BACKGROUND
since its first isolation from mice in  <dig>  efforts have been devoted to the studies of erysipelothrix rhusiopathiae .
e. rhusiopathiae is a gram-positive bacterium that has diverse cell morphology, remarkable chemical tolerance and poorly understood cellular structure and molecular mechanisms, especially those related to its virulence in a wide range of hosts, ranging from mammals and birds to reptiles and fish.
best known as the etiological agent of swine erysipelas– a disease that can cause acute symptoms such as septicaemia or lead to chronic syndromes like arthritis and endocarditis in pigs  <cit> , e. rhusiopathiae is generally believed to be transmitted via the gastrointestinal  tract by intake of contaminated water or food and causes great economical loss in husbandry worldwide.
given its ubiquitous nature in the environment and reservoir of asymptomatic carriers amongst both domestic and wild animals, the prevention and control of swine erysipelas is often challenging.
more than  <dig> serovars have so far been described  <cit> .
however, due to the considerable variance in their morphology, host specificity and/or pathogenicity, the practicality of serotyping remains debatable.
the heat labile capsule of e. rhusiopathiae has been associated with its resistance to phagocytosis and virulence by transposon mutagenesis study  <cit> .
surface proteins such as neuraminidase, spaa antigen, two adhesive surface proteins containing the c-terminal anchoring lpxtg motif  have shown positive correlation to virulence of the bacteria  <cit> – <cit> , however, little is known about the mechanisms of its pathogenicity.
in china, outbreaks of swine erysipelas have surfaced in recent years despite its relative tranquility in the past  <dig> decades, with an alarming trend of developing from scattered, domestic occurrences in a few farms to systemic outbursts in provincial scale.
three representative local reports, which are only available in chinese, are included in additional file  <dig>  in present study, we isolated an outbreak field strain sy <dig> from siyang, jiangsu and completed its bacterial genome sequencing and assembly.
the complete genome of e. rhusiopathiae strain fujisawa has just recently been published  <cit> .
together with the draft genome of strain atcc <dig>  data-mining of potential virulence factors, especially those related to capsular protein biosynthesis, would offer us a more reliable comparative genomic analysis and a better understanding of the common genomic structure and a more precise prediction of potential virulence factors for improvement of vaccine targets or strategy of disease control.
methods
isolation and total dna extraction of e. rhusiopathiae strain sy1027
e. rhusiopathiae strain sy <dig> was collected from a pig farm in siyang, jiangsu.
anticoagulant-treated blood was collected from diseased pigs by in-house veterinarians from pig farms following the nanjing agricultural university animal ethics committee guidelines  and sent to our laboratory.
the sample was inoculated in martin’s broth supplemented with 400 μg/ml neomycin and 70 μg/ml vancomycin.
a single colony was isolated by inoculation of the culture on streaked agar plate supplemented with horse serum.
the single colony of the bacterium was inoculated in modified feist broth , 5 g yeast extract,  <dig>  g l-arginine,  <dig>  ml tween  <dig>  in 1 l of  <dig>  m sodium phosphate buffer   <cit>  supplemented with 5%  fetal bovine serum  and 50 μg/ml kanamycin and grown to its exponential growth phase.
bacteria were harvested by centrifuge and its genomic dna was extracted according to the jgi bacterial dna isolation ctab protocol .
pyrosequencing and complete genome assembly of e. rhusiopathiae strain sy1027
to confirm the purity of the genomic dna of e. rhusiopathiae strain sy <dig>  16s rdna-specific region was amplified and  <dig> individual positive clones were sequenced by genetic analyzer  <dig> .
blastn analysis  <cit>  revealed that e. rhusiopathiae strain sy <dig> gdna sequences have high similarity to those from the other  <dig> strains of e. rhusiopathiae publicly accessible.
the quality and quantity of genomic dna were evaluated by  <dig> % agarose gel electrophoresis and nanodrop <dig> , and using the quant-it picogreen dsdna kit , respectively.
a whole genome shotgun library was generated with 500 ng of e. rhusiopathiae strain sy1027genomic dna.
the shotgun sequencing procedure was performed using  <dig> gs junior general library preparation kit, following the manufacturer’s instruction .
in addition, an 8 kb span paired end library was generated with 15 μg of e. rhusiopathiae strain sy <dig> genomic dna.
the paired end sequencing procedure was performed using  <dig> gs junior paired end library preparation kit, following the manufacturer’s instruction .
paired end reads were used as orientation guide for assembling the contigs into scaffolds.
the dna libraries were amplified by empcr and sequenced by flx titanium sequencing chemistry .
one shotgun run and one paired end run were performed on individual libraries prepared with same genomic dna sample.
after sequencing, the raw data were assembled by newbler  <dig>   with default parameters.
primer pairs were designed along the sequences flanking the gap regions for pcr gap filling.
the complete genome of e. rhusiopathiae strain sy <dig> was submitted to ncbi genbank and is publicly accessible .
genome annotation of e. rhusiopathiae strain sy1027
glimmer  <dig>   <cit>  was used for gene prediction in e. rhusiopathiae strain sy <dig> complete genome.
all predicted orf sequences were translated into amino acid sequences by in-house perl scripts.
blastp  <cit>  was applied to align the amino acid sequences against the ncbi non-redundant  database .
amino acid sequences with alignment length over 90% of its own length and over 40% match identity were chosen and the description of the best hit  was assigned as the annotation of predicted gene.
intergenic regions were annotated by repeatmasker  with default parameters.
for fair comparison, genome annotation was simultaneously conducted using pgaap .
functional gene analysis
blastp  <cit>  was applied to align the amino acid sequences against the cog database  <cit> .
amino acid sequences with alignment length over 90% of its own length and over 20% match identity were chosen and the description of the best hit  was assigned as the annotation of predicted gene.
all annotated genes were then classified based on their cog classes and compared to that of e. rhusiopathiae strain fujisawa.
pathway analysis
glimmer-predicted orf sequences of e. rhusiopathiae strain sy1027were translated into amino acid sequences by in-house perl scripts.
all sequences were submitted to kegg database  <cit>  for automatic pathway annotation  and then manually downloaded and curated by in-house perl scripts.
virulence gene analysis
blastp  <cit>  was applied to align the amino acid sequences against the vfdb database  <cit> .
amino acid sequences with alignment length over 90% of its own length and over 20% match identity were chosen and the description of the best hit  was assigned as the annotation of predicted gene.
genomic island  and pathogenicity island  analyses
genomic islands and pathogenicity islands were annotated using islandviewer   <cit>  and pai finder  on paidb  <cit>  respectively.
drug resistant gene analysis
blastp  <cit>  was applied to align the amino acid sequences against the ardb database  <cit> .
amino acid sequences with alignment length over 90% of its own length and over 40% match identity were chosen and the description of the best hit  was assigned as the annotation of predicted gene.
all annotated genes were designated by the antibiotics to which they render the bacteria resistant and compared to that of e. rhusiopathiae strains sy <dig>
annotation of plasmid/phage/prophage-derived genes using aclame database
blastp  <cit>  was applied to align the amino acid sequences against the aclame database  <cit> .
amino acid sequences with alignment length over 90% of its own length and over 40% match identity were chosen and the description of the best hit  was assigned as the annotation of predicted gene.
all annotated genes were classified according to their corresponding potential horizontal transferring vectors  and then compared to that identified in strain fujisawa.
comparative genomic analysis of e. rhusiopathiae strain sy <dig> and strain fujisawa
complete genomes of e. rhusiopathiae strain fujisawa  was downloaded from ncbi genbank.
orthologous genes were identified by reciprocal blat  <cit>  using glimmer-predicted e. rhusiopathiae strains sy <dig> and fujisawa genes.
predicted genes of e. rhusiopathiae strain sy <dig> which are found as single copies and with 90% minimum alignment length in strain fujisawa were designated as the core genes.
RESULTS
sequencing and assembly of erysipelothrix rhusiopathiae strain sy <dig> complete genome
erysipelothrix rhusiopathiae strain sy <dig> genome was sequenced and its complete de novo assembly was achieved by one shotgun run and one 8 kb-span paired end run followed by pcr gap filling.
a total of  <dig>  raw shotgun reads  and  <dig>  raw paired end reads  were generated, ~ <dig> % and ~ <dig> % of them, respectively, were aligned into  <dig> contigs and  <dig> scaffolds, yielding an average sequencing depth of ~23-fold.
average read lengths for the shotgun run and the paired end run are ~400 bp and ~300 bp, respectively.
the largest scaffold is  <dig> , <dig> base pairs  in size and contains  <dig> large contigs, while that of the n <dig> contig is  <dig>  bp, indicating that the raw assembly is highly continuous.
after pcr gap filling by sanger sequencing, the complete circular genome of e. rhusiopathiae strain sy <dig> is  <dig>  mb  with  <dig> % gc content, which is just ~30 kb smaller than and share the same gc content to that in strain fujisawa .
e. rhusiopathiae
strains sy <dig>  fujisawa and atcc <dig> genomes
e. rhusiopathiae
footnotes: #denotes the approximate number according to public database; ^denotes number of genes in percentage to total number of predicted open-reading frames by glimmer  <dig> ; *denotes length of sequence in percentage to total genome length; db – abbreviation for database; seq– abbreviation for sequence; n.a.
– non-applicable as strain atcc <dig> genome is only a draft assembly.
genome annotation of e. rhusiopathiae strain sy1027
<dig>  open reading frames  were predicted by glimmer version  <dig> , and  <dig>  of them  were annotated by blastp search against the ncbi non-redundant  database .
the full annotation result was attached as additional file  <dig>  simultaneous annotation using pgaap yielded  <dig>  cds .both numbers of annotated cds were similar to that in strain fujisawa .
<dig> rrna and  <dig> trna were identified via pgaap.
majority of them were arranged as large rna islands–  <dig> ribosomal rna  operons  and  <dig> trna islands .
the rrn operons are arranged in the typical order of 16s, 23s and 5s rrna genes, with the exception of rrn operon  <dig> which is composed of an additional copy of 5s rrna gene .
in comparison to strain fujisawa , a considerably fewer number of  <dig> rrn operons are present in strain sy <dig> genome .
the loci of pgaap-annotated cds, ncbi nr annotated cds and various rna islands were labeled in the circular representation of the sy <dig> genome .
comparative genomic analysis of e. rhusiopathiae strain sy1027
<dig>  core genes  were identified in the pan-genome  of the three e. rhusiopathiae strains .
<dig> genes are shared only between strains sy <dig> and fujisawa but not atcc <dig>  and only  <dig> between strains sy <dig> and atcc <dig>  whilst a far greater number of  <dig> genes are shared between strains fujisawa and atcc <dig>  similarly, a greater number of  <dig> strain-specific genes were identified in strain sy <dig>  in comparison to  <dig> in strain fujisawa and  <dig> in strain atcc <dig>  these figures imply that the strains fujisawa and atcc <dig> may be more similar to each other than to our strain sy <dig>  despite preliminary serotyping results that strain sy <dig> is of serotype 1a, same with the other two strains .synteny between the two complete genomes of strains sy <dig> and fujisawa is generally conserved, apart from a rearrangement observed at the ~850-875 kb region on strain sy <dig>  and an unmatched stretch between the two strains which is marked by a red box in figure  <dig>
as suggested by a previous phylogenetic analysis after completion of strain fujisawa genome sequencing  <cit> , erysipelothrix spp.
form a distinct secluded group sandwiched between the firmicutes and mollicutes.
since neither draft nor complete genomes of closely related species e.g.
e. tonsillarum and e. inopinata were available on genbank, while those less closely related ones have already been explored in the previous article, hence phylogenetic tree was not constructed in our present study.
cog annotation and comparative analysis of e. rhusiopathiae strain sy <dig> with strains fujisawa and atcc19414
<dig>   of all  <dig>  predicted orf were annotated by blastp search against ncbi cog database.
the top ten cog classes they were annotated in were  general function prediction only ,  translation, ribosomal structure and biogenesis ,  carbohydrate transport and metabolism ,  replication, recombination and repair ,  function unknown ,  transcription ,  inorganic ion transport and metabolism ,  amino acid transport and metabolism ,  defense mechanisms , and  cell wall/membrane/envelope biogenesis .
as expected, majority of the genes were involved in basic cellular functions, such as replication, transcription, translation and metabolism, however, up to ~ <dig> % of them only have predicted or unknown functions on cog database.
cog class distribution of strain sy <dig> was illustrated .
numbers and percentage of each cog class were tabulated as additional file  <dig> and their detailed annotations attached as additional file  <dig>
similar numbers of cog-annotated genes were found in strains fujisawa  and atcc  <dig>  .
percentage of gene annotation in atcc  <dig> is possibly higher due to incomplete sequencing coverage and assembly.
cog class distributions are highly similar between strains sy <dig> and fujisawa .
virulence gene/pathogenicity island-like gene annotation and comparative analysis of e. rhusiopathiae strain sy <dig> and with strains fujisawa and atcc19414
<dig> potential virulence genes  were identified by blastp search against vfdb.
categorized by their respective cog classes, their accession numbers and descriptions are listed in additional file  <dig>  many of these potential virulence genes are grouped under or partially involved in cell wall/membrane/envelope biogenesis  or inorganic ion transport and metabolism .
the former mainly includes enzymes or proteins involved in capsular polysaccharide and glycoprotein biosynthesis, and the latter include transporters for iron and magnesium uptake and manganese-dependent superoxide dismutase.
metal ions are scarce and limited in biological systems and their uptake by bacteria upon invasion of various host cells have shown positive correlation to bacterial virulence  <cit> – <cit> .
however, their functions and possible relevance to pathogenicity in e. rhusiopathiae remain unresolved.
a modestly higher number of virulence genes were identified in strain sy <dig> than in strain fujisawa in vfdb, while the number in strain atcc <dig> might be overestimated due to its draft genome nature .
<dig> genomic island-like regions were annotated via island viewer, while  <dig> pathogenicity island -like islands were identified in strain sy <dig> via pai finder in paidb .
in comparison to the search result in vfdb, only one pai-virulence gene  was annotated in pai-l <dig>  it may suggest strain sy <dig> genome has pai-like islands atypical of that found in strain fujisawa and the present entries in the paidb.
though groel is a chaperonin widely conserved in bacteria, its association to cell adherence and virulence has been described in clostridium spp.
and other bacteria  <cit> .
suggestion of a pai-island with groel in our strain may warrant further studies in elucidating its contribution to pathogenicity, as it may offer interesting insights into potential antigenic targets for vaccine development in e. rhusiopathiae.
in addition to the difference in gc content to the rest of the genome, trna were found to flank gi- <dig>  pai-l <dig> and pai-l <dig>  providing further support to these putative genomic islands .
nevertheless, the virulence of these putative regions in e. rhusiopathiae needs further elucidation, preferably by gene-knockout experiments.
since atcc <dig> genome was only completed to the level of scaffolds, search for gi/pai-like islands was unfeasible.
e. rhusiopathiae
strain sy <dig> genome
footnote: genomic island-like regions were predicted by islandpath-dimob on islandviewer   <cit> .
n.f.- not found.
e. rhusiopathiae
strain sy <dig> genome
ф
footnote: pathogenicity island-like regions were predicted by pai finder  on paidb  <cit> .
^representative pais from paidb hits; Фas annotated by vfdb; *homo.
pai – abbreviation for homologous pai; n.f.- not found.
apart from the public databases, a list of potential virulence factors previously suggested in e. rhusiopathiae were identified in the strain sy <dig> genome, and they span across various categories, including surface proteins, antioxidant proteins, phospholipases, hemolysins, capsular polysaccharide biosynthesis and extracellular proteins/enzymes .
surface proteins were annotated based on their lpxtg motifs, which act as sortase recognition sequence for the covalent binding of their carboxyl termini by sortases in gram-positive bacteria  <cit> , or gw  repeats for non-covalent interactions as observed in e. rhusiopathiae <cit> .
these include spaa antigen which is commonly used as vaccine target  <cit> , hyaluronidases and neuraminidases which may promote bacterial-host cell surface association  <cit> , rspa and rspb proteins which bind fibronectin and collagen i and ii and may participate in biofilm formation  <cit> .
interestingly, a premature stop codon  was found near the middle of putative rspa gene , leading to the annotation of  <dig> rspa-like proteins, each representing the n-terminally and c-terminally truncated proteins in strain sy <dig> genome , instead of  <dig> in strain fujisawa.
though our preliminary results suggest that these truncated proteins do not influence the serotyping of strain sy <dig> , their conformation, collagen binding capability and influence on host-bacterial interaction may need further elucidation.
e. rhusiopathiae was hypothesized to have an atypical capsule that renders it escape from phagocytosis and antioxidant proteins that facilitates its survival inside polymorphonuclear leukocytes and macrophages  <cit> .
transposon mutagenesis study showed that the loss of a cluster of genes, putatively a polycistronic mrna-coding operon, in strain fujisawa created a mutant deprived of capsule and pathogenicity  <cit> .
the operon was identified, including all  <dig> proteins potentially involved in capsular polysaccharide biosynthesis, in strain sy <dig>  similarly, superoxide dismutase, thioredoxins, thiol peroxidase, alkyl-hydroperoxide reductases and other proteins which confer resistance to oxidative stress, were also found in strain sy <dig>
phospholipases are often considered as potential virulence factors for intracellular pathogens, hypothesized via acquisition of host membrane lipids and disruption of phagosomal membrane.
in strain sy <dig> genome,  <dig> phospholipases were identified, same as in strain fujisawa .
as e. rhusiopathiae is highly tolerant and almost ubiquitous in nature, two-component regulatory systems which often regulate responses to changes in the environment were also searched for in strain sy <dig> genome.
<dig> pairs of histidine kinase-response regulator were identified–  <dig> shown similarity to phor-phob two-component regulatory system and the remaining one to agrc-agra two-component regulatory system .
e. rhusiopathiae
strain sy <dig> genome
similar to agrc-agra  two-component regulatory system
similar to phor-phob  two-component regulatory system
the highly potential virulence factors annotated both via vfdb and literature search - hlyb, cap5e, cap5f, cap5g, prra, soda and virr  – may require further elucidation on their correlation to pathogenicity, especially in intracellular survival of e. rhusiopathiae.
potential drug resistance gene in e. rhusiopathiae strain sy1027
<dig> potential drug resistance genes were annotated in e. rhusiopathiae strain sy <dig>  suggesting it may be resistant to macrolides, vancomycin and teicoplanin .
their orthologs were also found in strain fujisawa, sharing ~98-100% match id .
interestingly, a richer reservoir of potential drug resistance genes, both in number and variety, was annotated in strain atcc <dig> .
our preliminary antimicrobial results suggested that despite the presence of potential macrolide-resistant genes , the antimicrobial susceptibilities to different macrolide-class drugs  vary from moderate to minimal levels , hinting that further functional elucidation, preferably gene-knockout experiments, of the potential drug resistance genes are required to substantiate the in silico deduction.
in addition, a previous investigation on antimicrobial susceptibilities of e. rhusiopathiae suggested that it has partial or complete resistance to other classes of antibiotics  <cit>  which were not identified in our present search, hinting the presence of other potential drug resistance genes, either novel or unlisted in the ardb, in the bacteria.
e. rhusiopathiae
strains sy <dig>  fujisawa and atcc <dig> genomes
e. rhusiopathiae
e. rhusiopathiae
e. rhusiopathiae
potential horizontal transferring elements in e. rhusiopathiae strain sy1027
horizontal transferring elements are common in bacteria and generally believed to be a significant driving force in prokaryotic evolution  <cit> .
they are mainly divided into three types based on their respective vectors – plasmids, phages  and prophages  <cit> .
in strain sy <dig> genome,  <dig> potential horizontal transferring elements  were annotated via blastp against the aclame database .
amongst them, the majority  were putatively derived from plasmids, while the rest were from phages  and from prophages .
similar figures were observed in strain fujisawa genome, with a slightly fewer number of putative plasmid-derived genes and doubled number of putative phage-derived genes.
<dig> potential horizontal transferring elements were found in the draft genome of strain atcc <dig>  yet it might be an over-estimated figure due to incomplete or possible mis-assembly.
as observed in figure  <dig>  some of the potential virulence factors identified might have originated from horizontal gene transfer.
two of them –prra and soda–have high virulence potential owing to their identification via both vfdb and literature search.
the former is the transcriptional regulator in a prra-prrb two-component regulatory system related to oxygen control and intracellular replication in mycobacterium <cit> , while the latter encodes a superoxide dismutase which may offer a mean of antioxidant defense which is critical for intracellular survival and growth in e. rhusiopathiae <cit> .
kegg pathway analysis of e. rhusiopathiae strain sy1027
kegg pathway analysis was performed for e. rhusiopathiae strain sy <dig>  and the full list of kegg pathway annotation was listed in additional file  <dig>  strain sy <dig> genome contains glucose-specific iia component in phosphotransferase system , endoglucanase and glucose-6-phosphate isomerase, which suggest that the bacteria is capable in breaking down maltose and trehalose, cellulose and fructose, respectively.
the potential of utilizing other sugars besides glucose may partly explain the ubiquitous nature of e. rhusiopathiae in the environment.
similar to strain fujisawa, many critical genes in the tricarboxylic acid  cycle were found missing in strain sy <dig>  with the exception of fumarate hydratase, citrate coa-transferase, dihydrolipoamidesuccinyltransferase and dihydrolipoamide dehydrogenase.
genome reduction was previously hypothesized in e. rhusiopathiae strain fujisawa, given the partial loss of fatty acid biosynthesis and dna repair system similar in the mollicutes  <cit> .
likewise, strain sy <dig> seems to display these characteristics.
apart from the partial loss of tca cycle genes, only  <dig> genes related to dna repair were identified.
CONCLUSIONS
the complete genome of erysipelothrix rhusiopathiae strain sy <dig> was sequenced and assembled.
it is  <dig> , <dig> base pairs long, just  <dig> kilobases smaller than strain fujisawa, with the same gc level of  <dig> %.
it contains  <dig> open reading frames  predicted by glimmer  <dig> , of which  <dig>  are annotated by ncbi nr blast,  <dig> by cog database and  <dig> by kegg database.
<dig> potential virulence factors are annotated in strain sy <dig> by vfdb, while  <dig>  of them are common in the  <dig> strains,  <dig> of which are potentially related to antibiotic resistance and highly conserved  amongst the three strains of e. rhusiopathiae and only modestly homologous to other gastrointestinal tract-inhabiting firmicutes , e.g.
clostridium spp., enterococcus spp.
molecular identification of these genomic elements and potential virulence factors offer insights into testing prospective vaccine targets besides the widely used spaa antigens in swine erysipelas and development of more effective treatment or prevention in control of the disease.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
ahyk prepared the genomic dna for next-generation sequencing , post-ngs sequence alignment, genome assembly and annotation and comparative genomic analyses, and the drafting of the manuscript.
yl and pj isolated the bacteria from field sample and participated in design of study and coordination.
jj helped in writing in-house perl script and subsequent bioinformatics analyses.
fcl participated in the design of study and coordination.
all authors read and approved the final manuscript.
supplementary material
additional file 1
local chinese swine erysipelas outbreak reports.
three articles  on local swine erysipelas outbreaks published in chinese scientific journals.
click here for file
additional file 2
ncbi nr database annotation for
e. rhusiopathiae
strain.
the full list of orfs in e. rhusiopathiae strain sy <dig> annotated against ncbi non-redundant database.
click here for file
additional file 3
cog-annotated genes in
e. rhusiopathiae
strain sy <dig> genome.
tabulated summary of orfs in e. rhusiopathiae strain sy <dig> annotated against cog database under various cog classes.
click here for file
additional file 4
cog functional annotation for
e. rhusiopathiae
strain sy <dig> genome.
the full list of orfs in e. rhusiopathiae strain sy <dig> annotated against cog database.
click here for file
additional file 5
cog class annotation distribution between
e. rhusiopathiae
strains sy <dig> and fujisawa genomes.
a bar chart comparing the percentage of each single-letter cog class found between the  <dig> complete e. rhusiopathiae genomes.
click here for file
additional file 6
comparison of cog-annotated genes between
e. rhusiopathiae
strains sy <dig> and fujisawa.
tabulated comparison between cog-annotated genes under each single-letter cog class from the  <dig> complete e. rhusiopathiae genomes.
click here for file
additional file 7
vfdb-annotated genes between
e. rhusiopathiae
strain sy <dig> genome.
the list of vfdb-annotated genes in strain sy <dig> genome, classified by cog classes.
genes shared between strains sy <dig>  fujisawa and atcc <dig> were shaded in light gray while those shared between strains sy <dig> and fujisawa were highlighted in dark gray.
click here for file
additional file 8
other potential virulence factors in
e. rhusiopathiae
strain sy <dig> genome.
a panel of virulence factors mentioned in the literature was searched in strain sy <dig> genome and listed here.
^orf <dig>  orf <dig> and orf <dig> are pseudogenes with frame-shift or point mutation.
click here for file
additional file 9
ardb-annotated genes in
e. rhusiopathiae
strain sy <dig> genome.
list of ardb-annotated genes and their putative antibiotic resistance.
click here for file
additional file 10
aclame database-annotated genes in
e. rhusiopathiae
strain sy <dig> genome.
full list of orf annotated via aclame database in strain sy <dig> genome.
click here for file
additional file 11
kegg database-annotated genes in
e. rhusiopathiae
strain sy <dig> genome.
full list of kegg database-annotated genes in strain sy <dig> genome.
click here for file
acknowledgements
the authors would like to thank raymond k. h. hui for handling roche gs junior runs for this study.

 since its first isolation from mice in efforts have been devoted to the studies of erysipelothrix rhusiopathiae. e. rhusiopathiae is a gram-positive bacterium that has diverse cell morphology, remarkable chemical tolerance and poorly understood cellular structure and molecular mechanisms, especially those related to its virulence in a wide range of hosts, ranging from mammals and birds to reptiles and fish. best known as the etiological agent of swine erysipelas– a disease that can cause acute symptoms such as septicaemia or lead to chronic syndromes like arthritis and endocarditis in pigs, e. rhusiopathiae is generally believed to be transmitted via the gastrointestinal tract by intake of contaminated water or food and causes great economical loss in husbandry worldwide. given its ubiquitous nature in the environment and reservoir of asymptomatic carriers amongst both domestic and wild animals, the prevention and control of swine erysipelas is often challenging. more than serovars have so far been described. however, due to the considerable variance in their morphology, host specificity and/or pathogenicity, the practicality of serotyping remains debatable. the heat labile capsule of e. rhusiopathiae has been associated with its resistance to phagocytosis and virulence by transposon mutagenesis study. surface proteins such as neuraminidase, spaa antigen, two adhesive surface proteins containing the c-terminal anchoring lpxtg motif have shown positive correlation to virulence of the bacteria –, however, little is known about the mechanisms of its pathogenicity. in china, outbreaks of swine erysipelas have surfaced in recent years despite its relative tranquility in the past decades, with an alarming trend of developing from scattered, domestic occurrences in a few farms to systemic outbursts in provincial scale. three representative local reports, which are only available in chinese, are included in additional file in present study, we isolated an outbreak field strain sy from siyang, jiangsu and completed its bacterial genome sequencing and assembly. the complete genome of e. rhusiopathiae strain fujisawa has just recently been published. together with the draft genome of strain atcc data-mining of potential virulence factors, especially those related to capsular protein biosynthesis, would offer us a more reliable comparative genomic analysis and a better understanding of the common genomic structure and a more precise prediction of potential virulence factors for improvement of vaccine targets or strategy of disease control. methods isolation and total dna extraction of e. rhusiopathiae strain sy1027 e. rhusiopathiae strain sy was collected from a pig farm in siyang, jiangsu. anticoagulant-treated blood was collected from diseased pigs by in-house veterinarians from pig farms following the nanjing agricultural university animal ethics committee guidelines and sent to our laboratory. the sample was inoculated in martin’s broth supplemented with 400 μg/ml neomycin and 70 μg/ml vancomycin. a single colony was isolated by inoculation of the culture on streaked agar plate supplemented with horse serum. the single colony of the bacterium was inoculated in modified feist broth, 5 g yeast extract,  g l-arginine,  ml tween in 1 l of  m sodium phosphate buffer supplemented with 5% fetal bovine serum and 50 μg/ml kanamycin and grown to its exponential growth phase. bacteria were harvested by centrifuge and its genomic dna was extracted according to the jgi bacterial dna isolation ctab protocol. pyrosequencing and complete genome assembly of e. rhusiopathiae strain sy1027 to confirm the purity of the genomic dna of e. rhusiopathiae strain sy 16s rdna-specific region was amplified and individual positive clones were sequenced by genetic analyzer. blastn analysis revealed that e. rhusiopathiae strain sy gdna sequences have high similarity to those from the other strains of e. rhusiopathiae publicly accessible. the quality and quantity of genomic dna were evaluated by % agarose gel electrophoresis and nanodrop, and using the quant-it picogreen dsdna kit, respectively. a whole genome shotgun library was generated with 500 ng of e. rhusiopathiae strain sy1027genomic dna. the shotgun sequencing procedure was performed using gs junior general library preparation kit, following the manufacturer’s instruction. in addition, an 8 kb span paired end library was generated with 15 μg of e. rhusiopathiae strain sy genomic dna. the paired end sequencing procedure was performed using gs junior paired end library preparation kit, following the manufacturer’s instruction. paired end reads were used as orientation guide for assembling the contigs into scaffolds. the dna libraries were amplified by empcr and sequenced by flx titanium sequencing chemistry. one shotgun run and one paired end run were performed on individual libraries prepared with same genomic dna sample. after sequencing, the raw data were assembled by newbler with default parameters. primer pairs were designed along the sequences flanking the gap regions for pcr gap filling. the complete genome of e. rhusiopathiae strain sy was submitted to ncbi genbank and is publicly accessible. genome annotation of e. rhusiopathiae strain sy1027 glimmer was used for gene prediction in e. rhusiopathiae strain sy complete genome. all predicted orf sequences were translated into amino acid sequences by in-house perl scripts. blastp was applied to align the amino acid sequences against the ncbi non-redundant database. amino acid sequences with alignment length over 90% of its own length and over 40% match identity were chosen and the description of the best hit was assigned as the annotation of predicted gene. intergenic regions were annotated by repeatmasker with default parameters. for fair comparison, genome annotation was simultaneously conducted using pgaap. functional gene analysis blastp was applied to align the amino acid sequences against the cog database. amino acid sequences with alignment length over 90% of its own length and over 20% match identity were chosen and the description of the best hit was assigned as the annotation of predicted gene. all annotated genes were then classified based on their cog classes and compared to that of e. rhusiopathiae strain fujisawa. pathway analysis glimmer-predicted orf sequences of e. rhusiopathiae strain sy1027were translated into amino acid sequences by in-house perl scripts. all sequences were submitted to kegg database for automatic pathway annotation and then manually downloaded and curated by in-house perl scripts. virulence gene analysis blastp was applied to align the amino acid sequences against the vfdb database. amino acid sequences with alignment length over 90% of its own length and over 20% match identity were chosen and the description of the best hit was assigned as the annotation of predicted gene. genomic island and pathogenicity island analyses genomic islands and pathogenicity islands were annotated using islandviewer and pai finder on paidb respectively. drug resistant gene analysis blastp was applied to align the amino acid sequences against the ardb database. amino acid sequences with alignment length over 90% of its own length and over 40% match identity were chosen and the description of the best hit was assigned as the annotation of predicted gene. all annotated genes were designated by the antibiotics to which they render the bacteria resistant and compared to that of e. rhusiopathiae strains sy annotation of plasmid/phage/prophage-derived genes using aclame database blastp was applied to align the amino acid sequences against the aclame database. amino acid sequences with alignment length over 90% of its own length and over 40% match identity were chosen and the description of the best hit was assigned as the annotation of predicted gene. all annotated genes were classified according to their corresponding potential horizontal transferring vectors and then compared to that identified in strain fujisawa. comparative genomic analysis of e. rhusiopathiae strain sy and strain fujisawa complete genomes of e. rhusiopathiae strain fujisawa was downloaded from ncbi genbank. orthologous genes were identified by reciprocal blat using glimmer-predicted e. rhusiopathiae strains sy and fujisawa genes. predicted genes of e. rhusiopathiae strain sy which are found as single copies and with 90% minimum alignment length in strain fujisawa were designated as the core genes.  sequencing and assembly of erysipelothrix rhusiopathiae strain sy complete genome erysipelothrix rhusiopathiae strain sy genome was sequenced and its complete de novo assembly was achieved by one shotgun run and one 8 kb-span paired end run followed by pcr gap filling. a total of raw shotgun reads and raw paired end reads were generated, ~ % and ~ % of them, respectively, were aligned into contigs and scaffolds, yielding an average sequencing depth of ~23-fold. average read lengths for the shotgun run and the paired end run are ~400 bp and ~300 bp, respectively. the largest scaffold is, base pairs in size and contains large contigs, while that of the n contig is  bp, indicating that the raw assembly is highly continuous. after pcr gap filling by sanger sequencing, the complete circular genome of e. rhusiopathiae strain sy is  mb with % gc content, which is just ~30 kb smaller than and share the same gc content to that in strain fujisawa. e. rhusiopathiae strains sy fujisawa and atcc genomes e. rhusiopathiae footnotes: #denotes the approximate number according to public database; ^denotes number of genes in percentage to total number of predicted open-reading frames by glimmer; *denotes length of sequence in percentage to total genome length; db – abbreviation for database; seq– abbreviation for sequence; n.a. – non-applicable as strain atcc genome is only a draft assembly. genome annotation of e. rhusiopathiae strain sy1027 open reading frames were predicted by glimmer version, and of them were annotated by blastp search against the ncbi non-redundant database. the full annotation result was attached as additional file simultaneous annotation using pgaap yielded cds.both numbers of annotated cds were similar to that in strain fujisawa. rrna and trna were identified via pgaap. majority of them were arranged as large rna islands– ribosomal rna operons and trna islands. the rrn operons are arranged in the typical order of 16s, 23s and 5s rrna genes, with the exception of rrn operon which is composed of an additional copy of 5s rrna gene. in comparison to strain fujisawa, a considerably fewer number of rrn operons are present in strain sy genome. the loci of pgaap-annotated cds, ncbi nr annotated cds and various rna islands were labeled in the circular representation of the sy genome. comparative genomic analysis of e. rhusiopathiae strain sy1027 core genes were identified in the pan-genome of the three e. rhusiopathiae strains. genes are shared only between strains sy and fujisawa but not atcc and only between strains sy and atcc whilst a far greater number of genes are shared between strains fujisawa and atcc similarly, a greater number of strain-specific genes were identified in strain sy in comparison to in strain fujisawa and in strain atcc these figures imply that the strains fujisawa and atcc may be more similar to each other than to our strain sy despite preliminary serotyping results that strain sy is of serotype 1a, same with the other two strains.synteny between the two complete genomes of strains sy and fujisawa is generally conserved, apart from a rearrangement observed at the ~850-875 kb region on strain sy and an unmatched stretch between the two strains which is marked by a red box in figure  as suggested by a previous phylogenetic analysis after completion of strain fujisawa genome sequencing, erysipelothrix spp. form a distinct secluded group sandwiched between the firmicutes and mollicutes. since neither draft nor complete genomes of closely related species e.g. e. tonsillarum and e. inopinata were available on genbank, while those less closely related ones have already been explored in the previous article, hence phylogenetic tree was not constructed in our present study. cog annotation and comparative analysis of e. rhusiopathiae strain sy with strains fujisawa and atcc19414 of all predicted orf were annotated by blastp search against ncbi cog database. the top ten cog classes they were annotated in were general function prediction only, translation, ribosomal structure and biogenesis, carbohydrate transport and metabolism, replication, recombination and repair, function unknown, transcription, inorganic ion transport and metabolism, amino acid transport and metabolism, defense mechanisms, and cell wall/membrane/envelope biogenesis. as expected, majority of the genes were involved in basic cellular functions, such as replication, transcription, translation and metabolism, however, up to ~ % of them only have predicted or unknown functions on cog database. cog class distribution of strain sy was illustrated. numbers and percentage of each cog class were tabulated as additional file and their detailed annotations attached as additional file similar numbers of cog-annotated genes were found in strains fujisawa and atcc. percentage of gene annotation in atcc is possibly higher due to incomplete sequencing coverage and assembly. cog class distributions are highly similar between strains sy and fujisawa. virulence gene/pathogenicity island-like gene annotation and comparative analysis of e. rhusiopathiae strain sy and with strains fujisawa and atcc19414 potential virulence genes were identified by blastp search against vfdb. categorized by their respective cog classes, their accession numbers and descriptions are listed in additional file many of these potential virulence genes are grouped under or partially involved in cell wall/membrane/envelope biogenesis or inorganic ion transport and metabolism. the former mainly includes enzymes or proteins involved in capsular polysaccharide and glycoprotein biosynthesis, and the latter include transporters for iron and magnesium uptake and manganese-dependent superoxide dismutase. metal ions are scarce and limited in biological systems and their uptake by bacteria upon invasion of various host cells have shown positive correlation to bacterial virulence –. however, their functions and possible relevance to pathogenicity in e. rhusiopathiae remain unresolved. a modestly higher number of virulence genes were identified in strain sy than in strain fujisawa in vfdb, while the number in strain atcc might be overestimated due to its draft genome nature. genomic island-like regions were annotated via island viewer, while pathogenicity island -like islands were identified in strain sy via pai finder in paidb. in comparison to the search result in vfdb, only one pai-virulence gene was annotated in pai-l it may suggest strain sy genome has pai-like islands atypical of that found in strain fujisawa and the present entries in the paidb. though groel is a chaperonin widely conserved in bacteria, its association to cell adherence and virulence has been described in clostridium spp. and other bacteria. suggestion of a pai-island with groel in our strain may warrant further studies in elucidating its contribution to pathogenicity, as it may offer interesting insights into potential antigenic targets for vaccine development in e. rhusiopathiae. in addition to the difference in gc content to the rest of the genome, trna were found to flank gi- pai-l and pai-l providing further support to these putative genomic islands. nevertheless, the virulence of these putative regions in e. rhusiopathiae needs further elucidation, preferably by gene-knockout experiments. since atcc genome was only completed to the level of scaffolds, search for gi/pai-like islands was unfeasible. e. rhusiopathiae strain sy genome footnote: genomic island-like regions were predicted by islandpath-dimob on islandviewer. n.f.- not found. e. rhusiopathiae strain sy genome ф footnote: pathogenicity island-like regions were predicted by pai finder on paidb. ^representative pais from paidb hits; Фas annotated by vfdb; *homo. pai – abbreviation for homologous pai; n.f.- not found. apart from the public databases, a list of potential virulence factors previously suggested in e. rhusiopathiae were identified in the strain sy genome, and they span across various categories, including surface proteins, antioxidant proteins, phospholipases, hemolysins, capsular polysaccharide biosynthesis and extracellular proteins/enzymes. surface proteins were annotated based on their lpxtg motifs, which act as sortase recognition sequence for the covalent binding of their carboxyl termini by sortases in gram-positive bacteria, or gw repeats for non-covalent interactions as observed in e. rhusiopathiae. these include spaa antigen which is commonly used as vaccine target, hyaluronidases and neuraminidases which may promote bacterial-host cell surface association, rspa and rspb proteins which bind fibronectin and collagen i and ii and may participate in biofilm formation. interestingly, a premature stop codon was found near the middle of putative rspa gene, leading to the annotation of rspa-like proteins, each representing the n-terminally and c-terminally truncated proteins in strain sy genome, instead of in strain fujisawa. though our preliminary results suggest that these truncated proteins do not influence the serotyping of strain sy, their conformation, collagen binding capability and influence on host-bacterial interaction may need further elucidation. e. rhusiopathiae was hypothesized to have an atypical capsule that renders it escape from phagocytosis and antioxidant proteins that facilitates its survival inside polymorphonuclear leukocytes and macrophages. transposon mutagenesis study showed that the loss of a cluster of genes, putatively a polycistronic mrna-coding operon, in strain fujisawa created a mutant deprived of capsule and pathogenicity. the operon was identified, including all proteins potentially involved in capsular polysaccharide biosynthesis, in strain sy similarly, superoxide dismutase, thioredoxins, thiol peroxidase, alkyl-hydroperoxide reductases and other proteins which confer resistance to oxidative stress, were also found in strain sy phospholipases are often considered as potential virulence factors for intracellular pathogens, hypothesized via acquisition of host membrane lipids and disruption of phagosomal membrane. in strain sy genome, phospholipases were identified, same as in strain fujisawa. as e. rhusiopathiae is highly tolerant and almost ubiquitous in nature, two-component regulatory systems which often regulate responses to changes in the environment were also searched for in strain sy genome. pairs of histidine kinase-response regulator were identified– shown similarity to phor-phob two-component regulatory system and the remaining one to agrc-agra two-component regulatory system. e. rhusiopathiae strain sy genome similar to agrc-agra two-component regulatory system similar to phor-phob two-component regulatory system the highly potential virulence factors annotated both via vfdb and literature search - hlyb, cap5e, cap5f, cap5g, prra, soda and virr – may require further elucidation on their correlation to pathogenicity, especially in intracellular survival of e. rhusiopathiae. potential drug resistance gene in e. rhusiopathiae strain sy1027 potential drug resistance genes were annotated in e. rhusiopathiae strain sy suggesting it may be resistant to macrolides, vancomycin and teicoplanin. their orthologs were also found in strain fujisawa, sharing ~98-100% match id. interestingly, a richer reservoir of potential drug resistance genes, both in number and variety, was annotated in strain atcc. our preliminary antimicrobial results suggested that despite the presence of potential macrolide-resistant genes, the antimicrobial susceptibilities to different macrolide-class drugs vary from moderate to minimal levels, hinting that further functional elucidation, preferably gene-knockout experiments, of the potential drug resistance genes are required to substantiate the in silico deduction. in addition, a previous investigation on antimicrobial susceptibilities of e. rhusiopathiae suggested that it has partial or complete resistance to other classes of antibiotics which were not identified in our present search, hinting the presence of other potential drug resistance genes, either novel or unlisted in the ardb, in the bacteria. e. rhusiopathiae strains sy fujisawa and atcc genomes e. rhusiopathiae e. rhusiopathiae e. rhusiopathiae potential horizontal transferring elements in e. rhusiopathiae strain sy1027 horizontal transferring elements are common in bacteria and generally believed to be a significant driving force in prokaryotic evolution. they are mainly divided into three types based on their respective vectors – plasmids, phages and prophages. in strain sy genome, potential horizontal transferring elements were annotated via blastp against the aclame database. amongst them, the majority were putatively derived from plasmids, while the rest were from phages and from prophages. similar figures were observed in strain fujisawa genome, with a slightly fewer number of putative plasmid-derived genes and doubled number of putative phage-derived genes. potential horizontal transferring elements were found in the draft genome of strain atcc yet it might be an over-estimated figure due to incomplete or possible mis-assembly. as observed in figure  some of the potential virulence factors identified might have originated from horizontal gene transfer. two of them –prra and soda–have high virulence potential owing to their identification via both vfdb and literature search. the former is the transcriptional regulator in a prra-prrb two-component regulatory system related to oxygen control and intracellular replication in mycobacterium, while the latter encodes a superoxide dismutase which may offer a mean of antioxidant defense which is critical for intracellular survival and growth in e. rhusiopathiae. kegg pathway analysis of e. rhusiopathiae strain sy1027 kegg pathway analysis was performed for e. rhusiopathiae strain sy and the full list of kegg pathway annotation was listed in additional file strain sy genome contains glucose-specific iia component in phosphotransferase system, endoglucanase and glucose-6-phosphate isomerase, which suggest that the bacteria is capable in breaking down maltose and trehalose, cellulose and fructose, respectively. the potential of utilizing other sugars besides glucose may partly explain the ubiquitous nature of e. rhusiopathiae in the environment. similar to strain fujisawa, many critical genes in the tricarboxylic acid cycle were found missing in strain sy with the exception of fumarate hydratase, citrate coa-transferase, dihydrolipoamidesuccinyltransferase and dihydrolipoamide dehydrogenase. genome reduction was previously hypothesized in e. rhusiopathiae strain fujisawa, given the partial loss of fatty acid biosynthesis and dna repair system similar in the mollicutes. likewise, strain sy seems to display these characteristics. apart from the partial loss of tca cycle genes, only genes related to dna repair were identified.  the complete genome of erysipelothrix rhusiopathiae strain sy was sequenced and assembled. it is, base pairs long, just kilobases smaller than strain fujisawa, with the same gc level of %. it contains open reading frames predicted by glimmer, of which are annotated by ncbi nr blast, by cog database and by kegg database. potential virulence factors are annotated in strain sy by vfdb, while of them are common in the strains, of which are potentially related to antibiotic resistance and highly conserved amongst the three strains of e. rhusiopathiae and only modestly homologous to other gastrointestinal tract-inhabiting firmicutes, e.g. clostridium spp., enterococcus spp. molecular identification of these genomic elements and potential virulence factors offer insights into testing prospective vaccine targets besides the widely used spaa antigens in swine erysipelas and development of more effective treatment or prevention in control of the disease. competing interests the authors declare that they have no competing interests. authors’ contributions ahyk prepared the genomic dna for next-generation sequencing, post-ngs sequence alignment, genome assembly and annotation and comparative genomic analyses, and the drafting of the manuscript. yl and pj isolated the bacteria from field sample and participated in design of study and coordination. jj helped in writing in-house perl script and subsequent bioinformatics analyses. fcl participated in the design of study and coordination. all authors read and approved the final manuscript. supplementary material additional file 1 local chinese swine erysipelas outbreak reports. three articles on local swine erysipelas outbreaks published in chinese scientific journals. click here for file additional file 2 ncbi nr database annotation for e. rhusiopathiae strain. the full list of orfs in e. rhusiopathiae strain sy annotated against ncbi non-redundant database. click here for file additional file 3 cog-annotated genes in e. rhusiopathiae strain sy genome. tabulated summary of orfs in e. rhusiopathiae strain sy annotated against cog database under various cog classes. click here for file additional file 4 cog functional annotation for e. rhusiopathiae strain sy genome. the full list of orfs in e. rhusiopathiae strain sy annotated against cog database. click here for file additional file 5 cog class annotation distribution between e. rhusiopathiae strains sy and fujisawa genomes. a bar chart comparing the percentage of each single-letter cog class found between the complete e. rhusiopathiae genomes. click here for file additional file 6 comparison of cog-annotated genes between e. rhusiopathiae strains sy and fujisawa. tabulated comparison between cog-annotated genes under each single-letter cog class from the complete e. rhusiopathiae genomes. click here for file additional file 7 vfdb-annotated genes between e. rhusiopathiae strain sy genome. the list of vfdb-annotated genes in strain sy genome, classified by cog classes. genes shared between strains sy fujisawa and atcc were shaded in light gray while those shared between strains sy and fujisawa were highlighted in dark gray. click here for file additional file 8 other potential virulence factors in e. rhusiopathiae strain sy genome. a panel of virulence factors mentioned in the literature was searched in strain sy genome and listed here. ^orf orf and orf are pseudogenes with frame-shift or point mutation. click here for file additional file 9 ardb-annotated genes in e. rhusiopathiae strain sy genome. list of ardb-annotated genes and their putative antibiotic resistance. click here for file additional file 10 aclame database-annotated genes in e. rhusiopathiae strain sy genome. full list of orf annotated via aclame database in strain sy genome. click here for file additional file 11 kegg database-annotated genes in e. rhusiopathiae strain sy genome. full list of kegg database-annotated genes in strain sy genome. click here for file acknowledgements the authors would like to thank raymond k. h. hui for handling roche gs junior runs for this study.
it contains open reading frames predicted by glimmer, of which were annotated by pgaap, were annotated by ncbi nr blast, by cog database and by kegg database. genomic island- and pathogenicity island-like regions were also predicted, in which some showed association with trna and potential virulence factors. erysipelothrix rhusiopathiae is the causative agent of animal erysipelas and, to a fewer occurrences, human erysipeloid. complete genome sequencing of erysipelothrix rhusiopathiae, the causative agent of animal erysipelas, was performed. erysipelothrix rhusiopathiaecomplete genome assemblygenome characterizationerysipelasvirulence factors strain sy genome is, base pairs long, just kilobases smaller than strain fujisawa, with the same gc level of %. making use of the complete genome sequencing of e. rhusiopathiae strain sy and comparative genome analysis between the three highly pathogenic strains, the genomic structure and putative functional elements, such as pathogenicity island -like regions, potential virulence factors and horizontal transferring genes of the bacteria are identified. potential virulence factors were annotated in strain sy by vfdb, while of them are common in the strains, of which are potentially related to antibiotic resistance and highly conserved ) amongst the three strains of e. rhusiopathiae and modestly homologous to other gastrointestinal tract-inhabiting firmicutes, e.g. molecular identification of various genomic elements pave the way to the better understanding of mechanisms underlying metabolic capabilities, pathogenicity of swine erysipelas and prospective vaccine targets besides the widely used spaa antigens.
 erysipelothrix rhusiopathiae is the causative agent of animal erysipelas and, to a fewer occurrences, human erysipeloid. it is ubiquitous in nature and commensal in diverse species of animals, wild or domestic, from mammals and birds to reptiles and fish. mechanisms of its virulence and pathogenicity are poorly understood.  making use of the complete genome sequencing of e. rhusiopathiae strain sy and comparative genome analysis between the three highly pathogenic strains, the genomic structure and putative functional elements, such as pathogenicity island -like regions, potential virulence factors and horizontal transferring genes of the bacteria are identified. strain sy genome is, base pairs long, just kilobases smaller than strain fujisawa, with the same gc level of %. it contains open reading frames predicted by glimmer, of which were annotated by pgaap, were annotated by ncbi nr blast, by cog database and by kegg database. potential virulence factors were annotated in strain sy by vfdb, while of them are common in the strains, of which are potentially related to antibiotic resistance and highly conserved ) amongst the three strains of e. rhusiopathiae and modestly homologous to other gastrointestinal tract-inhabiting firmicutes, e.g. clostridium spp., enterococcus spp. genomic island- and pathogenicity island-like regions were also predicted, in which some showed association with trna and potential virulence factors.  complete genome sequencing of erysipelothrix rhusiopathiae, the causative agent of animal erysipelas, was performed. molecular identification of various genomic elements pave the way to the better understanding of mechanisms underlying metabolic capabilities, pathogenicity of swine erysipelas and prospective vaccine targets besides the widely used spaa antigens. erysipelothrix rhusiopathiaecomplete genome assemblygenome characterizationerysipelasvirulence factors 