peculiar to the species. instead, better knowledge of the genetic diversity of a bacterial species can be gained by comparative genomics. for example, comparative genomic analyses have established considerable intraspecies genetic diversity within the l. delbrueckii clade, but have also unraveled specific mechanisms of the host-microbe interaction that are common for all strains of the given species, suggesting species-specific rather than strain-specific host interaction properties. in the present study, we used comparative genomics to assess the overall genomic similarity of ten l. crispatus strains and defined their core and pan-genome. this global view on the gene content of l. crispatus provided an accurate account of features associated with vaginal health and represents the first effort to describe the genomic potential of this central urogenital species. specific focus was placed on the molecular mechanisms governing host-microbe and microbe-microbe interactions. these mechanisms involve genes encoding or implicated in the production of antimicrobial peptides, adhesion-associated compounds, exopolysaccharide, and s-layer proteins forming a paracrystalline structure on the cell surface. in addition, l. crispatus ortholog data was compared and contrasted with that of g. vaginalis, a frequent and predominant colonizer of the vagina of women with bv, implicated also in the development of the disease. these analyses revealed collective molecular factors in l. crispatus antagonistic to g. vaginalis, such as a counterpart to a g. vaginalis major subunit pilin. the detected factors provided an explanation for the previously reported ability of l. crispatus to reduce the adhesion of g. vaginalis to host cells and for the inverse association between l. crispatus and g. vaginalis colonization in the vagina. overall, this pan-genome study of l. crispatus broadens our knowledge of this central vaginal colonist and sheds light on the molecular mechanisms by which l. crispatus could prevent bv and protect the vagina from pathogens. materials and methods genome entries and strains all available genome sequences of l. crispatus in public databases as of january were included in this work. in addition, all available genome sequences of g. vaginalis with annotated coding sequences in their genome files as of may were included in the g. vaginalis genome analyses. to resolve the phylogenetic position of l. crispatus in respective to closely related lactobacilli, genomes of lactobacillus helveticus, lactobacillus acidophilus and bacillus subtilis were downloaded and analyzed together with the l. crispatus genomes. the set of l. helveticus, l. acidophilus and b. subtilis genomes included in this phylogenetic analysis is listed in additional file the annotated genomes were retrieved in genbank format from genbank or patric. for the draft genomes, supercontigs were preferred, if available.table overview of l. crispatus strains, properties and main findings *annotations are derived from patric. bv: bacterial vaginosis; ruti: recurrent urinary tract infection; apec: avian pathogenic escherichia coli. for adhesion assays, g. vaginalis strain isolated from a woman with bv and a vaginal lactobacillus crispatus strain ex533959vc isolated in the scope of the project “the vaginal microbiome: disease, genetics and the environment” of the human microbiome project were used. reference-based genome scaffolding the draft genomes of l. crispatus, l. helveticus, and l. acidophilus were subjected to reference-based genome scaffolding using progressive mauve genome alignment software with default settings. the genome sequences of the strains st dpc and ncfm served as references for the l. crispatus, l. helveticus, and l. acidophilus draft genomes, respectively. the contig order was confirmed through whole genome sequence comparisons that were generated using blastn, and visualized using the artemis comparison tool. putative plasmid-derived contigs among l. crispatus genomes were separated from chromosome derived sequence fragments using cbar with default settings. potential plasmid-derived contigs  kb or longer were then extracted and aligned to known plasmid sequences using patric’s blastn. contigs that aligned at ≥40% identity over ≥70% of their length were considered as plasmid-derived. phylogenetic analyses the organized scaffolds of the strains of l. crispatus, l. helveticus, and l. acidophilus were aligned using mauve progressive aligner. fully conserved columns with single nucleotide polymorphism were extracted with mauve genome alignment software, and used for the construction of the phylogenetic tree using phyml with default settings. maximum-likelihood trees were visualized with itol. for correct rooting of the phylogenetic tree, a snp-based phylogenetic tree including the b. subtilis genome as an out-group was constructed using the same approach. genome re-annotation in order to ensure the identical quality standards for all the investigated genomes, a functional annotation update was performed for l. crispatus cdss. additional annotation information for the cdss was obtained with blannotator, best blast, rast, the automatic annotation server, cog functional classification system, and by searching the predicted protein products against the pfam database release. for blannotator and best blast approach, blastp was run with default parameter settings, and hits that aligned with more than 40% amino-acid identity and 80% coverage threshold were retained. the rast and kaas and cog annotation was obtained using the services with default settings. pfam searches were performed locally using the hmmer package, relying on the pfam trusted cut-off for the score. the eps gene clusters were identified by manual examination of the annotation information. the presence of putative bacteriocin-encoding genes was determined with bagel with default settings. to identify genes associated with clustered regularly interspaced short palindromic repeats, cdss were screened for the presence of crispr-associated protein domains using the hmmscan program from the hmmer package. matches having scores exceeding the trusted cut-off values were considered significant. cas protein domain models were obtained from the tigrfam database. integration of annotation information was done using in-house perl scripts producing tab-delimited cdss information files. other bioinformatic analyses included identification of mobile genetic elements and crispr loci. genomic regions potentially obtained by horizontal gene transfer were predicted using islandpick, islandpath-dimob and sigi-hmm methods with the help of islandviewer meta-analysis tool with default settings. prophage-like gene-clusters were predicted with prohinder using default parameters. overlapping prophage-like genome regions were merged into single extended regions spanning a given genomic region and manually inspected. putative crispr loci were identified with pilercr run with default settings and manually adjusted. megablast was used for similarity searches between crispr-spacer sequences and virus and plasmid entries in the genbank database. only matches showing 100% identity over the complete crispr-spacer were retained. annotation of proteinaceous adhesion factors l. crispatus cdss potentially involved in binding to the host were identified by searching the predicted protein sequences against adhesion associated pfams. adhesion associated pfams were identified by searching the pfam database release entries with various keywords related to adhesion, host tissue components, and bacterial surface components, and by manual examination of the literature. the list of pfam domains is available in additional file in addition, non-adhesion related domains for the selected adhesion-related cdss were detected by searching the protein sequences against pfam release through the pfam website using gathering thresholds greater than or equal to the trusted cut-off. ortholog prediction ortholog groups among l. crispatus strains were identified using orthomcl. to estimate the development of the size of the core and pan-genome as a function of the number of sequenced l. crispatus strains, ortholog groups were determined iteratively for an increasing numbers of sequenced genomes. at each sample size, the analysis was repeated times with different random sets of l. crispatus genomes. orthomcl was run with default settings, except for a percent match threshold of and blastp set to print up to alignments. the protein products of the original cdss were used for the analysis. the same approach, but without the sampling procedure, was used to define the ortholog groups among g. vaginalis. because of the draft quality of most of the g. vaginalis genomes, ortholog groups present in ≥ 30 g. vaginalis genomes were considered as core groups. estimation of l. crispatuspan- and core genome sizes the estimation of the l. crispatus core and pan-genome sizes was based on the orthomcl results and was performed according to previously described approaches. the core genome was extrapolated by fitting an exponential decaying function y = κ exp + Ω to the median number of core ortholog groups with a weighted least square regression. in the equation, n is the number of sequenced strains and κ, τ, and Ω are free parameters optimized in the regression analysis. the Ω describes the estimated core genome size. the power law y = k nβ was fitted to pan-genome data with a weighted least square regression, where y is the median, n is the number of genomes, and k and β are free parameters. regression analyses were done using the nls function of the statistical software r. identification of significant enrichment of genes in cog-categories hypergeometric distribution was used to test the probability of the over-representation of core, strain-specific or variably conserved accessory genes in a given cluster of orthologous groups. the obtained p-values were subjected to bonferroni adjustment to reduce the number of false positives introduced by multiple hypothesis testing. only cog categories containing more than cdss were included in the analysis. statistical tests were performed using the statistical software r. identification of antagonistic factors against g. vaginalis virulence-related g. vaginalis cdss were inferred from a recent comparative genomic analysis and by comparison to the pfam database. the pfam search was done using the hmmersearch program from the hmmer package. hits were considered significant if their score was above a trusted cut-off value. virulence-related pfam models were identified based on a literature review. following the identification of g. vaginalis virulence factors, all the members of their ortholog groups were extracted, an alignment built using muscle with default settings, and a hidden markov model constructed using the hmmbuild command. the constructed hmms were then searched against the predicted l. crispatus proteomes with the hmmersearch program from the hmmer package in order to identify counterparts. hits with e-value greater than or equal to were accepted and manually inspected. detection of enzymes and metabolic pathway reconstructions using the automatic annotation server kaas, l. crispatus cdss were assigned with ec numbers describing enzymatic activity. each strain’s ability to ferment carbohydrates and synthetize bio-compounds was then tested by matching its ec complement against the sets of ecs of metabolic reactions providing the conversion of a given starting compound to a particular end product. a route was accepted as intact if at least one match was found for each enzyme-catalyzed reaction. metabolic routes between two given compounds were retrieved from the fmm server which connects different kegg reference reaction maps and reconstructs metabolic pathways between metabolites. for the analysis, the amino acids were paired with amino acid synthesis starting materials and with each other; carbohydrates were paired with selected key intermediates of the central carbon metabolism; the selected central carbon metabolism intermediates were paired with pyruvate or pyruvate, acetate and ethanol; and pyruvate was paired with various end products. the exact list of compound pairs screened is available in additional file to determine pathways encoded by the l. crispatus core genome, the above pathway reconstruction approach was repeated for the core genome-encoded ec complement. finally, the mode of carbohydrate fermentation was studied based on metacyc pathways for homolactic and heterolactic fermentation. hydrogen peroxide generating enzymes were detected by screening for ec numbers of the enzymes having the compound h2o as a product. adhesion assays bacteria were grown in supplemented brain heart infusion containing 2% gelatin, % yeast extract, % starch and % glucose, for 48 h at 37°c, in 10% co bacterial suspensions were collected by centrifugation at g at 4°c for 10 min and washed once with sterile phosphate buffered saline. bacteria were resuspended in pbs and the optical density at 600 nm was determined. correlations between od and colony forming units were made prior to the experiments, and the bacterial suspensions were adjusted to 1 × 108 cfus/ml, as optimized before. for the adhesion assays, hela cells were cultured in dmem supplemented with 10% fetal bovine serum and 1 iu penicillin-streptomycin/ml at 37°c and in 5% co cells were cultured in chamber slides until they reached a density of 2 ×  cells per well, at 37°c in 5% co before the adhesion assays, cells were washed twice with 200 μl of pbs to remove non-adherent cells and fixed with cold 4% paraformaldehyde in pbs for 10 min followed by washing three times with pbs. fab fragments prepared by papain treatment of purified igg against lea protein of l. crispatus st and flagellum of escherichia coli strain mg Δfima-h were available from a previous study. fab fragments in pbs supplemented with 5 mm phenylmethylsulfonyl fluoride were mixed in independent experiments with g. vaginalis or l. crispatus cells, at room temperature, for 30 min, with rotational agitation at  g. mixtures of fab fragments and bacteria or bacteria alone in pbs supplemented with 5 mm pmsf were incubated with pfa-fixed hela cells for 1 hour, at 37°c in 5% co each well was carefully washed twice with 200 μl of sterile pbs to remove non-adherent bacteria. bacterial quantification was done as previously described. briefly, after fixing with methanol, dapi was added to the wells. microscopic visualization was performed using an olympus bx epifluorescence microscope equipped with a ccd camera and filters capable of detecting the dapi staining. the number of adherent bacteria in randomly chosen microscope fields was determined using image j software. results were expressed as the bacteria per hela cells, according the mean ± standard deviation of the two independent experiments, with technical duplicates. the data were analyzed using the student’s t-test with the statistical software package spss. p-values of less than were considered significant.  general genomic features of l. crispatus the genome sequences of ten l. crispatus strains were compared and analyzed. these genomes contain cdss, of which had an assigned role in the original genome file. after the annotation update, cdss were functionally classified by at least one of the functional annotation tools. for each cds, the results of the different protein classification analyses were collected together and analyzed as a group. the obtained annotations are presented in additional file only one of the genomes is in one contig whereas the rest are in 5– super-contigs. putative plasmid-derived sequences, each with a length of bases or more were identified in three vaginal isolates, the rest having only chromosomal-associated super-contigs. using conserved genomic synteny, the orientation and order of the chromosomal-associated super-contigs of each draft genome was determined. analyses of the resulting architecture revealed that genomes were in general collinear and shared on average ~90% of each other’s content, comparable to conservation ratios seen in lactobacillus johnsonii, l. helveticus, and l. plantarum. the genomes of the strains 214- and sj-3c-us were most conserved, with ~97% of their sequences conserved in at least one strain, whereas only roughly 82% and 84% of the genomes of strains st and fb077- could be aligned against some other l. crispatus genome. these data indicate that each assembly presents a near complete chromosome, providing a solid foundation for inter-strain comparisons.figure whole-genome alignment of the l. crispatus genomes. the contigs of the draft genomes were ordered with mauve using the st genome as a reference. matching genome regions were identified with blastn and visualized using the artemis comparison tool. vertical bands represent the blastn matches. prophinder-predicted prophage-like genomic regions and islandviewer predicted gis are represented as blue boxes on the bottom and red boxes on the top strand of each genome, respectively. the l. crispatuspan-genome the microbial pan-genome is defined as the full complement of genes in a species. in total, this set of l. crispatus genomes comprised ortholog groups, including on average orthologs and co-orthologs per group. this current pan-genome was defined using orthomcl and was almost twice the average number of cdss and ortholog groups present in a single l. crispatus strain. the ortholog group accumulation curve describing the expansion of the pan-genome as a function of genomes added to the analysis fitted well a power law model and was far from saturated, indicating that the total gene pool accessible to the species has not yet been fully captured and suggesting yet-to-be discovered traits in l. crispatus, similar to that what has previously been reported for oenococcus oeni or lactobacillus paracasei. particularly, the regression model revealed an open pan-genome that grows by at least ten ortholog groups per every additional genome until isolates have had their genome defined.figure pan- and core genomes of l. crispatus. development of the pan- and core genomes as a function of the number of sequenced l. crispatus strains. the total number of genes found according to the pan- and core genome analysis is shown for increasing numbers of sequenced genomes. the dashed lines represent least squares fits to the medians and the r describes the suitability of the fit. the box plots present median, 25th and 75th percentiles, with the data extremes shown by whiskers outside the box. c) the distribution of core and accessory l. crispatus cdss within cog functional categories. for each category, the top and bottom bars show the percentage of the assigned core and accessory cdss relative to the entire core and the accessory l. crispatus cdss, respectively. the proportion of the strain-specific cdss is highlighted in the accessory bars. cogs significantly enriched in core, shared accessory, or strain-specific cdss are marked next to the cog identifiers. only cog functional categories with more than members are shown. the cog categories are given in the inset at the bottom of the figure. d) distribution of ortholog groups at different levels of conservation in each strain. the orthomcl-defined ortholog groups were classified into different levels of conservation according to the number of strains they were detected in. ortholog groups found in all the ten genomes represent the current core. conservation levels are represented by different colors. the l. crispatuscore genome the core genome is defined as the orthologous genes present in every strain of a species. we identified the current l. crispatus core genome to be comprised of ortholog groups that were conserved across all the ten analyzed strains. this common core captured ~57% of the ortholog groups of a given genome, which is slightly less than what orthologous grouping has revealed for another lactobacillus species. based on the examination of the cog functional categories and hypergeometric tests, the core was identified to be significantly enriched with genes belonging to cog categories j, t, and e. furthermore, ~10% of the ortholog groups in the core genome could not be assigned with a descriptive functional annotation, and thus may represent proteins with yet-to-be discovered housekeeping functions or other functions relevant to the basic aspects of the biology of the species. we also predicted the core genome to contain genes encoding features likely to contribute to cell envelope biogenesis, antimicrobial activity, and host-microbe interaction, as illustrated in detail below. to estimate the number of ortholog groups present in an infinite number of l. crispatus strains, the number of shared ortholog groups found on sequential addition of each new genome sequence was extrapolated by fitting an exponential decaying function to the medians of core genome sizes. as expected, the number of ortholog groups in the core genome initially decreased with the addition of each new genome sequence. the extrapolation of the curve designated that the core genome plateaus at 1116 ±  ortholog groups for an infinite number of l. crispatus strains. thus, the current l. crispatus core genome appears to be almost within the estimated error margin, indicating that the current core is nearly a perfect representation of the final core genome. however, it should be noted that gaps and sequencing errors in draft assemblies might have affected our estimate. the l. crispatusvariome we investigated the distribution of the l. crispatus pan-genome by assessing the number of strains sharing a particular ortholog group. in total, ortholog groups were present in some, but not in all the ten l. crispatus strains, forming the current l. crispatus accessory genome, suggested to provide selective advantages for different strain of a species. the overall composition of the cogs in the core and accessory genomes was mainly similar, the most notable over-representations of accessory genome-encoded genes being associated with cog categories l and q. enrichment in the l and q categories was driven by diversity in strain-specific transposon-associated classes and abc-type multidrug transporters, respectively. included in the accessory genome were also ortholog groups found only in a single strain. most of these ortholog groups belonged to the genomes of the strains fb077- and st, which also displayed the smallest and largest accessory gene pools, respectively. fewest strain-specific groups were present in the genome of the strain mv-1a-us. the mean number of the strain-specific ortholog groups found in the l. crispatus dataset was 131 ±  which forms a slightly bigger portion of the genome than what comparative analyses have previously detected in another lactobacillus species and less than in some other lactic acid bacteria such as o. oeni. as expected, the strain-specific gene pool is poorly characterized, close to 40% lacking a functional annotation. interestingly, transposase-related genes accounted for ~25% of all strain-specific genes with an informative functional annotation. protein homology searches revealed that ~30% of all strain-specific genes had the highest similarity to genes found in other strains of the l. delbrueckii clade. the species l. helveticus and lactobacillus kefiranofaciens were deduced to be the two most notable reservoirs of genetic variability, providing the best matching targets for about 10% and 5% of the strain-specific ortholog groups in l. crispatus. for example, up to 47% of strain-specific ortholog groups in strain sj-3c-us had the top match in l. helveticus. in addition, more distant lactobacillus species appear to have interacted with l. crispatus. specifically, the strain st seems to have received seven strain-specific ortholog groups from lactobacillus salivarius, which is only distantly related to l. crispatus st but known to exist in the same ecological niche. horizontal gene transfer hgt is a major force in bacterial evolution and can contribute to the fitness, metabolic versatility, and niche-adaptation of bacteria. for example, genomic islands harboring genes for carbohydrate utilization reflect to the lifestyle adaptation of lactobacillus plantarum. to determine the presence of gis and potentially horizontally acquired genes, the l. crispatus genomes were interrogated using islandviewer. this analysis identified between and gis in each genome. some of these gis agreed with the observed interruptions in the genomic synteny whereas others were conserved, highlighting the imprecision of the prediction methods or indicating the presence of ancient gi acquisition events in l. crispatus. the total span of gis was longest in l. crispatus 125-2-chn, shortest in the strain jv-v, and on average ~166 kb in a l. crispatus genome. based on cog and prophage-cluster analysis, over of the total of cdss in the gis encoded phage-related products or transposases, which is not surprising, given that many of the prophage-like genomic regions co-localize with the gis. in addition to the mobile elements, the gis were found to be rich in metabolism and biosynthesis-related genes. close to 20% of their gene content was predicted to be involved in sugar metabolism and amino acid biosynthesis, pointing a role for hgt in adaptation of l. crispatus to varying environments. for example, hgt events may have contributed to acquisition of cellobiose and fructose-specific transport systems as well as genes implicated in sialic acid utilization to certain l. crispatus strains. on the other hand, the more ancient gene acquisition events in l. crispatus provide an explanation for the observed presence of an additional copy of phosphoketolase genes missing in the closely related l. acidophilus and l. helveticus genomes included in the phylogenetic analysis. similarly, the investigated l. acidophilus and l. helveticus strains also lacked a gi-associated mannosylglycerate hydrolase encoding genes present in some l. crispatus strains. moreover, missing from l. acidophilus genomes were also a hydrogen peroxide producing glycolate oxidase gene that was present in all the l. crispatus and most l. helveticus genomes, further supporting the role of hgt in environmental adaptation. another hydrogen peroxide producing enzyme, puryvate oxidase, was in contrast predicted to be present in all except three l. crispatus, l. helveticus, and l. acidophilus genomes. the l. crispatus gis comprised also several putative eps biosynthesis genes in strains st 125-2-chn, and fb049- which is in accordance with the observation that eps gene clusters in lactobacilli often have abnormal gc content. finally, strain-specific genes were associated with gis. most of these were distributed somewhat randomly, but it was also possible to define eight long gis with considerably many strain-specific genes and probably thus acquired rather recently by hgt. in three of these gis, the majority of the cdss did not show significant similarities to proteins in the ncbi databases, suggesting a recent acquisition of yet-undiscovered traits. phages temperate phages are common in vaginal lactobacilli and can form a potential threat for lactobacillus populations maintaining a healthy vagina. some studies have even suggested that bacteriophage attack is the causative agent triggering the breakdown of the protective vaginal microbiota during bv. in this study, a total of prophage-like regions were identified comprising of cdss and accounting for more than a fifth of the ortholog groups in l. crispatus. markedly, this fraction of prophage-like ortholog groups in l. crispatus is substantially higher than the 9% reported for l. paracasei, indicating a large variation of prophage-related gene contents among different lactobacillus species. interestingly, the prophage-like clusters were enriched in the nine vaginal isolates of l. crispatus, whereas there was none in the chicken isolate st, possibly reflecting exposure to phage in the human vagina. specifically, the strains 125-2-chn, sj-3c-us, 214- jv-v fb077- and fb049- each contained between one and three prophage-like regions composed mostly of cdss with phage-related or non-informative annotations and with no or limited homology with the genome sequence of other l. crispatus strains. the remaining three vaginal isolates carried six candidate prophages, each consisting mostly of orphan cdss with phage-like or non-informative annotations. sequence analysis of l. crispatus st genome also revealed a prophage-like region, but this region was rejected, because associated with the strain’s own replication machinery. overall, the results are in accordance with the high degree of lysogeny, namely 77%, observed for vaginal l. crispatus strains. this suggests that temperate phages are widespread in vaginal lactobacilli and that transduction is an important mechanism for genome evolution in these bacteria. notably, the lack of common insertion sites between the isolates indicates that various sites of the l. crispatus genomes can serve as targets for phage integration. crispr/cas-systems crisprs are a family of dna repeats present in the genomes of many prokaryotes that are responsible for providing acquired immunity to exogenous dna from bacteriophages and plasmids. this system consists of a set of cas genes and an array of direct repeats separated by intervening sequence spacers derived from the invading dna. interestingly, distinct types of crispr/cas systems were identified for the vaginal l. crispatus isolates and the chicken isolated st. all the vaginal isolates but the strain 125-2-chn were predicted to have several genes that could be classified to belong to the previously described type ii crispr/cas system. analysis of the genome of 125-2-chn also revealed traces of the type ii system, but the presence of universal cas and cas core genes and the cas9/csn signature genes could not be verified, because the region next to csn is disrupted by a sequencing gap. nevertheless, the crispr arrays in each of the vaginal strains was composed of direct repeats with an identical consensus sequence of 36 bp and two to six spacer sequences each. homology searches between the identified spacers and public virus and plasmid sequences did not reveal the putative targets of these systems, which is in line with the previous spacer annotation survey identifying a plasmid or virus target only for 30% of the spacer sequences in lactic acid bacteria. the lack of identified targets of the l. crispatus spacers points to a pool of not yet sequenced vaginal phages and plasmids. interestingly, many spacers were identical across several of the vaginal strains, suggesting that these strains may share a recent ancestor or have encountered similar invading genetic elements in their past. it should be noted that these genome sequences are incomplete and that some spacers and repeats may have remained undetected. in addition, a type i crispr/cas system was identified in the st genome comprising eight cas genes and three crispr-arrays composed of direct repeats of 28 bp, and and spacers. the repeats were highly similar and resembled a repeat discovered in vaginal samples by rho et al.. however, the shortest array was positioned within a 423 bp long putative lcris_ gene and thus is most likely a false prediction. similarly to the vaginal isolates, the spacers of these systems did not match any known plasmid or virus sequence.table distribution of cas-proteins in l. crispatus the first number in the table cells describes the number of cdss part of the cas-protein associated ortholog group in a specific strain and the second number indicates the number of group members matching the cas-model. variation in crispr/cas locus in l. crispatus. the arrows represent different genes and their orientation within a locus. orthologous genes are positioned vertically. the cas genes with conserved function are of the same color, whereas grey describes genes not matching a cas model. outlines of genes orthologous to some cas gene, but not matching a cas model are color coded according to the ortholog groups. dashed lines represent contig breaks. diamonds represent direct repeats and boxes different spacer sequences. identical spacers are represented by the same color. the direct repeats of the top nine genomes are identical. prompted by the observed differences between the prevalence of crispr/cas systems in the vaginal strains and the chicken isolate, the distribution of the cas genes in publicly available lactobacillus genomes was tested. markedly, the type ii crispr/cas system hits were more frequent in vaginal than in non-vaginal lactobacilli, which suggests that the type ii system could be important in the vaginal environment. the prevalence of the other types of crispr/cas systems was not significantly different at alpha level. metabolic pathway reconstruction using the automatic annotation server kaas, we were able to assign ec numbers to the members of ortholog groups. surprisingly, the majority of the enzymes belonged to the core groups, which is somewhat different from the large intra-specific variation present within the metabolic contents of o. oeni or l. paracasei. in accordance with the high number of core genome-encoded enzymes, the in silico reconstruction of l. crispatus metabolic pathways suggests that the strains have a potential to utilize a rather same set of carbohydrates. the data supports the presence of metabolic routes in each strain for the conversion of a variety of sugars into the key intermediates of the pentose phosphate, embden–meyerhof–parnas, and tagatose-6-phosphate pathways. pathways for the conversion of the d-xylulose 5-phosphate and d-fructose -bisphosphate into several of their end products were also annotated for nine of the ten strains. the aforementioned indicates the presence of both embden–meyerhof–parnas and pentose phosphate pathways in the nine strains, which is typical for a heterofermentative species and contradictory to the previous classification of l. crispatus as a homofermentative species. the exception is the strain ctv- that had only partial pathways for many end product conversions, most likely because of sequencing gaps in the corresponding genomic loci. no routes were recorded for the conversion of tagatose-6-phosphate pathway intermediate into pyruvate in any of the strains. interestingly, the data also shows evidence for the presence of strain-specific glycerone conversions in l. crispatus 125-2-chn. regarding urogenital lifestyle, conserved pathways were annotated for the metabolism glucose and mannose, the former reported to be the major free monosaccharide and the latter a minor constituent of the vaginal fluid. although we did not detect complete routes for the metabolism of glycogen, seven vaginal strains were discovered to carry a gene coding for a type i pullulanase debranching enzyme, which could contribute to the degradation of glycogen. moreover, l. crispatus core appears to encode a sialic acid utilization regulator and an o-sialoglycoprotein endopeptidase that could contribute to the hydrolysis of o-sialoglycoproteins in the vaginal mucosa. notably, the manual examination of the enzyme contents revealed that each strain may generate hydrogen peroxide from pyruvate, of which the former acts as an antimicrobial compound. we also assessed the range of amino acids that l. crispatus has a potential to synthetize. based on the in silico analyses of the biosynthetic capabilities, all strains can synthesize seven amino acids either de novo or as derivatives using the same pathways, which is three and four amino acids more than l helveticus dpc or l. acidophilus ncfm, respectively. pathways for aspartate biosynthesis were also annotated in nine isolates, excluding the strain ctv- that did not share this property. we again speculate that the lack of biosynthesis route for aspartate is rather due to the draft nature of the genome sequence of this strain than a genuine loss. the other differences in amino-acid synthesis related to nuances in synthesis routes for cysteine, serine, and glycine, which seem to vary between isolates. overall, the in silico analyses predicted a dependency on external supplies of amino acids for l. crispatus similar to that described for closely related lactobacilli and shows that the strains are rather similar in their biosynthetic power. moreover, none of the detected conversions was deduced to be strain-specific, further highlighting the similarity. proteinaceous adhesins adhesion to host tissue has long been considered an important factor and a prerequisite for the long-term colonization of the human vagina, stimulation of the immune system, and antagonistic activity against harmful pathogens through competitive exclusion. we screened the l. crispatus proteomes for adhesion and host colonization related domains and identified proteins governing the ability of l. crispatus to colonize and interact with the host. these putative adhesins were associated with seven distinct types of adhesion-associated domains belonging to ortholog groups of which seven are part of the l. crispatus core genome. it should be noted, however, that members of the same ortholog group did not necessarily share adhesion domains. in addition, six strain-specific adhesins were identified, all of which were predicted to be mucus-binding proteins. interesting examples of the strain-specific adhesins include a sortase-anchored protein with multiple mucus-binding domains, and lcris_ being the only member of its ortholog group with adhesion-associated domains. one notable core adhesin was a putative fibronectin/fibrinogen-binding protein fbpa, which has recently been proposed to contribute to the fibronectin-binding properties of lactobacillus iners and to explain the stronger adhesion of l. iners to human fibronectin compared to other species of lactobacillus tested in the study. notably, our data does not support this hypothesis, since the presence of functional fbpa gene in the l. crispatus core genome should have resulted in equal adhesion abilities for the l. crispatus and l. iners strains tested in the study. markedly, the recently characterized lea protein of l. crispatus st belonging to lact was not identified, indicating that this adhesin binds to crop epithelium and epithelial cells from human vagina with some novel domain. in addition to the aforementioned putative adhesins, l. crispatus was predicted to harbor ~ putative s-layer protein-encoding genes that could potentially contribute to bacterial adhesion. however, these predicted s-layer proteins were different from the s-layer proteins of other related lactobacilli reportedly implicated in bacterial adhesion.table distribution of adhesion related proteins in l. crispatus the first number in the table cells describes the number of cdss part of the ortholog group in a specific strain and the second the number indicates the number of group members having the adhesion or colonization related pfam-domain. cell wall exopolysaccharide in the l. crispatus genomes, a highly variable genome region appears to be associated with eps biosynthesis. this eps gene cluster was observed in eight l. crispatus strains and noted to comprise eps biosynthesis genes, five of which were present within each operon. the five conserved genes were predicted to encode a transcriptional regulator, a polymerization and chain length determination protein, a tyrosine-protein kinase, a protein-tyrosine phosphatase, and the priming glycosyltransferase. the remaining genes coded for proteins with putative glycosyl transferase functions, indicating that the strains produce epss with different sugar monomers and glycosidic linkages. markedly, eps gene clusters were not detected in the genomes of l. crispatus jv-v and 214- figure variation in eps gene cluster in l. crispatus. the organization and conservation of the exopolysaccharide synthesis regions in l. crispatus. orthologous genes are represented with the same color and stars indicate genes found in different loci. dashed lines represent contig breaks in the mv-1a-us and sj-3c-us clusters. antimicrobial potential in l. crispatus lactobacillus species can maintain the vaginal ecosystem in a healthy condition by the production of antimicrobial substances such as lactic acid, hydrogen peroxide and bacteriocin-like substances. lactic acid is the main end product of the carbohydrate fermentation in lactobacilli and can contribute to the vaginal acidity and thereby inhibit the colonization and proliferation of harmful micro-organisms in the vagina. the l. crispatus strains studied here appeared to possess between three to four l-lactate dehydrogenases for the conversion of puryvate into lactic acid. interestingly, one specific ldh locus found in five l. crispatus strains was flanked by a transposase enzyme gene that may affect its expression. we also discovered hydrogen peroxide producing enzymes in each l. crispatus, which correlates well with the experimental data showing that hydrogen peroxide generation is common among vaginal l. crispatus. using bagel, the bacteriocin content of l. crispatus was investigated. this method was able classify several sets of putative bacteriocin gene clusters in each strain, including at least two regions encoding bacteriolysins. in addition, regions implicated in the production of class ii bacteriocins were revealed in the vaginal isolates. a pediocin-like bacteriocin that inhibits the growth of pathogenic listeria and clostridium species was present in five vaginal isolates and all nine encoded a two-component bacteriocin ls that inhibits the growth of isolates belonging to genera listeria, shigella, and yersinia. notably, the pediocin-like bacteriocin encoding genes were found in the vicinity of cdss encoding proteins harboring a domain for enterocin a immunity.table distribution of predicted bacteriocin related proteins in l. crispatus for the bacteriocin-like molecules with pfam-domain, the first number in the table cells describes the number of cdss part of the ortholog group in a specific strain, and the second number indicates the number of group members having the pfam-domain. helveticin j refers to the antimicrobial molecule first identified in l. helveticus and bacteriocin helveticin j to one identified in l. acidophilus. antagonistic activities against g. vaginalis bv is the most common vaginal disorders, affecting up to a third of women. it has been associated with increased risk for preterm birth, urinary tract infections, and hiv infection, and represents a condition in which the normal protective lactobacilli community is replaced by an overgrowth of anaerobic bacteria. although the etiology of bv is not known, g. vaginalis is present in up to 95% of all bv cases, indicating that it could have a role in bv. in our efforts to decipher the genetic basis of the inhibitory actions of the species l. crispatus against g. vaginalis, we performed ortholog grouping of the available g. vaginalis data and used comparative genomics to identify shared common molecular mechanisms between g. vaginalis and l. crispatus. importantly, our analyses revealed several components by which l. crispatus could interfere with the attachment of g. vaginalis in the vagina. firstly, fibronectin-binding could play a role in this process, given that proteins with fivar domains related to hyaluronate or fibronectin-binding were encoded in the core genomes of both g. vaginalis and l. crispatus. secondly, searching l. crispatus proteins against the g. vaginalis hmm database suggested another l. crispatus protein, which could play a role in preventing the cell adhesion of g. vaginalis to fibronectin. intriguingly, this counterpart of the g. vaginalis fivar-proteins was distributed in nine l. crispatus strains, but had no known adhesion domains. another interesting core orholog group of g. vaginalis was gvag many members of this ortholog group contained a bacterial ig-like domain, which is distantly related to the interaction domains, namely fn and big_, associated with several l. crispatus core adhesins. moreover, searches against the g. vaginalis hmms revealed two additional l. crispatus adhesins that could act as counterparts of gvag although having mucin-binding domains. finally, of the three g. vaginalis pilus-encoding gene clusters that were identified based on the pilus-encoding genes listed by yeoman et al., the one associated with most isolates had borderline counterparts in the l. crispatus core genome. its major subunit pilin appears to have two potential antagonists in the l. crispatus core genome encoding a -kilodalton protein and the lea protein. in addition, the long cds located next to the major subunit component in the cluster and showing similarity to known adhesins and surface antigens, could be inhibited by the members of the lact and lact based on the g. vaginalis hmm searches. taken together, these findings indicate that l. crispatus could interfere with fibronectin-binding and pilus components of g. vaginalis. of the other listed virulence-related factors in g. vaginalis, the invasion-associated hydrolase, protein with two g-related albumin-binding modules, nlpa lipoprotein, and endothelin-converting enzyme have potential antagonists encoded by the l. crispatus core based on the g. vaginalis hmm searches. a noteworthy finding is that the g-related albumin-binding module protein present in 17 g. vaginalis isolates shared similarity with l. crispatus proteins, including all nine fivar-domain associated proteins of the lact. adhesion inhibition assays to hela cells our comparative analysis described several species-wide factors by which l. crispatus could compete with g. vaginalis in the vagina. for example, the lea protein was identified as a prominent counterpart of one of the g. vaginalis core adhesins and was thereby predicted to participate in the adherence inhibition of this pathogen. to validate the role of lea in the antagonism against g. vaginalis, the adhesion capacity of a vaginal l. crispatus isolate ex533959vc and bv-associated g. vaginalis to hela cells was tested using the previously described approach with and without the pretreatment with fab fragments prepared against lea. markedly, the anti-lea fab fragments significantly reduced the adhesion level of both bacterial species to hela cells whereas the unrelated anti-flagellum fab fragments showed no inhibitory effect. the reduction in adherence was most evident for the strain ex533959vc06; the anti-lea fab fragment pretreatment resulting in % and % reduction in adhesion to hela cells compared with the untreated or anti-flagellum fab fragment pretreated bacteria, respectively. intriguingly, pretreating g. vaginalis with the anti-lea fab fragments caused also a significant reduction in adherence compared with the untreated bacterial cells or bacteria pretreated with the control anti-flagellum fab fragments. these observations validated the predicted competitive character between lea and g. vaginalis, suggesting a role for lea in the previously identified ability of l. crispatus to exclude and displace g. vaginalis from hela cells. the results also provide an explanation to the inverse association between l. crispatus and g. vaginalis colonization in the vagina. based on our comparative genomic analyses, the lea protein achieves its inhibitory effect by competing with the same attachment sites as the pili of g. vaginalis. of note, our adhesion assay provided a further support for the species-wide distribution of lea among l. crispatus, since the strain ex533959vc has not yet been sequenced. furthermore, since lea has previously been studied only in the chicken isolate st, our results serve as the first record of the functionality of lea in vaginal l. crispatus.figure inhibition of l. crispatus or g. vaginalis adhesion to hela cells by lea-specific fab fragments. cells of l. crispatus ex533959vc or g. vaginalis were pretreated with lea-specific igg fab fragments or unrelated anti-flagellum fab fragments or left untreated in pbs supplemented with 5 mm pmsf before the adhesion assays. the number of adherent bacteria per epithelial cell in randomly chosen microscopic fields was determined. the assay was performed twice with duplicate samples and the results show mean values of adherent bacteria. the asterisk indicates p <  as calculated by student’s t test. phylogenetic relations phylogentic relations between the selected l. crispatus strains and strains of closely related species l. acidophilus and l. helveticus were examined based on a maximum-likelihood tree built from the snps of the core genome. altogether conserved polymorphic sites were identified from the genome alignments and used for the construction of a phylogenetic tree. the phylogenetic tree clearly shows that strains of the same species cluster together and that each lactobacillus species has differentiated as a distinct entity. the species l. crispatus and l. helveticus share the most recent common ancestor and form a sister group to species l. acidophilus, which is accordance with previously reported phylogenetic trees. among the l. crispatus cluster, the chicken isolated st branches off first from the vaginal isolates.figure phylogenetic tree. phylogenetic relations of the selected l. crispatus, l. helveticus and l. acidophilus strains based on the snps of the core genome. the b. subtilis genome was used as the out-group to root the tree, but is not shown in the figure. in the inset, the branching pattern of the l. crispatus strains is highlighted.  the rapidly increasing number of complete microbial genomes offers previously unimaginable possibilities to understand the phenotypic and genomic diversity in a particular species. in this study, we have taken advantage of publicly available l. crispatus genomes and present the genetic landscape of this important urogenital lactic acid bacterium. we assessed the overall genomic similarity of ten strains and defined the l. crispatus pan- and core genomes. these analyses depicted high sequence identity and extensive synteny punctuated by several gis, and revealed a current pan-genome that is nearly two times larger than the number of ortholog groups present in an average l. crispatus strain. about one third of all ortholog groups were assigned to all strains, constituting the current l. crispatus core genome and encoding the basic aspects of l. crispatus biology. importantly, these core features comprised several cdss for the production of antimicrobial molecules and competitive exclusion of the bv associated species g. vaginalis, shedding light on the molecular mechanisms by which l. crispatus could maintain vaginal health. the pan-genome analysis also revealed singleton ortholog groups associated with only one strain. the enrichment of functions related to replication and repair among these genes indicates the influence of transposons in genome evolution in this species. a third of the strain-specific ortholog groups had the highest similarity to genes found in the other strains of the l. delbrueckii clade, suggesting notable sequence influx from closely related lactobacilli. our regression analysis indicates that the genetic diversity present within l. crispatus has not yet been comprehensively captured. specifically, we estimate that over ten new ortholog groups will be discovered per every additional genome until almost 300 l. crispatus strains have had their genomes defined. this estimation may be compromised by the uncertainty caused by the draft genomes that have up to sequence gaps. nevertheless, the data implies the presence of large repertoires of undiscovered l. crispatus genes to be sequenced in the future. the phylogenetic tree based on core genome snps among the ten isolates revealed that the chicken isolated st branches off first from the l. crispatus cluster and that the l. acidophilus cluster is a sister taxon to l. helveticus and l. crispatus, as suggested earlier. from the perspective of vaginal health, the most interesting genomic diversity regions in l. crispatus include the loci related to eps biosynthesis, prophages and adaptive immunity, of which the latter two may play a role in bv. firstly, the genetic differences in the composition of the eps gene region may participate in the l. crispatus adhesion, biofilm formation and competitive exclusion of pathogens. the eps-deficient strains jv-v and 214- are particularly interesting, as the deprivation of eps has been reported to promote bacterial adhesion in other lactobacilli. secondly, the presence of prophage-like clusters in the vaginal l. crispatus genomes is in accordance with the previously observed high level of lysogeny in vaginal l. crispatus strains. if truly inducible, the spontaneous release of the prophages could contribute to the development of bv. finally, a relationship was depicted between the life environment of the strains and their adaptive immunity systems, suggesting that different types crispr/cas systems could be beneficial in different environments. this hypothesis is further supported by the analysis of the cas gene contents of lactobacillus genomes that revealed higher rates of the type ii crispr/cas systems in vaginal than in non-vaginal lactobacilli. in addition, the crispr-arrays of the vaginal l. crispatus strains carry evidence of encounters with common invaders, as several of the spacer sequences were identical between several strains. the defined l. crispatus core genome helps to explain how this species can thrive in the vaginal environment and benefit vaginal health. in the vaginal epithelium of reproductive age females, large quantities of glycogen are broken down and then metabolized into lactic acid, which is thought to result in acidification of the vagina. although l. crispatus lacks complete enzymatic machinery for glycogen degradation, the core genome encodes enzymatic pathways for the utilization of a range of carbohydrates available in the vaginal fluid, which could support the urogenital commensal lifestyle of l. crispatus. encoded in the core are also several features potentially governing host-interactions and displaying an antagonistic activity against other micro-organisms. interestingly, the bacteriocin-like molecules encoded by the l. crispatus genomes could inhibit biofilm integrated g. vaginalis cells, shown to be more resistant to hydrogen peroxide and lactic acid than the cells in planktonic state. specifically, as g. vaginalis is known to develop an adherent biofilm on the vaginal epithelium in bv this property could provide attractive means to restore the normal vaginal flora. in addition to the antimicrobial properties, l. crispatus was detected to contain several proteins that could mediate the previously reported competitive exclusion of g. vaginalis from epithelial cells and explain the inverse association between l. crispatus and g. vaginalis colonization in the vagina. most notably, these specific interference mechanisms might include blocking the attachment of g. vaginalis by disturbing the pilus-mediated adhesion of the pathogen. this mechanism could involve lea, shown here to be universally present in all l. crispatus strains, and demonstrated using lea-specific fab fragments to inhibit the adhesion of g. vaginalis adhesion to hela cells. although lea showed sequence similarity to a pilus component of g. vaginalis, further studies are still needed to decipher whether the counterpart of lea is indeed the pilin subunit or some other adhesion associated molecule of g. vaginalis. in addition, we cannot rule out that surface molecules other than the ones recognized by the anti-lea fab fragments have participated in the contact between g. vaginalis and the host cell, since the fab fragments did not abolish the adhesion completely. nevertheless, the lea protein appears to be a key mediator of the competitive exclusion of g. vaginalis. in summary, we have presented a comparative analysis of ten l. crispatus genomes available within the public databases at the time of this study and provided a comprehensive look on the pan-genomic structure of this important urogenital species. furthermore, our analyses revealed a list of core genes implicated in protecting the urogenital tract from g. vaginalis colonization, providing new insights into the treatment and prevention of bv. electronic supplementary material additional file 1: overview of g. vaginalis strains and properties. in the table, the genomic properties of the g. vaginalis strains used in this study are given. hmp refers to the human microbiome project. additional file 2: list of l. helveticus, l. acidophilus and b. subtilis genomes included in the phylogenetic analysis. the accession is given for each genome. additional file 3: list of pfam domains used in the annotation of putative l. crispatus adhesins. the accession, id, and description are given for each pfam matching an adhesion or colonization related keyword. ones in the remaining columns indicate that the pfam matched some l. crispatus cds, passed the manual curation process and was included in the final list of adhesion or colonization related domains. additional file 4: the start and end compounds used in the metabolism screens. this table describes the compound pairs related to the de novo synthesis and interconversion of amino acids and carbohydrate metabolism. additional file 5: the l. crispatus data table. results of the different bioinformatic analyses for each l. crispatus cds. additional file 6: reservoirs of genetic variability. this table describes the distribution of best blast hits of strain-specific l. crispatus cdss. additional file 7: prevalence of different types of crispr/cas systems in vaginal and non-vaginal lactobacilli. additional file 8: variation in metabolism related enzymes in l. crispatus. the horizontal lines represent the orthologous groups with assigned ec numbers and the different strains are indicated at the bottom of the picture. the presence of a given ortholog group in a specific strain is indicated with grey or dark grey. the absence of a given ortholog group is indicated with light grey. the colored bar on the left describes the conservation level of the ortholog groups and follows that of the figure . the dendrogram was generated using ward linkage clustering of the presence/absence data. additional file 9: detected metabolic routes between different carbohydrate metabolism compounds and between different amino acid biosynthesis related compounds. the presence of intact metabolic route from one compound to another is indicated for each l. crispatus genome. pathways found in the core genome analysis are marked with yellow color. additional file 10: domain organization of l. crispatus adhesion and colonization factors. a representative member of each orthomcl-group is presented graphically. the larger colored blocks represent adhesion or colonization related pfam-domains and the thinner blocks other domains. the names of each color-coded domain are given at the bottom of the picture. additional file 11: overview of g. vaginalis ortholog groups. for each group, the associated proteins, pfam domains, matching proteins in l. crispatus, the total number of matching proteins in l. crispatus, and total number of l. crispatus genomes providing the matches are given in the table. abbreviations bvbacterial vaginosis cascrispr-associated cdscoding sequence cfuscolony forming units cogcluster of orthologous groups crisprclustered regularly interspaced short palindromic repeat epsexopolysaccharide gigenomic island gitgastrointestinal tract gutgenitourinary tract hgthorizontal gene transfer hmmhidden markov model lealactobacillus epithelium adhesin odoptical density pbsphosphate buffered saline pfaparaformaldehyde pmsfphenylmethylsulfonyl fluoride rutirecurrent urinary tract infection snpsingle nucleotide polymorphism. competing interests the authors declare that they have no competing interests. authors’ contributions to designed experiments and the adhesion experiments, analyzed the genomic data and prepared the manuscript. mk designed experiments, analyzed the genomic data and prepared the manuscript. jc performed the adhesion assays, analyzed the adhesion data and participated in preparing the manuscript. nc designed the adhesion experiments, analyzed the adhesion data and participated in preparing the manuscript. se provided the fab fragments. bww designed the adhesion experiments and participated in preparing the manuscript. lp participated in preparing the manuscript. lh participated in preparing the manuscript. pa participated in preparing the manuscript. all authors read, commented and approved the final manuscript. 
moreover, l. crispatus can contribute to the urogenital health of the host through competitive exclusion and the production of antimicrobial agents. notably, the conserved genetic backbone of l. crispatus accounted for close to 60% of the ortholog groups of an average l. crispatus strain and included factors for the competitive exclusion of g. vaginalis, providing an explanation on how this urogenital species could improve vaginal health. comparative genomicslactobacillus crispatuspan-genomecore genomenormal florabacterial vaginosisgardnerella vaginaliscompetitive exclusionissue-copyright-statement© the author 2014 in order to investigate the genetic diversity of this important urogenital species, we performed a comparative genomic analysis of l. crispatus. these findings thereby suggest that l. crispatus core proteins could protect the vagina from g. vaginalis and bacterial vaginosis. it can be recovered from the gastrointestinal tract of animals and it is a common constituent of the vaginal microbiota of humans. using antibody fragments specific for one of the conserved l. crispatus adhesins, we demonstrated that the l. crispatus core proteins have a potential to reduce the ability of gardnerella vaginalis to adhere to epithelial cells. importantly, the current core was observed to encode bacterial components potentially promoting urogenital health. utilizing the completed genome sequence of a strain st and the draft genome sequences of nine other l. crispatus isolates, we defined the scale and scope of the pan- and core genomic potential of l. crispatus. our comparative analysis identified and ortholog groups present in all or only some of the ten strains, respectively. lactobacillus crispatus is a ubiquitous micro-organism encountered in a wide range of host-associated habitats. based on mathematical modeling, sequencing of additional l. crispatus isolates would result in the identification of new genes and functions, whereas the conserved core of the ten strains was a good representation of the final l. crispatus core genome, estimated to level at about ortholog groups. our pan-genome analysis provides insights into the intraspecific genome variability and the collective molecular mechanisms of the species l. crispatus.
 lactobacillus crispatus is a ubiquitous micro-organism encountered in a wide range of host-associated habitats. it can be recovered from the gastrointestinal tract of animals and it is a common constituent of the vaginal microbiota of humans. moreover, l. crispatus can contribute to the urogenital health of the host through competitive exclusion and the production of antimicrobial agents. in order to investigate the genetic diversity of this important urogenital species, we performed a comparative genomic analysis of l. crispatus.  utilizing the completed genome sequence of a strain st and the draft genome sequences of nine other l. crispatus isolates, we defined the scale and scope of the pan- and core genomic potential of l. crispatus. our comparative analysis identified and ortholog groups present in all or only some of the ten strains, respectively. based on mathematical modeling, sequencing of additional l. crispatus isolates would result in the identification of new genes and functions, whereas the conserved core of the ten strains was a good representation of the final l. crispatus core genome, estimated to level at about ortholog groups. importantly, the current core was observed to encode bacterial components potentially promoting urogenital health. using antibody fragments specific for one of the conserved l. crispatus adhesins, we demonstrated that the l. crispatus core proteins have a potential to reduce the ability of gardnerella vaginalis to adhere to epithelial cells. these findings thereby suggest that l. crispatus core proteins could protect the vagina from g. vaginalis and bacterial vaginosis.  our pan-genome analysis provides insights into the intraspecific genome variability and the collective molecular mechanisms of the species l. crispatus. using this approach, we described the differences and similarities between the genomes and identified features likely to be important for urogenital health. notably, the conserved genetic backbone of l. crispatus accounted for close to 60% of the ortholog groups of an average l. crispatus strain and included factors for the competitive exclusion of g. vaginalis, providing an explanation on how this urogenital species could improve vaginal health. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords comparative genomicslactobacillus crispatuspan-genomecore genomenormal florabacterial vaginosisgardnerella vaginaliscompetitive exclusionissue-copyright-statement© the author 2014 