BACKGROUND
pais play important roles in the adaptation and increased virulence of pathogens.
bacterial pai often encode both effector molecules responsible for disease and secretion systems that deliver these effectors to host cells
<cit> .
pais are a distinct type of genomic island.
pais contain mobile genetic elements , which were acquired by the bacteria through hgt.
bacterial genomes contain various types of mges, such as transposons, plasmids, and bacteriophages.
all of these elements may be acquired by hgt.
many mges serve as shuttles for genes that are beneficial to bacteria during their proliferation in a host environment.
several mges have been found in the genomes of pathogenic bacteria that contain genes conferring antibiotic resistance and genes encoding virulence factors, such as exotoxins, adhesins, and secretion systems
<cit> .
pathogenic bacteria often make use of suicide mechanisms, in which the death of individual cells benefits the survival of the population.
this mechanism is regulated by the toxin-antitoxin system , which is related to dna replication, mrna stability, protein synthesis, cell-wall biosynthesis and atp synthesis
<cit> .
the ϵ antitoxin-ζ toxin system  is a type ii ta system.
it is distributed over plasmids and chromosomes of various pathogenic bacteria
<cit> .
these systems benefit the stability of the genomic island in the bacterial genome.
s. zooepidemicus is the ancestor of streptococcus equi ssp.
equi  and these two strains express many of the same proteins and virulence factors.
however, unlike s. equi, which is host-restricted and only infects horses, s. zooepidemicus has no host preference.
s. zooepidemicus is primarily an opportunistic pathogen infecting a wide variety of animal species, including important domestic species, which makes it a pathogen of veterinary concern.
s. zooepidemicus causes mastitis in cows and mares, and is the most frequently isolated opportunistic pathogen of horses
<cit> .
occasionally, s. zooepidemicus can infect humans via zoonotic transmission from infected animals and causes invasive infections in humans such as septicemia and meningitis
<cit> .
in  <dig>  sichuan province experienced an s. zooepidemicus outbreak that resulted in the death of  <dig>  pigs and great economic losses.
s. zooepidemicus is an important pathogen of streptococcal diseases in swine
<cit>  and it remains a great threat to chinese swine breeding.
in the present study, we used comparative genomic analyses between s. zooepidemicus atcc <dig> and other published s. zooepidemicus strains
<cit>  to investigate the mechanisms underlying the differing pathogenicities of streptococcus equi ssp.
in particular, we tried to ascertain how s. zooepidemicus atcc <dig> is able to cause such a serious disease in pig.
we determined the complete genome sequence of s. zooepidemicus atcc <dig> , a virulent strain isolated from a dead pig in china.
the complete genome sequence not only permitted detailed analysis of the phylogenic relationship between species, but also provided insights into the biology and pathogenic capacity of this streptococcus.
RESULTS
genomic features and basic transcriptomic structure
the  <dig> ,264-bp genome of sz <dig> comprises a single circular chromosome with a gc content of  <dig> %  and the genome information have been reported previously
<cit> .
the gc content is similar to that of streptococcus equi subsp.
zooepidemicus mgcs <dig>
<cit> , streptococcus equi subsp.
equi  <dig>  and streptococcus equi subsp.
zooepidemicus h <dig>
<cit> .
the genome contains  <dig>  protein-encoding genes,  <dig> trna genes, and five 5s-16s-23s rrna operon gene clusters.
among the protein coding genes,  <dig>  are predicted to encode conserved hypothetical proteins that are similar to proteins of unknown factions in other genomes, and  <dig> hypothetical genes  have no matches in the nr protein database .
the remaining  <dig> genes were assigned putative functions.
eighty-one genes were identified as mobile elements, including those encoding a competence protein, a phage associated protein, a conjugation protein, a transposase and a site-specific recombinase, suggesting that these elements are used to take up and incorporate foreign dna and are involved in reconstructing the genome architecture.
furthermore, global transcriptome analysis of sz <dig> using rna-seq confirmed that  <dig> of the  <dig>  orfs are expressed, but with different sequence coverages in vitro and in vivo .
comparative gene expression analysis reveals that  <dig> genes are upregulated and  <dig> genes are downregulated  by more than a 2-fold change in reads per kilo base per million  values  in vivo.
the upregulated genes include  <dig> hypothetical protein coding genes and  <dig> response regulator, transcription regulator genes and chaperone protein encoding genes, suggesting that the differential expression of these orfs plays an important role in survival of sz <dig> within the different host environments.
additionally, we found that some genes, including mala , mald , male , malf , malg  and malq  were upregulated when sz <dig> infected mice.
these genes are related to maltose transport and metabolism and utilization of carbohydrates, which is essential for the ability of pathogenic bacteria to cause disease.
group a streptococcus  strains express male on their surface, and the transcript levels of the male gene were significantly increased during growth in human saliva compared to common medium.
male may contribute to the ability of gas to colonize the oropharynx by utilizing maltose
<cit> .
in addition, studies in s. pneumoniae have shown that deletions in carbon metabolism genes, including the maltose operon, lead to decreased production of known virulence factors, such as capsular polysaccharide and cholera toxin
<cit> .
male of sz <dig> is a maltodextrin-binding protein, which also binds longer maltodextrins .
the upregulation of this protein and other maltose utilization-related proteins may contribute to the infection of sz <dig>  further investigation into these carbohydrate transport and metabolism pathways genes may yield novel insights into the pathogenesis of sz <dig>  we also observed that certain known virulence factors were upregulated during sz <dig> infection, for example, streptokinase  and fibronectin-binding protein .
the upregulation of bacteriocin  could help sz <dig> compete with other bacteria that colonize the host.
comparative genomic analysis and pathogenicity islands
comparative analysis of sz <dig> genome with three other genomes revealed that the evolution of sz <dig> has been driven by genomic rearrangements and hgt.
x-alignment analysis of sz <dig> versus sz10565
<cit> , se <dig> and sz70
<cit>  revealed that small and large scale chromosome inversions have occurred during replication termination between sz <dig> and se <dig> and between sz <dig> and sz <dig> .
these genome rearrangements may influence the transcription of surrounding genes after the hgt process, which has contributed to the shaping of the sz <dig> genome.
the comparative analysis of the sz <dig> genome with the three other genomes identified  <dig>  orthologous genes that are shared by all four strains .
in addition,  <dig>   <dig> and  <dig> genes are shared between sz <dig> and sz <dig>  sz <dig> and se <dig>  respectively, suggesting that sz <dig> and sz <dig> are more closely related than the other strains.
furthermore, x-alignment analysis of sz <dig> versus sz10565
<cit> , se <dig> and sz70
<cit>  also suggested that sz <dig> is closer to sz <dig> than to the other two species.
phylogenetic trees of the four strains were constructed based on the sequences of the  <dig>  orthologous genes using minimum evolution and neighbor joining phylogenic reconstruction methods available in the mega package .
the phylogenic trees also indicated that sz <dig> is much closer to sz <dig> than to the other two species, which is consistent with the r genome-scale alignment analysis.
further comparative analysis showed that  <dig> genes are specific to sz <dig>  which include  <dig>  that were annotated as “hypothetical protein”, among which  <dig> encode small proteins with lengths of no more than  <dig> amino acids .
these small proteins are annotated as hypothetical proteins; however, certain highly conserved hypothetical proteins may play important roles in response to specific environmental stresses and host adaptation.
for example, these small proteins have been reported to have evolved in response to specific environmental stress and to participate in the suppression of the type iii secretion system
<cit> .
the remaining functional genes encode  <dig> virulence proteins,  <dig> phage-associated proteins, eight transposases, five site-specific recombinases, a conjugation protein, a phage integrase, a phage recombinase, an is transposase and a relaxase.
these results suggest that the sz <dig> genome acquired these virulence genes through hgt, either by transduction with phages or by conjugation with plasmids or chromosomal fragments.
furthermore, these sz35246-specific genes are tightly clustered into four regions, varying in length from about  <dig> kb to  <dig> kb, which were as termed pais  .
an orthologous genes analysis between sz <dig> and sz <dig>  sz <dig>  se <dig> confirmed these genomic islands are present in the sz <dig> genome only .
the genes located in these four pais might be involved in sz35246’s pathogenesis in causing swine streptococcosis and its strong virulence.
these islands were further confirmed the annotation information and the co-linearity comparison of the sz <dig> genome with those of the three other genomes.
significantly, sequence and annotation analyses of these islands revealed that sesecisland_ <dig>  sesecisland_ <dig> and sesecisland_ <dig> contain the same type of virulence genes involved in the bacterial ta systems that have been reported to play subtle roles in the survival of bacteria under harsh natural environments
<cit> .
based on previous analyses of ta systems in escherichia coli k12
<cit> , mycobacterium tuberculosis <cit>  and mycobacterium smegmatis <cit> , we speculated that acquired-ta systems might play a positive role in survival of sz <dig> under different host environments.
the rm system is used by bacteria to protect themselves from foreign dna, such as bacteriophages and other viruses.
genes encoding rm system proteins, which include a restriction endonuclease and a restriction endonuclease control protein, were identified in a cluster in sesecisland_ <dig>  based on these results, we speculated that the acquired rm system might be involved in defense against infection by foreign dna such as prophages and viruses.
thus, the pais may allow sz <dig> to adapt to various host stress conditions and to defend itself against infection by prophages, other bacteria and viruses.
the expression and potential impact of these islands on the physiology, pathogenesis and host adaptation of sz <dig> are discussed below.
i. sesecisland_1: phd/doc ta system
sesecisland_ <dig> contains  <dig> genes ,  <dig> of which are sz35246-specific genes, including mobile elements resembling the is <dig> family transposase , a prophage site-specific recombinase resolvase family protein , a putative conjugal transfer protein , a conjugation protein  and a tnpx site-specific recombinase family protein , suggesting that this island is an integrative conjugative element .
sesecisland_ <dig> contains  <dig> structural phage loci, indicating that mges, such as phages, are also implicated in hgt in streptococcus species.
further analysis showed that the island has an abnormal gc skew and that the island-located genes have an average g+c content of  <dig> % , which is significantly different from the mean value for the genome  .
a major feature within sesecisland_ <dig> is the presence of two genes  encoding proteins homologous to addiction module antitoxin phd protein and killer doc protein, respectively.
the phd-doc ta system is the type ii ta system that first identified in bacteriophage p1
<cit> .
phylogenetic trees based on phd/doc proteins also suggested that phd/doc proteins are highly homologous to those of streptococcus phage phi-m <dig> and phi-ssud <dig> .
the phd/doc mrnas are co-expressed from the same promoter and both are translated into proteins.
these proteins form a stable ta complex to block the functions of the doc toxin.
doc is a toxin that kills plasmid-free segregants, and phd is an unstable antidote that neutralizes the toxin.
doc inhibits translation elongation by association with the 30s ribosomal subunit
<cit> .
under stress conditions or host change, phd is degraded by atp-dependent serine proteases, such as clpxp protease
<cit> , resulting in freeing of the toxin from the ta system and inducing cell growth inhibition and cell death
<cit> .
interestingly, a gene  encoding a protein homologous to the e. coli clpxp protein is present in this island.
doc toxins are expressed in two conditions  , which agrees with previous reports that doc causes cell growth and death by inhibiting translation without affecting transcription and replication.
the observations and results reported here support the hypothesis that sesecisland_ <dig> helps sz <dig> to adapt to environmental and host changes.
ii.
sesecisland_2: fic/doc ta system
sesecisland_ <dig> contains an important open reading frame , sesec_ <dig>  which encodes a protein with a filamentation induced by camp  domain .
the fic domain is classified together with a second family of sequences, doc , in the pfam protein families database
<cit> .
the fic/doc family protein sequences are aligned against this protein present inside other bacteria.
interestingly, phylogenetic analysis revealed that the fic/doc protein is homologous to that of fusobacterium nucleatum subsp fusiforme .
fic/doc family proteins are known as members of a ta system, the functional sites are common to both families
<cit> .
the fic protein has been reported to be involved in cell division and synthesis of folate, indicating that the fic protein and camp are involved in a regulatory mechanism of cell division via folate metabolism
<cit> .
fic family virulence proteins may be important in many bacterial pathogens.
for example, the immunoglobulin-binding protein a  of histophilus somni contains a direct repeat of two fic domains, and mutation of ibpa or just the fic domain of ibpa decreased the virulence of this bacteria.
the fic domain has been shown to covalently modify host rho gtpases with amp, which may explain how the fic domain influences bacterial virulence
<cit> .
thus, the fic family protein in sesecisland_ <dig> may be involved in the pathogenicity of sz <dig>
<dig>
sesecisland_ <dig> encodes  <dig> genes ,  <dig> of which are sz35246-specific.
sesecisland_ <dig> also contains certain mobile elements, including an endonuclease relaxase , a bacterial mobilization protein  integrase/recombinase  and transposase protein .
thus, we speculate that this region also plays important roles in bacterial adaptation, virulence and physiology.
iii.
sesecisland_3: ϵ/ζ ta system
sesecisland_ <dig> contains  <dig> sz35246-specific genes , the most notable of which are two genes annotated as type ii ta system genes encoding a ζ toxin protein  and an ϵ antitoxin protein .
virb4/virb6/vird <dig> components  from the type iv secretion system  are also present.
additionally, virulence-associated factors, such as glucan-binding protein and abortive infection protein, are also encoded by this region.
all the virulence genes are expressed under in vitro and in vivo conditions.
several mges such as site-specific recombinases  and transposases  were also identified in this island.
the bioinformatics analysis showed that a type ii ta system, a type iv secretion system and other virulence genes were present in this island, which may contribute directly to the bacterium’s pathogenicity and host adaption.
ϵ/ζ systems ensure stable plasmid inheritance by inducing death in plasmid-deprived offspring cells.
members of the ϵ/ζ systems have been found on resistance plasmids in major human pathogens
<cit> .
by contrast, chromosomally encoded ϵ/ζ systems were reported to contribute to virulence of pathogenic bacteria.
brown et al.
compared clinical serotype  <dig> isolates with ζ toxin gene knockout strains in mixed systemic and respiratory infections of mice, and thus connected the ζ toxin with virulence in streptococcus pneumonia <cit> .
the ϵ/ζ system also exists in the  <dig> k pathogenicity island of streptococcus suis serotype  <dig>  this bacterium is an important zoonotic pathogen, causing more than  <dig> cases of severe human infection worldwide
<cit> .
the ζ toxin is inhibited by its cognate antitoxin, ϵ. the structure of the complex of ζ toxin inactivated by ϵ antitoxin  was solved by x-ray crystallography
<cit> .
upon degradation of ϵ, the ζ toxin is released, allowing this enzyme to inhibit bacterial cell wall synthesis, which eventually triggers autolysis
<cit> .
the toxic effect of the ζ toxin has also been demonstrated in a diverse array of organisms, including saccharomyces cerevisiae <cit> .
phylogenetic analysis of ζ proteins and ϵ antitoxin proteins showed that the proteins from sz <dig> are highly homologous to those of streptococcus urinalis 2285– <dig>  streptococcus intermedius f <dig> and streptococcus vestibularis f <dig> and widely distributed in many bacteria .
this broad distribution has been reported that the zeta toxin family on plasmids
<cit> , bacterial chromosomes
<cit>  and in streptococcus pneumonia and streptococcus suis serotype  <dig> pais.
the broad distribution of this system within the bacterial kingdom suggests that it uses a ubiquitous bacteriotoxic mechanism to overcome host defenses and environmental changes.
on the other hand, we hypothesized that horizontal transfer of this island may occur through t4ss-mediated conjugation process, because four genes products display similarities to streptococcus t4ss components.
<dig>
iv sesecisland_4: rm system and virulence island
sesecisland_ <dig> contains eight sz35246-specific genes , the two mobile elements  are transposase is <dig> and phage integrase , suggesting that this island has been acquired by hgt from another microorganism.
the major feature of this island is three strain specific genes  that were annotated as rm system proteins, which protect bacteria from foreign dna, such as bacteriophages.
the rm system is strategy that permits bacteria to live in difference environments
<cit> , allowing bacteria erect a barrier to gene transfer and making them resistant to phage infection
<cit> .
taken together, these data suggest that the rm systems is a remarkable characteristic of sz <dig> and is probably involved in the adaptation of these bacteria to different environmental conditions.
relationship between pais and sz <dig> virulence
to prove that the genes located within the pais affect the virulence of sz <dig>  we deleted part of sesecisland_ <dig> from sesec_ <dig> to sesec_ <dig>  pcr was used to confirm the deletion , sequencing results showed that exactly  <dig>  bp of sesecisland_ <dig> was deleted, including the genes belong to the ϵ /ζ ta system .
the deleted region started with tn <dig> transposon gene , and two repeat sequences, including transposase genes , were located at the flank of the deleted region.
these two repeat sequences and the tn <dig> transposon gene formed the structural basis for deleting such a long fragment.
the mutant strain ∆island3-sz <dig> and wild-type sz <dig>  were used to infect icr mice to evaluate the influence of partial pai deletion on bacterial virulence.
the percent survival significantly increased in ∆island3-sz <dig> infected mice ,  <dig> days post-infection, only one of the ten mice was dead; however, of the mice infected with wild-type sz <dig>  only one was alive.
the survival curve indicated that partial deletion of a pai did affect the virulence of sz <dig>  and that some of these genes in the pai are important for bacterial pathogenicity.
genes located in the other three pais require further study to determine their role in bacterial virulence.
8: table s <dig>   genomic organization of the partial sesecisland_ <dig> deletion locus and its flanking repeat sequences in sz <dig>  the fragment from  <dig> to  <dig> was knocked out, including the zeta toxin  and epsilon antitoxin  genes.
the deleted region started with a tn <dig> transposon protein gene and is flanked by two repeated regions.
after the reciprocal recombination, only one repeat region remained.
other potential virulence genes dispersed in the sz <dig> genome
strain sz <dig> was isolated from a nasal swab taken from a healthy thoroughbred racehorse in newmarket, england, in 2000
<cit> .
a genome wide comparison of sz <dig> with sz <dig> identified sz35246-specific genes, some of which may be involved in the virulence of sz <dig> and may provide clues as to why sz <dig> causes such a serious swine streptococcosis but other strains do not.
virulence-associated protein e , which was originally identified in dichelobacter nodosus <cit> , is part of a vap region of d. nodosus that is associated with virulence
<cit> .
the mechanism by which vape affects virulence has not been determined yet, but the presence of an integrase gene xerc  immediately upstream of vape, suggested a role for bacteriophages in the evolution and transfer of these bacterial virulence determinants; i.e., it is possible that exchange of this putative virulence factor with other bacteriophages could take place
<cit> .
moreover, a vape-like gene has also been identified in a pathogenicity island of staphylococcus aureus <cit> .
the pathogens of a footrot outbreak in a debre zeit swine farm were identified as staphylococcus aureus and dichelobacter nodosus, both bacteria contain the vape gene.
vape has not been identified in other strains of s. zooepidemicus, but only in sz <dig>  this gene may be related to sz <dig> pathogenicity towards pig.
the role of the vape gene in the virulence of sz <dig> remains to be clarified.
adherence is an essential requirement for invasion of cells by bacterial pathogens.
long extracellular structures resembling fimbriae mediate adhesion to components of the host extracellular matrix, such as collagen and fibronectin.
we identified seven sz <dig> unique proteins that contain an lpxtg motif , including collagen-like protein sclz.
<dig> , fibrinogen- and ig-binding protein precursor , cell surface protein , t-antigen-like fimbrial structural subunit protein , putative cell surface protein , inla-like domain containing cell surfaced-anchored protein  and collagen-like surface-anchored protein scle .
all of these proteins are anchored on the bacterial surface and may be involved in bacterial adhesion and invasion.
fibronectin -binding proteins have been reported to mediate the invasion of host cells without the need for other bacterial factors
<cit> .
fn, which has received much attention as a target of bacterial adhesins, it is a glycoprotein found in the extracellular matrix and body fluids of vertebrates.
fn-binding proteins are found in streptococcus pyogenes , staphylococcus aureus , streptococcus dysgalactiae , and other bacterial species
<cit> .
in previous research, an fnz gene was found in s. zooepidemicus and a sfs gene was only found in s. equi, both of which genes encode a cell surface protein that binds fn
<cit> .
the sfs gene  was identified in sz <dig> for the first time.
the transcriptome data showed that the sfs gene was upregulated infection of a host by sz <dig> .
presumably, the expression of this gene promoted bacterial pathogenicity by inhibiting the binding between collagen and fn.
the sz <dig> and sz <dig> genomes both have the fim iii operon  .
the structural proteins of type iii fimbriae have an amino-terminal secretion signal and a carboxy-terminal sorting signal, and their assembly into fimbriae is dependent on the adjacently encoded dedicated sortases
<cit> .
sz <dig> contains two loci that encode genes putatively required for pilus expression, but lacks this putative pilus locus.
recent studies of salmonella enterica revealed that the presence of fimbriae increases the ability of host-restricted bacteria to invade normally restrictive cells
<cit> .
thus, we hypothesize that the presence of the fim iii operon might be associated with breaking host-restriction by s. zooepidemicus.
CONCLUSIONS
the genome and expression analysis of sz <dig> provided fundamental information on the physiology and potential pathogenic capacity of this bacterium.
the comparison of the genomes of sz <dig>  se <dig>  sz <dig> and sz <dig> identified gens that are specific to sz <dig>  these genes may be related to the bacterium’s pathogenic function, including causing swine streptococcosis and breaking host-restriction.
we identified novel mges, which may have been involved in the evolution of sz <dig>  the presence of the elements and the phylogenetic analysis indicated that this genome has been shaped by chromosomal inversion, recombination and hgt events.
sz <dig> probably acquired its pais and certain specific genes through hgt.
the presence of ta systems exists in three of genomic islands of sz <dig> may be related to this strains pathogenicity.
study of these systems will form the basis of our future research.
the availability of the complete sz <dig> genome sequence will facilitate further studies of this pathogen and the development of diagnostics and vaccines.

 pais play important roles in the adaptation and increased virulence of pathogens. bacterial pai often encode both effector molecules responsible for disease and secretion systems that deliver these effectors to host cells. pais are a distinct type of genomic island. pais contain mobile genetic elements, which were acquired by the bacteria through hgt. bacterial genomes contain various types of mges, such as transposons, plasmids, and bacteriophages. all of these elements may be acquired by hgt. many mges serve as shuttles for genes that are beneficial to bacteria during their proliferation in a host environment. several mges have been found in the genomes of pathogenic bacteria that contain genes conferring antibiotic resistance and genes encoding virulence factors, such as exotoxins, adhesins, and secretion systems. pathogenic bacteria often make use of suicide mechanisms, in which the death of individual cells benefits the survival of the population. this mechanism is regulated by the toxin-antitoxin system, which is related to dna replication, mrna stability, protein synthesis, cell-wall biosynthesis and atp synthesis. the ϵ antitoxin-ζ toxin system is a type ii ta system. it is distributed over plasmids and chromosomes of various pathogenic bacteria. these systems benefit the stability of the genomic island in the bacterial genome. s. zooepidemicus is the ancestor of streptococcus equi ssp. equi and these two strains express many of the same proteins and virulence factors. however, unlike s. equi, which is host-restricted and only infects horses, s. zooepidemicus has no host preference. s. zooepidemicus is primarily an opportunistic pathogen infecting a wide variety of animal species, including important domestic species, which makes it a pathogen of veterinary concern. s. zooepidemicus causes mastitis in cows and mares, and is the most frequently isolated opportunistic pathogen of horses. occasionally, s. zooepidemicus can infect humans via zoonotic transmission from infected animals and causes invasive infections in humans such as septicemia and meningitis. in sichuan province experienced an s. zooepidemicus outbreak that resulted in the death of pigs and great economic losses. s. zooepidemicus is an important pathogen of streptococcal diseases in swine and it remains a great threat to chinese swine breeding. in the present study, we used comparative genomic analyses between s. zooepidemicus atcc and other published s. zooepidemicus strains to investigate the mechanisms underlying the differing pathogenicities of streptococcus equi ssp. in particular, we tried to ascertain how s. zooepidemicus atcc is able to cause such a serious disease in pig. we determined the complete genome sequence of s. zooepidemicus atcc, a virulent strain isolated from a dead pig in china. the complete genome sequence not only permitted detailed analysis of the phylogenic relationship between species, but also provided insights into the biology and pathogenic capacity of this streptococcus.  genomic features and basic transcriptomic structure the,264-bp genome of sz comprises a single circular chromosome with a gc content of % and the genome information have been reported previously. the gc content is similar to that of streptococcus equi subsp. zooepidemicus mgcs, streptococcus equi subsp. equi and streptococcus equi subsp. zooepidemicus h. the genome contains protein-encoding genes, trna genes, and five 5s-16s-23s rrna operon gene clusters. among the protein coding genes, are predicted to encode conserved hypothetical proteins that are similar to proteins of unknown factions in other genomes, and hypothetical genes have no matches in the nr protein database. the remaining genes were assigned putative functions. eighty-one genes were identified as mobile elements, including those encoding a competence protein, a phage associated protein, a conjugation protein, a transposase and a site-specific recombinase, suggesting that these elements are used to take up and incorporate foreign dna and are involved in reconstructing the genome architecture. furthermore, global transcriptome analysis of sz using rna-seq confirmed that of the orfs are expressed, but with different sequence coverages in vitro and in vivo. comparative gene expression analysis reveals that genes are upregulated and genes are downregulated by more than a 2-fold change in reads per kilo base per million values in vivo. the upregulated genes include hypothetical protein coding genes and response regulator, transcription regulator genes and chaperone protein encoding genes, suggesting that the differential expression of these orfs plays an important role in survival of sz within the different host environments. additionally, we found that some genes, including mala, mald, male, malf, malg and malq were upregulated when sz infected mice. these genes are related to maltose transport and metabolism and utilization of carbohydrates, which is essential for the ability of pathogenic bacteria to cause disease. group a streptococcus strains express male on their surface, and the transcript levels of the male gene were significantly increased during growth in human saliva compared to common medium. male may contribute to the ability of gas to colonize the oropharynx by utilizing maltose. in addition, studies in s. pneumoniae have shown that deletions in carbon metabolism genes, including the maltose operon, lead to decreased production of known virulence factors, such as capsular polysaccharide and cholera toxin. male of sz is a maltodextrin-binding protein, which also binds longer maltodextrins. the upregulation of this protein and other maltose utilization-related proteins may contribute to the infection of sz further investigation into these carbohydrate transport and metabolism pathways genes may yield novel insights into the pathogenesis of sz we also observed that certain known virulence factors were upregulated during sz infection, for example, streptokinase and fibronectin-binding protein. the upregulation of bacteriocin could help sz compete with other bacteria that colonize the host. comparative genomic analysis and pathogenicity islands comparative analysis of sz genome with three other genomes revealed that the evolution of sz has been driven by genomic rearrangements and hgt. x-alignment analysis of sz versus sz10565, se and sz70 revealed that small and large scale chromosome inversions have occurred during replication termination between sz and se and between sz and sz. these genome rearrangements may influence the transcription of surrounding genes after the hgt process, which has contributed to the shaping of the sz genome. the comparative analysis of the sz genome with the three other genomes identified orthologous genes that are shared by all four strains. in addition, and genes are shared between sz and sz sz and se respectively, suggesting that sz and sz are more closely related than the other strains. furthermore, x-alignment analysis of sz versus sz10565, se and sz70 also suggested that sz is closer to sz than to the other two species. phylogenetic trees of the four strains were constructed based on the sequences of the orthologous genes using minimum evolution and neighbor joining phylogenic reconstruction methods available in the mega package. the phylogenic trees also indicated that sz is much closer to sz than to the other two species, which is consistent with the r genome-scale alignment analysis. further comparative analysis showed that genes are specific to sz which include that were annotated as “hypothetical protein”, among which encode small proteins with lengths of no more than amino acids. these small proteins are annotated as hypothetical proteins; however, certain highly conserved hypothetical proteins may play important roles in response to specific environmental stresses and host adaptation. for example, these small proteins have been reported to have evolved in response to specific environmental stress and to participate in the suppression of the type iii secretion system. the remaining functional genes encode virulence proteins, phage-associated proteins, eight transposases, five site-specific recombinases, a conjugation protein, a phage integrase, a phage recombinase, an is transposase and a relaxase. these results suggest that the sz genome acquired these virulence genes through hgt, either by transduction with phages or by conjugation with plasmids or chromosomal fragments. furthermore, these sz35246-specific genes are tightly clustered into four regions, varying in length from about kb to kb, which were as termed pais. an orthologous genes analysis between sz and sz sz se confirmed these genomic islands are present in the sz genome only. the genes located in these four pais might be involved in sz35246’s pathogenesis in causing swine streptococcosis and its strong virulence. these islands were further confirmed the annotation information and the co-linearity comparison of the sz genome with those of the three other genomes. significantly, sequence and annotation analyses of these islands revealed that sesecisland_ sesecisland_ and sesecisland_ contain the same type of virulence genes involved in the bacterial ta systems that have been reported to play subtle roles in the survival of bacteria under harsh natural environments. based on previous analyses of ta systems in escherichia coli k12, mycobacterium tuberculosis and mycobacterium smegmatis, we speculated that acquired-ta systems might play a positive role in survival of sz under different host environments. the rm system is used by bacteria to protect themselves from foreign dna, such as bacteriophages and other viruses. genes encoding rm system proteins, which include a restriction endonuclease and a restriction endonuclease control protein, were identified in a cluster in sesecisland_ based on these results, we speculated that the acquired rm system might be involved in defense against infection by foreign dna such as prophages and viruses. thus, the pais may allow sz to adapt to various host stress conditions and to defend itself against infection by prophages, other bacteria and viruses. the expression and potential impact of these islands on the physiology, pathogenesis and host adaptation of sz are discussed below. i. sesecisland_1: phd/doc ta system sesecisland_ contains genes, of which are sz35246-specific genes, including mobile elements resembling the is family transposase, a prophage site-specific recombinase resolvase family protein, a putative conjugal transfer protein, a conjugation protein and a tnpx site-specific recombinase family protein, suggesting that this island is an integrative conjugative element. sesecisland_ contains structural phage loci, indicating that mges, such as phages, are also implicated in hgt in streptococcus species. further analysis showed that the island has an abnormal gc skew and that the island-located genes have an average g+c content of %, which is significantly different from the mean value for the genome. a major feature within sesecisland_ is the presence of two genes encoding proteins homologous to addiction module antitoxin phd protein and killer doc protein, respectively. the phd-doc ta system is the type ii ta system that first identified in bacteriophage p1. phylogenetic trees based on phd/doc proteins also suggested that phd/doc proteins are highly homologous to those of streptococcus phage phi-m and phi-ssud. the phd/doc mrnas are co-expressed from the same promoter and both are translated into proteins. these proteins form a stable ta complex to block the functions of the doc toxin. doc is a toxin that kills plasmid-free segregants, and phd is an unstable antidote that neutralizes the toxin. doc inhibits translation elongation by association with the 30s ribosomal subunit. under stress conditions or host change, phd is degraded by atp-dependent serine proteases, such as clpxp protease, resulting in freeing of the toxin from the ta system and inducing cell growth inhibition and cell death. interestingly, a gene encoding a protein homologous to the e. coli clpxp protein is present in this island. doc toxins are expressed in two conditions, which agrees with previous reports that doc causes cell growth and death by inhibiting translation without affecting transcription and replication. the observations and results reported here support the hypothesis that sesecisland_ helps sz to adapt to environmental and host changes. ii. sesecisland_2: fic/doc ta system sesecisland_ contains an important open reading frame, sesec_ which encodes a protein with a filamentation induced by camp domain. the fic domain is classified together with a second family of sequences, doc, in the pfam protein families database. the fic/doc family protein sequences are aligned against this protein present inside other bacteria. interestingly, phylogenetic analysis revealed that the fic/doc protein is homologous to that of fusobacterium nucleatum subsp fusiforme. fic/doc family proteins are known as members of a ta system, the functional sites are common to both families. the fic protein has been reported to be involved in cell division and synthesis of folate, indicating that the fic protein and camp are involved in a regulatory mechanism of cell division via folate metabolism. fic family virulence proteins may be important in many bacterial pathogens. for example, the immunoglobulin-binding protein a of histophilus somni contains a direct repeat of two fic domains, and mutation of ibpa or just the fic domain of ibpa decreased the virulence of this bacteria. the fic domain has been shown to covalently modify host rho gtpases with amp, which may explain how the fic domain influences bacterial virulence. thus, the fic family protein in sesecisland_ may be involved in the pathogenicity of sz sesecisland_ encodes genes, of which are sz35246-specific. sesecisland_ also contains certain mobile elements, including an endonuclease relaxase, a bacterial mobilization protein integrase/recombinase and transposase protein. thus, we speculate that this region also plays important roles in bacterial adaptation, virulence and physiology. iii. sesecisland_3: ϵ/ζ ta system sesecisland_ contains sz35246-specific genes, the most notable of which are two genes annotated as type ii ta system genes encoding a ζ toxin protein and an ϵ antitoxin protein. virb4/virb6/vird components from the type iv secretion system are also present. additionally, virulence-associated factors, such as glucan-binding protein and abortive infection protein, are also encoded by this region. all the virulence genes are expressed under in vitro and in vivo conditions. several mges such as site-specific recombinases and transposases were also identified in this island. the bioinformatics analysis showed that a type ii ta system, a type iv secretion system and other virulence genes were present in this island, which may contribute directly to the bacterium’s pathogenicity and host adaption. ϵ/ζ systems ensure stable plasmid inheritance by inducing death in plasmid-deprived offspring cells. members of the ϵ/ζ systems have been found on resistance plasmids in major human pathogens. by contrast, chromosomally encoded ϵ/ζ systems were reported to contribute to virulence of pathogenic bacteria. brown et al. compared clinical serotype isolates with ζ toxin gene knockout strains in mixed systemic and respiratory infections of mice, and thus connected the ζ toxin with virulence in streptococcus pneumonia. the ϵ/ζ system also exists in the k pathogenicity island of streptococcus suis serotype this bacterium is an important zoonotic pathogen, causing more than cases of severe human infection worldwide. the ζ toxin is inhibited by its cognate antitoxin, ϵ. the structure of the complex of ζ toxin inactivated by ϵ antitoxin was solved by x-ray crystallography. upon degradation of ϵ, the ζ toxin is released, allowing this enzyme to inhibit bacterial cell wall synthesis, which eventually triggers autolysis. the toxic effect of the ζ toxin has also been demonstrated in a diverse array of organisms, including saccharomyces cerevisiae. phylogenetic analysis of ζ proteins and ϵ antitoxin proteins showed that the proteins from sz are highly homologous to those of streptococcus urinalis 2285– streptococcus intermedius f and streptococcus vestibularis f and widely distributed in many bacteria. this broad distribution has been reported that the zeta toxin family on plasmids, bacterial chromosomes and in streptococcus pneumonia and streptococcus suis serotype pais. the broad distribution of this system within the bacterial kingdom suggests that it uses a ubiquitous bacteriotoxic mechanism to overcome host defenses and environmental changes. on the other hand, we hypothesized that horizontal transfer of this island may occur through t4ss-mediated conjugation process, because four genes products display similarities to streptococcus t4ss components. iv sesecisland_4: rm system and virulence island sesecisland_ contains eight sz35246-specific genes, the two mobile elements are transposase is and phage integrase, suggesting that this island has been acquired by hgt from another microorganism. the major feature of this island is three strain specific genes that were annotated as rm system proteins, which protect bacteria from foreign dna, such as bacteriophages. the rm system is strategy that permits bacteria to live in difference environments, allowing bacteria erect a barrier to gene transfer and making them resistant to phage infection. taken together, these data suggest that the rm systems is a remarkable characteristic of sz and is probably involved in the adaptation of these bacteria to different environmental conditions. relationship between pais and sz virulence to prove that the genes located within the pais affect the virulence of sz we deleted part of sesecisland_ from sesec_ to sesec_ pcr was used to confirm the deletion, sequencing results showed that exactly bp of sesecisland_ was deleted, including the genes belong to the ϵ /ζ ta system. the deleted region started with tn transposon gene, and two repeat sequences, including transposase genes, were located at the flank of the deleted region. these two repeat sequences and the tn transposon gene formed the structural basis for deleting such a long fragment. the mutant strain ∆island3-sz and wild-type sz were used to infect icr mice to evaluate the influence of partial pai deletion on bacterial virulence. the percent survival significantly increased in ∆island3-sz infected mice, days post-infection, only one of the ten mice was dead; however, of the mice infected with wild-type sz only one was alive. the survival curve indicated that partial deletion of a pai did affect the virulence of sz and that some of these genes in the pai are important for bacterial pathogenicity. genes located in the other three pais require further study to determine their role in bacterial virulence. 8: table s genomic organization of the partial sesecisland_ deletion locus and its flanking repeat sequences in sz the fragment from to was knocked out, including the zeta toxin and epsilon antitoxin genes. the deleted region started with a tn transposon protein gene and is flanked by two repeated regions. after the reciprocal recombination, only one repeat region remained. other potential virulence genes dispersed in the sz genome strain sz was isolated from a nasal swab taken from a healthy thoroughbred racehorse in newmarket, england, in 2000. a genome wide comparison of sz with sz identified sz35246-specific genes, some of which may be involved in the virulence of sz and may provide clues as to why sz causes such a serious swine streptococcosis but other strains do not. virulence-associated protein e, which was originally identified in dichelobacter nodosus, is part of a vap region of d. nodosus that is associated with virulence. the mechanism by which vape affects virulence has not been determined yet, but the presence of an integrase gene xerc immediately upstream of vape, suggested a role for bacteriophages in the evolution and transfer of these bacterial virulence determinants; i.e., it is possible that exchange of this putative virulence factor with other bacteriophages could take place. moreover, a vape-like gene has also been identified in a pathogenicity island of staphylococcus aureus. the pathogens of a footrot outbreak in a debre zeit swine farm were identified as staphylococcus aureus and dichelobacter nodosus, both bacteria contain the vape gene. vape has not been identified in other strains of s. zooepidemicus, but only in sz this gene may be related to sz pathogenicity towards pig. the role of the vape gene in the virulence of sz remains to be clarified. adherence is an essential requirement for invasion of cells by bacterial pathogens. long extracellular structures resembling fimbriae mediate adhesion to components of the host extracellular matrix, such as collagen and fibronectin. we identified seven sz unique proteins that contain an lpxtg motif, including collagen-like protein sclz., fibrinogen- and ig-binding protein precursor, cell surface protein, t-antigen-like fimbrial structural subunit protein, putative cell surface protein, inla-like domain containing cell surfaced-anchored protein and collagen-like surface-anchored protein scle. all of these proteins are anchored on the bacterial surface and may be involved in bacterial adhesion and invasion. fibronectin -binding proteins have been reported to mediate the invasion of host cells without the need for other bacterial factors. fn, which has received much attention as a target of bacterial adhesins, it is a glycoprotein found in the extracellular matrix and body fluids of vertebrates. fn-binding proteins are found in streptococcus pyogenes, staphylococcus aureus, streptococcus dysgalactiae, and other bacterial species. in previous research, an fnz gene was found in s. zooepidemicus and a sfs gene was only found in s. equi, both of which genes encode a cell surface protein that binds fn. the sfs gene was identified in sz for the first time. the transcriptome data showed that the sfs gene was upregulated infection of a host by sz. presumably, the expression of this gene promoted bacterial pathogenicity by inhibiting the binding between collagen and fn. the sz and sz genomes both have the fim iii operon. the structural proteins of type iii fimbriae have an amino-terminal secretion signal and a carboxy-terminal sorting signal, and their assembly into fimbriae is dependent on the adjacently encoded dedicated sortases. sz contains two loci that encode genes putatively required for pilus expression, but lacks this putative pilus locus. recent studies of salmonella enterica revealed that the presence of fimbriae increases the ability of host-restricted bacteria to invade normally restrictive cells. thus, we hypothesize that the presence of the fim iii operon might be associated with breaking host-restriction by s. zooepidemicus.  the genome and expression analysis of sz provided fundamental information on the physiology and potential pathogenic capacity of this bacterium. the comparison of the genomes of sz se sz and sz identified gens that are specific to sz these genes may be related to the bacterium’s pathogenic function, including causing swine streptococcosis and breaking host-restriction. we identified novel mges, which may have been involved in the evolution of sz the presence of the elements and the phylogenetic analysis indicated that this genome has been shaped by chromosomal inversion, recombination and hgt events. sz probably acquired its pais and certain specific genes through hgt. the presence of ta systems exists in three of genomic islands of sz may be related to this strains pathogenicity. study of these systems will form the basis of our future research. the availability of the complete sz genome sequence will facilitate further studies of this pathogen and the development of diagnostics and vaccines. 
the present study used comparative genomics to examine the different pathogenicities of s. zooepidemicus. zooepidemicus is an important pathogen causing swine streptococcosis in china. genome of s. zooepidemicus atcc comprises,264-bp of a single circular chromosome, with a gc content of %. genes of the fim iii operon were presumed to be involved in breaking the host-restriction of sz further analysis of the ta and rm systems in the pais will improve our understanding of the pathogenicity of this bacterium and could lead to the development of diagnostics and vaccines. genome wide comparisons of sz with three other strains and transcriptome analysis revealed novel genes related to bacterial virulence and breaking the host-restriction. pathogenicity islands of s. zooepidemicus have been transferred among bacteria through horizontal gene transfer and play important roles in the adaptation and increased virulence of s. zooepidemicus. comparative genome analysis of sz s. zooepidemicus mgcs, streptococcus equi. and s. zooepidemicus h identified sz35246-specific genes, clustered into three toxin-antitoxin systems pais and one restriction modification system pai.
 streptococcus equi ssp. zooepidemicus is an important pathogen causing swine streptococcosis in china. pathogenicity islands of s. zooepidemicus have been transferred among bacteria through horizontal gene transfer and play important roles in the adaptation and increased virulence of s. zooepidemicus. the present study used comparative genomics to examine the different pathogenicities of s. zooepidemicus.  genome of s. zooepidemicus atcc comprises,264-bp of a single circular chromosome, with a gc content of %. comparative genome analysis of sz s. zooepidemicus mgcs, streptococcus equi. ssp. equi. and s. zooepidemicus h identified sz35246-specific genes, clustered into three toxin-antitoxin systems pais and one restriction modification system pai. these four acquired pais encode proteins that may contribute to the overall pathogenic capacity and fitness of this bacterium to adapt to different hosts. analysis of the in vivo and in vitro transcriptomes of this bacterium revealed differentially expressed pai genes and non-pai genes, suggesting that sz possess mechanisms for infecting animals and adapting to a wide range of host environments. analysis of the genome identified potential sz virulence genes. genes of the fim iii operon were presumed to be involved in breaking the host-restriction of sz  genome wide comparisons of sz with three other strains and transcriptome analysis revealed novel genes related to bacterial virulence and breaking the host-restriction. four specific pais, which were judged to have been transferred into sz genome through hgt, were identified for the first time. further analysis of the ta and rm systems in the pais will improve our understanding of the pathogenicity of this bacterium and could lead to the development of diagnostics and vaccines. 