BACKGROUND
the gastrointestinal  tract of vertebrates is colonised by a complex microbial community dominated by bacteria referred to as the gut microbiota.
by having a profound influence on vertebrate physiology, metabolism, and immune functions, the gut microbiota plays important roles in the health of the host  <cit> .
these associations open avenues for the development of therapies that aim to restore the ecosystem, but their implementation requires a mechanistic understanding about the ecological principles that shape and regulate microbial communities  <cit> .
in contrast to microbial symbiosis in invertebrates, little is known about the basic principles that underlie symbiotic interactions in vertebrates and how they evolve  <cit> .
lactobacillus reuteri, a gram-positive bacterial species that colonises the gut of a variety of vertebrate species, has been used as a model to determine the ecology and evolution of vertebrate gut symbionts  <cit> .
the ecological strategies of l. reuteri are fundamentally different in humans and animals  <cit> .
in rodents, pigs, chickens and horses, lactobacilli form large populations in proximal regions of the gi tract, and they adhere directly to the stratified squamous epithelium present at these sites .
in mice and rats, adherence occurs in the forestomach  <cit> , and this process appears to be important with regards to the ecological fitness of the bacteria  <cit> .
the epithelial associations formed can be considered biofilms as the bacteria are arranged in multiple layers and are encased in a polysaccharide matrix  <cit> .
in contrast, stratified squamous epithelia are absent in the human gut, and epithelial cell layers rich in lactobacilli equivalent to those found in the above-mentioned animals have not been described  <cit> .
rather, a more transient colonisation of the human gi tract by l. reuteri is likely to be mediated by mucus-binding adhesins , resulting in a relatively low prevalence in the human population even although this species is still considered to be autochthonous in humans  <cit> .
using a combination of population genetics and comparative genomics, we have recently demonstrated that l. reuteri is composed of host-specific clades with lineage-specific genomic differences that reflect the niche characteristics in the gi tract of the respective hosts.
host adaptation of this species is supported by genetic clustering of strains originating from common or related hosts.
amplified-fragment length polymorphism  and multi-locus sequence analysis  with more than 100 l. reuteri strains isolated from humans, pigs, rats, mice, chickens and turkeys revealed that considerable genetic heterogeneity exists within the l. reuteri population, with distinct phylogenetic clades that reflect host origin of the strains  <cit> .
experiments in lactobacillus-free mice to measure the ecological fitness of strains originating from different hosts supported host adaptation, as only rodent strains colonised mice efficiently  <cit> .
furthermore the ability of l. reuteri to form epithelial biofilms in the mouse forestomach of mono-associated mice was strictly dependent on the strain’s host origin  <cit> .
genome comparisons of l. reuteri strains originating from different hosts identified lineage-specific genomic content that reflects the niche differences in the gi tract of rodents and humans.
the ecological significance of a subset of rodent-specific l. reuteri 100- <dig> genes was demonstrated in the context of the murine gut  <cit> .
this mutational analysis revealed that genes encoding proteins involved in epithelial adherence, specialised protein transport, cell aggregation, environmental sensing and cell lysis contributed to biofilm formation and colonisation.
in particular, the inactivation of a serine-rich repeat protein  surface adhesin with a devoted transport system  completely abrogated colonisation of the mouse forestomach, indicating that initial adhesion represented the most significant step in biofilm formation, likely conferring host specificity  <cit> .
similarly, host-strain specific adhesins have been recently reported in other l. reuteri strains as playing a key role in the interaction of the bacteria with their host.
these include the mucus-binding proteins, cmba from l. reuteri human strains  <cit>  and mub from l. reuteri atcc  <dig>  a strain isolated from pig .
comparative genomics of l. reuteri strains also revealed distinct levels of genetic heterogeneity in different phylogenetic lineages.
while human l. reuteri strains from the lineage ii, f <dig> , atcc pta- <dig> , and atcc pta- <dig>  possess a closed pan-genome with > <dig>  % average nucleotide identity , strains from rodents possessed a larger pan-genome with a variable gene content and an ani of around 96–97 %, and the majority of the rodent-specific genes detected by comparative genomic hybridisation  were not conserved among rodent strains  <cit> .
the work described above has established l. reuteri as an excellent model to elucidate mechanisms for host-microbial symbiosis in vertebrates.
however, the mechanisms by which l. reuteri strains specifically colonise the porcine gi tract and the evolutionary processes that resulted in host-specific phylogenetic clusters have not been elucidated.
such knowledge is relevant as l. reuteri is one of the most dominant species in the porcine gi tract  <cit>  and pig-derived strains of l. reuteri are used as probiotics to improve pig health and well-being .
previous mlsa indicated that porcine isolates fall within two distinct phylogenetic groups, clades iv and v, that are to a large degree host-specific, suggesting adaptation and specialisation towards the porcine host  <cit> .
here we performed a pan-genome analysis of six l. reuteri strains isolated from pigs  with the goal to gain insight into the molecular basis for colonization of the porcine gut, identify genes marking host-specific adaptations, and determine differences between the two l. reuteri lineages present in pigs.
further comparison with the genomes from human and rodent l. reuteri strains was carried out providing novel insights into the genomic events in l. reuteri that occurred during specialisation to the porcine host.
RESULTS
complete genome sequence of the pig l. reuteri strain atcc 53608
the l. reuteri atcc  <dig> genome was sequenced by  <dig> pyrosequencing and previously published as a draft assembly  <cit> .
according to previous phylogenetic analyses, the atcc  <dig> strain falls into the pig-specific mlst cluster iv  <cit>  .
here the genome was completed and fully assembled as described in methods to create a model reference backbone for genome comparisons of l. reuteri strains from pig.table  <dig>
l. reuteri strains used in this study for genome sequencing and for comparative genomics
amulti-locus sequence type analysis  <cit>
bmlst determined from genome sequence
genome organisation
the genome of l. reuteri atcc  <dig> consists of three circular molecules , a chromosome of 1943635 bp and two plasmids of 138515 bp  and 9093 bp , respectively.
the two plasmids were confirmed to be closed circular molecules by pcr with primer pairs b40/b <dig>  and sc10-for/sc10-rev  that flanked each single gap.
the chromosome has an average gc content of  <dig>  % compared with  <dig>  % for the small and  <dig>  % for the large plasmid.
the oriloc software  <cit>  in combination with the r software package  <cit>  was used to identify the putative oric and ter regions based on the cumulated 3rd codon position skews.
in the case of the chromosome, the positive peak for the combined skew is located immediately upstream of the dnaa gene and we predict the origin of replication to be located in the at rich  intergenic region between the rpmh  and dnaa  genes.
allowing for one mismatch, we detected  <dig> dnaa boxes  in this stretch of the genome, which were equally distributed between the forward and the reverse strands and we therefore designated the first base pair of dnaa as position  <dig> of the chromosome.
the negative peak of the combined skew, indicating the putative terminus region, is located in lratcc53608_ <dig> .
a total of  <dig> cdss were identified on the chromosome,  <dig>  % of which were encoded on the leading strand of chromosome replication.
this strong leading strand coding preference is a typical value observed in many other gram-positive organisms.
plasmid pi is a megaplasmid which carries a repa/para type replicative system and—like the chromosome—displays strong 3rd codon position skews.
this plasmid carries a total of  <dig> cdss,  <dig>  % of which are encoded on the leading strand of replication.
besides an incomplete prophage, the plasmid also carries genes encoding enzymes for purine and pyrimidine metabolism , a nicotinamide mononucleotide transporter, a dnae and trna genes .
though none of these can explain the stable maintenance of this megaplasmid, resulting in a considerable burden on the cell’s metabolism, as they are all additional copies of genes that are also present on the chromosome.
a toxin–antitoxin system encoded on this plasmid  is the most likely explanation for its stability.
plasmid pi also encodes a large number of hypothetical proteins.
plasmid pii carries six cdss and is potentially mobilisable by means of a predicted relaxase/mobilase.
a putative replication protein shares low sequence similarity with replication proteins from other lactobacilli.
interestingly, the completed genome sequence confirmed plasmid isolation data that strain atcc  <dig> lacks the 10251 bp plasmid plul <dig>  which is present in parental strain  <dig>  <cit>   and which encodes resistance to both erythromycin  and streptogramin a .fig.
<dig> multireplicon genome of l. reuteri strain atcc  <dig>  circles from outside to the centre: genes on forward strand , genes on reverse strand , pseudogenes , alien genes , alien genes , phast bacteriophage remnants , rrna genes , trna genes , gc content , gc skew
predicted primary metabolism
based on the genome annotation, l. reuteri atcc  <dig> has very limited amino acid synthesis capabilities.
this strain should be able to synthesise serine and glycine de novo from pyruvate using serine dehydratase , which catalyses pyruvate-serine interconversion.
the α and β chains of this protein are encoded by sdaaa and sdaab , respectively.
serine could subsequently be converted to glycine by serine hydroxymethyltransferase encoded by glya.
l. reuteri cannot carry out de novo synthesis of the aspartate family of amino acids as it does not carry a gene for pyruvate carboxylase, however it should be able to synthesise lysine and asparagine from aspartate.
the strain also lacks the gene for glutamate synthase but can interconvert glutamate and glutamine and carries a probac operon enabling the synthesis of proline from glutamate.
most pathways for vitamin biosynthesis are incomplete .
complete pathways were only found for the biosynthesis of folate and possibly riboflavin.
l. reuteri atcc  <dig> cannot synthesise lipoic acid, but appears able to produce coenzyme a from pantothenate and nad+ and nadp+ from nicotinamide mononucleotide, for whose uptake the genome encodes two transport proteins .
purine and pyrimidine biosynthetic pathways are present in l. reuteri atcc  <dig> and the formation of deoxyribonucleotides is not hampered by oxygen, as the strain possesses an oxygen-requiring class ib enzyme encoded by nrde <dig> and nrdf <dig>  and the necessary auxiliary proteins nrdh and nrdi , in addition to oxygen-sensitive class iii ribonucleotide reductases encoded by nrdd and nrdg .
the genes encoding these different ribonucleotide reductases appear organised in two operons , the first gene of which is preceded by one or two nrdr boxes, respectively, that according to the regprecise database  <cit>  are typical for lactobacillaceae, indicating that these genes are regulated by the negative transcriptional regulator nrdr .
horizontal gene transfer : pseudogenes, alien genes, is elements and bacteriophages
the genome of l. reuteri atcc  <dig> contains  <dig> pseudogenes in total, representing around  <dig>  % of the total gene number, thus significantly higher than the 1–5 % average figure reported for other bacterial genomes  <cit> .
the colombo sigi-hmm software  <cit>  predicts  <dig>  % of the total gene content of strain atcc  <dig> to originate from hgt .
the genome of atcc  <dig> was found to be moderately repetitive, containing  <dig> different insertion sequence  elements from seven is element families, particularly of the is <dig> family  contributing a total of  <dig> genes  or  <dig>  % of all genes encoded in the genome of l. reuteri atcc  <dig>  using the is element sequences we tried to identify the insertion sites and in many cases were able to create target site sequence logos using weblogo  <dig>  <cit>  on the basis of the direct repeats flanking the is elements .
the phast software  <cit>  identified three extended regions of bacteriophage origin in the genome of l. reuteri atcc  <dig>  the first is located on the chromosome  and the latter two are located in close vicinity to each other on plasmid i  .
assembly and chromosomal features of four additional pig strains of l. reuteri
we selected four additional l. reuteri strains isolated from pigs for genome sequencing, choosing strains from both pig-specific mlst clades iv  and v   <cit>  .
based on mlst analysis of seven housekeeping genes, type v strains are located phylogenetically close to, although distinct from, l. reuteri strains of human/avian origin type vi, whereas those from type iv fall between human and rodent isolates of types ii and iii, respectively  <cit> .
high coverage illumina data were obtained for these four l. reuteri strains and draft assemblies were generated as described in methods.
here we assigned taxon data to each separate contig and plotted % gc against the % coverage.
during the assembly process, a very low proportion of the reads was assigned to a few contaminating contigs of eukaryotic origin; additionally, in one of the assemblies , it was possible to discern a related lactobacillus sequence at a very low coverage, which gave rise to many short contigs that were removed from the final assembly.
the length of the final assembly and representation of expected genes is presented in table  <dig>  each of the assemblies possessed ≤ <dig> contigs of >100 bp at high coverage with a median contig length of  <dig>   <dig>   <dig> and 5386 bp for pg-3b, 3c <dig>  20- <dig> and lp167- <dig>  respectively.
the chromosomes of the completed and draft assembly strains showed syntenic conservation as aligned by mauve  <cit>  and by mummer v <dig>   <cit>  with relatively few regions of difference observed .
each draft genome was compared to the atcc  <dig> genome by measuring the % ani—lp167- <dig> , pg-3b , 3c <dig>  and 20- <dig> .
all their ani values were >95 %, confirming their identities as members of the same species  <cit> , viz.
l. reuteri.
the slightly lower ani values of just over 95 % for strains 3c <dig> and 20- <dig> reflected their presence in a separate phylogenetic clade to the others.table  <dig> draft genome assembly statistics for four l. reuteri strains isolated from pig gi tract
fig.
<dig> genome conservation in the six pig strains of l. reuteri.
from top to bottom, the mauve-alignment backbone of the draft genome sequences is shown as follows: i <dig>  atcc  <dig>  lp167- <dig>  pg-3b, 20- <dig> and 3c <dig>  blocks of sequence conservation are denoted by burgundy lines, whilst blocks as calculated by mauve are shown in alternating colours
the pig l. reuteri pan-genome
the complete atcc  <dig> genome was used as a model reference backbone to compare the assemblies of the additional four draft genomes of l. reuteri from pigs .
in addition, the recently completed genome of another pig l. reuteri strain, i <dig>   <cit> , was included in genome comparisons.
the combination of these six strains was referred to as the pig l. reuteri pan-genome.
analysis of the pig chromosomal core and accessory genomes
the backbone genetic information is defined as the genetic information common to all strains tested, which is also referred to as the ‘core genome’ and in this case represents genes present in all of the above l. reuteri strains isolated from pig.
the core set from these six genomes contained  <dig> orthologous gene clusters as determined by orthomcl analysis  <cit>  and  <dig> gene clusters, comprising  <dig> “unique” genes that occurred at least once in individual genomes—the difference between the latter two values being due to the presence of at least two gene copies in some genomes.
another  <dig> gene clusters were shared by at least two genomes, giving a total of  <dig> orthologous gene clusters in the current pig strain pan-genome .
very short predicted genes  were excluded from the analysis.
genes variable among pig strains make up the accessory genome.
these regions were parsed from the pig isolates at the dna level using the panseq program  <cit> .
a successive round of bioinformatic purification of dna sequence from the output of the panseq pipeline was carried out using mummer  <cit>  to further refine the data.
the accessory regions were annotated separately and viewed functionally as a pie chart .
compared to the functions of the whole genomes, these regions are rich in genes encoding cell wall-associated proteins, capsule/exopolysaccharide  biosynthetic enzymes, phage-related functions, mobile elements and dna metabolic enzymes, whilst being low in essential metabolism genes.
this is similar to the situation with rodent strains of l. reuteri where many cell surface proteins, proteins involved in surface polysaccharide biosynthesis and prophages fall within the accessory genome  <cit> .fig.
<dig> pan-genome of the six pig l. reuteri strains.
each genome is represented by a coloured oval.
the number in the centre represents the core gene set shared among the six pig strain genomes expressed as orthologous gene clusters per genome.
the numbers of genes unique to each genome are indicated by the outer values.
in addition,  <dig> orthologous gene clusters were shared by at least two genomes, contributing to a current pig strain pan-genome of  <dig> orthologous gene clusters.
short genes  were excluded from the analysis
fig.
<dig> predicted functions encoded by genes of the accessory genome from six l. reuteri porcine isolates.
functions listed are:  <dig>  cofactors;  <dig>  cell wall and capsule;  <dig>  virulence and defence;  <dig>  phages and mobile elements;  <dig>  rna metabolism;  <dig>  nucleosides and nucleotides;  <dig>  protein metabolism;  <dig>  regulation;  <dig>  dna metabolism;  <dig>  fatty acids and lipids;  <dig>  respiration;  <dig>  stress response;  <dig>  amino acids and derivatives;  <dig>  sulphur metabolism; and  <dig>  carbohydrates
the six genome sequences from porcine origin were examined further for the presence of clustered regularly interspaced short palindromic repeats  against the crisprdb database  <cit>  using the crisprfinder tool  <cit>  and any such sequences compared using the crisprcompar website  <cit> .
however, although questionable crisprs were identified for all genomes, no confirmed crisprs were uncovered by this analysis.
questionable crisprs include small crisprs of two to three direct repeats and structures where the repeats are not 100 % identical.
in the atcc  <dig> finished genome, questionable crisprs were located at 893116–893231 bp and comprised a set of two virtually identical direct repeats.
an almost identical situation occurs at 868686–868801 bp in strain i <dig>  however, no crispr-associated  gene hits were uncovered upstream or downstream of that region in either atcc  <dig> or i <dig>  which is located directly downstream from the accessory secretion system, a genetic region likely acquired by hgt.
analysis of plasmids in porcine l. reuteri strains
the putative plasmid dna sequences of the four pig draft genomes were aligned to plasmid sequences of pi and pii from l. reuteri atcc  <dig>  the sequence of plasmid plul <dig> from parental strain  <dig> and the six plasmid sequences from strain i <dig> and compared.
this method was used to extract potential plasmid sequences as contigs from the total assembly of the four strains.
without exception, all of the contigs extracted from each of the strains were present within the respective accessory regions that had been identified previously in comparison to strain atcc  <dig>  efforts to compare the gene annotations from the l. reuteri atcc  <dig> plasmids to those of the newly identified sequences were unsuccessful, in line with the lack of dna sequence alignment, and supporting the findings that the plasmid dna is heterogeneous and relatively unrelated in each of these strains.
further comparisons of the plasmids of atcc  <dig> with those from the other l. reuteri pig strains did not indicate large conservation amongst the plasmids of this species.
rather the plasmids appear isolate-dependent, which correlates with the results of plasmid isolation from these strains .
comparative genomics of l. reuteri genomes from different host origin
in order to gain insights into the subset of genes involved in adaptation to the porcine gut, we performed a comparative genomic analysis of 20 l. reuteri strains that are currently available, which originate from the pig , human  and rodent  gi tracts and from sourdough .
these strains were lp167- <dig>  pg-3b, 3c <dig>  20- <dig>  atcc  <dig>  i <dig>  jcm1112t, dsm20016t, atcc pta- <dig>  , atcc pta- <dig>  , cf48-3a <dig>  atcc  <dig>  , td <dig>  lpuph <dig>  mlc <dig>  100- <dig>  tmw <dig> , tmw <dig> , lth <dig> and lth <dig>  a set of  <dig> of the available  <dig> genomes was used in the analysis, whereby jcm1112t and dsm20016t were considered as a single genome  since these two strains are derived from the same isolate .
the core set of these  <dig> genomes contained  <dig> orthologous gene clusters by orthomcl  <cit>  analysis, while there were  <dig> gene clusters, comprising  <dig> “unique” genes that occurred at least once in individual genomes – the difference between the latter two values again being due to the presence of at least two gene copies in some genomes.
another  <dig> gene clusters were shared by at least two genomes, giving a total l. reuteri pan-genome of  <dig> orthologous gene clusters .
a circular comparison of the genomes of the six strains isolated from pig with genomes from a selection of seven other l. reuteri strains from human and rodent is shown in additional file 1: figure s <dig>
homologous genes were calculated using l. reuteri atcc  <dig> as the reference strain by reciprocal best match analysis requiring a 40 % match by fasta searches over 80 % of the target protein length.
this required the target protein to be predicted almost in entirety using current gene prediction software prodigal  <cit>  and glimmer <dig>  <cit> .
with this method, unpredicted proteins, either due to unusual base composition or to fragmentation at the contig level, would lead to a negative result.
using protein sequences derived from the genomic data, three additional analyses  using the joint genome institute’s integrated microbial genomes  phylogenetic profiler tool  <cit> , reciprocal fasta with the annotation software artemis  <cit> , and orthomcl  <cit>  all indicated that there were no genes conserved in all six pig-derived genomes that were absent in the genomes of l. reuteri strains from other hosts.
therefore our analyses showed that there are no pig-specific genes.
previous mlsa revealed that pig isolates fall into two distinct clades  <cit> .
a phylogeny based on the core genes of  <dig> strains of l. reuteri  confirmed the phylogenetic clusters and their membership.
clade iv contains strains i <dig>  atcc  <dig>  lp167- <dig> and pg-3b, and is more closely related to the rodent cluster iii, while clade v, which is more closely related to human/poultry strains of clade vi, contains strains 20- <dig> and 3c <dig>  as reported previously  <cit> , the geographical origin of the strains was not related to the phylogenetic clades since l. reuteri strain i <dig> was isolated in china  <cit>  but found as a close relative of atcc  <dig> , isolated in sweden  <cit> .
these findings indicate that the co-evolution of l. reuteri with pigs has resulted in the formation of two vastly distinct phylogenetic clusters, which suggests that evolution of these two populations, although driven by the porcine host , was subjected to different selective pressures that led to their separation.
the distinct evolutionary trajectories could also have resulted in the absence of common genes that reflect host adaptation among porcine strains.
the relatedness of pig clade iv with rodent clade iii strains, and of pig clade v with human/poultry clade vi strains may reflect their evolution from ancestral strains of rodent and human origin, respectively, following contact with pig populations.fig.
<dig> a maximum likelihood phylogenetic tree of the core genome alignment of 20 l. reuteri genomes.
concatenated core gene sequences were compared.
the tree was bootstrapped with  <dig> replicates, shown as percentages at each branch.
numbers i-vi represent the original mlsa-based groupings.
the host origin of each strain is denoted in parenthesis after the strain name as follows:  human,  pig,  rodent and  sourdough
to gain insight into this diversification process, we identified genes that were specific to the two individual clades, and conserved among the strains.
this analysis revealed  <dig> lineage iv-specific genes encoding proteins > <dig> amino acids.
among these genes was one  encoding a surface protein of  <dig> amino acids in length that contained five mucus-binding  domains .
in addition, cluster iv strains contained three linked genes  encoding a tetr family transcriptional regulator, a conserved hypothetical protein with a dhs-like nad/fad-binding domain and an nad-dependent protein deacetylase of the sir <dig> family whose functions are as yet unknown.
the two clade iv-specific esterase-lipases  could be involved in the utilization of lipids in the pig’s diet but on closer examination appeared to be two parts of the same pseudogene.
an interesting feature of the clade-iv-specific genes was that they had very high levels of homology  among the four different strains, which supports that they are highly conserved among porcine isolates of this clade.table  <dig> genes specific to l. reuteri pig clades iv and v
of the  <dig> genes encoding proteins > <dig> amino acids that were specific to pig cluster v , several represented mobile genetic elements, including transposons and phage-related sequences.
however, four lineage-specific surface proteins were detected, three of which contained a sec10/pgra surface exclusion domain, found in gram-positive adhesins such as spyad from group a streptococci and implicated in cell adhesion  <cit> .
two of these sec10/pgra proteins  also contained a rib/alpha-like repeat from the rib adhesin of group b streptococci  <cit> .
two genes encoding glycosyltransferases  were also identified.
in addition, a putative copper oxidase and a magnesium-transporting atpase were detected, suggesting interactions of this l. reuteri lineage with metal ions.
several hypothetical proteins were found with high homology among the two strains.
interestingly, one of these proteins  showed 97 % aa identity to a protein from lactobacillus amylovorus, which is indicative of hgt from another dominant member of the pig microbiota  <cit> , suggesting an important function in the pig gut.
previous pan-genome analysis of l. reuteri rodent and human strains revealed that the pdu-cbi-cob-hem cluster was conserved within human strains, and the cluster was absent in most rodent strains  <cit> .
this cluster codes for cobalamin  biosynthesis, glycerol utilization, propanediol fermentation and production of the antimicrobial compound reuterin  <cit> .
the genetic region encompassing the reuterin and vitamin b <dig> biosynthetic pathways is located at 293889–338218 bp on porcine reference isolate l. reuteri atcc  <dig>  as in rodent strains, it appears that this pdu-cbi-cob-hem genomic island is present only in a subset of porcine strains, confirming previous findings  <cit> .
out of the four strains from clade iv, only atcc  <dig> and lp167- <dig> possess this genomic island, while it is present in both clade v strains 20- <dig> and 3c <dig> studied ─ although this island is not conserved in other members of pig clade v  <cit> .
thus it appears that the possession of the reuterin genomic island does not correlate directly with the relationship of the genetic backbone.
as with the rat isolate 100- <dig>  there is evidence that the cluster has been deleted from the genome of pig isolate i <dig> through the action of mobile elements.
molecular determinants of host recognition
in order to gain further insight into the molecular determinants of host-strain specificity, we focused on the genomic analysis of the major cell surface components that have been implicated in the interaction of l. reuteri strains with the host in vivo.
cell surface proteins
several surface proteins of l. reuteri that are involved in colonisation by binding to epithelia, epithelial cells, or mucus have been functionally characterised .
proteins such as mub and the large surface protein  contain lpxtg cell wall anchoring motifs, are extremely large, contain multiple repeated motifs and resemble adhesins of pathogenic microbes  <cit> .
here, a total of  <dig> surface protein-encoding genes and three conserved pseudogenes were predicted from the completed genome of strain atcc  <dig> .
potential homologues were identified in the other pig strains from the annotation and sequence read data from the draft assemblies by alignment to atcc  <dig> using bowtie  <dig>  <cit>  and checked separately by burrows-wheeler aligner software, bwa  <cit> .
using bedtools  <cit> , the percentage of each protein coding sequence covered was determined in the four pig strain draft genomes and results are displayed in a heatmap  with the characteristics of the proteins.
many of these surface proteins are predicted to be involved in epithelial adhesion and biofilm formation.
mub was found only in strain atcc  <dig> but a few other mucbp  domain-containing proteins were identified, some as conserved pseudogenes, and two were found to be pig clade iv-specific .
other putative surface proteins found only in atcc  <dig> were lratcc53608_ <dig>  _ <dig> and _ <dig> although the significance of these is unknown.
several proteins with one or more lysin motif , a domain involved in peptidoglycan binding  <cit> , were present in all five pig strains analysed, including homologues of lratcc53608_ <dig>  which are identical to lr_ <dig> from rodent strain 100- <dig>  lysm-containing proteins lr_ <dig> and lr_ <dig> are putative aggregation promoting factor proteins that have been implicated in biofilm formation and colonisation of strain 100- <dig> in mice  <cit> , suggesting a similar role in the pig host.
in the heatmap of additional file 1: figure s <dig>  there is therefore a clear difference between the two representatives of clade iv, pg-3b and lp167- <dig>  and the two representatives of clade v, 20- <dig> and 3c <dig>  with respect to the presence or absence of certain surface proteins.
on the other hand, the other member of clade iv that was used as the reference strain, atcc  <dig>  possessed additional surface proteins not found in strains pg-3b and lp167- <dig>
one interesting putative surface protein, not included in the above analysis because of its lack of a consensus secretion signal, was the duf <dig> repeat-containing protein encoded by lratcc53608_ <dig> and its homologues in pig clade iv and in three sourdough strains .
although a putative pseudogene in these strains, it may still be translated as a protein ranging in size from about 880– <dig> aa and exported by a non-classical secretion pathway .
duf <dig> repeats are found in many gram-positive cell surface adhesins, such as maba, a modulator of adhesion and biofilm formation, from lactobacillus rhamnosus gg  <cit> .
homologues of this protein were not found in the two pig clade v strains and were also absent in all human strains but functional counterparts with a typical secretion signal were present in all rodent strains and one sourdough strain .
the significance of this duf <dig> repeat-containing surface protein in l. reuteri is as yet unknown but it appears to be conserved phylogenetically as either putative pseudogenes or functional genes in different host-derived strains.
the accessory seca2-secy <dig> secretion system
in streptococci and staphylococci, the accessory seca2-secy <dig> system facilitates the selective export of glycosylated serine-rich repeat proteins  that often function as adhesins such as gspb, fap <dig> and srap .
this auxiliary protein secretion system is present in a limited number of gram-positive bacteria and mycobacteria and other members of the class bacilli  <cit> ; its sparse distribution among different species of lactobacilli implies that this system was horizontally acquired by only a few lactobacillus lineages.
in the l. reuteri rodent strain 100- <dig>  an srrp  was identified as the only protein secreted by seca2-secy <dig> and shown to be essential for the formation of biofilm in germ-free mice  <cit> .
the gene content within the accessory sec cluster is conserved in l. reuteri atcc  <dig> when compared with that of rat strain 100- <dig> .
furthermore, the seca2-secy <dig> cluster is also conserved in the other five pig strains .
it is of note that on the phylogenetic tree of fig.
<dig>  the seca2-secy <dig> clusters of atcc  <dig> and of i <dig> fell between those of clade v strains 20- <dig> and 3c <dig> and the other clade iv strains pg-3b and lp167- <dig>  with which atcc  <dig> and i <dig> share the most backbone genetic material.
this may indicate that the seca2-secy <dig> region of atcc  <dig> has undergone hgt from clade iv or that some amount of introgression is underway.
hgt of the seca2-secy <dig> cluster in l. reuteri pig strains is supported by the presence of mobile genetic elements within the cluster of atcc  <dig> , a low gc content and by results from the analysis with alien_hunter  <cit>  confirming that this region is likely to have been acquired by hgt, although it cannot be excluded that the presence of mobile elements is a prelude to deletion or modification of the cluster.
by analogy to the 100- <dig> srrp and to srrps from other bacteria, the srrp homologue in atcc  <dig>  and the other pig strains is likely to be secreted through this accessory pathway and indeed, this protein is found in extracellular extracts of atcc  <dig> .
lratcc53608_ <dig>  like lr_ <dig> from 100- <dig>  is unusually serine rich  and contains  <dig> of the predominant 10-amino acid repeat slsnsvsmse, compared with  <dig> such repeats in lr_ <dig>  however, each srrp from the pig and rodent strains differs with respect to the number and sequences of their serine-rich repeats .
comparing these repeat sequences, the srrps from clade iv strains atcc  <dig>  lp167- <dig> and i <dig> are more similar, while that from the other clade iv strain, pg-3b, is more like the srrp from clade v strain 20- <dig>  the serine residues of the srrps are likely to be glycosylated by glycosyltransferases associated with the seca2-secy <dig> cluster .fig.
<dig> phylogeny and blast comparisons of seca2-secy <dig> regions from the six pig strains of l. reuteri.
for the four draft genomes of lp167- <dig>  pg-3b, 20- <dig> and 3c <dig>  the differently coloured gene bars represent separate contigs that were brought together after alignment to the completed reference atcc  <dig> genome using mummer.
genome comparisons are shown as blast matches, with the darker bars being of higher match.
the phylogenetic tree was generated from the sequences only of the seca2-secy <dig> regions
no other functional large surface proteins are linked to the seca2-secy <dig> cluster in these pig strains, except for two lpxtg-containing pseudogenes that flank the cluster at either end .
lratcc53608_0902-_ <dig> is a mucbp-containing lpxtg pseudogene whereas lratcc53608_0916-_ <dig> is an srrp pseudogene that possibly arose from gene duplication of the functional srrp, lratcc53608_ <dig>  in the completed genome of strain i <dig> the srrp itself is a pseudogene  but not annotated as such .
interestingly, this strain possesses a homologue  of the cmba  mucus-binding protein that is found in many human isolates of l. reuteri  <cit>  but is absent in other pig strains.
also of interest, the mucbp-lpxtg pseudogenes from the pig strains have intact homologues in rodent and sourdough isolates of l. reuteri, suggesting their loss of function during evolution in the pig host .
the srrps showed high sequence conservation among pig strains, particularly in the n-terminal putative binding region .
homology between the n-terminal domains of srrps from pig and rodent strains was lower , suggesting that they may share a common structure but bind to different target ligands in their respective hosts.
as expected, the pseudo-srrps showed more sequence diversity and were only about 30–40 % identical at the amino acid level to their respective functional counterparts .
conservation of the srrps and seca2-secy <dig> cluster in all six l. reuteri strains from pig mlsa lineages iv and v  and in most strains from rodents  but not in isolates from human and poultry hosts  may explain host-specific differences in l. reuteri biofilm formation  <cit> .
the eps cluster
l. reuteri strains can produce eps in several forms depending on the strain - levan  catalysed by the enzyme levansucrase , inulin  catalysed by the enzyme inulosucrase   <cit>  and reuteran  catalysed by glucosyltransferases  gtfa or gtfo .
ftf and inu share 86 % aa similarity and are therefore difficult to distinguish at the sequence level.
some l. reuteri strains possess both types of enzyme but others have only one.
mutation of the single ftf gene in rodent strain 100- <dig> resulted in loss of eps production and although the ftf mutant was able to colonise the murine gi tract of lactobacillus-free mice in the absence of competition, colonisation was impaired in competition with the wild type, indicating a role in host interaction  <cit> .
genomic analyses of l. reuteri atcc  <dig> and the other five pig strains identified ftf/inu homologues in all six strains with approximately 80 % similarity at the protein level to that from rat strain 100- <dig> , suggesting that the ftf/inu gene was acquired by hgt early in the evolution of the species and subsequently diversified at accelerated rates.
homologues of ftf/inu are also present in human isolates of l. reuteri, atcc  <dig>  and cf48-3a <dig>  recently, a cluster of more than  <dig> genes, including several encoding gtf’s, that may contribute to eps synthesis and is similarly organised compared to eps-related genes described in other lactobacillus strains, including l. rhamnosus gg  <cit> , was reported in the l. reuteri human strain atcc  <dig>   <cit> .
although the start of this operon was similar in the human strain atcc pta- <dig> , the presence of an insertional element in strain atcc pta- <dig>  after the first  <dig> genes indicated that genomic rearrangements and gene loss had occurred  <cit> .
regions relating to these putative eps gene clusters were found in the accessory genomes of the six pig strains .
despite the close phylogenetic relationships between strains of the atcc  <dig> clade, the predicted eps gene clusters showed poor conservation, with only the epsa, c and d encoding genes strictly conserved, suggesting large-scale eps gene cluster differences at the isolate level between these strains.
this is in accordance with the eps cluster in rodent l. reuteri strains, where the two eps gene clusters show high variability  <cit> .
like the rodent strains, all six pig strains also possessed a second cluster of genes unlinked to the putative eps cluster in fig.
7—lratcc53608_0648-_ <dig>  lri_0601-_ <dig>  lr3c6_00497-_ <dig>  lr202_01632-_ <dig>  lrlp167_00566-_ <dig> and lrpg3b_01609-_ <dig> – four of which included a second epsc-encoding gene and were therefore capable of producing other extracellular polysaccharides.fig.
<dig> predicted eps gene clusters in the six l. reuteri pig strains.
a, pg-3b, b, lp167- <dig>  c, 3c <dig>  d, 20- <dig>  e, atcc  <dig> and f, i <dig>  colour scheme: yellow, gtf; green, other protein involved in polysaccharide biosynthesis; blue, membrane protein; orange, hypothetical protein; pink, transposase/mobile element protein; brown, pseudogene; red, rna polymerase σ subunit
the glucansucrase/reuteransucrase gtfa responsible for reuteran biosynthesis was originally described in probiotic strain  <dig>  <cit>  and an ncbi blastp analysis revealed the presence of homologues in sourdough strains tmw <dig>  and tmw <dig>  with 93 % aa identity to gtfa.
the homologous gtfo  <cit>  from human probiotic strains cf48-3a <dig> and atcc  <dig>  are 68 % identical to gtfa.
other l. reuteri homologues gtf <dig> and gtfml <dig> with 78–79 % aa identity to gtfa have been described in probiotic strain  <dig> and mouse strain ml <dig>  synthesising α- <dig> -linked dextran and α- <dig> -linked mutan, respectively  <cit> .
interestingly, no homologues with significant  aa identity to the above-mentioned four gtf’s over the entire protein length were found in pig isolates atcc  <dig>  3c <dig> and 20- <dig>  however, homologues from the other pig strains i <dig>  pg-3b and lp167- <dig>  had 95–99 % aa identity to gtf <dig>  suggesting that these three clade iv strains most likely synthesise an α- <dig> -linked dextran-type polymer.
like the ftf and inu genes, the gtf <dig> homologues are unlinked to the putative eps clusters displayed in fig.
<dig>  indicating that the latter are involved in the biosynthesis of other types of eps.
thus, the ability of different strains to produce distinct types of eps in the extracellular matrix in the host’s git may be a contributing factor in determining host specificity.
CONCLUSIONS
this comparative genomics study provides novel insights into the ecology and evolution of the species l. reuteri with the porcine host.
together with previous phylogenetic analyses based on a small set of housekeeping genes, the genomic analysis highlights two divergent clades within l. reuteri pig strains that can be differentiated from other l. reuteri lineages.
although no genes were identified that were conserved among all pig isolates and specific to this host, we identified genes specific for and conserved among strains from the two pig phylogenetic lineages iv and v, with high rates of homology among strains.
the two populations of porcine l. reuteri appear to have evolved separately, through a process driven by distinct selective pressures, e.g.
different pig host genotypes or environmental factors such as dietary components, which resulted in the absence of common pig-specific genes among these two populations.
however, from the phylogenetic analysis, the closeness of rodent clade iii with pig clade iv strains, and of human clade vi with pig clade v strains could reflect an evolution of ancestral strains following contact of rodents and humans, respectively, with pig populations.
the identification of several surface proteins, some with mucus-binding mucbp domains or other repeated domains found associated with adhesins, as being pig clade-specific might contribute to host-specificity, and ultimately to the clustering of strains away from other l. reuteri lineages.
clearly, these genes provide a basis for future functional studies on the ecology of l. reuteri in the pig git.

 the gastrointestinal tract of vertebrates is colonised by a complex microbial community dominated by bacteria referred to as the gut microbiota. by having a profound influence on vertebrate physiology, metabolism, and immune functions, the gut microbiota plays important roles in the health of the host. these associations open avenues for the development of therapies that aim to restore the ecosystem, but their implementation requires a mechanistic understanding about the ecological principles that shape and regulate microbial communities. in contrast to microbial symbiosis in invertebrates, little is known about the basic principles that underlie symbiotic interactions in vertebrates and how they evolve. lactobacillus reuteri, a gram-positive bacterial species that colonises the gut of a variety of vertebrate species, has been used as a model to determine the ecology and evolution of vertebrate gut symbionts. the ecological strategies of l. reuteri are fundamentally different in humans and animals. in rodents, pigs, chickens and horses, lactobacilli form large populations in proximal regions of the gi tract, and they adhere directly to the stratified squamous epithelium present at these sites. in mice and rats, adherence occurs in the forestomach, and this process appears to be important with regards to the ecological fitness of the bacteria. the epithelial associations formed can be considered biofilms as the bacteria are arranged in multiple layers and are encased in a polysaccharide matrix. in contrast, stratified squamous epithelia are absent in the human gut, and epithelial cell layers rich in lactobacilli equivalent to those found in the above-mentioned animals have not been described. rather, a more transient colonisation of the human gi tract by l. reuteri is likely to be mediated by mucus-binding adhesins, resulting in a relatively low prevalence in the human population even although this species is still considered to be autochthonous in humans. using a combination of population genetics and comparative genomics, we have recently demonstrated that l. reuteri is composed of host-specific clades with lineage-specific genomic differences that reflect the niche characteristics in the gi tract of the respective hosts. host adaptation of this species is supported by genetic clustering of strains originating from common or related hosts. amplified-fragment length polymorphism and multi-locus sequence analysis with more than 100 l. reuteri strains isolated from humans, pigs, rats, mice, chickens and turkeys revealed that considerable genetic heterogeneity exists within the l. reuteri population, with distinct phylogenetic clades that reflect host origin of the strains. experiments in lactobacillus-free mice to measure the ecological fitness of strains originating from different hosts supported host adaptation, as only rodent strains colonised mice efficiently. furthermore the ability of l. reuteri to form epithelial biofilms in the mouse forestomach of mono-associated mice was strictly dependent on the strain’s host origin. genome comparisons of l. reuteri strains originating from different hosts identified lineage-specific genomic content that reflects the niche differences in the gi tract of rodents and humans. the ecological significance of a subset of rodent-specific l. reuteri 100- genes was demonstrated in the context of the murine gut. this mutational analysis revealed that genes encoding proteins involved in epithelial adherence, specialised protein transport, cell aggregation, environmental sensing and cell lysis contributed to biofilm formation and colonisation. in particular, the inactivation of a serine-rich repeat protein surface adhesin with a devoted transport system completely abrogated colonisation of the mouse forestomach, indicating that initial adhesion represented the most significant step in biofilm formation, likely conferring host specificity. similarly, host-strain specific adhesins have been recently reported in other l. reuteri strains as playing a key role in the interaction of the bacteria with their host. these include the mucus-binding proteins, cmba from l. reuteri human strains and mub from l. reuteri atcc a strain isolated from pig. comparative genomics of l. reuteri strains also revealed distinct levels of genetic heterogeneity in different phylogenetic lineages. while human l. reuteri strains from the lineage ii, f, atcc pta-, and atcc pta- possess a closed pan-genome with >  % average nucleotide identity, strains from rodents possessed a larger pan-genome with a variable gene content and an ani of around 96–97 %, and the majority of the rodent-specific genes detected by comparative genomic hybridisation were not conserved among rodent strains. the work described above has established l. reuteri as an excellent model to elucidate mechanisms for host-microbial symbiosis in vertebrates. however, the mechanisms by which l. reuteri strains specifically colonise the porcine gi tract and the evolutionary processes that resulted in host-specific phylogenetic clusters have not been elucidated. such knowledge is relevant as l. reuteri is one of the most dominant species in the porcine gi tract and pig-derived strains of l. reuteri are used as probiotics to improve pig health and well-being. previous mlsa indicated that porcine isolates fall within two distinct phylogenetic groups, clades iv and v, that are to a large degree host-specific, suggesting adaptation and specialisation towards the porcine host. here we performed a pan-genome analysis of six l. reuteri strains isolated from pigs with the goal to gain insight into the molecular basis for colonization of the porcine gut, identify genes marking host-specific adaptations, and determine differences between the two l. reuteri lineages present in pigs. further comparison with the genomes from human and rodent l. reuteri strains was carried out providing novel insights into the genomic events in l. reuteri that occurred during specialisation to the porcine host.  complete genome sequence of the pig l. reuteri strain atcc 53608 the l. reuteri atcc genome was sequenced by pyrosequencing and previously published as a draft assembly. according to previous phylogenetic analyses, the atcc strain falls into the pig-specific mlst cluster iv. here the genome was completed and fully assembled as described in methods to create a model reference backbone for genome comparisons of l. reuteri strains from pig.table l. reuteri strains used in this study for genome sequencing and for comparative genomics amulti-locus sequence type analysis bmlst determined from genome sequence genome organisation the genome of l. reuteri atcc consists of three circular molecules, a chromosome of 1943635 bp and two plasmids of 138515 bp and 9093 bp, respectively. the two plasmids were confirmed to be closed circular molecules by pcr with primer pairs b40/b and sc10-for/sc10-rev that flanked each single gap. the chromosome has an average gc content of  % compared with  % for the small and  % for the large plasmid. the oriloc software in combination with the r software package was used to identify the putative oric and ter regions based on the cumulated 3rd codon position skews. in the case of the chromosome, the positive peak for the combined skew is located immediately upstream of the dnaa gene and we predict the origin of replication to be located in the at rich intergenic region between the rpmh and dnaa genes. allowing for one mismatch, we detected dnaa boxes in this stretch of the genome, which were equally distributed between the forward and the reverse strands and we therefore designated the first base pair of dnaa as position of the chromosome. the negative peak of the combined skew, indicating the putative terminus region, is located in lratcc53608_. a total of cdss were identified on the chromosome,  % of which were encoded on the leading strand of chromosome replication. this strong leading strand coding preference is a typical value observed in many other gram-positive organisms. plasmid pi is a megaplasmid which carries a repa/para type replicative system and—like the chromosome—displays strong 3rd codon position skews. this plasmid carries a total of cdss,  % of which are encoded on the leading strand of replication. besides an incomplete prophage, the plasmid also carries genes encoding enzymes for purine and pyrimidine metabolism, a nicotinamide mononucleotide transporter, a dnae and trna genes. though none of these can explain the stable maintenance of this megaplasmid, resulting in a considerable burden on the cell’s metabolism, as they are all additional copies of genes that are also present on the chromosome. a toxin–antitoxin system encoded on this plasmid is the most likely explanation for its stability. plasmid pi also encodes a large number of hypothetical proteins. plasmid pii carries six cdss and is potentially mobilisable by means of a predicted relaxase/mobilase. a putative replication protein shares low sequence similarity with replication proteins from other lactobacilli. interestingly, the completed genome sequence confirmed plasmid isolation data that strain atcc lacks the 10251 bp plasmid plul which is present in parental strain and which encodes resistance to both erythromycin and streptogramin a.fig. multireplicon genome of l. reuteri strain atcc circles from outside to the centre: genes on forward strand, genes on reverse strand, pseudogenes, alien genes, alien genes, phast bacteriophage remnants, rrna genes, trna genes, gc content, gc skew predicted primary metabolism based on the genome annotation, l. reuteri atcc has very limited amino acid synthesis capabilities. this strain should be able to synthesise serine and glycine de novo from pyruvate using serine dehydratase, which catalyses pyruvate-serine interconversion. the α and β chains of this protein are encoded by sdaaa and sdaab, respectively. serine could subsequently be converted to glycine by serine hydroxymethyltransferase encoded by glya. l. reuteri cannot carry out de novo synthesis of the aspartate family of amino acids as it does not carry a gene for pyruvate carboxylase, however it should be able to synthesise lysine and asparagine from aspartate. the strain also lacks the gene for glutamate synthase but can interconvert glutamate and glutamine and carries a probac operon enabling the synthesis of proline from glutamate. most pathways for vitamin biosynthesis are incomplete. complete pathways were only found for the biosynthesis of folate and possibly riboflavin. l. reuteri atcc cannot synthesise lipoic acid, but appears able to produce coenzyme a from pantothenate and nad+ and nadp+ from nicotinamide mononucleotide, for whose uptake the genome encodes two transport proteins. purine and pyrimidine biosynthetic pathways are present in l. reuteri atcc and the formation of deoxyribonucleotides is not hampered by oxygen, as the strain possesses an oxygen-requiring class ib enzyme encoded by nrde and nrdf and the necessary auxiliary proteins nrdh and nrdi, in addition to oxygen-sensitive class iii ribonucleotide reductases encoded by nrdd and nrdg. the genes encoding these different ribonucleotide reductases appear organised in two operons, the first gene of which is preceded by one or two nrdr boxes, respectively, that according to the regprecise database are typical for lactobacillaceae, indicating that these genes are regulated by the negative transcriptional regulator nrdr. horizontal gene transfer: pseudogenes, alien genes, is elements and bacteriophages the genome of l. reuteri atcc contains pseudogenes in total, representing around  % of the total gene number, thus significantly higher than the 1–5 % average figure reported for other bacterial genomes. the colombo sigi-hmm software predicts  % of the total gene content of strain atcc to originate from hgt. the genome of atcc was found to be moderately repetitive, containing different insertion sequence elements from seven is element families, particularly of the is family contributing a total of genes or  % of all genes encoded in the genome of l. reuteri atcc using the is element sequences we tried to identify the insertion sites and in many cases were able to create target site sequence logos using weblogo on the basis of the direct repeats flanking the is elements. the phast software identified three extended regions of bacteriophage origin in the genome of l. reuteri atcc the first is located on the chromosome and the latter two are located in close vicinity to each other on plasmid i. assembly and chromosomal features of four additional pig strains of l. reuteri we selected four additional l. reuteri strains isolated from pigs for genome sequencing, choosing strains from both pig-specific mlst clades iv and v. based on mlst analysis of seven housekeeping genes, type v strains are located phylogenetically close to, although distinct from, l. reuteri strains of human/avian origin type vi, whereas those from type iv fall between human and rodent isolates of types ii and iii, respectively. high coverage illumina data were obtained for these four l. reuteri strains and draft assemblies were generated as described in methods. here we assigned taxon data to each separate contig and plotted % gc against the % coverage. during the assembly process, a very low proportion of the reads was assigned to a few contaminating contigs of eukaryotic origin; additionally, in one of the assemblies, it was possible to discern a related lactobacillus sequence at a very low coverage, which gave rise to many short contigs that were removed from the final assembly. the length of the final assembly and representation of expected genes is presented in table  each of the assemblies possessed ≤ contigs of >100 bp at high coverage with a median contig length of and 5386 bp for pg-3b, 3c 20- and lp167- respectively. the chromosomes of the completed and draft assembly strains showed syntenic conservation as aligned by mauve and by mummer v with relatively few regions of difference observed. each draft genome was compared to the atcc genome by measuring the % ani—lp167-, pg-3b, 3c and 20-. all their ani values were >95 %, confirming their identities as members of the same species, viz. l. reuteri. the slightly lower ani values of just over 95 % for strains 3c and 20- reflected their presence in a separate phylogenetic clade to the others.table draft genome assembly statistics for four l. reuteri strains isolated from pig gi tract fig. genome conservation in the six pig strains of l. reuteri. from top to bottom, the mauve-alignment backbone of the draft genome sequences is shown as follows: i atcc lp167- pg-3b, 20- and 3c blocks of sequence conservation are denoted by burgundy lines, whilst blocks as calculated by mauve are shown in alternating colours the pig l. reuteri pan-genome the complete atcc genome was used as a model reference backbone to compare the assemblies of the additional four draft genomes of l. reuteri from pigs. in addition, the recently completed genome of another pig l. reuteri strain, i, was included in genome comparisons. the combination of these six strains was referred to as the pig l. reuteri pan-genome. analysis of the pig chromosomal core and accessory genomes the backbone genetic information is defined as the genetic information common to all strains tested, which is also referred to as the ‘core genome’ and in this case represents genes present in all of the above l. reuteri strains isolated from pig. the core set from these six genomes contained orthologous gene clusters as determined by orthomcl analysis and gene clusters, comprising “unique” genes that occurred at least once in individual genomes—the difference between the latter two values being due to the presence of at least two gene copies in some genomes. another gene clusters were shared by at least two genomes, giving a total of orthologous gene clusters in the current pig strain pan-genome. very short predicted genes were excluded from the analysis. genes variable among pig strains make up the accessory genome. these regions were parsed from the pig isolates at the dna level using the panseq program. a successive round of bioinformatic purification of dna sequence from the output of the panseq pipeline was carried out using mummer to further refine the data. the accessory regions were annotated separately and viewed functionally as a pie chart. compared to the functions of the whole genomes, these regions are rich in genes encoding cell wall-associated proteins, capsule/exopolysaccharide biosynthetic enzymes, phage-related functions, mobile elements and dna metabolic enzymes, whilst being low in essential metabolism genes. this is similar to the situation with rodent strains of l. reuteri where many cell surface proteins, proteins involved in surface polysaccharide biosynthesis and prophages fall within the accessory genome.fig. pan-genome of the six pig l. reuteri strains. each genome is represented by a coloured oval. the number in the centre represents the core gene set shared among the six pig strain genomes expressed as orthologous gene clusters per genome. the numbers of genes unique to each genome are indicated by the outer values. in addition, orthologous gene clusters were shared by at least two genomes, contributing to a current pig strain pan-genome of orthologous gene clusters. short genes were excluded from the analysis fig. predicted functions encoded by genes of the accessory genome from six l. reuteri porcine isolates. functions listed are: cofactors; cell wall and capsule; virulence and defence; phages and mobile elements; rna metabolism; nucleosides and nucleotides; protein metabolism; regulation; dna metabolism; fatty acids and lipids; respiration; stress response; amino acids and derivatives; sulphur metabolism; and carbohydrates the six genome sequences from porcine origin were examined further for the presence of clustered regularly interspaced short palindromic repeats against the crisprdb database using the crisprfinder tool and any such sequences compared using the crisprcompar website. however, although questionable crisprs were identified for all genomes, no confirmed crisprs were uncovered by this analysis. questionable crisprs include small crisprs of two to three direct repeats and structures where the repeats are not 100 % identical. in the atcc finished genome, questionable crisprs were located at 893116–893231 bp and comprised a set of two virtually identical direct repeats. an almost identical situation occurs at 868686–868801 bp in strain i however, no crispr-associated gene hits were uncovered upstream or downstream of that region in either atcc or i which is located directly downstream from the accessory secretion system, a genetic region likely acquired by hgt. analysis of plasmids in porcine l. reuteri strains the putative plasmid dna sequences of the four pig draft genomes were aligned to plasmid sequences of pi and pii from l. reuteri atcc the sequence of plasmid plul from parental strain and the six plasmid sequences from strain i and compared. this method was used to extract potential plasmid sequences as contigs from the total assembly of the four strains. without exception, all of the contigs extracted from each of the strains were present within the respective accessory regions that had been identified previously in comparison to strain atcc efforts to compare the gene annotations from the l. reuteri atcc plasmids to those of the newly identified sequences were unsuccessful, in line with the lack of dna sequence alignment, and supporting the findings that the plasmid dna is heterogeneous and relatively unrelated in each of these strains. further comparisons of the plasmids of atcc with those from the other l. reuteri pig strains did not indicate large conservation amongst the plasmids of this species. rather the plasmids appear isolate-dependent, which correlates with the results of plasmid isolation from these strains. comparative genomics of l. reuteri genomes from different host origin in order to gain insights into the subset of genes involved in adaptation to the porcine gut, we performed a comparative genomic analysis of 20 l. reuteri strains that are currently available, which originate from the pig, human and rodent gi tracts and from sourdough. these strains were lp167- pg-3b, 3c 20- atcc i jcm1112t, dsm20016t, atcc pta-, atcc pta-, cf48-3a atcc, td lpuph mlc 100- tmw, tmw, lth and lth a set of of the available genomes was used in the analysis, whereby jcm1112t and dsm20016t were considered as a single genome since these two strains are derived from the same isolate. the core set of these genomes contained orthologous gene clusters by orthomcl analysis, while there were gene clusters, comprising “unique” genes that occurred at least once in individual genomes – the difference between the latter two values again being due to the presence of at least two gene copies in some genomes. another gene clusters were shared by at least two genomes, giving a total l. reuteri pan-genome of orthologous gene clusters. a circular comparison of the genomes of the six strains isolated from pig with genomes from a selection of seven other l. reuteri strains from human and rodent is shown in additional file 1: figure s homologous genes were calculated using l. reuteri atcc as the reference strain by reciprocal best match analysis requiring a 40 % match by fasta searches over 80 % of the target protein length. this required the target protein to be predicted almost in entirety using current gene prediction software prodigal and glimmer. with this method, unpredicted proteins, either due to unusual base composition or to fragmentation at the contig level, would lead to a negative result. using protein sequences derived from the genomic data, three additional analyses using the joint genome institute’s integrated microbial genomes phylogenetic profiler tool, reciprocal fasta with the annotation software artemis, and orthomcl all indicated that there were no genes conserved in all six pig-derived genomes that were absent in the genomes of l. reuteri strains from other hosts. therefore our analyses showed that there are no pig-specific genes. previous mlsa revealed that pig isolates fall into two distinct clades. a phylogeny based on the core genes of strains of l. reuteri confirmed the phylogenetic clusters and their membership. clade iv contains strains i atcc lp167- and pg-3b, and is more closely related to the rodent cluster iii, while clade v, which is more closely related to human/poultry strains of clade vi, contains strains 20- and 3c as reported previously, the geographical origin of the strains was not related to the phylogenetic clades since l. reuteri strain i was isolated in china but found as a close relative of atcc, isolated in sweden. these findings indicate that the co-evolution of l. reuteri with pigs has resulted in the formation of two vastly distinct phylogenetic clusters, which suggests that evolution of these two populations, although driven by the porcine host, was subjected to different selective pressures that led to their separation. the distinct evolutionary trajectories could also have resulted in the absence of common genes that reflect host adaptation among porcine strains. the relatedness of pig clade iv with rodent clade iii strains, and of pig clade v with human/poultry clade vi strains may reflect their evolution from ancestral strains of rodent and human origin, respectively, following contact with pig populations.fig. a maximum likelihood phylogenetic tree of the core genome alignment of 20 l. reuteri genomes. concatenated core gene sequences were compared. the tree was bootstrapped with replicates, shown as percentages at each branch. numbers i-vi represent the original mlsa-based groupings. the host origin of each strain is denoted in parenthesis after the strain name as follows: human, pig, rodent and sourdough to gain insight into this diversification process, we identified genes that were specific to the two individual clades, and conserved among the strains. this analysis revealed lineage iv-specific genes encoding proteins > amino acids. among these genes was one encoding a surface protein of amino acids in length that contained five mucus-binding domains. in addition, cluster iv strains contained three linked genes encoding a tetr family transcriptional regulator, a conserved hypothetical protein with a dhs-like nad/fad-binding domain and an nad-dependent protein deacetylase of the sir family whose functions are as yet unknown. the two clade iv-specific esterase-lipases could be involved in the utilization of lipids in the pig’s diet but on closer examination appeared to be two parts of the same pseudogene. an interesting feature of the clade-iv-specific genes was that they had very high levels of homology among the four different strains, which supports that they are highly conserved among porcine isolates of this clade.table genes specific to l. reuteri pig clades iv and v of the genes encoding proteins > amino acids that were specific to pig cluster v, several represented mobile genetic elements, including transposons and phage-related sequences. however, four lineage-specific surface proteins were detected, three of which contained a sec10/pgra surface exclusion domain, found in gram-positive adhesins such as spyad from group a streptococci and implicated in cell adhesion. two of these sec10/pgra proteins also contained a rib/alpha-like repeat from the rib adhesin of group b streptococci. two genes encoding glycosyltransferases were also identified. in addition, a putative copper oxidase and a magnesium-transporting atpase were detected, suggesting interactions of this l. reuteri lineage with metal ions. several hypothetical proteins were found with high homology among the two strains. interestingly, one of these proteins showed 97 % aa identity to a protein from lactobacillus amylovorus, which is indicative of hgt from another dominant member of the pig microbiota, suggesting an important function in the pig gut. previous pan-genome analysis of l. reuteri rodent and human strains revealed that the pdu-cbi-cob-hem cluster was conserved within human strains, and the cluster was absent in most rodent strains. this cluster codes for cobalamin biosynthesis, glycerol utilization, propanediol fermentation and production of the antimicrobial compound reuterin. the genetic region encompassing the reuterin and vitamin b biosynthetic pathways is located at 293889–338218 bp on porcine reference isolate l. reuteri atcc as in rodent strains, it appears that this pdu-cbi-cob-hem genomic island is present only in a subset of porcine strains, confirming previous findings. out of the four strains from clade iv, only atcc and lp167- possess this genomic island, while it is present in both clade v strains 20- and 3c studied ─ although this island is not conserved in other members of pig clade v. thus it appears that the possession of the reuterin genomic island does not correlate directly with the relationship of the genetic backbone. as with the rat isolate 100- there is evidence that the cluster has been deleted from the genome of pig isolate i through the action of mobile elements. molecular determinants of host recognition in order to gain further insight into the molecular determinants of host-strain specificity, we focused on the genomic analysis of the major cell surface components that have been implicated in the interaction of l. reuteri strains with the host in vivo. cell surface proteins several surface proteins of l. reuteri that are involved in colonisation by binding to epithelia, epithelial cells, or mucus have been functionally characterised. proteins such as mub and the large surface protein contain lpxtg cell wall anchoring motifs, are extremely large, contain multiple repeated motifs and resemble adhesins of pathogenic microbes. here, a total of surface protein-encoding genes and three conserved pseudogenes were predicted from the completed genome of strain atcc. potential homologues were identified in the other pig strains from the annotation and sequence read data from the draft assemblies by alignment to atcc using bowtie and checked separately by burrows-wheeler aligner software, bwa. using bedtools, the percentage of each protein coding sequence covered was determined in the four pig strain draft genomes and results are displayed in a heatmap with the characteristics of the proteins. many of these surface proteins are predicted to be involved in epithelial adhesion and biofilm formation. mub was found only in strain atcc but a few other mucbp domain-containing proteins were identified, some as conserved pseudogenes, and two were found to be pig clade iv-specific. other putative surface proteins found only in atcc were lratcc53608_ _ and _ although the significance of these is unknown. several proteins with one or more lysin motif, a domain involved in peptidoglycan binding, were present in all five pig strains analysed, including homologues of lratcc53608_ which are identical to lr_ from rodent strain 100- lysm-containing proteins lr_ and lr_ are putative aggregation promoting factor proteins that have been implicated in biofilm formation and colonisation of strain 100- in mice, suggesting a similar role in the pig host. in the heatmap of additional file 1: figure s there is therefore a clear difference between the two representatives of clade iv, pg-3b and lp167- and the two representatives of clade v, 20- and 3c with respect to the presence or absence of certain surface proteins. on the other hand, the other member of clade iv that was used as the reference strain, atcc possessed additional surface proteins not found in strains pg-3b and lp167- one interesting putative surface protein, not included in the above analysis because of its lack of a consensus secretion signal, was the duf repeat-containing protein encoded by lratcc53608_ and its homologues in pig clade iv and in three sourdough strains. although a putative pseudogene in these strains, it may still be translated as a protein ranging in size from about 880– aa and exported by a non-classical secretion pathway. duf repeats are found in many gram-positive cell surface adhesins, such as maba, a modulator of adhesion and biofilm formation, from lactobacillus rhamnosus gg. homologues of this protein were not found in the two pig clade v strains and were also absent in all human strains but functional counterparts with a typical secretion signal were present in all rodent strains and one sourdough strain. the significance of this duf repeat-containing surface protein in l. reuteri is as yet unknown but it appears to be conserved phylogenetically as either putative pseudogenes or functional genes in different host-derived strains. the accessory seca2-secy secretion system in streptococci and staphylococci, the accessory seca2-secy system facilitates the selective export of glycosylated serine-rich repeat proteins that often function as adhesins such as gspb, fap and srap. this auxiliary protein secretion system is present in a limited number of gram-positive bacteria and mycobacteria and other members of the class bacilli; its sparse distribution among different species of lactobacilli implies that this system was horizontally acquired by only a few lactobacillus lineages. in the l. reuteri rodent strain 100- an srrp was identified as the only protein secreted by seca2-secy and shown to be essential for the formation of biofilm in germ-free mice. the gene content within the accessory sec cluster is conserved in l. reuteri atcc when compared with that of rat strain 100-. furthermore, the seca2-secy cluster is also conserved in the other five pig strains. it is of note that on the phylogenetic tree of fig.  the seca2-secy clusters of atcc and of i fell between those of clade v strains 20- and 3c and the other clade iv strains pg-3b and lp167- with which atcc and i share the most backbone genetic material. this may indicate that the seca2-secy region of atcc has undergone hgt from clade iv or that some amount of introgression is underway. hgt of the seca2-secy cluster in l. reuteri pig strains is supported by the presence of mobile genetic elements within the cluster of atcc, a low gc content and by results from the analysis with alien_hunter confirming that this region is likely to have been acquired by hgt, although it cannot be excluded that the presence of mobile elements is a prelude to deletion or modification of the cluster. by analogy to the 100- srrp and to srrps from other bacteria, the srrp homologue in atcc and the other pig strains is likely to be secreted through this accessory pathway and indeed, this protein is found in extracellular extracts of atcc. lratcc53608_ like lr_ from 100- is unusually serine rich and contains of the predominant 10-amino acid repeat slsnsvsmse, compared with such repeats in lr_ however, each srrp from the pig and rodent strains differs with respect to the number and sequences of their serine-rich repeats. comparing these repeat sequences, the srrps from clade iv strains atcc lp167- and i are more similar, while that from the other clade iv strain, pg-3b, is more like the srrp from clade v strain 20- the serine residues of the srrps are likely to be glycosylated by glycosyltransferases associated with the seca2-secy cluster.fig. phylogeny and blast comparisons of seca2-secy regions from the six pig strains of l. reuteri. for the four draft genomes of lp167- pg-3b, 20- and 3c the differently coloured gene bars represent separate contigs that were brought together after alignment to the completed reference atcc genome using mummer. genome comparisons are shown as blast matches, with the darker bars being of higher match. the phylogenetic tree was generated from the sequences only of the seca2-secy regions no other functional large surface proteins are linked to the seca2-secy cluster in these pig strains, except for two lpxtg-containing pseudogenes that flank the cluster at either end. lratcc53608_0902-_ is a mucbp-containing lpxtg pseudogene whereas lratcc53608_0916-_ is an srrp pseudogene that possibly arose from gene duplication of the functional srrp, lratcc53608_ in the completed genome of strain i the srrp itself is a pseudogene but not annotated as such. interestingly, this strain possesses a homologue of the cmba mucus-binding protein that is found in many human isolates of l. reuteri but is absent in other pig strains. also of interest, the mucbp-lpxtg pseudogenes from the pig strains have intact homologues in rodent and sourdough isolates of l. reuteri, suggesting their loss of function during evolution in the pig host. the srrps showed high sequence conservation among pig strains, particularly in the n-terminal putative binding region. homology between the n-terminal domains of srrps from pig and rodent strains was lower, suggesting that they may share a common structure but bind to different target ligands in their respective hosts. as expected, the pseudo-srrps showed more sequence diversity and were only about 30–40 % identical at the amino acid level to their respective functional counterparts. conservation of the srrps and seca2-secy cluster in all six l. reuteri strains from pig mlsa lineages iv and v and in most strains from rodents but not in isolates from human and poultry hosts may explain host-specific differences in l. reuteri biofilm formation. the eps cluster l. reuteri strains can produce eps in several forms depending on the strain - levan catalysed by the enzyme levansucrase, inulin catalysed by the enzyme inulosucrase and reuteran catalysed by glucosyltransferases gtfa or gtfo. ftf and inu share 86 % aa similarity and are therefore difficult to distinguish at the sequence level. some l. reuteri strains possess both types of enzyme but others have only one. mutation of the single ftf gene in rodent strain 100- resulted in loss of eps production and although the ftf mutant was able to colonise the murine gi tract of lactobacillus-free mice in the absence of competition, colonisation was impaired in competition with the wild type, indicating a role in host interaction. genomic analyses of l. reuteri atcc and the other five pig strains identified ftf/inu homologues in all six strains with approximately 80 % similarity at the protein level to that from rat strain 100-, suggesting that the ftf/inu gene was acquired by hgt early in the evolution of the species and subsequently diversified at accelerated rates. homologues of ftf/inu are also present in human isolates of l. reuteri, atcc and cf48-3a recently, a cluster of more than genes, including several encoding gtf’s, that may contribute to eps synthesis and is similarly organised compared to eps-related genes described in other lactobacillus strains, including l. rhamnosus gg, was reported in the l. reuteri human strain atcc. although the start of this operon was similar in the human strain atcc pta-, the presence of an insertional element in strain atcc pta- after the first genes indicated that genomic rearrangements and gene loss had occurred. regions relating to these putative eps gene clusters were found in the accessory genomes of the six pig strains. despite the close phylogenetic relationships between strains of the atcc clade, the predicted eps gene clusters showed poor conservation, with only the epsa, c and d encoding genes strictly conserved, suggesting large-scale eps gene cluster differences at the isolate level between these strains. this is in accordance with the eps cluster in rodent l. reuteri strains, where the two eps gene clusters show high variability. like the rodent strains, all six pig strains also possessed a second cluster of genes unlinked to the putative eps cluster in fig. 7—lratcc53608_0648-_ lri_0601-_ lr3c6_00497-_ lr202_01632-_ lrlp167_00566-_ and lrpg3b_01609-_ – four of which included a second epsc-encoding gene and were therefore capable of producing other extracellular polysaccharides.fig. predicted eps gene clusters in the six l. reuteri pig strains. a, pg-3b, b, lp167- c, 3c d, 20- e, atcc and f, i colour scheme: yellow, gtf; green, other protein involved in polysaccharide biosynthesis; blue, membrane protein; orange, hypothetical protein; pink, transposase/mobile element protein; brown, pseudogene; red, rna polymerase σ subunit the glucansucrase/reuteransucrase gtfa responsible for reuteran biosynthesis was originally described in probiotic strain and an ncbi blastp analysis revealed the presence of homologues in sourdough strains tmw and tmw with 93 % aa identity to gtfa. the homologous gtfo from human probiotic strains cf48-3a and atcc are 68 % identical to gtfa. other l. reuteri homologues gtf and gtfml with 78–79 % aa identity to gtfa have been described in probiotic strain and mouse strain ml synthesising α- -linked dextran and α- -linked mutan, respectively. interestingly, no homologues with significant aa identity to the above-mentioned four gtf’s over the entire protein length were found in pig isolates atcc 3c and 20- however, homologues from the other pig strains i pg-3b and lp167- had 95–99 % aa identity to gtf suggesting that these three clade iv strains most likely synthesise an α- -linked dextran-type polymer. like the ftf and inu genes, the gtf homologues are unlinked to the putative eps clusters displayed in fig.  indicating that the latter are involved in the biosynthesis of other types of eps. thus, the ability of different strains to produce distinct types of eps in the extracellular matrix in the host’s git may be a contributing factor in determining host specificity.  this comparative genomics study provides novel insights into the ecology and evolution of the species l. reuteri with the porcine host. together with previous phylogenetic analyses based on a small set of housekeeping genes, the genomic analysis highlights two divergent clades within l. reuteri pig strains that can be differentiated from other l. reuteri lineages. although no genes were identified that were conserved among all pig isolates and specific to this host, we identified genes specific for and conserved among strains from the two pig phylogenetic lineages iv and v, with high rates of homology among strains. the two populations of porcine l. reuteri appear to have evolved separately, through a process driven by distinct selective pressures, e.g. different pig host genotypes or environmental factors such as dietary components, which resulted in the absence of common pig-specific genes among these two populations. however, from the phylogenetic analysis, the closeness of rodent clade iii with pig clade iv strains, and of human clade vi with pig clade v strains could reflect an evolution of ancestral strains following contact of rodents and humans, respectively, with pig populations. the identification of several surface proteins, some with mucus-binding mucbp domains or other repeated domains found associated with adhesins, as being pig clade-specific might contribute to host-specificity, and ultimately to the clustering of strains away from other l. reuteri lineages. clearly, these genes provide a basis for future functional studies on the ecology of l. reuteri in the pig git. 
furthermore, we generated draft genomes of four additional l. reuteri strains isolated from pig faeces or lower gi tract, lp167- pg-3b, 20- and 3c and subjected all five genomes to a comparative genomic analysis together with the previously completed genome of strain i a phylogenetic analysis based on whole genomes showed that porcine l. reuteri strains fall into two distinct clades, as previously suggested by multi-locus sequence analysis. however, how l. reuteri strains colonise the gastrointestinal tract of pigs is unknown. to gain insight into the ecology of l. reuteri in the pig gut, the genome sequence of the porcine small intestinal isolate l. reuteri atcc was completed and consisted of a chromosome of mbp and two plasmids of kbp and kbp, respectively. these six pig l. reuteri genomes contained a core set of orthologous gene clusters, as determined by orthomcl analysis, that contributed to a pan-genome totalling gene clusters. genome comparisons of the six pig l. reuteri strains with 14 l. reuteri strains from other host origins gave a total pan-genome of gene clusters that included a core genome of gene clusters but revealed that there were no pig-specific genes per se. this study extends the phylogenetic analysis of l. reuteri strains at a genome-wide level, pointing to distinct evolutionary trajectories of porcine l. reuteri lineages, and providing new insights into the genomic events in l. reuteri that occurred during specialisation to their hosts. lactobacillus reuteri is a gut symbiont of a wide variety of vertebrate species that has diversified into distinct phylogenetic clades which are to a large degree host-specific. lactobacillus reuteripighost-specificitycomparative genomicsclade-specific genessurface adhesinsserine-rich repeat proteinsauxiliary secretion systemissue-copyright-statement© the author 2015 previous work demonstrated host specificity in mice and begun to determine the mechanisms by which gut colonisation and host restriction is achieved. however, genes specific for and conserved among strains of the two pig phylogenetic lineages were detected, some of which encoded cell surface proteins that could contribute to the diversification of the two lineages and their observed host specificity. the occurrence of two distinct pig-derived clades may reflect differences in host genotype, environmental factors such as dietary components or to evolution from ancestral strains of human and rodent origin following contact with pig populations.
 lactobacillus reuteri is a gut symbiont of a wide variety of vertebrate species that has diversified into distinct phylogenetic clades which are to a large degree host-specific. previous work demonstrated host specificity in mice and begun to determine the mechanisms by which gut colonisation and host restriction is achieved. however, how l. reuteri strains colonise the gastrointestinal tract of pigs is unknown.  to gain insight into the ecology of l. reuteri in the pig gut, the genome sequence of the porcine small intestinal isolate l. reuteri atcc was completed and consisted of a chromosome of mbp and two plasmids of kbp and kbp, respectively. furthermore, we generated draft genomes of four additional l. reuteri strains isolated from pig faeces or lower gi tract, lp167- pg-3b, 20- and 3c and subjected all five genomes to a comparative genomic analysis together with the previously completed genome of strain i a phylogenetic analysis based on whole genomes showed that porcine l. reuteri strains fall into two distinct clades, as previously suggested by multi-locus sequence analysis. these six pig l. reuteri genomes contained a core set of orthologous gene clusters, as determined by orthomcl analysis, that contributed to a pan-genome totalling gene clusters. genome comparisons of the six pig l. reuteri strains with 14 l. reuteri strains from other host origins gave a total pan-genome of gene clusters that included a core genome of gene clusters but revealed that there were no pig-specific genes per se. however, genes specific for and conserved among strains of the two pig phylogenetic lineages were detected, some of which encoded cell surface proteins that could contribute to the diversification of the two lineages and their observed host specificity.  this study extends the phylogenetic analysis of l. reuteri strains at a genome-wide level, pointing to distinct evolutionary trajectories of porcine l. reuteri lineages, and providing new insights into the genomic events in l. reuteri that occurred during specialisation to their hosts. the occurrence of two distinct pig-derived clades may reflect differences in host genotype, environmental factors such as dietary components or to evolution from ancestral strains of human and rodent origin following contact with pig populations. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords lactobacillus reuteripighost-specificitycomparative genomicsclade-specific genessurface adhesinsserine-rich repeat proteinsauxiliary secretion systemissue-copyright-statement© the author 2015 