Shotgun sequencing of Yersinia enterocolitica strain W22703 (biotype 2, serotype O:9): genomic evidence for oscillation between invertebrates and mammals
© Fuchs et al; licensee BioMed Central Ltd. 2011
Received: 28 October 2010
Accepted: 31 March 2011
Published: 31 March 2011
Yersinia enterocolitica strains responsible for mild gastroenteritis in humans are very diverse with respect to their metabolic and virulence properties. Strain W22703 (biotype 2, serotype O:9) was recently identified to possess nematocidal and insecticidal activity. To better understand the relationship between pathogenicity towards insects and humans, we compared the W22703 genome with that of the highly pathogenic strain 8081 (biotype1B; serotype O:8), the only Y. enterocolitica strain sequenced so far.
We used whole-genome shotgun data to assemble, annotate and analyse the sequence of strain W22703. Numerous factors assumed to contribute to enteric survival and pathogenesis, among them osmoregulated periplasmic glucan, hydrogenases, cobalamin-dependent pathways, iron uptake systems and the Yersinia genome island 1 (YGI-1) involved in tight adherence were identified to be common to the 8081 and W22703 genomes. However, sets of ~550 genes revealed to be specific for each of them in comparison to the other strain. The plasticity zone (PZ) of 142 kb in the W22703 genome carries an ancient flagellar cluster Flg-2 of ~40 kb, but it lacks the pathogenicity island YAPIYe, the secretion system ysa and yts1, and other virulence determinants of the 8081 PZ. Its composition underlines the prominent variability of this genome region and demonstrates its contribution to the higher pathogenicity of biotype 1B strains with respect to W22703. A novel type three secretion system of mosaic structure was found in the genome of W22703 that is absent in the sequenced strains of the human pathogenic Yersinia species, but conserved in the genomes of the apathogenic species. We identified several regions of differences in W22703 that mainly code for transporters, regulators, metabolic pathways, and defence factors.
The W22703 sequence analysis revealed a genome composition distinct from other pathogenic Yersinia enterocolitica strains, thus contributing novel data to the Y. enterocolitica pan-genome. This study also sheds further light on the strategies of this pathogen to cope with its environments.
The genus Yersinia currently comprises three human pathogens (Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica), and at least 14 species considered harmless for humans, namely Y. aldovae, Y. bercovieri, Y. frederiksenii, Y. intermedia, Y. kristensenii, Y. mollaretii, Y. rohdei, Y. ruckeri, Y. aleksiciae, Y. similis, Y. massiliensis, Y. entomophaga, Y. nurmii and Y. pekkanenii. Y. enterocolitica infection causes diarrhea, terminal ileitis, and mesenteric lymphadenitis, but not systemic infection, and often leads to secondary immunologically induced sequelae including erythema nodosum, reactive arthritis and Reiter's syndrome . The heterogenous species Y. enterocolitica encompass six biotypes as differentiated upon biochemical tests . Biotype 1A strains are considered avirulent due to the lack of the Yersinia virulence plasmid pYV , whereas biotype 1B strains are highly pathogenic and lethal for mice. They form a geographically distinct group predominately isolated in North America and carry a high-pathogenicity island (HPI) . Biotype 2-5 strains mainly found in Europe and Japan compose a weakly pathogenic group unable to kill mice .
We have recently shown that strain W22703 (biotype 2, serotype O:9) confers lethality towards nematodes and Manduca sexta larvae upon oral infection, and that this insecticidal activity is correlated with the presence of the so-called pathogenicity island TC-PAIYe[13, 14]. This 20 kb-fragment is present in the biotype 2-5 strains, but absent in most biotype 1A and B strains, and carries the toxin complex (TC) genes tcaA, tcaB, tcaC and tccC with homology to TC genes of Photorhabdus luminescens. However, the absence of TC-PAIYe is not reflected by a loss of toxicity in case of subcutaneous infection, indicating the presence of yet unknown insecticidal determinants in Y. enterocolitica.
To investigate the genomic heterogeneity of the species Y. enterocolitica, we have chosen to sequence the genome of the low-pathogenicity strain W22703. We report the annotation of this second genome sequence of a Y. enterocolitica strain, and a detailed comparative genome analysis of the W22703 genome with that of strain 8081, a representative of the highly pathogenic biotype 1B group. The data obtained provide novel insights into the biology, metabolism, adaptation strategies and evolutionary relationships of Y. enterocolitica.
Y. enterocolitica 8081
G + C content [%]
Number of contigs >1000 bp
2 (linear chromosome, plasmid pYV)
Number of CDs
Coding density [%]
Average gene size
Common IS elements (numbers)
IS911A, ISRM3, IS1328 (3), IS1329, IS1400, IS1666, IS1667 (4), IS1668, IS1669
IS elements not shared
IS2A/D/F/H/I/K; iso-IS1N; IS1328; IS911B
IS3, IS4, IS1111, IS1222, IS1330, IS1541, IS1660 (6), IS1664, IS1665 (6), IS1669 (4), IS1664 (2), IS1222, IS3, IS Sod4
Genome comparison with strain 8081
Regions of difference
Virulence genes or cluster present or absent in W22703 compared with 8081
Predicted functions of genes present in W22703 and absent in 8081
amino acids ABC transporters
1812, 1658§, 1393, 1802*, 1162*, 1660§, 1186, 1757
major facilitator superfamily
1802, 1240, 1885§, 1996
sodium/bile acid symporter family
efflux transporter, RND family
divalent cation transporter
iron complex transport system substrate-binding protein*
conjugation transfer protein, TraD family§
type IV prepilin§
chromosome segregation ATPase
amidase, hydantoinase/carbamoylase family protein
multisensor hybrid histidine kinase
iron-sulfur cluster repair di-iron protein
thiol-disulfid exchange protein
purine catabolism protein
Cell wall biogenesis or surface proteins
flagella gene cluster II*
outer membrane protease
small integrale membrane protein§
outer membrane protein
TonB-dependent heme receptor
pili biogenesis and assembly
1847, 1393, 1882
cell wall-associated hydrolases
integral membrane protein CcmA
extracellular ligand-binding receptor
bile salt/choloylglycin hydrolase (bsh)
Rtx cluster rtxH, rtxC, rtxA
insecticidal toxins (TC-PAIYe)
1579, 1975, 1854, 1855, 1856
small toxic polypeptide
hemolysin; hemolysin activator protein
colicin-E2/pyocin S2 immunity protein operon
Proteins for DNA/RNA regulation and processing
1088*, 1894, 1885, 1825§, 1835§(2), 1764, 1557§, 1200§, 1171§, 1885(2) §, 1891, 1803, 1802
RpiR-like transcriptional regulator
CRP/FNR family transcriptional regulator*
LuxR-like transcriptional regulator
negative regulator of β-lactamase expression
response regulator receiver protein
helix destabilizing protein
superfamily I DNA helicase
filamentation induced by cAMP protein Fic
sensor protein with MASE1 domain
1116, 1170, 1897, 1882, 1763, 1760, 1837
plasmid stabilisation system*
To survive within its host organisms, Y. enterocolitica needs to overcome both cellular (hemocytic) and peptide-mediated components. A candidate of the former group is the exported repeats-in-toxin RtxA, a cytolytic toxin of approximately 3200 amino acids length encoded by contig 1867. It clusters with a putative Rtx activating protein, RtxC, and a peptide chain release factor 1, RtxH (Table 2). Among the yersiniae, only one Y. enterocolitica serotype O:3 strain and Y. kristensenii carry homologs of these proteins. The genome of W22703 encodes three two-partner secretion (TCS) systems involved in hemolysin release, one of which is absent in the 8081 genome. Three peptidases of W22703 might play a role in resistance towards antimicrobial peptides. W22703 also produces an antibacterial protein or bacteriocin that is absent in 8081. The pyocin locus on contig 1216 probably encodes killer proteins and a dual type immunity protein with domains similar to pyocin-E2 and colicin (S2). Its biological function, as well as that of a small toxic polypeptide (contig 1810), is yet unknown.
Secretion/transfer systems and transporters
Two distinct, chromosomally located type three secretion systems (T3SS) mark one of the most striking differences between the two genomes. While ysa of 8081 is absent in W22703, this strain harbours another T3SS, which we termed ysa2, located on contigs 1804 and 1807 (Figure 2B). PCR targeting the flanking regions resulted in a ~200 bp fragment, justifying the link of both contigs. Sequence analysis of ysa2 revealed a mosaic structure with a G/C content of 49.4% between the flanking genes YE0315 and YE0312, and a G/C content of 40.9% between YE0312 and YE0311, indicating two independent LGT events. The whole cluster is collinear to respective regions in apathogenic yersiniae such as Y. frederikseni and Y. intermedia. We found homologs of the plasmid-encoded T3SS of Y. pestis and Y. pseudotuberculosis, and partial collinearity, with respect to the left part of this 29 kb island. The right part carries yscC and yscD homologs, but no homologs of YopB, YopD or LcrV involved in translocon formation . The functionality of ysa2 is unknown. Of the two type 2 secretion system (T2SS) cluster yst1 and yst2 in 8081, only yst2 responsible for a general secretion pathway (GSP) is present in strain W22703. Contig 1170 encodes factors involved in conjugal DNA-transfer, namely TraD, a MobA/MobL protein, and a putative type IV prepilin that might contribute to LGT.
As iron is often a rate-limiting factor for pathogenic bacteria during infection, W22703 requires iron-scavenging systems for survival in the host. Beside the two iron and enterobactin transport systems within the PZ, those comprise a hemophore cluster for heme binding and uptake (Table 2), and a putative iron binding protein (contig 1360), the latter one absent in 8081.
We also identified eight ABC transporters in W22703, two phosphotransferase systems (PTS), four permeases, two major facilitators, a sodium:bile acid symporter, and other transporters listed in Table 2 or mentioned in the sections on metabolism and virulence. All of these are without counterparts in the genome of strain 8081. A glucitol/sorbitol-specific transporter (contig 1884; YE1093-YE1098) and a sorbose uptake system are also present in 8081, but not in Y. pestis and Y. pseudotuberculosis, and have homologs in all or most non-pathogenic species sequenced so far. A cellobiose uptake system was also identified (contig 1882). In total, our analysis identified a higher number of putative transporters with respect to strain 8081.
Plasticity zone (PZ)
The recently identified flagellar cluster Flg-2  within the PZ of W22703 is absent in the genome of 8081. It comprises 44 genes encoding factors for the flagellar motility apparatus and for flagellar biosynthesis, but lacks chemotaxis genes (Figure 3). A region of approximately 11,300 bp flanked by a transposase gene and the replicon of an IncF plasmid RepFIB comprises an ABC transporter and a regulatory gene; however, the functionality of this region, which is unique with respect to all Yersinia sequences available so far, is in doubt due to its low coding density.
The main flagella and chemotaxis gene cluster I (flg-1) of 8081 is present in W22703 (contigs 1361, 1428, 1469 and 1890), but only one of two type-1 fimbrial operons was found (contig 1271; YE0782-YE0786). The surface-exposed lipopolysaccharide (LPS) molecule constituting the serotype O:9 O-antigen is synthesized by the O-polysaccharide gene cluster (contig 1162) ; a second glycosyltransferase gene cluster is located on contig 1878 (Figure 2A). The role of the O-polysaccharide and the outer core hexasaccharide in resistance of Y. enterocolitica to human complement and polymyxin B has been described recently .
Several enzymatic activities common to both Y. enterocolitica genomes compared here are involved in nitrogen metabolism. Examples are the capability to catalyse urease that is encoded by seven genes on contig 1225. The assimilation of the urease product ammonia for amino acid and nucleotide synthesis is then achieved by glutamine synthase. Two ornithine decarboxylases forming putrescine, and a putrescine/ornithine antiporter are encoded on the PZ and also contribute to amino acid metabolism.
Like 8081, strain W22703 carries the cel gene cluster for cellulose production (contig 1798) and the genes mdoC, mdoG and mdoH for osmoregulated periplasmic glucan (OPG) biosynthesis (contig 1967). Both the capability to produce cellulose and to synthesize OPGs have been lost or inactivated in Y. pestis and Y. pseudotuberculosis. OPG mutants exhibit deficiencies in virulence, biofilm formation and antibiotic resistance, as well as hypersensitivity towards bile salts .
The capability to utilize propanediol in a cobalamin (vitamin B12)-dependent manner is encoded on contigs 2012, 1667, 1476, 1555, 1999, and 1235, and the respective cob/cbi/pdu genes are collinear to the 8081 genes YE2707-YE2750. In line with the yersiniae core genome, ttr genes responsible for tetrathionate reduction are present (contig 1975), and the eut genes allowing B12-dependent ethanolamine utilization are absent. The mtn genes located on contig 1812 are involved in methionine salvage. This cobalamin-dependent pathway recycles methylthioadenosine derived from sperimidine, spermin and N-acylhomoserine lactone synthesis. The hydrogenases Hyd-4 encoded within the hyf locus (contigs 1162, 1947) and Hyd-2 within the hyb locus (PZ; contig 1891, Figure 3) are also present in W22703.
Distinct metabolic properties of W22703
W22703 is endowed with several metabolic enzymes that are unique in comparison to strain 8081 (Figure 2A), among them a serine-pyruvate transaminase involved in glycine-, serine- and threonine-metabolism (contig 1812). The reductases encoded on contigs 1186 and 1976 suggest that W22703, but not 8081, is able to use nitrate and dimethylsulfoxide (DMSO) as alternative electron acceptor under anaerobic conditions. In contrast, pathways for trimethylamine and thiosulfate oxidation are present in both genomes. Beside the DMSO reductase, contig 1186 harbours another ROD encoding an ABC transporter, a putative nitrilase or cyanide hydratase that catalyzes nitrile into amino acids and ammonium or hydrogen cyanid into formamide, and a putative acetamidase or formamidase. Contig 1973 carries a gene cluster enabling W22703 to uptake N-acetylgalactosamine that is then isomerized to tagatose. In addition to YE0550A-YE0555, we identified a second operon for sucrose utilization on contig 1240.
Metabolic pathways lost in W22703
Predicted functions of genes present in 8081 and absent in W22703
copper/silver efflux system
multidrug efflux protein
phosphohistidinoprotein-hexose phosphotransferase component of PTS system (Hpr)
glucoside specific PTS system
membrane transport protein
molybdate ABC transporter permease protein
cation efflux system
sulfite reductase subunit protein
NADH:flavin oxidoreductase/NADH oxidase family protein
conjugal transfer protein
type 4 prepilin-like proteins leader peptide processing enzyme
integrating conjugative element protein
UDP-sugar hydrolase/5'-nucleotidase periplasmic precursor
fucose isomerase fucI
Cell wall biogenesis or surface proteins
YGI-2 (putative glycolipoprotein)
type-1 fimbrial operon
YE0982, YE1234-YE1236, YE1871, YE3483, YE4156
YE3576 (chiY), YE2830, YE3411, YE3494A
acyl-UDP-N-acetylglucosamine O-acyltransferase, lpxA2
high pathogenicity island HPI (Yersiniabactin)
HlyA-like hemolysin and transporter (TPS)
heme acquisition system
T3SS effector protein
ccdA/ccdB, toxin/antitoxin system
bifunctional antitoxin/transcriptional repressor RelB
arsenic resistance operon
metal resistance protein
iron-sulfur cluster assembly protein
metallo-β-lactamase superfamily protein
cytonecrosis factor-like toxin
antibiotic biosynthesis monooxygenase
Rho-activating domain of cytotoxic necrotizing factor
Proteins for DNA/RNA regulation and processing
LuxR family transcription regulatory protein
YE0039, YE0343, YE1026, YE1165, YE2050-YE2051, YE3033
transcriptional regulatory protein
YE1102, YE1172, YE1825, YE3496A, YE4129
Cro/CI family transcriptional regulator
plasmid-related transcriptional repressor protein
GntR-family regulatory protein
YGI-3, putative integrated plasmid
YGI-4, putative integrated plasmid
prophage region YE98
prophage region YE185
prophage region YE200
prophage region YE250
YE0986, YE0990, YE3513
plasmid stabilisation protein
chromosome partitioning system
Dynamic genomes: further regions absent in W22703 or 8081
Whole genome comparison allows to follow the dynamic processes by which genomes separate from a common ancestor. In addition to genomic islands or clusters already mentioned above, the genomes of the two Y. enterocolitica strains compared here differ by a set of regions, indicating the dynamic of sequence acquisition and loss. The prophage regions YE98 (YE0854-YE0888), YE185 (YE1667-YE1693), YE200 (YE1799-YE1819) and YE250 (YE2292-YE2363) of strain 8081 are absent in strain W22703 that, however, carries another prophage of 37 CDS in contig 1796. Then, the Yersinia genome islands YGI-2, YGI-3 and YGI-4  are missing in W22703. YGI-2 carries genes for the synthesis, modification, and export of an outer membrane anchored glycolipoprotein. Of this island, only homologs of YE0912 encoding a 2,5 diketo-D-gluconic acid reductase B and of YE0911 encoding a 3-oxo-acyl-(acyl carrier protein) synthase II are present in W22703. On contig 1854, we identified two homologs of YE0979, which encodes a DNA-binding protein, and the hypothetical gene YE0980 from YGI3 harbouring a putative integrated plasmid.
The genome analysis and genome comparison performed here intended to contribute to a better understanding of the ecology, pathogenicity and evolution of Y. enterocolitica. Adding, rearranging and reducing or losing DNA has been proposed as the general recipe for Yersinia genome evolution when eight less-pathogenic strains had been compared .
Several ROD shown in Figure 2A-B and Figure 3 might be recent acquisitions due to the significant deviation of their G/C content, while others might have been acquired early after separation of both strains, or indicate regions that have been lost or substituted in 8081. Beside the virulence plasmid, the PZ is a second example for the acquisition of virulence genes. Remarkably, this region is approximately 55 kb shorter in W22703 and lacks several determinants proposed or known to contribute to pathgenicity towards humans. Together with the presence of a large ancient flagellar gene cluster and a region of obvious genetic degeneration, this finding strongly reflects the lower virulence potential of W22703 in comparison to 8081 , and confirms the importance of this region for the manifestation of virulence properties of Y. enterocolitica.
Other (virulence) regions absent in W22703 might have been acquired by 8081 after separation of both strains. The novel T3SS (ysa2) that is absent in the pathogenic species Y. pestis and Y. pseudotuberculosis, but present in apathogenic species such as Y. intermedia, Y. frederiksenii and Y. kristensenii, might play a role in the interaction with non-mammalian hosts. The TC proteins have been shown to be secreted upon activity of the plasmid-encoded T3SS of Y. pestis. Since we used a pYV-free W22703 derivative to demonstrate TC-based insecticidal activity of this strain , ysa2 is a candidate for TcaA secretion by W22703. This finding supports the assumption that T3SS are not unique vehicles for delivering anti-vertebrate factors but ancient secretion systems for the transport of effector molecules across host membranes, with the potential to play a role in a wide range of bacteria-host interactions . Together with a number of putative virulence factors of W22703 (Table 2), ysa2 contributes to the pathosphere of yersiniae, a concept hypothesizing that all of the pathogenic genes shared by enteric bacteria form a "pool" .
The pathogen Y. enterocolitica has a complex life cycle encompassing aquatic and biological environments. Due to its known capability to interact with invertebrates and mammals, it exhibits a multiphasic phenotype upon colonizing and potentially killing more than one host species . Although little is known about putative signals and regulatory circuits required to switch or modulate necessary changes from one state to the other, candidates are genes listed in Table 2 or induced at low temperature .
Although some of their functions remain to be experimentally confirmed, the metabolic pathways present in strain W22703 confirm the relevance of several metabolic traits for gut-adapted Y. enterocolitica. Examples are cobalamin-dependent utilization of propanediol, hydrogenase activities, cellulose production, tetrathionate reduction and ornithine decarboxylase activity, all of which are absent, lost or inactivated in systemic Y. pestis and Y. pseudotuberculosis[17, 18]. Interestingly, tetrathionate that acts as a terminal electron acceptor during anaerobic degradation of 1,2-propanediol or ethanolamine is formed in the inflamed gut upon infection . The hydrogenases Hyd-4 and Hyd-2 might contribute to the adaptation of yersiniae to gut environments [39, 40]. Two additional reductases of W22703 allowing to use nitrate and DMSO are in line with this assumption. A potentially insect-specific resistance mechanism and/or catabolic trait of strain W22703 is provided by the ROD of contig 1186 (Figure 2A) inserted between YE0815 and YE0816. It encodes an acetamidase/formamidase, a branched chain amino acid transporter, an ABC transporter and a putative nitrilase/cyanide hydratase. These predicted functions point to a role of this chromosomal region in the acquisition of nitrogen sources. Indeed, insects such as Zygaena filipendulae produce cyanogenic glucosides that might then be used by W22703 as nitrogen source via a metabolic route of bacteria that includes cyanide nitrilase, hydratase and formate dehydrogenase activities [41–43]. Although speculative so far, the functions of contig 1186 might represent a further determinant contributing to invertebrate host adaptation of strain W22703. The operon on contig 1973 including a PTS is responsible for tagatose utilization; this metabolic trait is not only common to human intestinal bacteria, but was found to be specifically induced during insect infection by P. luminescens[44, 45]. Interestingly, the PZ gene bsh encodes a chologlycin hydrolase or bile salt hydrolase (Figure 3) that catalyzes the deconjugation of conjugated bile salts to liberate amino acids and free primary bile acids .
Recently, a genome comparison between the genomes of the insect pathogen P. luminescens and Y. enterocolitica 8081 revealed a huge number of common genes that might contribute to the adaptation of yersiniae to invertebrate hosts . Interestingly, we identified nearly all of these factors also in the genome of W22703, underlining the assumption that Y. enterocolitica strains share the capability to interact with nematodes or insects.
Although further genome sequences are required to learn more about the evolution of Y. enterocolitica strains, this study indicates that beside the Yersinia virulence plasmids, the highly flexible PZ indeed contributes to the acquisition of determinants that might increase the pathogenicity towards humans. On the other hand, insecticidal toxins, the novel T3SS or specific metabolic properties might play a crucial role for the adaptation of Y. enterocolitica strains to non-mammalian hosts.
Bacterial strains and growth conditions
Y. enterocolitica W22703(pYVe227) is a nalidixic acid-resistant (NalR) restriction mutant (Res-Mod') isolated from strain W227 . A plasmidless isogenic derivative (W22703 pYV-) was used. To avoid contaminations and to validate the strain cultured for DNA isolation, strain W22703 pYV- was streaked from a glycerol stock on Yersinia selective agar plates (CIN agar base; Becton Dickison, Heidelberg, Germany). A single colony was used for inoculation of Luria-Bertani (LB) broth (10 g l-1 tryptone, 5 g l-1 yeast extract, and 5 g l-1 NaCl) containing 20 μg ml-1 nalidixic acid, and the culture was grown for twelve hours at a selective temperature of 15°C. When the culture had reached stationary phase, aliquots were plated in parallel on LB and Yersinia seletive agar plates. PCRs targeting W22703-specific genes tcaA, tcaC and two genes of Flg-2 were performed as a further control.
General molecular techniques
DNA and RNA manipulation was performed according to standard procedures . To isolate chromosomal DNA, 1.5 ml of a bacterial culture was centrifuged, and the sediment was resuspended in 400 μl of lysis buffer (100 mM Tris pH 8.0, 5 mM EDTA, 200 mM NaCl). After incubation for 15 min on ice, 10 μl of 10% SDS and 5 μl of proteinase K (10 mg/ml) were added, and the sample was incubated overnight at 55°C. The chromosomal DNA was then precipitated with 500 μl of isopropanol, washed in ethanol, dried, and dissolved in 500 μl of TE buffer (10 mM Tris-HCl, 1 mM Na2EDTA, pH 7.4) containing 1 μl of RNase (10 mg/ml). Polymerase chain reactions (PCR) were carried out with Taq polymerase (Fermentas, Vilnius, Lithunia) and the following programme: one cycle at 95°C for 2 min; 30 cycles at 95°C for 10 sec, at the appropriate annealing temperature for 30 sec, at 72°C for 45 sec to 180 sec depending on the expected fragment length; one cycle at 72°C for 10 min. 4 μl of chromosomal DNA (100 ng ml-1) was used as template for PCR amplification, and the GeneRuler DNA mix (Fermentas) served as DNA ladder. For gap closure, the following oligonucleotides were used (targeted contigs): 5'-CAACATTAAATCACGAAGG-3'/5'-TTAGTACAAATACCGATGG-3' (1804/1807); 5'-GTTTCTTTATGGGCGCG-3'/5'-TTGGCATGGAGGCCTG-3' (1891/1803); 5'-TAACCTCTAGCGCGG-3'/5'-CCCCGATAGTTCTGG-3' (1088/1891).
Genome sequencing and accession
High throughput sequencing of a shotgun library was done on the GS FLX system (Roche, 454 Life Sciences, Branford, USA) using the Titanium series with approximately 20-fold coverage, and assembly were performed by Eurofins MWG GmbH, Ebersberg, Germany. According to the newly defined standards for classification of genome sequences , the Y. enterocolitica genome sequence belongs to the category "Annotation-Directed Improvement". The EMBL accession numbers for the sequences reported in this paper are FR718488-FR718797. The raw sequence data files are deposited in the ENA trace archive as ERP000495. The annotated sequence is available under the URL address http://pedant.gsf.de.
Genome annotation and analyses
The PEDANT software system (http://pedant.gsf.de; ) was used for automatic genome sequence analysis and annotation . Protein coding genes were predicted using the GeneMarkS software program using default settings . Biochemical pathway prediction and reconstruction were performed using the KEGG , BRENDA , and Microbes online  databases. tRNAs were identified using tRNAscanSE , rRNA homologs with blastn . Additional manual homology searches of predicted proteins were performed by BLAST analysis (http://www.ncbi.nlm.nih.gov/BLAST/) to ascribe a protein function or domain.
Comparison with the genome sequence of Y. enterocolitica 8081 (EMBL accession numbers are AM286415 for the chromosome and AM286416 for the virulence plasmid pYV; PEDANT database name is p3_p190_Yer_enter) was performed using the Y. enterocolitica Blast Server from the Sanger Institute (http://www.sanger.ac.uk/cgi-bin/blast/submitblast/yersinia). The criterion applied was an 80% identity of the amino acid sequence. Incomplete proteins encoded on contig ends were considered to be present in strain W22703 if the lacking sequence could be identified on another contig. Genome sequences of Yersinia strains were obtained from the NCBI database and compared using the homepage http://www.microbesonline.org/. Protein sequence alignment was done with the ClustalW program . Phylogenetic trees for ROD and T3SS have been automatically calculated using the the software PhyloGenie  and the default parameters according to its documentation. We used NCBI nr  as reference database and excluded proteins of unclassified taxa.
- ABC transporter:
ATP-binding cassette transporter
lateral gene transfer
region(s) of difference
two-partner secretion system
type three secretion system
type two secretion system
two component system
Yersinia genome island.
We thank Siegfried Scherer for generous support of this study. This work was in part funded by the Deutsche Forschungsgemeinschaft (FU 375/4-1).
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