BACKGROUND
comparative genomics of strains belonging to species with a large spectrum of hosts, such as staphylococcus aureus, have highlighted two main evolutionary trends linked to the adaptation to a new host: acquisition of new functions through lateral gene transfer facilitating colonization of new niches and gene loss associated with the host specialization  <cit> .
these trends were also recognized when considering the emergence of highly virulent host-specialized pathogens from bacterial species with a broader host range.
for instance massive gene losses were associated to the emergence of human or equine pathogens such as salmonella enterica sv typhi and paratyphi, bordetella pertussis and parapertussishom and burkholderia mallei that respectively derive from salmonella typhimurium, bordetella bronchiseptica and burkholderia pseudomallei <cit> .
during these transitions, it was postulated that gene inactivation and deletions were probably favored by genetic drift and evolutionary bottlenecks.
in line with this model the host-specialized pathogens often showed a much higher number of insertions sequences  than their parental strains and is expansion was proposed to be largely responsible for gene deletions and genome rearrangements observed in these species  <cit> .
streptococcus agalactiae also referred to as group b streptococcus  is a gram-positive bacterium that has emerged as a leading cause of neonatal infections during the sixties and represents an increasing cause of infections in the elderly and in adults with underlying diseases  <cit> .
as a commensal it colonizes the digestive and genitourinary tracts of up to 30% of the human adult population  <cit> .
however, s. agalactiae was initially described as an animal pathogen causing mastitis in ruminant  <cit> .
since the 70’s, s. agalactiae was found to be responsible for epidemic events of invasive diseases in fish farms, leading to a mortality of up to 30%  <cit> .
cases of infection were also reported for other aquatic poikilotherms such as frogs  <cit>  and aquatic mammals such as dolphins  <cit> .
how gbs is able to adapt to its different hosts remains poorly understood.
the genetic diversity of gbs populations has been studied using different methods including multilocus sequence typing   <cit> , which led to the recognition of different clonal complexes .
some of these clonal complexes display host preference.
for instance, cc <dig> is essentially associated with the bovine host and the hypervirulent sequence type   <dig> strains are mainly isolated from humans.
however incidentally strains belonging to human-associated clonal complexes are also isolated from bovines  <cit>  suggesting that relationships between clonal complexes and host specificity are not so strict.
further analysis of the complete genome sequences of eight isolates of human origin and one of bovine origin has highlighted the composite organization of s. agalactiae genomes with a conserved backbone  and a dispensable genome composed of genomic islands that are highly variable between the different strains  <cit> .
the st <dig> strain 2- <dig>  was initially isolated as responsible for several epidemics in fish farms in israel  <cit> .
this strain, which showed a restricted metabolic pattern, thermosensitivity and lack of ß hemolytic and camp activities was first classified as a different species, streptococcus difficile, but proved later to be a genuine serotype ib s. agalactiae strain  <cit> .
s. agalactiae strains were repeatedly isolated from fish infections and found to cluster into two main groups  <cit> .
the first group corresponds to strains belonging to the clonal complex  <dig>  also displaying strains isolated from human and bovine hosts.
the other strains share two or more common mlst alleles with strain 2- <dig> and are classified in st <dig>   <dig>   <dig>   <dig>   <dig> and  <dig>  as st <dig> strains, these sts were until now never isolated from humans  <cit> .
in addition, a third group of strains belonging to clonal complex  <dig> has recently been described in fish and humans  <cit> .
the draft genome sequences of one st <dig> strain, strain stir-cd- <dig>  and two st <dig> strains, strain zq <dig> and gd201008- <dig>  respectively isolated from disease outbreaks affecting farmed tilapia in honduras and nile tilapia in china were recently published  <cit> .
to decipher the phylogenomic relationships between s. agalactiae strains isolated from fish or other poikilotherm animals and strains isolated from human or bovine, we analyzed the genomic sequence of seven isolates belonging to st260- <dig> and st6- <dig>  including the strain 2- <dig>  comparative analysis confirmed that the two groups of gbs strains responsible for fish epidemic diseases are distantly related.
we found that adaptation to fish does not involve any specific function compared to human cc <dig> isolates.
conversely, specialization to the fish host of the st260- <dig> strains was associated with massive gene inactivation and deep remodeling of metabolic and regulatory networks that we also characterized at the transcriptome level.
this genome reduction likely occurred through reca independent recombination.
RESULTS
fish st <dig> strains are closely related to human strains
we first compared the gene content of s. agalactiae strains isolated from fish but grouped into the same cc as the human strain a <dig> by sequencing the genome of strains cf <dig> and ss <dig>  isolated in usa and uk respectively .
whole genome sequence comparison showed that strain cf <dig> differed from strain a <dig> by only  <dig> snps .
cf <dig> was also closely related to the recently described strains zq <dig> and gd201008- <dig>  <cit>  isolated from diseased fish in china, which differ by only  <dig> and  <dig> snps respectively.
in contrast the st <dig> strain ss <dig> was more distant , and proved to be closer to the st <dig> strain h36b  isolated from human  <cit>  .
analysis of snp distributions along the genome sequence showed a uniform distribution when strains of the same st were compared , with a mean polymorphism of  <dig> - <dig>  snp per  <dig> nt except in the sequence of an inserted prophage.
in contrast alignment of st <dig> versus st <dig> strains revealed a mosaic pattern of regions of low polymorphism  interrupted by several regions of higher polymorphism  that were probably gained by recombination with distantly related gbs strains, as previously suggested  <cit> .
one of these regions corresponds to the capsule locus that encodes a serotype ia capsule in st <dig> strains and a serotype ib capsule in st <dig> strains.
therefore st <dig> and st <dig> strains probably shared a common ancestor and recently diverged by recombination with other gbs strains, modifying the capsular serotype between both st.
gene content was similar between strains cf <dig> and a <dig> except for  <dig> genes that were disrupted in cf <dig> and five in a <dig> .
seven genes were specifically disrupted in strain ss <dig>  in addition, we found that the three st <dig> strains cf <dig>  zq <dig> and gd201008- <dig> isolated from fish shared one short genomic island that was absent from other gbs and probably resulted from lateral gene transfer.
this genomic island encodes proteins 70-95% identical with proteins of streptococcus anginosus, including a protein with a lpxtg-motif cell-wall anchor domain .
sharing of this island in addition to the low number of snps between strains cf <dig>  zq <dig> and gd201008- <dig> suggests that the three strains have a common recent ancestor that propagated worldwide.
in contrast, emergence of the st <dig> strain ss <dig> can be considered as an independent event.
overall, the low level of polymorphism with human strain indicates that cc <dig> strains infecting fish recently diverged from strains isolated in humans and bovines.
since at least two independent events of emergence were observed, this suggests that cc <dig> strains might be more amenable to fish colonization/infection than other gbs clonal complexes isolated from humans or bovines.
in agreement with this hypothesis, it was recently shown that a s. agalactiae st <dig> strain isolated from human was able to cause disease in nile tilapia  <cit> .
st260- <dig> strains form an independent lineage which underwent reductive evolution
to explore the phylogenomics relationships between the second group of strains isolated from fish, and cc <dig> strains, we sequenced the genome of five strains belonging to st <dig> and st <dig>  while the genomes of four of these strains were obtained as draft sequences, the genome of strain 2- <dig> was sequenced to completion.
strain 2- <dig> genome consists of a single circular chromosome of  <dig> , <dig> bp ; this is  <dig> to 25% smaller than the genome sizes of other sequenced gbs strains, which range from  <dig>  kb  to  <dig>  kb .
the g + c content  is similar to that of other gbs strains.
compared to human gbs genomes, the genome of strain 2- <dig> lacks one rdna cluster and  <dig> trna genes .
the deletion of this rdna cluster is associated with the translocation of a  <dig> kb genomic region probably caused by recombination between flanking ribosomal rna operons, as also observed in salmonella typhi  <cit> .
except for this region the genome of strain 2- <dig> is syntenic to the genomes of human strains .
the four other strains have a similar genome size as strain 2- <dig> .
whole-genome sequence comparison of the five strains showed that they clustered into two distinct subgroups correlating with the mlst classification .
the st <dig> strain ss <dig> isolated from frog in louisiana differed from the strain 2- <dig> by only  <dig> snps.
strains 90- <dig> isolated in louisiana in  <dig> and 05-108a isolated in  <dig> in honduras, with  <dig> snps can be considered as variants of the same clone.
strain ss <dig> isolated from frog in taiwan diverged from the  <dig> former st <dig> strains by  <dig> snps.
on average, the st <dig> strains showed  <dig>  snps with st <dig> strains.
st260- <dig> strains were also related to the st <dig> strain sa20- <dig> .
in contrast, st260- <dig> displayed  <dig>  snp with cc <dig> strains.
analysis of the snp distribution along the genome sequences of st <dig> and st <dig> strains revealed a uniform pattern of  <dig> snps per kb , suggesting that no recombination occurred in this lineage.
a phylogenetic analysis of strains of human, bovine and fish or frog origins confirmed that st260-261- <dig> strains constitute a distinct lineage.
separation of this lineage from other s. agalactiae clonal complexes, including cc <dig> strains, was probably ancient, pre-dating the separation between the three strains of human origin  .
while the comparison of the whole genome sequence showed the same mean identity between st260- <dig> strains and the three human strains, a higher proportion of nucleotides was found to align with the genome of strain a <dig>  than with other gbs genomes  .
the low proportion of nucleotides that did not align with the genomes of human gbs strains suggested that st260- <dig> strains encode only few specific functions.
indeed, only four genomic islands were characterized in strain 2- <dig>  that represented  <dig> kb in total .
however these four regions essentially contained pseudogenes and one of them was absent from the st <dig> strains.
we also identified in st260- <dig> strains eleven to twenty copies of issag <dig>  an insertion sequence previously described in the genomes of human isolates and other streptococci  <cit>  .
in addition, the genomes of st260- <dig> strains contained  <dig> regions categorized as genomic islands in human s. agalactiae genomes  <cit> , but shared by most gbs characterized so far .
they did not contain any integrative and conjugative elements  or prophages and only two inactivated copies of integrases were identified.
they also lacked two mobile genetic elements encoding important virulence loci: the cis-mobilizable element encoding the major surface antigen alpha like protein/rib  <cit>  and the composite transposon coding for the c5a peptidase and the laminin binding protein  <cit> .
in total the difference in genomic island content between st260- <dig> strains and gbs strains isolated from humans or bovines accounted for about 70-80% of the genome reduction .
an interesting exception was the genomic island  <dig>  that was previously described only in strains a <dig>  and h36b  and therefore is also shared by cc <dig> strains isolated from fish.
this conservation is in favor of a role in fish colonization.
this gi encodes two phosphotransferase systems  for galactitol, sugar abc transporters and genes for galactose utilization  .
sharing of this genomic island explained the higher number of aligned nucleotides between strains 2- <dig> and a <dig>
genome annotation predicted  <dig>   <dig>   <dig> and  <dig> protein coding genes for strains 2- <dig>  90- <dig>  ss <dig> and 05-108a respectively, which is significantly less compared to  <dig> in nem <dig>   <dig> in a <dig> and  <dig> in 2603v/r.
furthermore, 190- <dig> pseudogenes were identified in each strain  compared to  <dig> to  <dig> in the human strains.
this revealed a massive reduction of the functional genome during the time-course of adaptation to fish in the st260- <dig> lineage.
reductive evolution is an ongoing process in the st260- <dig> lineage
to get more insights into the evolution of the st260- <dig> lineage, we further analyzed the nucleotide changes leading to gene disruption and genome size reduction compared to human strains.
strain a <dig>  belonging to cc <dig>  was used as a reference to compare the genome sequences of the five st260- <dig> strains.
we also aligned the genomes of two human and one bovine isolates to identify nucleotide changes specific to the fish lineage.
genomic islands and insertion sequences were excluded from the analysis as well as thirty sequences whose evolution involved both insertions and deletions of nucleotides.
in these conditions, sequence alignment between strain 2- <dig> and a <dig> genomes revealed  <dig> simple insertion/deletion events ranging from  <dig> to  <dig>  nt .
using the sequences of human strains as outgroups, we found that, among these indels,  <dig> corresponded to deletions and  <dig> to insertions that specifically occurred in the st <dig> sublineage, after st260- <dig> divergence.
the mean size of deletions largely exceeded that of insertions since only  <dig> nucleotides were gained while  <dig>  were lost.
deletions, insertions and nucleotide replacements were respectively responsible for  <dig>   <dig> and  <dig> gene disruptions.
in addition, 15% of the indels led to minor protein size modifications  and their consequences on protein functions were more difficult to predict.
finally 30% of the indels were characterized in intergenic regions.
altogether mutations leading to gain or loss of nucleotides represented approximately 12% of total mutations after the divergence from st <dig> strains and constituted the major process of gene disruption.
this evolution was not specific to the st <dig> sublineage as a similar number of insertions and deletions occurred in the st <dig> sublineage accounting for  <dig> gene disruptions .
in addition,  <dig> indels and  <dig> snps relative to strain a <dig> sequence were common to the five st260- <dig> strains but were not observed in a <dig> vs other gbs strains.
forty per cent of these indels were intragenic and led to  <dig> gene disruptions .
finally,  <dig> genes disrupted by a small indel in one sublineage were deleted in the other, suggesting that gene disruption could be a preliminary step to gene loss and genome reduction by secondary longer deletions.
as a consequence some loci were observed under different states of decay in the two sublineages.
this is the case for instance for the cyl locus, the crispr <dig> locus and the pil <dig> pilus locus .
furthermore, the average size of the indels was found to increase following the divergence of the two sublineages  compared to the common branch.
therefore, while the process of reductive evolution was already evident in the ancestor of the st260- <dig> strains, it was even more pronounced after divergence of the two sublineages.
to better evaluate the specificity of the evolutionary process occurring in st260- <dig> strains, we compared the core genome sequence of strains a <dig> and nem <dig>  two strains of human origin differing by approximately  <dig>  snp.
we detected  <dig> indels, which represents half the number of indels in the 2- <dig> vs a <dig> comparison.
these indels were essentially short and as much as 80% of them occurred in intergenic regions while less than 4% led to gene inactivations.
therefore, gain or loss of nucleotides was also frequent in other s. agalactiae strains, but most indels in coding sequences were probably eliminated by purifying selection.
fitting with this hypothesis, we observed one indel per 5- <dig> snps during the recent evolution of st <dig> strains where purifying selection is probably not effective.
a similar proportion of indels vs snp was also reported for two strains of s. enterica sv paratyphi  <cit> .
finally, to gain further insights into the mechanism of the observed insertions and deletions, we analyzed the sequences flanking the indels in strain 2- <dig>  among the  <dig> indels of  <dig> nt,  <dig>  occurred in homopolymeric tracts longer than four nucleotides and likely resulted from dna polymerase slippage during replication.
interestingly,  <dig> of the  <dig>  indels larger than four nucleotides apparently also involved recombination between repeated sequences.
the median size of the repeats was  <dig> nucleotides, with  <dig> sequences larger than  <dig> nucleotides, the largest being  <dig> nt long .
since the threshold of repeat length for reca-dependent homologous recombination is 23- <dig> nt  <cit> , most of the indels probably occurred by reca independent recombination.
it has been shown that, under laboratory conditions, the efficiency of illegitimate recombination is highly dependent on the size of the repeats  <cit>  and inversely dependent on the distance between repeats  <cit> .
our results suggest that reca independent recombination between repeats of moderate sizes may also lead to long deletions.
furthermore we did not find any evidence that deletions of long sequences may depend on longer repeats .
this may indicate that the lower efficiency of illegitimate recombination between short repeats is buffered by their higher frequency in the genome.
some of the large deletions might also correspond to several consecutive shorter ones.
from this analysis we propose that accumulation of small and easily reversible indels is common in s. agalactiae, mostly occurring as a consequence of reca independent recombination.
most of these indels are probably removed from the population due to purifying selection during long-term evolution.
alternatively they may be compensated by a second loss or gain of nucleotides.
in the st260- <dig> lineages host specialization led to the relaxation of the negative selection on genes that became dispensable, allowing the accumulation of larger deletions.
functional adaptation to a host restricted way of living
st260- <dig> strains differ from other gbs strains by their host specificity and high pathogenicity.
as mentioned, this lineage has only few specific genes compared to other strains and most of them are carried by genomic islands in the process of being eliminated.
in particular, we did not identify any specific surface proteins or putative virulence factors.
therefore, the specific properties of the st260- <dig> strains more likely rely on functions shared with human isolates and on the loss of some functions.
the  <dig> genes common to the two sublineages were grouped into functional categories according to kegg classification .
compared to functional classification of a <dig> genes, this analysis revealed a dramatic drop in the proportions of genes involved in mobile and extrachromosomal functions  and in cellular processes, including the pathogenesis subcategory .
the other major functions submitted to evolutionary erosion were energy metabolism, transport and binding, regulation and signal transduction .
conversely the basal functions of the cell such as transcription and protein synthesis were nearly not affected.
bacterial-host interactions and virulence factors
surface components play an important role for tissue colonization and infection by mediating interactions between the pathogen and the host cells and evasion from immune defense.
gbs strains possess two distinct polysaccharide antigens, the highly sialylated capsule polysaccharide and the group b-antigen.
st260- <dig> strains harbor the  <dig> genes involved in the type ib capsule synthesis  similarly to the st <dig> strains h36b and ss <dig>  they also possess the  <dig> genes responsible for the synthesis of the group b antigen .
only seven proteins with a lpxtg signal for cell-wall anchor are conserved  including three important s. agalactiae virulence factors of human strains: the fibrinogen-binding protein a, the serine-rich protein srr <dig> and the biba/hvga protein  <cit> .
however, while in human strains these three proteins carry a variable number of a repeated motif, these repeats have been lost in the st260- <dig> strains.
this might decrease their accessibility to host immune system and cellular receptors by tethering them to the cell wall.
1: proteins anchored to the peptidoglycan by an lpxtg c-terminal motif.
<dig> : the gene has been inactivated.
<dig> : amino-acid repeats have been lost in the corresponding protein.
among the other proteins that are virulence factors in human gbs isolates, the five strains lack the c5a peptidase, the laminin binding protein and components of the pilus.
the cyl locus has also been inactivated in the two sublineages  in agreement with the absence of detectable hemolytic activity on blood agar plates.
another major test for s. agalactiae identification is the detection of camp activity  <cit> .
while both st <dig> and  <dig> strains were reported to be negative for the camp factor reaction  <cit>  we found that the gene encoding the camp factor was disrupted only in st <dig> strains.
we experimentally confirmed that strain ss <dig> was negative for the camp test detection, probably because of a lower level of gene expression .
finally, the five st260- <dig> strains express a fibrinogen/fibronectin binding protein of the pava family and a hyaluronidase that were proposed to be virulence factors in gbs or other streptococcus species  <cit>  and could also have a role in fish infection.
altogether, approximately 60% of the genes for proteins involved in pathogenesis and considered as important virulence genes in human stains  <cit>  were affected by genome reduction.
in this context the capsule could be a major virulence factor in the fish host, as also observed for streptococcus iniae, another streptococcus species pathogenic for fish  <cit> .
metabolism
analysis of the missing functions revealed a profound remodeling of the metabolism of strains of this lineage.
numerous transport systems for carbon sources  and enzymes for degradation of polysaccharides  were missing or inactivated, reflecting a reduced capacity to utilize diverse carbon sources.
in addition st260- <dig> strains seem unable to utilize glycerol and glycerol phosphate as genes encoding the glycerol kinase, the glycerol dehydrogenase and the glycerol-phosphate permease are missing or mutated.
fermentative pathways utilizing pyruvate/acetate conversion are also altered, as the phosphotransacetylase gene is missing.
in mammals, gbs is primarily considered as a commensal of the digestive tract, an environment rich in diverse c-sources.
in contrast, reduction in the catabolic capacities observed in st260- <dig> strains is in favor of a transition to an obligate pathogen style-of-life.
transport functions
approximately 20% of the genes associated with transport systems were missing or inactivated.
the targeted functions were mostly the import of nutrients, in particular of c-sources and the transport of inorganic and metal ions.
the na+/h + antiporter, the k + uptake permease were inactivated in both sublineages.
therefore st260- <dig> strains may be affected in ionic exchange and would have a reduced capacity to maintain their homeostasis in the face of a changing external environment.
transcriptional networks
globally, gene disruption and deletion events affected  <dig> out of the  <dig> transcriptional regulators predicted in human strains .
as much as  <dig> out of the  <dig> two-component systems   found in gbs were inactivated, either in one  or in both  sublineages .
interestingly, analysis of the genome sequence of strain 2- <dig> revealed that the rgf tcs, which is involved in the control of virulence in the human st <dig> strains  <cit> , was associated with two putative bacteriocins with a double glycine leader peptide and with a bacteriocin export transporter .
this suggests that this tcs originates from a bacteriocin operon that has been partially deleted in human strains.
altogether our observations show that s. agalactiae strains adapted to fish may have a reduced capacity to respond to environmental changes compared to human strains and only eight tcs, including the two major systems, ciarh and covrs, may be sufficient to allow gbs adaptation to the different environments encountered in fish.
interestingly, both ciarh and covrs were also involved in the regulation of virulence genes in gbs human strains  <cit> .
the higher virulence of st260- <dig> strains might also be due to the deregulated expression of some virulence genes, as observed in the transition from local to systemic infections in group a streptococci  <cit> .
adaptation to fish is associated with broad changes in gene expression
as a first step to explore changes in gene expression linked to host adaptation, we performed a comparative analysis at the transcriptome level of strains a <dig> , cf <dig> , 2- <dig>  and ss <dig> .
<dig> genes present in the four strains and with 100% identity matches with probes of the array were taken into account in this analysis .
profound modifications in gene expression were observed in st260- <dig> strains and to a lesser extent in strain cf <dig> compared to strain a <dig> .
although strains cf <dig> and a <dig> are closely related, the expression of more than  <dig> genes varied by a two-fold factor between the two strains.
in particular 40% of the genes involved in energy metabolism were expressed at a lower level in the fish isolate than in strain a <dig>  in contrast expression of the gene encoding the virulence protein srr <dig> was more than  <dig> fold increased compared to strain a <dig>  although up-regulation of the srr <dig> gene is not conserved in the st260- <dig> fish isolates, in strain cf <dig>  it might facilitate penetration of the fish blood brain barrier, leading to an increased tendency to cause meningitis, as shown for the srr <dig> protein of human isolates  <cit> .
both the number of genes for which expression was modified and the amplitude of these modifications were even larger in st260- <dig> isolates .
for instance two genes encoding putative glyoxalases are expressed at 6- <dig> higher levels in st260- <dig> strains than in strain a <dig>  suggesting higher needs in methylglyoxal detoxification.
genes encoding the enzymes for the synthesis of arginine  were 10- <dig> fold more expressed.
four functional categories were more affected by variations in gene expression in both st260- <dig> sublineages: metabolism, transport, regulation and signal transduction .
interestingly these functional categories were also particularly affected by gene erosion, suggesting a remodeling of metabolic networks.
in addition to these global tendencies, st <dig> and st <dig> strains also harbor sublineage-specific variations in gene expression that probably result from differential gene decay and might lead to specific adaptations.
for instance, in strain ss <dig>  genes encoding phosphoglycerate kinase, phosphoglyceromutase and pyruvate kinase involved in glycolysis and neoglucogenesis are expressed at a lower level than in strain a <dig>  in parallel, genes coding for the arginine deiminase, ornithine carbamoylkinase and carbamate kinase involved in the deiminase pathway are up-regulated, suggesting a shift towards arginine fermentation as the main pathway for energy production in st <dig> strains  <cit> .
this up-regulation of the deiminase pathway is associated with a decreased expression of the negative regulator argr and the increased expression of the arginine/ornithine antiporter.
in contrast, in the same growth conditions, upregulation of the genes for hpr-kinase and cody involved in catabolic repression and for lactate dehydrogenase suggests that strain 2- <dig> might generate energy essentially through glucose utilization and lactic fermentation.
iron import might be more efficient in st <dig> than in st <dig> strains since strain ss <dig> expresses genes for iron abc transporter and ferrichrome abc transporter at higher levels than strain a <dig>  while these genes are down-regulated or unchanged in strain 2- <dig>  both sublineages express lower amounts of atp synthase, the enzyme responsible for atp-proton motive force interconversion, than strain a <dig>  indicating that they may be impaired in ph homeostasis in acidic conditions  <cit> .
since expression of pseudogenes is energetically costly and may generate deleterious products, we took advantage of the massive genome erosion in the st260- <dig> lineage to look for evidence for negative selective pressure acting on pseudogene expression.
among the  <dig> pseudogenes represented by at least one probe on the array, only 22% were down-regulated compared to strain a <dig>  whereas 64% did not present a significant change in mrna levels and 14% were even up-regulated.
furthermore silencing of pseudogenes in the st260- <dig> lineage did not seem to markedly increase with time.
indeed the proportion of down-regulated pseudogenes was similar among pseudogenes arising before  and after the differentiation of sublineages .
altogether this indicates that no strong evolutionary pressure acts to silence pseudogene expression.
although modifications in gene expression generally affected different genes in st260- <dig> and cc <dig> strains,  <dig> genes were found to vary in the same way in the  <dig> strains isolated from fish.
interestingly, these  <dig> genes included the operon for the capsule synthesis, the genes for zoocin and hyaluronidase, two response regulators of tcs systems  and three targets of the ciarh tcs that were up-regulated.
sixteen genes were downregulated or inactivated in the three fish strains compared to a <dig>  among which  <dig> were involved in energy metabolism or in carbohydrate uptake.
whether these regulations reflect a common mechanism of adaptation to fish environment remains to be established.
however, the higher expression of the genes for the capsule synthesis might favor resistance to environmental conditions and to fish immune system, as it has also been reported for s. iniae <cit> .
similarly the high hyaluronidase expression may help s. agalactiae strains to break through fish tissues and be involved in virulence to fish  <cit> .
CONCLUSIONS
our results show that s. agalactiae strains leading to epidemic diseases in fish farms and cold blood animals belong to at least two distinct groups that differ by their strategies of host adaptation.
cc <dig> strains have the potency to colonize and infect multiple hosts such as fish, human and cattle.
from a genomic point of view, these cc <dig> fish strains are not distinguished from their human counterparts by any significant genomic island.
however, contrasting with this genomic relatedness, large differences in gene expression were observed and could participate to the adaptation to the fish host.
conversely, our genome analysis indicates that strains of the st260- <dig> complex diverged anciently from human and cattle strains and subsequently accumulated specific adaptations leading to the emergence of sublineages.
st260- <dig> strains exhibit a striking pattern of genome reduction and we took advantage of the emergence of sublineages to reconstruct the different steps involved in this process.
we found that accumulation of short indels can be observed all along the evolution of the gbs species, participating to strain-specific gene disruptions.
therefore even in the core genome of human gbs strains some genes are dispensable.
nevertheless the number of inactivated genes greatly increased during specialization of st260- <dig> strains to fish.
these gene inactivations mainly result from the ongoing accumulation of short indels, but a tendency to eliminate inactivated genes by deleting longer sequences is more noticeable in the sublineage specific branches.
in contrast with what has previously been observed in the course of genomic reduction associated to host specialization or to intracellular symbiosis, deletion events are not correlated with an amplification of insertion sequences.
neither could complex genome rearrangements be noticed, suggesting that recombination between is is not a general mechanism for genome reduction.
indeed our results point to non-homologous recombination as an alternative mechanism of genome reductive evolution.

 comparative genomics of strains belonging to species with a large spectrum of hosts, such as staphylococcus aureus, have highlighted two main evolutionary trends linked to the adaptation to a new host: acquisition of new functions through lateral gene transfer facilitating colonization of new niches and gene loss associated with the host specialization. these trends were also recognized when considering the emergence of highly virulent host-specialized pathogens from bacterial species with a broader host range. for instance massive gene losses were associated to the emergence of human or equine pathogens such as salmonella enterica sv typhi and paratyphi, bordetella pertussis and parapertussishom and burkholderia mallei that respectively derive from salmonella typhimurium, bordetella bronchiseptica and burkholderia pseudomallei. during these transitions, it was postulated that gene inactivation and deletions were probably favored by genetic drift and evolutionary bottlenecks. in line with this model the host-specialized pathogens often showed a much higher number of insertions sequences than their parental strains and is expansion was proposed to be largely responsible for gene deletions and genome rearrangements observed in these species. streptococcus agalactiae also referred to as group b streptococcus is a gram-positive bacterium that has emerged as a leading cause of neonatal infections during the sixties and represents an increasing cause of infections in the elderly and in adults with underlying diseases. as a commensal it colonizes the digestive and genitourinary tracts of up to 30% of the human adult population. however, s. agalactiae was initially described as an animal pathogen causing mastitis in ruminant. since the 70’s, s. agalactiae was found to be responsible for epidemic events of invasive diseases in fish farms, leading to a mortality of up to 30%. cases of infection were also reported for other aquatic poikilotherms such as frogs and aquatic mammals such as dolphins. how gbs is able to adapt to its different hosts remains poorly understood. the genetic diversity of gbs populations has been studied using different methods including multilocus sequence typing, which led to the recognition of different clonal complexes. some of these clonal complexes display host preference. for instance, cc is essentially associated with the bovine host and the hypervirulent sequence type strains are mainly isolated from humans. however incidentally strains belonging to human-associated clonal complexes are also isolated from bovines suggesting that relationships between clonal complexes and host specificity are not so strict. further analysis of the complete genome sequences of eight isolates of human origin and one of bovine origin has highlighted the composite organization of s. agalactiae genomes with a conserved backbone and a dispensable genome composed of genomic islands that are highly variable between the different strains. the st strain 2- was initially isolated as responsible for several epidemics in fish farms in israel. this strain, which showed a restricted metabolic pattern, thermosensitivity and lack of ß hemolytic and camp activities was first classified as a different species, streptococcus difficile, but proved later to be a genuine serotype ib s. agalactiae strain. s. agalactiae strains were repeatedly isolated from fish infections and found to cluster into two main groups. the first group corresponds to strains belonging to the clonal complex also displaying strains isolated from human and bovine hosts. the other strains share two or more common mlst alleles with strain 2- and are classified in st and as st strains, these sts were until now never isolated from humans. in addition, a third group of strains belonging to clonal complex has recently been described in fish and humans. the draft genome sequences of one st strain, strain stir-cd- and two st strains, strain zq and gd201008- respectively isolated from disease outbreaks affecting farmed tilapia in honduras and nile tilapia in china were recently published. to decipher the phylogenomic relationships between s. agalactiae strains isolated from fish or other poikilotherm animals and strains isolated from human or bovine, we analyzed the genomic sequence of seven isolates belonging to st260- and st6- including the strain 2- comparative analysis confirmed that the two groups of gbs strains responsible for fish epidemic diseases are distantly related. we found that adaptation to fish does not involve any specific function compared to human cc isolates. conversely, specialization to the fish host of the st260- strains was associated with massive gene inactivation and deep remodeling of metabolic and regulatory networks that we also characterized at the transcriptome level. this genome reduction likely occurred through reca independent recombination.  fish st strains are closely related to human strains we first compared the gene content of s. agalactiae strains isolated from fish but grouped into the same cc as the human strain a by sequencing the genome of strains cf and ss isolated in usa and uk respectively. whole genome sequence comparison showed that strain cf differed from strain a by only snps. cf was also closely related to the recently described strains zq and gd201008- isolated from diseased fish in china, which differ by only and snps respectively. in contrast the st strain ss was more distant, and proved to be closer to the st strain h36b isolated from human. analysis of snp distributions along the genome sequence showed a uniform distribution when strains of the same st were compared, with a mean polymorphism of - snp per nt except in the sequence of an inserted prophage. in contrast alignment of st versus st strains revealed a mosaic pattern of regions of low polymorphism interrupted by several regions of higher polymorphism that were probably gained by recombination with distantly related gbs strains, as previously suggested. one of these regions corresponds to the capsule locus that encodes a serotype ia capsule in st strains and a serotype ib capsule in st strains. therefore st and st strains probably shared a common ancestor and recently diverged by recombination with other gbs strains, modifying the capsular serotype between both st. gene content was similar between strains cf and a except for genes that were disrupted in cf and five in a. seven genes were specifically disrupted in strain ss in addition, we found that the three st strains cf zq and gd201008- isolated from fish shared one short genomic island that was absent from other gbs and probably resulted from lateral gene transfer. this genomic island encodes proteins 70-95% identical with proteins of streptococcus anginosus, including a protein with a lpxtg-motif cell-wall anchor domain. sharing of this island in addition to the low number of snps between strains cf zq and gd201008- suggests that the three strains have a common recent ancestor that propagated worldwide. in contrast, emergence of the st strain ss can be considered as an independent event. overall, the low level of polymorphism with human strain indicates that cc strains infecting fish recently diverged from strains isolated in humans and bovines. since at least two independent events of emergence were observed, this suggests that cc strains might be more amenable to fish colonization/infection than other gbs clonal complexes isolated from humans or bovines. in agreement with this hypothesis, it was recently shown that a s. agalactiae st strain isolated from human was able to cause disease in nile tilapia. st260- strains form an independent lineage which underwent reductive evolution to explore the phylogenomics relationships between the second group of strains isolated from fish, and cc strains, we sequenced the genome of five strains belonging to st and st while the genomes of four of these strains were obtained as draft sequences, the genome of strain 2- was sequenced to completion. strain 2- genome consists of a single circular chromosome of, bp; this is to 25% smaller than the genome sizes of other sequenced gbs strains, which range from kb to kb. the g + c content is similar to that of other gbs strains. compared to human gbs genomes, the genome of strain 2- lacks one rdna cluster and trna genes. the deletion of this rdna cluster is associated with the translocation of a kb genomic region probably caused by recombination between flanking ribosomal rna operons, as also observed in salmonella typhi. except for this region the genome of strain 2- is syntenic to the genomes of human strains. the four other strains have a similar genome size as strain 2-. whole-genome sequence comparison of the five strains showed that they clustered into two distinct subgroups correlating with the mlst classification. the st strain ss isolated from frog in louisiana differed from the strain 2- by only snps. strains 90- isolated in louisiana in and 05-108a isolated in in honduras, with snps can be considered as variants of the same clone. strain ss isolated from frog in taiwan diverged from the former st strains by snps. on average, the st strains showed snps with st strains. st260- strains were also related to the st strain sa20-. in contrast, st260- displayed snp with cc strains. analysis of the snp distribution along the genome sequences of st and st strains revealed a uniform pattern of snps per kb, suggesting that no recombination occurred in this lineage. a phylogenetic analysis of strains of human, bovine and fish or frog origins confirmed that st260-261- strains constitute a distinct lineage. separation of this lineage from other s. agalactiae clonal complexes, including cc strains, was probably ancient, pre-dating the separation between the three strains of human origin. while the comparison of the whole genome sequence showed the same mean identity between st260- strains and the three human strains, a higher proportion of nucleotides was found to align with the genome of strain a than with other gbs genomes. the low proportion of nucleotides that did not align with the genomes of human gbs strains suggested that st260- strains encode only few specific functions. indeed, only four genomic islands were characterized in strain 2- that represented kb in total. however these four regions essentially contained pseudogenes and one of them was absent from the st strains. we also identified in st260- strains eleven to twenty copies of issag an insertion sequence previously described in the genomes of human isolates and other streptococci. in addition, the genomes of st260- strains contained regions categorized as genomic islands in human s. agalactiae genomes, but shared by most gbs characterized so far. they did not contain any integrative and conjugative elements or prophages and only two inactivated copies of integrases were identified. they also lacked two mobile genetic elements encoding important virulence loci: the cis-mobilizable element encoding the major surface antigen alpha like protein/rib and the composite transposon coding for the c5a peptidase and the laminin binding protein. in total the difference in genomic island content between st260- strains and gbs strains isolated from humans or bovines accounted for about 70-80% of the genome reduction. an interesting exception was the genomic island that was previously described only in strains a and h36b and therefore is also shared by cc strains isolated from fish. this conservation is in favor of a role in fish colonization. this gi encodes two phosphotransferase systems for galactitol, sugar abc transporters and genes for galactose utilization. sharing of this genomic island explained the higher number of aligned nucleotides between strains 2- and a genome annotation predicted and protein coding genes for strains 2- 90- ss and 05-108a respectively, which is significantly less compared to in nem in a and in 2603v/r. furthermore, 190- pseudogenes were identified in each strain compared to to in the human strains. this revealed a massive reduction of the functional genome during the time-course of adaptation to fish in the st260- lineage. reductive evolution is an ongoing process in the st260- lineage to get more insights into the evolution of the st260- lineage, we further analyzed the nucleotide changes leading to gene disruption and genome size reduction compared to human strains. strain a belonging to cc was used as a reference to compare the genome sequences of the five st260- strains. we also aligned the genomes of two human and one bovine isolates to identify nucleotide changes specific to the fish lineage. genomic islands and insertion sequences were excluded from the analysis as well as thirty sequences whose evolution involved both insertions and deletions of nucleotides. in these conditions, sequence alignment between strain 2- and a genomes revealed simple insertion/deletion events ranging from to nt. using the sequences of human strains as outgroups, we found that, among these indels, corresponded to deletions and to insertions that specifically occurred in the st sublineage, after st260- divergence. the mean size of deletions largely exceeded that of insertions since only nucleotides were gained while were lost. deletions, insertions and nucleotide replacements were respectively responsible for and gene disruptions. in addition, 15% of the indels led to minor protein size modifications and their consequences on protein functions were more difficult to predict. finally 30% of the indels were characterized in intergenic regions. altogether mutations leading to gain or loss of nucleotides represented approximately 12% of total mutations after the divergence from st strains and constituted the major process of gene disruption. this evolution was not specific to the st sublineage as a similar number of insertions and deletions occurred in the st sublineage accounting for gene disruptions. in addition, indels and snps relative to strain a sequence were common to the five st260- strains but were not observed in a vs other gbs strains. forty per cent of these indels were intragenic and led to gene disruptions. finally, genes disrupted by a small indel in one sublineage were deleted in the other, suggesting that gene disruption could be a preliminary step to gene loss and genome reduction by secondary longer deletions. as a consequence some loci were observed under different states of decay in the two sublineages. this is the case for instance for the cyl locus, the crispr locus and the pil pilus locus. furthermore, the average size of the indels was found to increase following the divergence of the two sublineages compared to the common branch. therefore, while the process of reductive evolution was already evident in the ancestor of the st260- strains, it was even more pronounced after divergence of the two sublineages. to better evaluate the specificity of the evolutionary process occurring in st260- strains, we compared the core genome sequence of strains a and nem two strains of human origin differing by approximately snp. we detected indels, which represents half the number of indels in the 2- vs a comparison. these indels were essentially short and as much as 80% of them occurred in intergenic regions while less than 4% led to gene inactivations. therefore, gain or loss of nucleotides was also frequent in other s. agalactiae strains, but most indels in coding sequences were probably eliminated by purifying selection. fitting with this hypothesis, we observed one indel per 5- snps during the recent evolution of st strains where purifying selection is probably not effective. a similar proportion of indels vs snp was also reported for two strains of s. enterica sv paratyphi. finally, to gain further insights into the mechanism of the observed insertions and deletions, we analyzed the sequences flanking the indels in strain 2- among the indels of nt, occurred in homopolymeric tracts longer than four nucleotides and likely resulted from dna polymerase slippage during replication. interestingly, of the indels larger than four nucleotides apparently also involved recombination between repeated sequences. the median size of the repeats was nucleotides, with sequences larger than nucleotides, the largest being nt long. since the threshold of repeat length for reca-dependent homologous recombination is 23- nt, most of the indels probably occurred by reca independent recombination. it has been shown that, under laboratory conditions, the efficiency of illegitimate recombination is highly dependent on the size of the repeats and inversely dependent on the distance between repeats. our results suggest that reca independent recombination between repeats of moderate sizes may also lead to long deletions. furthermore we did not find any evidence that deletions of long sequences may depend on longer repeats. this may indicate that the lower efficiency of illegitimate recombination between short repeats is buffered by their higher frequency in the genome. some of the large deletions might also correspond to several consecutive shorter ones. from this analysis we propose that accumulation of small and easily reversible indels is common in s. agalactiae, mostly occurring as a consequence of reca independent recombination. most of these indels are probably removed from the population due to purifying selection during long-term evolution. alternatively they may be compensated by a second loss or gain of nucleotides. in the st260- lineages host specialization led to the relaxation of the negative selection on genes that became dispensable, allowing the accumulation of larger deletions. functional adaptation to a host restricted way of living st260- strains differ from other gbs strains by their host specificity and high pathogenicity. as mentioned, this lineage has only few specific genes compared to other strains and most of them are carried by genomic islands in the process of being eliminated. in particular, we did not identify any specific surface proteins or putative virulence factors. therefore, the specific properties of the st260- strains more likely rely on functions shared with human isolates and on the loss of some functions. the genes common to the two sublineages were grouped into functional categories according to kegg classification. compared to functional classification of a genes, this analysis revealed a dramatic drop in the proportions of genes involved in mobile and extrachromosomal functions and in cellular processes, including the pathogenesis subcategory. the other major functions submitted to evolutionary erosion were energy metabolism, transport and binding, regulation and signal transduction. conversely the basal functions of the cell such as transcription and protein synthesis were nearly not affected. bacterial-host interactions and virulence factors surface components play an important role for tissue colonization and infection by mediating interactions between the pathogen and the host cells and evasion from immune defense. gbs strains possess two distinct polysaccharide antigens, the highly sialylated capsule polysaccharide and the group b-antigen. st260- strains harbor the genes involved in the type ib capsule synthesis similarly to the st strains h36b and ss they also possess the genes responsible for the synthesis of the group b antigen. only seven proteins with a lpxtg signal for cell-wall anchor are conserved including three important s. agalactiae virulence factors of human strains: the fibrinogen-binding protein a, the serine-rich protein srr and the biba/hvga protein. however, while in human strains these three proteins carry a variable number of a repeated motif, these repeats have been lost in the st260- strains. this might decrease their accessibility to host immune system and cellular receptors by tethering them to the cell wall. 1: proteins anchored to the peptidoglycan by an lpxtg c-terminal motif.: the gene has been inactivated.: amino-acid repeats have been lost in the corresponding protein. among the other proteins that are virulence factors in human gbs isolates, the five strains lack the c5a peptidase, the laminin binding protein and components of the pilus. the cyl locus has also been inactivated in the two sublineages in agreement with the absence of detectable hemolytic activity on blood agar plates. another major test for s. agalactiae identification is the detection of camp activity. while both st and strains were reported to be negative for the camp factor reaction we found that the gene encoding the camp factor was disrupted only in st strains. we experimentally confirmed that strain ss was negative for the camp test detection, probably because of a lower level of gene expression. finally, the five st260- strains express a fibrinogen/fibronectin binding protein of the pava family and a hyaluronidase that were proposed to be virulence factors in gbs or other streptococcus species and could also have a role in fish infection. altogether, approximately 60% of the genes for proteins involved in pathogenesis and considered as important virulence genes in human stains were affected by genome reduction. in this context the capsule could be a major virulence factor in the fish host, as also observed for streptococcus iniae, another streptococcus species pathogenic for fish. metabolism analysis of the missing functions revealed a profound remodeling of the metabolism of strains of this lineage. numerous transport systems for carbon sources and enzymes for degradation of polysaccharides were missing or inactivated, reflecting a reduced capacity to utilize diverse carbon sources. in addition st260- strains seem unable to utilize glycerol and glycerol phosphate as genes encoding the glycerol kinase, the glycerol dehydrogenase and the glycerol-phosphate permease are missing or mutated. fermentative pathways utilizing pyruvate/acetate conversion are also altered, as the phosphotransacetylase gene is missing. in mammals, gbs is primarily considered as a commensal of the digestive tract, an environment rich in diverse c-sources. in contrast, reduction in the catabolic capacities observed in st260- strains is in favor of a transition to an obligate pathogen style-of-life. transport functions approximately 20% of the genes associated with transport systems were missing or inactivated. the targeted functions were mostly the import of nutrients, in particular of c-sources and the transport of inorganic and metal ions. the na+/h + antiporter, the k + uptake permease were inactivated in both sublineages. therefore st260- strains may be affected in ionic exchange and would have a reduced capacity to maintain their homeostasis in the face of a changing external environment. transcriptional networks globally, gene disruption and deletion events affected out of the transcriptional regulators predicted in human strains. as much as out of the two-component systems found in gbs were inactivated, either in one or in both sublineages. interestingly, analysis of the genome sequence of strain 2- revealed that the rgf tcs, which is involved in the control of virulence in the human st strains, was associated with two putative bacteriocins with a double glycine leader peptide and with a bacteriocin export transporter. this suggests that this tcs originates from a bacteriocin operon that has been partially deleted in human strains. altogether our observations show that s. agalactiae strains adapted to fish may have a reduced capacity to respond to environmental changes compared to human strains and only eight tcs, including the two major systems, ciarh and covrs, may be sufficient to allow gbs adaptation to the different environments encountered in fish. interestingly, both ciarh and covrs were also involved in the regulation of virulence genes in gbs human strains. the higher virulence of st260- strains might also be due to the deregulated expression of some virulence genes, as observed in the transition from local to systemic infections in group a streptococci. adaptation to fish is associated with broad changes in gene expression as a first step to explore changes in gene expression linked to host adaptation, we performed a comparative analysis at the transcriptome level of strains a, cf, 2- and ss. genes present in the four strains and with 100% identity matches with probes of the array were taken into account in this analysis. profound modifications in gene expression were observed in st260- strains and to a lesser extent in strain cf compared to strain a. although strains cf and a are closely related, the expression of more than genes varied by a two-fold factor between the two strains. in particular 40% of the genes involved in energy metabolism were expressed at a lower level in the fish isolate than in strain a in contrast expression of the gene encoding the virulence protein srr was more than fold increased compared to strain a although up-regulation of the srr gene is not conserved in the st260- fish isolates, in strain cf it might facilitate penetration of the fish blood brain barrier, leading to an increased tendency to cause meningitis, as shown for the srr protein of human isolates. both the number of genes for which expression was modified and the amplitude of these modifications were even larger in st260- isolates. for instance two genes encoding putative glyoxalases are expressed at 6- higher levels in st260- strains than in strain a suggesting higher needs in methylglyoxal detoxification. genes encoding the enzymes for the synthesis of arginine were 10- fold more expressed. four functional categories were more affected by variations in gene expression in both st260- sublineages: metabolism, transport, regulation and signal transduction. interestingly these functional categories were also particularly affected by gene erosion, suggesting a remodeling of metabolic networks. in addition to these global tendencies, st and st strains also harbor sublineage-specific variations in gene expression that probably result from differential gene decay and might lead to specific adaptations. for instance, in strain ss genes encoding phosphoglycerate kinase, phosphoglyceromutase and pyruvate kinase involved in glycolysis and neoglucogenesis are expressed at a lower level than in strain a in parallel, genes coding for the arginine deiminase, ornithine carbamoylkinase and carbamate kinase involved in the deiminase pathway are up-regulated, suggesting a shift towards arginine fermentation as the main pathway for energy production in st strains. this up-regulation of the deiminase pathway is associated with a decreased expression of the negative regulator argr and the increased expression of the arginine/ornithine antiporter. in contrast, in the same growth conditions, upregulation of the genes for hpr-kinase and cody involved in catabolic repression and for lactate dehydrogenase suggests that strain 2- might generate energy essentially through glucose utilization and lactic fermentation. iron import might be more efficient in st than in st strains since strain ss expresses genes for iron abc transporter and ferrichrome abc transporter at higher levels than strain a while these genes are down-regulated or unchanged in strain 2- both sublineages express lower amounts of atp synthase, the enzyme responsible for atp-proton motive force interconversion, than strain a indicating that they may be impaired in ph homeostasis in acidic conditions. since expression of pseudogenes is energetically costly and may generate deleterious products, we took advantage of the massive genome erosion in the st260- lineage to look for evidence for negative selective pressure acting on pseudogene expression. among the pseudogenes represented by at least one probe on the array, only 22% were down-regulated compared to strain a whereas 64% did not present a significant change in mrna levels and 14% were even up-regulated. furthermore silencing of pseudogenes in the st260- lineage did not seem to markedly increase with time. indeed the proportion of down-regulated pseudogenes was similar among pseudogenes arising before and after the differentiation of sublineages. altogether this indicates that no strong evolutionary pressure acts to silence pseudogene expression. although modifications in gene expression generally affected different genes in st260- and cc strains, genes were found to vary in the same way in the strains isolated from fish. interestingly, these genes included the operon for the capsule synthesis, the genes for zoocin and hyaluronidase, two response regulators of tcs systems and three targets of the ciarh tcs that were up-regulated. sixteen genes were downregulated or inactivated in the three fish strains compared to a among which were involved in energy metabolism or in carbohydrate uptake. whether these regulations reflect a common mechanism of adaptation to fish environment remains to be established. however, the higher expression of the genes for the capsule synthesis might favor resistance to environmental conditions and to fish immune system, as it has also been reported for s. iniae. similarly the high hyaluronidase expression may help s. agalactiae strains to break through fish tissues and be involved in virulence to fish.  our results show that s. agalactiae strains leading to epidemic diseases in fish farms and cold blood animals belong to at least two distinct groups that differ by their strategies of host adaptation. cc strains have the potency to colonize and infect multiple hosts such as fish, human and cattle. from a genomic point of view, these cc fish strains are not distinguished from their human counterparts by any significant genomic island. however, contrasting with this genomic relatedness, large differences in gene expression were observed and could participate to the adaptation to the fish host. conversely, our genome analysis indicates that strains of the st260- complex diverged anciently from human and cattle strains and subsequently accumulated specific adaptations leading to the emergence of sublineages. st260- strains exhibit a striking pattern of genome reduction and we took advantage of the emergence of sublineages to reconstruct the different steps involved in this process. we found that accumulation of short indels can be observed all along the evolution of the gbs species, participating to strain-specific gene disruptions. therefore even in the core genome of human gbs strains some genes are dispensable. nevertheless the number of inactivated genes greatly increased during specialization of st260- strains to fish. these gene inactivations mainly result from the ongoing accumulation of short indels, but a tendency to eliminate inactivated genes by deleting longer sequences is more noticeable in the sublineage specific branches. in contrast with what has previously been observed in the course of genomic reduction associated to host specialization or to intracellular symbiosis, deletion events are not correlated with an amplification of insertion sequences. neither could complex genome rearrangements be noticed, suggesting that recombination between is is not a general mechanism for genome reduction. indeed our results point to non-homologous recombination as an alternative mechanism of genome reductive evolution. 
in this lineage, specialization to the fish host was correlated with a massive gene inactivation and broad changes in gene expression. while strains belonging to the clonal complex cannot be distinguished from their human cc counterparts according to their gene content, strains belonging to the st260- types probably diverged a long time ago. to decipher the genetic basis for the emergence of these highly virulent gbs strains and of their adaptation to fish, we have analyzed the genomic sequence of seven strains isolated from fish and other poikilotherms. comparative analysis shows that the two groups of gbs strains responsible for fish epidemic diseases are only distantly related. we took advantage of the low level of sequence divergence between gbs strains and of the emergence of sublineages to reconstruct the different steps involved in this process. non-homologous recombination was found to have played a major role in the genome erosion. during host specialization, inactivation of genes whose function is no more required is favored by changes in selective constraints and evolutionary bottlenecks. our results show that the early phase of genome reduction during host specialization mostly involves accumulation of small and likely reversible indels, followed by a second evolutionary step marked by a higher frequency of large deletions. the gram positive bacteria streptococcus agalactiae, responsible for septicemia and meningitis in neonates also emerged during the seventies as a cause of severe epidemics in fish farms.
 during host specialization, inactivation of genes whose function is no more required is favored by changes in selective constraints and evolutionary bottlenecks. the gram positive bacteria streptococcus agalactiae, responsible for septicemia and meningitis in neonates also emerged during the seventies as a cause of severe epidemics in fish farms. to decipher the genetic basis for the emergence of these highly virulent gbs strains and of their adaptation to fish, we have analyzed the genomic sequence of seven strains isolated from fish and other poikilotherms.  comparative analysis shows that the two groups of gbs strains responsible for fish epidemic diseases are only distantly related. while strains belonging to the clonal complex cannot be distinguished from their human cc counterparts according to their gene content, strains belonging to the st260- types probably diverged a long time ago. in this lineage, specialization to the fish host was correlated with a massive gene inactivation and broad changes in gene expression. we took advantage of the low level of sequence divergence between gbs strains and of the emergence of sublineages to reconstruct the different steps involved in this process. non-homologous recombination was found to have played a major role in the genome erosion.  our results show that the early phase of genome reduction during host specialization mostly involves accumulation of small and likely reversible indels, followed by a second evolutionary step marked by a higher frequency of large deletions. streptococcus agalactiaehost-adaptationnon-homologous recombinationgene inactivationvirulence 