BACKGROUND
bifidobacteria are a common component of the microbiota of the gastrointestinal tract  of a broad range of hosts, and their presence is associated with a positive health status of the gut  <cit> .
however, little is known about the precise molecular mechanisms that explain these probiotic effects .
for this reason a considerable number of ongoing scientific efforts aim to precisely explain how these benefits are being provided, and in many cases such efforts involve comparative and functional genome analyses.
sequenced bifidobacterial genomes range in size from  <dig>  to  <dig>  mbp , and their genomic organization is in line with that of a typical bacterial chromosome  <cit> .
b. longum subsp.
infantis, bifidobacterium bifidum and bifidobacterium breve are typical inhabitants of the infant intestine, which is presumed sterile at birth but becomes rapidly colonized by bacteria immediately following  delivery  <cit> .
functional analyses conducted on bifidobacterial genomes have also revealed how they adapt to a certain niche.
for example, the presence of enzymes dedicated to the metabolism of human milk oligosaccharides  in b. longum subsp.
infantis showed how this species is specialized in colonizing the infant gut  <cit> .
in vivo gene expression analyses conducted on b. breve ucc <dig> and b. bifidum prl <dig> have revealed genes that encode functions required for gut colonization and persistence  <cit> .
furthermore, comparative genome hybridization  analyses on various b. breve isolates has highlighted the existence of a high level of sequence homology among members of this species, and it also identified genetic functions that appear to be more variable within this bifidobacterial taxon  <cit> .
such variable functions are associated with bifidobacterial adaptation to the host environment and defence against invasion of foreign dna.
they include, among others, crispr  sequences,  restriction/modification  systems and genes involved in the production of particular extracellular structures, such as capsule exopolysaccharides  and sortase-dependent pili  <cit> .
however, cgh analysis is not sufficient to describe the genetic diversity of a species, as it can only detect genes present in the reference genome, but cannot identify genes that are present in tested genomes yet absent in the reference genome.
for this reason we decided to investigate the genome variability within the b. breve taxon by performing whole genome sequencing and comparative analysis of thirteen b. breve strains.
the generated genome data sets were used to perform a pan-genomic analysis which allowed the definition of the total number of different genes encoded by the entire b. breve group , as well as the total number of common genes present in all isolates   <cit> .
corresponding pan- and core-genome information, as obtained by an increasing number of genome sequences, can be used to determine if sequenced representatives of a certain species have provided all expected gene diversity present in that taxon , or if additional sequencing is still necessary before essentially all genes of the species have been identified  .
as this approach takes the overall collection of genetic functions assigned to a certain species  in consideration, rather than conducting individual analyses for each strain, it is believed to represent an accurate and advanced method to explore genomic diversity of a particular bacterial taxon.
RESULTS
general genome features
in order to assess the chromosomal features of representative members of the b. breve species, we analyzed the genome sequences of thirteen different b. breve strains , eight of which were sequenced in the framework of this study , and which had previously been isolated from different human environments, such as infant feaces , human milk  or adult faeces .table  <dig>
list of
bifidobacterium breve
representatives
strain name
ecological origin
affilation
accession number
sequencing status
b. breve
ucc2003
b. breve
s27
b. breve
689b
b. breve
ncfb 2258
b. breve
jcm 7017
b. breve
dsm 20213
b. breve
12l
b. breve
2l
b. breve
31l
b. breve
cect 7263
b. breve
jcm 7019
b. breve
dpc 6330
b. breve
acs-071-v-sch8b
our sequencing efforts resulted in fully sequenced genomes for six strains , while the assembly of the two remaining genome sequences resulted in multiple contigs .
furthermore, five complete and draft b. breve genomes  were retrieved from the ncbi public database.
genome alignment conducted on the eight complete genomes, using b. breve ucc <dig> as reference sequence  <cit> , established an average sequence length of  <dig> ,100 bp, where b. breve jcm  <dig> represents the strain with the smallest chromosome , while b. breve ucc <dig> possessed the largest chromosome .
all b. breve genomes here analyzed displayed an average g+c content of 58%, which is consistent with the range of g+c mol% content of genomes of the bifidobacterium genus  <cit> .
in order to facilitate a coherent comparative analysis, we performed a consistent open reading frame  prediction for all available b. breve  genome sequences.
in this way, a comparable number of genes was obtained for each genome, with an average value of  <dig> open reading frames  per genome .
notably, a  functional in silico prediction could be made for 74% of the identified orfs, while the remaning 26% were predicted to encode hypothetical proteins.table  <dig>
general features of eight complete genomes of
bifidobacterium breve
b. breve
b. breve
b. breve
b. breve
b. breve
b. breve
b. breve
b. breve
genome length
number of genes
trna
rrna
hypothetical proteins
genes with assigned function
is elements/ transposases
plasmid
prophage/integrated episome
crispr
as displayed in table  <dig>  all fully sequenced genomes were observed and experimentally verified to encompass two identical rrna loci located at non-adjacent positions in the genome with the exception of b. breve s <dig> which contains three of such loci; an average of  <dig> dispersed trna genes were noted per b. breve genome.
as previously observed in other bifidobacterial genomes , the atg sequence appears to be the preferred start codon , while gtg, ttg and ctg seem to be less frequently used, with a calculated frequency percentage of  <dig> %,  <dig> % and  <dig> %, respectively.
blastp comparisons performed between all orfs identified on the eight fully sequenced genomes  revealed the presence of  <dig> orthologues  in addition to  <dig> gene families whose presence was observed in some but not all of the eight investigated b. breve members .
the comparative analyses of the eight fully sequenced b. breve genomes also allowed the identification of an average of  <dig> unique genes per genome, mostly representing hypothetical functions or mobile elements .
the cluster of orthologous group  classification performed for the identified orthologous genes showed that the majority of these genes are predicted to be involved in various housekeeping functions, especially those related to carbohydrate and amino acid metabolism, and associated transport activities, in agreement with the general features previously observed for bifidobacteria  .figure  <dig>
comparative genomics of fully sequenced
b. breve
genomes and phylogenetic supertree.
a) venn diagram representing the orthologous and unique gene families as based on blastp comparison  and mcl clustering algorithm analyses.
b) cluster of orthologues  classification of the  <dig> families of orthologues.
for each cog entry the average percentage of hits among b. breve has been indicated.
the most abundant families have also been indicated and they are assigned to housekeeping functions.
from outer to inner circle: b. breve ucc <dig>  b. breve jcm  <dig>  b. breve 689b, b. breve ncfb  <dig>  b. breve s <dig>  b. breve jcm  <dig>  b. breve 12l, b. breve acs-071-v-sch8b.
c) phylogenetic supertree showing the relationship between thirteen b. breve strains , b. longum subsp.
longum ncc <dig>  b. longum subsp.
infantis atcc  <dig>  b. bifidum prl <dig>  b. adolescentis atcc  <dig>  b. dentium bd <dig>  b. animalis subsp.
animalis atcc  <dig>  b. animalis subsp.
lactis dsm  <dig> and b. asteroides prl2011), three actinobacteria  and a single member of the firmicutes as an outlier .
the predicted mobilome of b. brevespecies
all complete genome sequences were investigated for the presence of mobile elements such as is elements and genes specifying transposases, and this analysis revealed that the b. breve jcm  <dig> genome contains the largest number  of such mobile elements, while the b. breve acs-071-v-sch8b genome encompasses just  <dig> is elements and transposase-encoding genes.
the is classification according to the isfinder database  <cit>  also showed that is <dig> is the most frequently occurring insertion family in b. breve.
the complete chromosomes were also examined for the presence of prophages and plasmids.
the prophage-like dna element bbr- <dig> of b. breve ucc <dig> was previously analysed and represents a likely prophage-remnant  <cit> .
notably, in our analysis, we identified two other prophage like-elements , 689b- <dig> in b. breve 689b , which appears to be incomplete, and 7019- <dig> in b. breve jcm  <dig> , which appears to represent a complete prophage.
notably, the b. breve s <dig> genome appears to contain an integrated episome s27- <dig> , which is predicted to specify several hypothetical proteins, an integrase , a dna transfer protein , and a cell wall anchor domain protein .
an extra-chromosomal  sequence was confirmed to be present in b. breve ncfb  <dig>  and this plasmid is 100% identical to the previously published pcibb <dig>  <cit>  in full length blastn alignment.
whole-genome alignments and phylogenetic analysis
the eight fully sequenced b. breve genomes were also aligned using b. breve ucc <dig> as the reference chromosome.
the observed degree of alignment as displayed in a dot-plot exhibited a near-continuous straight line, indicating that all these genomes are highly syntenic, with the only exception of apparent inversions in the middle of the genome sequences of b. breve acs-071-v-sch8b  and b. breve jcm  <dig>  .
in the case of b. breve jcm  <dig> we confirmed this genomic inversion by pcr, demonstrating that this was not due to an assembly error .
furthermore, analysis of the dna that directly flanks these two inversions revealed the presence of sequences specifying mobile elements/transposases, which may have acted as mechanistic drivers for this genomic reshuffling through homologous recombination  <cit> .
b. breve acs-071-v-sch8b contains truncated integrases and transposases flanking the inversion , while the b. breve jcm  <dig> genome contains an hypothetical protein and a putative conjugative transposon at the left end of the inversion and sequences encoding a replication initiation factor, excisionase and integrase at the other inversion end .
a blast alignment performed on the above mentioned genes for b. breve jcm  <dig> revealed high identity  with mobile elements found in clostridium difficile  <dig>  <cit> , which suggests their acquisition by means of horizontal gene transfer .
in order to investigate the phylogenetic relationship between b. breve and other bifidobacteria, a phylogenetic supertree was computed based on  <dig> orthologues, selected on the basis of the comparison of the thirteen b. breve genomes , other sequenced bifidobacterium species , and three additional actinobacterial genomes, , combined with a member of firmicutes as a representative outgroup .
as shown in the resulting consensus tree  all thirteen b. breve members fall into the b. longum phylogenetic group, which is consistent with a previous assignment based on a multilocus approach  <cit> .
as shown in a previous study the colonization of the infant gut by representatives of b. breve and b. longum occurs immediately after birth, with a correlation being observed between strains present in mother and progeny, thus suggesting that such bifidobacteria are transmitted from mother to child during vaginal delivery and/or breast feeding  <cit> .
the strains analyzed in this study possess very similar ecological origins and it was therefore not surprising that no clear separation of these strains was observed within the tree.
however, b. breve jcm  <dig>  an isolate from adult faeces, clustered in a separate branch, while the b. breve milk isolates also cluster together.
additionally, most of the infant stool isolates were shown to cluster in the same group at the bottom of the tree.
b. brevecore and dispensable genome
comparative genomic analysis based on blastp comparisons and mcl clustering algorithm between the eight complete b. breve genomes  allowed the definition of a set of  <dig> gene families, representing the core genome for the b. breve species, defined as a pool of gene families that is present in all of the considered genomes  <cit> , and representing the  <dig> orthologues mentioned above plus an additional  <dig> paralogues.
inspection of corresponding cog assignments  revealed that many components of this core genome represent functions related to cellular housekeeping.
it is also worth mentioning that this set of core families is composed of common functions which can be present in single copy , but also functions present in multiple copy -type transporters represent a typical example).
variability among the b. breve genomes is due to a specific set of functions also called dispensable genes which are present in more than one of the examined b. breve genomes, yet not present in all, as well as genes that are specific for just one strain  <cit> .
our analysis revealed a total of  <dig> families of variable genes,  <dig> of which are classified as unique.
of these  <dig> gene families, 49% encode hypothetical proteins, while the remainder is assigned to more informative features, such as genes predicted to encode proteins involved in capsular exopolysaccharide  synthesis, in phage resistance , in the production of sortase-dependent pili, and in carbohydrate metabolism, including various carbohydrate transporters .
notably, our in silico data corroborate and extend previously published cgh analyses, that had been performed to explore the genomic diversity of b. breve <cit> .
furthermore, the total gene pool  extracted from the eight b. breve complete genomes was compared with that of six complete and publicly available chromosome sequences of b. longum subsp.
longum , which is phylogenetically the closest related taxon to b. breve <cit> .
this comparison showed that  <dig> gene families  are specifically present in b. breve, yet absent in b. longum subsp.
longum.
of these  <dig> gene families, approximately 50% encode unknown or hypothetical functions, while the other 50% represent functions similar to the ones observed in the variable regions of b. breve .
the analysis also showed that  <dig> genes families are specifically present in b. longum subsp.
longum, yet absent in b. breve, where approximately 68% are coding unknown or hypothetical functions, while the remaining 32% specify mobile elements, abc transporters and glycosyl hydrolases .
variability among b. brevegenomes
in order to determine the presence of regions containing clusters of genes putatively acquired by hgt  in b. breve, the g+c mol% for each orf was calculated and only genes with a significantly different g+c content  were plotted .
this analysis established that 10% of the total of gene families present in b. breve displays a deviating g+c content , the majority of which  are mapped in eight variable genomic regions  that were concordant with the regions of variability identified by a blast-based comparative analysis  and the variable regions previously detected by cgh  <cit> .
as shown by the outcomes of a hierarchical clustering analysis that was performed to scrutinize the presence or absence of genes in the eight complete b. breve representatives, the overall of variability accounts for approximately 30% of the total of genes predicted for this species , while the remaining 70% represents the core genes .figure  <dig>
regions of variability among the
b. breve
genomes.
a) blast-based genome atlas showing the presence of each orf from b. breve ucc <dig> and the other b. breve complete representatives.
from outer to inner circle: b. breve ucc <dig>  b. breve jcm  <dig>  b. breve 689b, b. breve ncfb  <dig>  b. breve s <dig>  b. breve jcm  <dig>  b. breve 12l, b. breve acs-071-v-sch8b.
b) alignment showing in red the orfs with significant g+c mol% in b. breve ; highlighted in grey are regions of variability  that were identified based on gene presence/absence and g+c mol% deviation in each strain.
c) hierarchical clustering heatmap representing the variability of b. breve in terms of presence/absence of genes for eight complete genomes of b. breve with associated percentage of core and variable gene families represented as pie chart.
these variable regions were shown to include the eps cluster  <dig>  containing two opposite orientated operons , conjugative transposon, excisionase and integrase of b. breve jcm  <dig> , a crispr locus , pilus-encoding genes , clusters encoding enzymes involved in the metabolism of carbohydrates  and hypothetical proteins  .
comparative analysis conducted on the eight fully sequenced b. breve chromosomes revealed the presence of an apparently intact eps cluster  <dig> in b. breve jcm  <dig>  b. breve jcm  <dig>  b. breve 689b and b. breve s <dig>  positioned in an identical genomic location as b. breve ucc <dig> with genes ranging between 100–50% of similarity in blastp alignments.
in contrast, b. breve acs-071-v-sch8b, b. breve ncfb  <dig>  and b. breve 12l appear to only contain a remnant eps biosynthesis cluster, where the gene encoding a presumed priming glycosyl transferase is present, while many other genes related to eps biosynthesis are absent .
interestingly, two eps clusters were observed in the draft genome b. breve 31l , one of which containing several transposases and located in the same genomic position as the eps cluster  <dig> of b. breve ucc <dig>  a second putative exopolysaccharide biosynthesis cluster named eps cluster  <dig> , was also shown to be present in the other analysed b. breve genomes and it appears to be more conserved than eps cluster  <dig> among the strains analyzed .
a crispr locus, previously described for b. breve ucc <dig>  <cit> , was found in the chromosome of b. breve s <dig>  b. breve acs-071-v-sch8b, b. breve ncfb  <dig>  b. breve jcm  <dig> and b. breve jcm  <dig>  where variability was observed in the spacer region of each strain-specific crispr .
in contrast, a crispr locus appears to be absent from the chromosomes of b. breve 689b and the b. breve milk isolates .table  <dig>
bifidobacterium breve
variable regions
b. breve
b. breve
b. breve
b. breve
b. breve
b. breve
b. breve
b. breve
eps cluster 2
eps cluster 1
crispr
r/m system 1
r/m system 2
r/m system 3
pil1
pil2
pil3
a varying number of predicted type ii dna r/m systems were identified in each of the eight completed b. breve genomes.
the chromosomes of b. breve ucc <dig> and b. breve jcm  <dig> are each predicted to encode three r/m systems, while the chromosomes of b. breve jcm  <dig> and b. breve jcm  <dig> each encompass two such systems, and the chromosomes of the remaining strains b. breve 12l, b. breve 689b, b. breve acs-071-v-sch8b, b. breve s <dig> are each predicted to contain a single r/m system .
regarding genes that encode  adhesion factors, a type ivb tight adherence  locus was previously characterized in b. breve ucc <dig>  <cit>  and its presence was also observed in all other b. breve strains with an high degree of similarity .
in contrast to the tad-like genes, the analyzed b. breve genomes were shown to contain a varying number of sortase-dependent pilus-encoding loci: b. breve ucc <dig>  b. breve ncfb  <dig> and b. breve jcm  <dig> contain three sortase-dependent pili loci , while b. breve jcm  <dig>  b. breve 12l, b. breve 689b, b. breve acs-071-v-sch8b, b. breve s <dig> only contain  <dig>  .
in most cases , an apparent frameshift within a 10– <dig> guanine nucleotide stretch in the first surface protein-encoding gene of pil <dig> and pil <dig> was present, a phenomenon previously observed for b. breve ucc <dig>  <cit> , as well as for b. bifidum prl <dig>  <cit> .
pan-genome analysis
in order to estimate the total number of different genes present in representatives of the b. breve species, and the expected number of new genes for every inclusion of an additional b. breve genome, we applied a pan-genome analysis pipeline named pgap v <dig>   <cit> .
a total number of thirteen b. breve genomes  were included in this analysis, and from the results as displayed in figure  <dig> the pan-genome curve displays an asymptotic trend, growing with an average rate of  <dig> genes per genome in the first nine iterations, after which the number of new genes rapidly decreases and where the resulting curve leads to a value of  <dig> genes.
this suggests that following the inclusion of nine genomes, the incorporation of additional genomes only leads to a minor increase in the pan genome size.
in fact, in silico analysis of such ‘new’ genes show that they mostly encode small and hypothetical proteins.
similar results were achieved using the core-genome function, where the asymptotic trend is even more evident and after the 9th genome iteration the total number of genes in the core genome stabilizes to a value of  <dig>  which is comparable with the number of gene families extracted in our comparative analysis .
both trends observed in the pan-genome and core-genome functions indicate that b. breve displays an essentially closed pan-genome, and that the number of genomes analyzed here is sufficient to describe the complete gene repertoire of this bifidobacterial species.figure  <dig>
pan-genome and core-genome of
b. breve.
a) accumulated number of new genes in the b. breve pan-genome plotted against the number of genomes added.
the deduced mathematical function is also indicated.
b) accumulated number of genes attributed to the core-genome plotted against the number of added genomes.
the deduced mathematical function is also reported.
carbohydrate utilization
as from the previous cgh work performed on  <dig> b. breve isolates  <cit> , genetic variability among the analysed representatives of this group was observed for genes previously characterized as being involved in the utilization of the carbohydrates ribose  <cit> , sucrose  <cit>  and raffinose  <cit> , as well as the plant-derived polysaccharides starch  <cit> , galactan  <cit>  and cellodextrin  <cit> .
interestingly, in all analysed strains, genes are present that are predicted to encode enzymes involved in the uptake and utilization of host-derived mono/oligosaccharides, in particular mucin and human milk oligosaccharides .
examples of this include gene clusters predicted to be involved in the metabolism of sialic acid , lacto-n-biose through a leloir-like metabolic pathway  <cit>  , fucose  and n-linked glycans  <cit>  .
although b. breve is not known to be able to grow on mucin or hmos  <cit> , host-derived mono/oligosaccharides may become available through hydrolytic activities of other  bacteria present in the gut , allowing b. breve strains to utilize such liberated carbohydrates through a phenomenon called cross-feeding  <cit> .
in order to extend our knowledge on carbohydrate fermentation capabilities of b. breve, an in silico prediction of all glycosyl hydrolases  was first performed on the eight complete b. breve genomes.
this analysis essentially confirmed an abundance of members of gh family  <dig> , which was previously defined as a distinctive characteristic of b. breve <cit> .
it also highlighted the presence of a small number of ghs that appear to be present in just a single member of b. breve , and their annotation suggests that they may encode novel hydrolytic activities.figure  <dig>
b. breve
carbohydrate profiling.
a) hierarchical clustering analysis performed on the phenotype observed in b. breve ucc <dig>  b. breve ncfb  <dig>  b. breve 12l, b. breve 2l, b. breve 31l, b. breve 689b, b. breve jcm  <dig> and b. breve jcm  <dig>  b. breve s <dig> tested for growing on  <dig> sugars.
b) the in silico prediction and numerical presence of all identified gh family members according to the cazy classification in the eight fully sequenced b. breve strains.
c) heatmap showing the in silico gene-trait matching exercise performed on the  <dig> clusters that were derived from the hierarchical clustering analysis.
the relative matching distance of each cluster is indicated with colour gradient and the main results are highlighted.
comparing carbohydrate fermentation profiles of nine b. breve strains  revealed that all strains are able to ferment a common set of sugars, such as glucose, lactose, lactulose, maltose and raffinose.
in contrast, fermentation capabilities for the other sugars such as galactan, sucrose, pullulan, amylopectin, starch, maltodextrins, sorbitol, mannitol, fructose, melezitose, cellobiose, xylose and ribose, were shown to be variable among the strains tested.
none of the b. breve strains assayed here was shown to be capable of utilizing inulin, arabinose, maltulose, mannose, trehalose and galactose .
in bifidobacteria, genes involved in the utilization of a given sugar are frequently organized in gene clusters containing genes that encode one or more specific ghs and associated transport system, and are frequently placed under the transcriptional control of a laci-type regulator specified by a gene that is also located adjacent to or within such a gene cluster .
a gene-trait matching analysis performed on these b. breve strains, based on the association between the  <dig> presence/absence clusters of genes  obtained by hierarchical clustering  analysis and growth/no growth phenotype, allowed an in silico assessment of the role of certain genes associated with carbohydrate metabolism and carried by the b. breve chromosomes sequenced here, several of which had previously been characterized in b. breve ucc <dig> .
this analysis was carried out on the sugars that generated differential carbohydrate profiles among the tested b. breve strains , and allowed the identification of  <dig> genes that correspond with a strain’s ability to grow on ribose, galactan, sucrose, melezitose, cellobiose, mannitol and sorbitol .table  <dig>
gene-trait matching with functions resulting from hierarchical clustering analysis
ribose
cluster45
ribose transport system permease protein rbsd
pfkb family carbohydrate kinase
ribokinase
galactan/sucrose
cluster6
amylosucrase or alpha-glucosidase
glycosyl hydrolases family  <dig>  endogalactanase, galactan metabolism
melezitose
cluster10
raffinose synthase or seed imbibition protein sip1/alpha-galactosidase
cellobiose
cluster36
cellodextrin binding protein
mannitol/sorbitol
cluster23
alcohol dehydrogenase
putative glyoxalase family protein
alcohol dehydrogenase
for example, growth on ribose was shown to correspond with cluster <dig>  which contains genes that have previously been described to be required for ribose utilization  <cit> , such as rbsd, encoding a component of the presumed ribose transport system, and rbsk, encoding a ribokinase.
the obtained strain-specific growth profile distribution on galactan corresponds with cluster <dig>  which encompasses a gene specifying an endogalactanase , which has been previously shown to be involved in galactan metabolism in b. breve ucc <dig>  <cit> .
also, strain-specific growth on melezitose corresponds to cluster <dig>  which includes a gene encoding an alpha-galactosidase  shown to be required for the utilization of this carbohydrate  <cit> .
furthermore, this analysis allowed the identification of a gene cluster in b. breve jcm  <dig> , which is predicted to encode elements for the regulation, transport and metabolism of the sugar alcohol sorbitol by some members of the b. breve taxon .
notably, this locus encompasses an alcohol dehydrogenase-encoding gene , which was targeted for insertion mutagenesis, and followed by a gene  predicted to specify an abc transporter, and presumed to be responsible for internalization of this carbohydrate, and gene  encoding a predicted a transcriptional regulator.
as compared to the wild type b. breve jcm  <dig>  the insertion mutant strain, designated here as b. breve jcm 7017– <dig>  was incapable of using sorbitol as a sole carbohydrate source, thereby confirming the role of this gene cluster in sorbitol metabolism .figure  <dig>
sorbitol gene cluster in
b. breve
and insertion mutant growth curve.
a) locus map showing the comparative analysis of the gene cluster putatively involved in the utilization of the sugar alcohol sorbitol in certain b. breve chromosomes.
all genes are coloured coded based on their function.
the percentage of similarity based on blastp alignment and the alcohol dehydrogenase-encoding gene targeted for the genetic insertion experiment are indicated.
b) diagram showing the growth curves of b. breve jcm  <dig> wild-type and b. breve jcm 7017– <dig> insertion mutant on rogosa modified mrs  with the addition of lactose and sorbitol  <dig> % over 24 hours.
CONCLUSIONS
genome sequencing of eight b. breve strains and comparative analysis of these genomes, combined with five additional, publicly available b. breve genomes, allowed the description of the pan-genome of the b. breve species, which was shown to follow an essentially closed trend.
as pan-genomic analysis was only recently introduced for the description of bacterial species  <cit> , its application is still somewhat controversial and subject to scientific scrutiny.
one clear limitation of pan-genome analysis is the difficulty of assessing whether a pan-genome is really closed or not, given the dynamic nature of a given bacterial population and its associated tendency to evolve and exchange genetic material.
in general it can be said that a closed pan-genome implies that the gene exchange within a species is low and this certainly seems to be the case for the thirteen b. breve genomes analyzed here.
moreover, the in silico prediction of orfs with a deviating g+c mol% content coupled with a comparative genomics analysis, allowed the identification of eight genomic regions of variability in the b. breve pangenome representing approximately 30% of the total of gene content within the b. breve species, containing a large fraction of orfs that have been acquired by hgt .
apart from hypothetical proteins and mobile elements, the gene functions contained within these variable regions are predicted to be required for environmental niche adaptation of this group.
for a gut commensal the process of colonization involves cell-cell and cell-host interactions  and evasion of the host adaptive immune response  <cit>  , as well as metabolic flexibility to acquire energy from a variety of carbon sources independent of the age of the host .
furthermore, evolutionary pressures to resist invasion of foreign dna  also appears to provide an explanation for the presence of crispr, crispr-associated genes, as well as r/m systems in the variable regions of b. breve.
the comparative genomics approach used in this study also facilitated the explanation of certain differences previously observed in the carbon sources utilization in some b. breve members and allowed the definition of a new cluster responsible for the fermentation of the sugar alcohol sorbitol.
for this reason the in silico analysis presented in this study represents a robust starting point for future functional genomics investigations focusing on  this bifidobacterial species, in order to elucidate the spectrum of functions and mechanisms of interaction with the host environment to explain the presence of these bacteria in the human gut and the reported beneficial effects on their host.

 bifidobacteria are a common component of the microbiota of the gastrointestinal tract of a broad range of hosts, and their presence is associated with a positive health status of the gut. however, little is known about the precise molecular mechanisms that explain these probiotic effects. for this reason a considerable number of ongoing scientific efforts aim to precisely explain how these benefits are being provided, and in many cases such efforts involve comparative and functional genome analyses. sequenced bifidobacterial genomes range in size from to mbp, and their genomic organization is in line with that of a typical bacterial chromosome. b. longum subsp. infantis, bifidobacterium bifidum and bifidobacterium breve are typical inhabitants of the infant intestine, which is presumed sterile at birth but becomes rapidly colonized by bacteria immediately following delivery. functional analyses conducted on bifidobacterial genomes have also revealed how they adapt to a certain niche. for example, the presence of enzymes dedicated to the metabolism of human milk oligosaccharides in b. longum subsp. infantis showed how this species is specialized in colonizing the infant gut. in vivo gene expression analyses conducted on b. breve ucc and b. bifidum prl have revealed genes that encode functions required for gut colonization and persistence. furthermore, comparative genome hybridization analyses on various b. breve isolates has highlighted the existence of a high level of sequence homology among members of this species, and it also identified genetic functions that appear to be more variable within this bifidobacterial taxon. such variable functions are associated with bifidobacterial adaptation to the host environment and defence against invasion of foreign dna. they include, among others, crispr sequences, restriction/modification systems and genes involved in the production of particular extracellular structures, such as capsule exopolysaccharides and sortase-dependent pili. however, cgh analysis is not sufficient to describe the genetic diversity of a species, as it can only detect genes present in the reference genome, but cannot identify genes that are present in tested genomes yet absent in the reference genome. for this reason we decided to investigate the genome variability within the b. breve taxon by performing whole genome sequencing and comparative analysis of thirteen b. breve strains. the generated genome data sets were used to perform a pan-genomic analysis which allowed the definition of the total number of different genes encoded by the entire b. breve group, as well as the total number of common genes present in all isolates. corresponding pan- and core-genome information, as obtained by an increasing number of genome sequences, can be used to determine if sequenced representatives of a certain species have provided all expected gene diversity present in that taxon, or if additional sequencing is still necessary before essentially all genes of the species have been identified. as this approach takes the overall collection of genetic functions assigned to a certain species in consideration, rather than conducting individual analyses for each strain, it is believed to represent an accurate and advanced method to explore genomic diversity of a particular bacterial taxon.  general genome features in order to assess the chromosomal features of representative members of the b. breve species, we analyzed the genome sequences of thirteen different b. breve strains, eight of which were sequenced in the framework of this study, and which had previously been isolated from different human environments, such as infant feaces, human milk or adult faeces.table list of bifidobacterium breve representatives strain name ecological origin affilation accession number sequencing status b. breve ucc2003 b. breve s27 b. breve 689b b. breve ncfb 2258 b. breve jcm 7017 b. breve dsm 20213 b. breve 12l b. breve 2l b. breve 31l b. breve cect 7263 b. breve jcm 7019 b. breve dpc 6330 b. breve acs-071-v-sch8b our sequencing efforts resulted in fully sequenced genomes for six strains, while the assembly of the two remaining genome sequences resulted in multiple contigs. furthermore, five complete and draft b. breve genomes were retrieved from the ncbi public database. genome alignment conducted on the eight complete genomes, using b. breve ucc as reference sequence, established an average sequence length of,100 bp, where b. breve jcm represents the strain with the smallest chromosome, while b. breve ucc possessed the largest chromosome. all b. breve genomes here analyzed displayed an average g+c content of 58%, which is consistent with the range of g+c mol% content of genomes of the bifidobacterium genus. in order to facilitate a coherent comparative analysis, we performed a consistent open reading frame prediction for all available b. breve genome sequences. in this way, a comparable number of genes was obtained for each genome, with an average value of open reading frames per genome. notably, a functional in silico prediction could be made for 74% of the identified orfs, while the remaning 26% were predicted to encode hypothetical proteins.table general features of eight complete genomes of bifidobacterium breve b. breve b. breve b. breve b. breve b. breve b. breve b. breve b. breve genome length number of genes trna rrna hypothetical proteins genes with assigned function is elements/ transposases plasmid prophage/integrated episome crispr as displayed in table  all fully sequenced genomes were observed and experimentally verified to encompass two identical rrna loci located at non-adjacent positions in the genome with the exception of b. breve s which contains three of such loci; an average of dispersed trna genes were noted per b. breve genome. as previously observed in other bifidobacterial genomes, the atg sequence appears to be the preferred start codon, while gtg, ttg and ctg seem to be less frequently used, with a calculated frequency percentage of %, % and %, respectively. blastp comparisons performed between all orfs identified on the eight fully sequenced genomes revealed the presence of orthologues in addition to gene families whose presence was observed in some but not all of the eight investigated b. breve members. the comparative analyses of the eight fully sequenced b. breve genomes also allowed the identification of an average of unique genes per genome, mostly representing hypothetical functions or mobile elements. the cluster of orthologous group classification performed for the identified orthologous genes showed that the majority of these genes are predicted to be involved in various housekeeping functions, especially those related to carbohydrate and amino acid metabolism, and associated transport activities, in agreement with the general features previously observed for bifidobacteria.figure comparative genomics of fully sequenced b. breve genomes and phylogenetic supertree. a) venn diagram representing the orthologous and unique gene families as based on blastp comparison and mcl clustering algorithm analyses. b) cluster of orthologues classification of the families of orthologues. for each cog entry the average percentage of hits among b. breve has been indicated. the most abundant families have also been indicated and they are assigned to housekeeping functions. from outer to inner circle: b. breve ucc b. breve jcm b. breve 689b, b. breve ncfb b. breve s b. breve jcm b. breve 12l, b. breve acs-071-v-sch8b. c) phylogenetic supertree showing the relationship between thirteen b. breve strains, b. longum subsp. longum ncc b. longum subsp. infantis atcc b. bifidum prl b. adolescentis atcc b. dentium bd b. animalis subsp. animalis atcc b. animalis subsp. lactis dsm and b. asteroides prl2011), three actinobacteria and a single member of the firmicutes as an outlier. the predicted mobilome of b. brevespecies all complete genome sequences were investigated for the presence of mobile elements such as is elements and genes specifying transposases, and this analysis revealed that the b. breve jcm genome contains the largest number of such mobile elements, while the b. breve acs-071-v-sch8b genome encompasses just is elements and transposase-encoding genes. the is classification according to the isfinder database also showed that is is the most frequently occurring insertion family in b. breve. the complete chromosomes were also examined for the presence of prophages and plasmids. the prophage-like dna element bbr- of b. breve ucc was previously analysed and represents a likely prophage-remnant. notably, in our analysis, we identified two other prophage like-elements, 689b- in b. breve 689b, which appears to be incomplete, and 7019- in b. breve jcm, which appears to represent a complete prophage. notably, the b. breve s genome appears to contain an integrated episome s27-, which is predicted to specify several hypothetical proteins, an integrase, a dna transfer protein, and a cell wall anchor domain protein. an extra-chromosomal sequence was confirmed to be present in b. breve ncfb and this plasmid is 100% identical to the previously published pcibb in full length blastn alignment. whole-genome alignments and phylogenetic analysis the eight fully sequenced b. breve genomes were also aligned using b. breve ucc as the reference chromosome. the observed degree of alignment as displayed in a dot-plot exhibited a near-continuous straight line, indicating that all these genomes are highly syntenic, with the only exception of apparent inversions in the middle of the genome sequences of b. breve acs-071-v-sch8b and b. breve jcm. in the case of b. breve jcm we confirmed this genomic inversion by pcr, demonstrating that this was not due to an assembly error. furthermore, analysis of the dna that directly flanks these two inversions revealed the presence of sequences specifying mobile elements/transposases, which may have acted as mechanistic drivers for this genomic reshuffling through homologous recombination. b. breve acs-071-v-sch8b contains truncated integrases and transposases flanking the inversion, while the b. breve jcm genome contains an hypothetical protein and a putative conjugative transposon at the left end of the inversion and sequences encoding a replication initiation factor, excisionase and integrase at the other inversion end. a blast alignment performed on the above mentioned genes for b. breve jcm revealed high identity with mobile elements found in clostridium difficile, which suggests their acquisition by means of horizontal gene transfer. in order to investigate the phylogenetic relationship between b. breve and other bifidobacteria, a phylogenetic supertree was computed based on orthologues, selected on the basis of the comparison of the thirteen b. breve genomes, other sequenced bifidobacterium species, and three additional actinobacterial genomes,, combined with a member of firmicutes as a representative outgroup. as shown in the resulting consensus tree all thirteen b. breve members fall into the b. longum phylogenetic group, which is consistent with a previous assignment based on a multilocus approach. as shown in a previous study the colonization of the infant gut by representatives of b. breve and b. longum occurs immediately after birth, with a correlation being observed between strains present in mother and progeny, thus suggesting that such bifidobacteria are transmitted from mother to child during vaginal delivery and/or breast feeding. the strains analyzed in this study possess very similar ecological origins and it was therefore not surprising that no clear separation of these strains was observed within the tree. however, b. breve jcm an isolate from adult faeces, clustered in a separate branch, while the b. breve milk isolates also cluster together. additionally, most of the infant stool isolates were shown to cluster in the same group at the bottom of the tree. b. brevecore and dispensable genome comparative genomic analysis based on blastp comparisons and mcl clustering algorithm between the eight complete b. breve genomes allowed the definition of a set of gene families, representing the core genome for the b. breve species, defined as a pool of gene families that is present in all of the considered genomes, and representing the orthologues mentioned above plus an additional paralogues. inspection of corresponding cog assignments revealed that many components of this core genome represent functions related to cellular housekeeping. it is also worth mentioning that this set of core families is composed of common functions which can be present in single copy, but also functions present in multiple copy -type transporters represent a typical example). variability among the b. breve genomes is due to a specific set of functions also called dispensable genes which are present in more than one of the examined b. breve genomes, yet not present in all, as well as genes that are specific for just one strain. our analysis revealed a total of families of variable genes, of which are classified as unique. of these gene families, 49% encode hypothetical proteins, while the remainder is assigned to more informative features, such as genes predicted to encode proteins involved in capsular exopolysaccharide synthesis, in phage resistance, in the production of sortase-dependent pili, and in carbohydrate metabolism, including various carbohydrate transporters. notably, our in silico data corroborate and extend previously published cgh analyses, that had been performed to explore the genomic diversity of b. breve. furthermore, the total gene pool extracted from the eight b. breve complete genomes was compared with that of six complete and publicly available chromosome sequences of b. longum subsp. longum, which is phylogenetically the closest related taxon to b. breve. this comparison showed that gene families are specifically present in b. breve, yet absent in b. longum subsp. longum. of these gene families, approximately 50% encode unknown or hypothetical functions, while the other 50% represent functions similar to the ones observed in the variable regions of b. breve. the analysis also showed that genes families are specifically present in b. longum subsp. longum, yet absent in b. breve, where approximately 68% are coding unknown or hypothetical functions, while the remaining 32% specify mobile elements, abc transporters and glycosyl hydrolases. variability among b. brevegenomes in order to determine the presence of regions containing clusters of genes putatively acquired by hgt in b. breve, the g+c mol% for each orf was calculated and only genes with a significantly different g+c content were plotted. this analysis established that 10% of the total of gene families present in b. breve displays a deviating g+c content, the majority of which are mapped in eight variable genomic regions that were concordant with the regions of variability identified by a blast-based comparative analysis and the variable regions previously detected by cgh. as shown by the outcomes of a hierarchical clustering analysis that was performed to scrutinize the presence or absence of genes in the eight complete b. breve representatives, the overall of variability accounts for approximately 30% of the total of genes predicted for this species, while the remaining 70% represents the core genes.figure regions of variability among the b. breve genomes. a) blast-based genome atlas showing the presence of each orf from b. breve ucc and the other b. breve complete representatives. from outer to inner circle: b. breve ucc b. breve jcm b. breve 689b, b. breve ncfb b. breve s b. breve jcm b. breve 12l, b. breve acs-071-v-sch8b. b) alignment showing in red the orfs with significant g+c mol% in b. breve; highlighted in grey are regions of variability that were identified based on gene presence/absence and g+c mol% deviation in each strain. c) hierarchical clustering heatmap representing the variability of b. breve in terms of presence/absence of genes for eight complete genomes of b. breve with associated percentage of core and variable gene families represented as pie chart. these variable regions were shown to include the eps cluster containing two opposite orientated operons, conjugative transposon, excisionase and integrase of b. breve jcm, a crispr locus, pilus-encoding genes, clusters encoding enzymes involved in the metabolism of carbohydrates and hypothetical proteins. comparative analysis conducted on the eight fully sequenced b. breve chromosomes revealed the presence of an apparently intact eps cluster in b. breve jcm b. breve jcm b. breve 689b and b. breve s positioned in an identical genomic location as b. breve ucc with genes ranging between 100–50% of similarity in blastp alignments. in contrast, b. breve acs-071-v-sch8b, b. breve ncfb and b. breve 12l appear to only contain a remnant eps biosynthesis cluster, where the gene encoding a presumed priming glycosyl transferase is present, while many other genes related to eps biosynthesis are absent. interestingly, two eps clusters were observed in the draft genome b. breve 31l, one of which containing several transposases and located in the same genomic position as the eps cluster of b. breve ucc a second putative exopolysaccharide biosynthesis cluster named eps cluster, was also shown to be present in the other analysed b. breve genomes and it appears to be more conserved than eps cluster among the strains analyzed. a crispr locus, previously described for b. breve ucc, was found in the chromosome of b. breve s b. breve acs-071-v-sch8b, b. breve ncfb b. breve jcm and b. breve jcm where variability was observed in the spacer region of each strain-specific crispr. in contrast, a crispr locus appears to be absent from the chromosomes of b. breve 689b and the b. breve milk isolates.table bifidobacterium breve variable regions b. breve b. breve b. breve b. breve b. breve b. breve b. breve b. breve eps cluster 2 eps cluster 1 crispr r/m system 1 r/m system 2 r/m system 3 pil1 pil2 pil3 a varying number of predicted type ii dna r/m systems were identified in each of the eight completed b. breve genomes. the chromosomes of b. breve ucc and b. breve jcm are each predicted to encode three r/m systems, while the chromosomes of b. breve jcm and b. breve jcm each encompass two such systems, and the chromosomes of the remaining strains b. breve 12l, b. breve 689b, b. breve acs-071-v-sch8b, b. breve s are each predicted to contain a single r/m system. regarding genes that encode adhesion factors, a type ivb tight adherence locus was previously characterized in b. breve ucc and its presence was also observed in all other b. breve strains with an high degree of similarity. in contrast to the tad-like genes, the analyzed b. breve genomes were shown to contain a varying number of sortase-dependent pilus-encoding loci: b. breve ucc b. breve ncfb and b. breve jcm contain three sortase-dependent pili loci, while b. breve jcm b. breve 12l, b. breve 689b, b. breve acs-071-v-sch8b, b. breve s only contain. in most cases, an apparent frameshift within a 10– guanine nucleotide stretch in the first surface protein-encoding gene of pil and pil was present, a phenomenon previously observed for b. breve ucc, as well as for b. bifidum prl. pan-genome analysis in order to estimate the total number of different genes present in representatives of the b. breve species, and the expected number of new genes for every inclusion of an additional b. breve genome, we applied a pan-genome analysis pipeline named pgap v. a total number of thirteen b. breve genomes were included in this analysis, and from the results as displayed in figure  the pan-genome curve displays an asymptotic trend, growing with an average rate of genes per genome in the first nine iterations, after which the number of new genes rapidly decreases and where the resulting curve leads to a value of genes. this suggests that following the inclusion of nine genomes, the incorporation of additional genomes only leads to a minor increase in the pan genome size. in fact, in silico analysis of such ‘new’ genes show that they mostly encode small and hypothetical proteins. similar results were achieved using the core-genome function, where the asymptotic trend is even more evident and after the 9th genome iteration the total number of genes in the core genome stabilizes to a value of which is comparable with the number of gene families extracted in our comparative analysis. both trends observed in the pan-genome and core-genome functions indicate that b. breve displays an essentially closed pan-genome, and that the number of genomes analyzed here is sufficient to describe the complete gene repertoire of this bifidobacterial species.figure pan-genome and core-genome of b. breve. a) accumulated number of new genes in the b. breve pan-genome plotted against the number of genomes added. the deduced mathematical function is also indicated. b) accumulated number of genes attributed to the core-genome plotted against the number of added genomes. the deduced mathematical function is also reported. carbohydrate utilization as from the previous cgh work performed on b. breve isolates, genetic variability among the analysed representatives of this group was observed for genes previously characterized as being involved in the utilization of the carbohydrates ribose, sucrose and raffinose, as well as the plant-derived polysaccharides starch, galactan and cellodextrin. interestingly, in all analysed strains, genes are present that are predicted to encode enzymes involved in the uptake and utilization of host-derived mono/oligosaccharides, in particular mucin and human milk oligosaccharides. examples of this include gene clusters predicted to be involved in the metabolism of sialic acid, lacto-n-biose through a leloir-like metabolic pathway, fucose and n-linked glycans. although b. breve is not known to be able to grow on mucin or hmos, host-derived mono/oligosaccharides may become available through hydrolytic activities of other bacteria present in the gut, allowing b. breve strains to utilize such liberated carbohydrates through a phenomenon called cross-feeding. in order to extend our knowledge on carbohydrate fermentation capabilities of b. breve, an in silico prediction of all glycosyl hydrolases was first performed on the eight complete b. breve genomes. this analysis essentially confirmed an abundance of members of gh family, which was previously defined as a distinctive characteristic of b. breve. it also highlighted the presence of a small number of ghs that appear to be present in just a single member of b. breve, and their annotation suggests that they may encode novel hydrolytic activities.figure b. breve carbohydrate profiling. a) hierarchical clustering analysis performed on the phenotype observed in b. breve ucc b. breve ncfb b. breve 12l, b. breve 2l, b. breve 31l, b. breve 689b, b. breve jcm and b. breve jcm b. breve s tested for growing on sugars. b) the in silico prediction and numerical presence of all identified gh family members according to the cazy classification in the eight fully sequenced b. breve strains. c) heatmap showing the in silico gene-trait matching exercise performed on the clusters that were derived from the hierarchical clustering analysis. the relative matching distance of each cluster is indicated with colour gradient and the main results are highlighted. comparing carbohydrate fermentation profiles of nine b. breve strains revealed that all strains are able to ferment a common set of sugars, such as glucose, lactose, lactulose, maltose and raffinose. in contrast, fermentation capabilities for the other sugars such as galactan, sucrose, pullulan, amylopectin, starch, maltodextrins, sorbitol, mannitol, fructose, melezitose, cellobiose, xylose and ribose, were shown to be variable among the strains tested. none of the b. breve strains assayed here was shown to be capable of utilizing inulin, arabinose, maltulose, mannose, trehalose and galactose. in bifidobacteria, genes involved in the utilization of a given sugar are frequently organized in gene clusters containing genes that encode one or more specific ghs and associated transport system, and are frequently placed under the transcriptional control of a laci-type regulator specified by a gene that is also located adjacent to or within such a gene cluster. a gene-trait matching analysis performed on these b. breve strains, based on the association between the presence/absence clusters of genes obtained by hierarchical clustering analysis and growth/no growth phenotype, allowed an in silico assessment of the role of certain genes associated with carbohydrate metabolism and carried by the b. breve chromosomes sequenced here, several of which had previously been characterized in b. breve ucc. this analysis was carried out on the sugars that generated differential carbohydrate profiles among the tested b. breve strains, and allowed the identification of genes that correspond with a strain’s ability to grow on ribose, galactan, sucrose, melezitose, cellobiose, mannitol and sorbitol.table gene-trait matching with functions resulting from hierarchical clustering analysis ribose cluster45 ribose transport system permease protein rbsd pfkb family carbohydrate kinase ribokinase galactan/sucrose cluster6 amylosucrase or alpha-glucosidase glycosyl hydrolases family endogalactanase, galactan metabolism melezitose cluster10 raffinose synthase or seed imbibition protein sip1/alpha-galactosidase cellobiose cluster36 cellodextrin binding protein mannitol/sorbitol cluster23 alcohol dehydrogenase putative glyoxalase family protein alcohol dehydrogenase for example, growth on ribose was shown to correspond with cluster which contains genes that have previously been described to be required for ribose utilization, such as rbsd, encoding a component of the presumed ribose transport system, and rbsk, encoding a ribokinase. the obtained strain-specific growth profile distribution on galactan corresponds with cluster which encompasses a gene specifying an endogalactanase, which has been previously shown to be involved in galactan metabolism in b. breve ucc. also, strain-specific growth on melezitose corresponds to cluster which includes a gene encoding an alpha-galactosidase shown to be required for the utilization of this carbohydrate. furthermore, this analysis allowed the identification of a gene cluster in b. breve jcm, which is predicted to encode elements for the regulation, transport and metabolism of the sugar alcohol sorbitol by some members of the b. breve taxon. notably, this locus encompasses an alcohol dehydrogenase-encoding gene, which was targeted for insertion mutagenesis, and followed by a gene predicted to specify an abc transporter, and presumed to be responsible for internalization of this carbohydrate, and gene encoding a predicted a transcriptional regulator. as compared to the wild type b. breve jcm the insertion mutant strain, designated here as b. breve jcm 7017– was incapable of using sorbitol as a sole carbohydrate source, thereby confirming the role of this gene cluster in sorbitol metabolism.figure sorbitol gene cluster in b. breve and insertion mutant growth curve. a) locus map showing the comparative analysis of the gene cluster putatively involved in the utilization of the sugar alcohol sorbitol in certain b. breve chromosomes. all genes are coloured coded based on their function. the percentage of similarity based on blastp alignment and the alcohol dehydrogenase-encoding gene targeted for the genetic insertion experiment are indicated. b) diagram showing the growth curves of b. breve jcm wild-type and b. breve jcm 7017– insertion mutant on rogosa modified mrs with the addition of lactose and sorbitol % over 24 hours.  genome sequencing of eight b. breve strains and comparative analysis of these genomes, combined with five additional, publicly available b. breve genomes, allowed the description of the pan-genome of the b. breve species, which was shown to follow an essentially closed trend. as pan-genomic analysis was only recently introduced for the description of bacterial species, its application is still somewhat controversial and subject to scientific scrutiny. one clear limitation of pan-genome analysis is the difficulty of assessing whether a pan-genome is really closed or not, given the dynamic nature of a given bacterial population and its associated tendency to evolve and exchange genetic material. in general it can be said that a closed pan-genome implies that the gene exchange within a species is low and this certainly seems to be the case for the thirteen b. breve genomes analyzed here. moreover, the in silico prediction of orfs with a deviating g+c mol% content coupled with a comparative genomics analysis, allowed the identification of eight genomic regions of variability in the b. breve pangenome representing approximately 30% of the total of gene content within the b. breve species, containing a large fraction of orfs that have been acquired by hgt. apart from hypothetical proteins and mobile elements, the gene functions contained within these variable regions are predicted to be required for environmental niche adaptation of this group. for a gut commensal the process of colonization involves cell-cell and cell-host interactions and evasion of the host adaptive immune response, as well as metabolic flexibility to acquire energy from a variety of carbon sources independent of the age of the host. furthermore, evolutionary pressures to resist invasion of foreign dna also appears to provide an explanation for the presence of crispr, crispr-associated genes, as well as r/m systems in the variable regions of b. breve. the comparative genomics approach used in this study also facilitated the explanation of certain differences previously observed in the carbon sources utilization in some b. breve members and allowed the definition of a new cluster responsible for the fermentation of the sugar alcohol sorbitol. for this reason the in silico analysis presented in this study represents a robust starting point for future functional genomics investigations focusing on this bifidobacterial species, in order to elucidate the spectrum of functions and mechanisms of interaction with the host environment to explain the presence of these bacteria in the human gut and the reported beneficial effects on their host. 
bifidobacteria are commonly found as part of the microbiota of the gastrointestinal tract of a broad range of hosts, where their presence is positively correlated with the host’s health status. in silico analysis of genome sequences from thirteen b. breve strains isolated from different environments shows that the genetic variability of this species principally consists of hypothetical genes and mobile elements, but, interestingly, also genes correlated with the adaptation to host environment and gut colonization. in this study, we assessed the genomes of thirteen representatives of bifidobacterium breve, which is not only a frequently encountered component of the human gut microbiota, but can also be isolated from human milk and vagina. genome analysis of thirteen representatives of the b. breve species revealed that the deduced pan-genome exhibits an essentially close trend. bifidobacterium breveevolutionary genomicscore genomedispensable genomepan-genomeissue-copyright-statement© the author 2014 gene-trait matching analysis showed clear correlations between known metabolic capabilities and characterized genes, and it also allowed the identification of a gene cluster involved in the utilization of the alcohol-sugar sorbitol. for this reason our analyses suggest that this number of b. breve representatives is sufficient to fully describe the pan-genome of this species. comparative genomics also facilitated the genetic explanation for differential carbon source utilization phenotypes previously observed in different strains of b. breve. these latter genes specify the biosynthetic machinery for sortase-dependent pili and exopolysaccharide production, as well as genes that provide protection against invasion of foreign dna, and genes that encode enzymes responsible for carbohydrate fermentation.
 bifidobacteria are commonly found as part of the microbiota of the gastrointestinal tract of a broad range of hosts, where their presence is positively correlated with the host’s health status. in this study, we assessed the genomes of thirteen representatives of bifidobacterium breve, which is not only a frequently encountered component of the human gut microbiota, but can also be isolated from human milk and vagina.  in silico analysis of genome sequences from thirteen b. breve strains isolated from different environments shows that the genetic variability of this species principally consists of hypothetical genes and mobile elements, but, interestingly, also genes correlated with the adaptation to host environment and gut colonization. these latter genes specify the biosynthetic machinery for sortase-dependent pili and exopolysaccharide production, as well as genes that provide protection against invasion of foreign dna, and genes that encode enzymes responsible for carbohydrate fermentation. gene-trait matching analysis showed clear correlations between known metabolic capabilities and characterized genes, and it also allowed the identification of a gene cluster involved in the utilization of the alcohol-sugar sorbitol.  genome analysis of thirteen representatives of the b. breve species revealed that the deduced pan-genome exhibits an essentially close trend. for this reason our analyses suggest that this number of b. breve representatives is sufficient to fully describe the pan-genome of this species. comparative genomics also facilitated the genetic explanation for differential carbon source utilization phenotypes previously observed in different strains of b. breve. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords bifidobacterium breveevolutionary genomicscore genomedispensable genomepan-genomeissue-copyright-statement© the author 2014 