Shotgun sequencing of Yersinia enterocolitica strain W22703 (biotype 2, serotype O:9): genomic evidence for oscillation between invertebrates and mammals

  • Thilo M Fuchs1Email author,

    Affiliated with

    • Katharina Brandt1,

      Affiliated with

      • Mandy Starke1 and

        Affiliated with

        • Thomas Rattei2

          Affiliated with

          BMC Genomics201112:168

          DOI: 10.1186/1471-2164-12-168

          Received: 28 October 2010

          Accepted: 31 March 2011

          Published: 31 March 2011

          Abstract

          Background

          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.

          Results

          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.

          Conclusion

          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.

          Background

          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 [1], Y. aleksiciae [2], Y. similis [3], Y. massiliensis [4], Y. entomophaga [5], Y. nurmii [6] and Y. pekkanenii [7]. 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 [8]. The heterogenous species Y. enterocolitica encompass six biotypes as differentiated upon biochemical tests [9]. Biotype 1A strains are considered avirulent due to the lack of the Yersinia virulence plasmid pYV [10], 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) [11]. Biotype 2-5 strains mainly found in Europe and Japan compose a weakly pathogenic group unable to kill mice [12].

          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 [15].

          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.

          Results

          General features

          The shotgun sequencing of the Y. enterocolitica strain W22703 genome revealed a total number of 243656 reads with an average read length of 363. Assembly of 232502 reads resulted in 305 contigs larger and 705 contigs shorter than 1,000 base pairs (bp) with a median level of coverage in contigs > 5 kb of 16.49 (Additional file 1); one contig (1796) exceeds this coverage level more than twice (40x). The genome has an average G + C content of 46.9% (Table 1). Upon PEDANT based annotation [16] and search against a non-redundant protein database, 4003 genes corresponding to a coding density of 84.4% could be identified, but an unknown number of genes might have been missed due the short contigs not assembled (Additional file 2). The analysis also revealed at least 68 tRNA genes. The fewer number of tRNA genes compared to finished Yersinia genomes is probably due to collapsing of reads of the repeat sequences into fewer contigs [17]. The exact number of rRNA operons could not be estimated from this draft assembly, as reads from identical copies probably assemble into the same contigs. The risk of frameshifts due to sequencing errors in longer homo-oligomers was reduced by the high coverage of the assembly. We have determined 111 pairs of consecutive ORFs having best similarity to the same protein. However, this number also includes real pseudogenes not affected by any sequencing error.
          Table 1

          Genome features

          Property

          W22703 pYV-

          Y. enterocolitica 8081 [18]

          Size [bp]

          <4,754,619

          4,615,899

          G + C content [%]

          46.9

          47.3

          Number of contigs >1000 bp

          305

          2 (linear chromosome, plasmid pYV)

          Number of CDs

          >4,003

          4,037

          Coding density [%]

          84.4

          83.8

          Average gene size

          unknown

          968

          rRNA operons

          unknown

          7

          tRNAs

          ≥ 68

          81

          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

          Prophage regions

          2

          4

          Sequence coverage

          16-fold

          9-fold

          Genome comparison with strain 8081

          Y. enterocolitica 8081 is one of three strains of this species whose sequences were available until February 2011 [1820]. It belongs to the biotype 1B group with higher pathogenicity potential to humans than the biotype 2-5 group. To delineate the most relevant features of the W22703 genome, we decided to base our further analysis on a genome comparison between the shotgun sequence of strain W22703 and the linear genome sequence of Y. enterocolitica strain 8081. The alignment of both genomes using Mauve [21] shows long syntenic regions with few rearrangements and a general high sequence conservation, but also regions in both genomes that are not shared with the other (Additional file 3). Upon automatic and manual BLAST analysis, we revealed 550 genes present in the 8081 genome but absent in that of W22703, and 551 genes that are specific for W22703 with respect to strain 8081. The virulence plasmid pYV [22] was not considered here. Figure 1 shows the categories under which the W22703 genes absent in 8081 are summarized. Besides hypothetical genes and those of unknown function, the largest numbers of gene-encoded factors fall into the functional groups transporter, metabolism and DNA/RNA processing. The latter group comprises 18 regulatory genes. The motility and phage sections are mainly composed of the ancestral flagellar locus flgII (see below) and one specific prophage.
          http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-12-168/MediaObjects/12864_2010_3302_Fig1_HTML.jpg
          Figure 1

          Functional categories of genes present in strain W22703 and absent in strain 8081

          Regions of difference

          We then searched for regions of difference (ROD) between the genome sequences of 8081 and W22703. By definition, those ROD genes do not belong to the core genome of the two Y. enterocolitica strains compared here, but might constitute additional metabolic or virulence-associated properties contributing to the overall strain fitness. Twelve ROD present in strain W22703 are shown in Figure 2A. While the average GC-content of the W22703 genome sequence is 46.9%, the ROD on contigs 1240, 1162, 1764, 1812, and 1280 show an at least 2% higher or lower GC-content, suggesting their acquisition by lateral gene transfer (LGT) [23]. Phylogenetic tree analysis, however, revealed closely related genes of these contigs in other Yersinia strains, with the exception of contig 1280 that harbours phage related genes. The 8081 genes flanking the ROD might give additional information about the underlying recombination events. For example, a glycosyltransferase operon of 8081 (YE3070-YE3087) might have been replaced by a related operon on contig 1878 (or vice versa) that possibly contributes to O-antigen synthesis. A substitution of hypothetical genes by a non-homologous cluster of functionally unknown genes is observed in contig 1764. The ROD on contigs 1186, 1240, 1280 and 1973 obviously interrupt gene linearity with respect to the 8081 genome, indicating that loss and substitution, or insertion, of genes might have taken place in these cases. Contig 1280 harbours several phage-related genes and is therefore assumed to represent a second prophage region. Transposase genes were found in the 8081 genome between YE2773 and YE2779, a region covered by the LPS synthesis in W22703 (contig 1162), and a similar observation was made for the PTS encoding cluster on contig 1884. More functional details on these ROD are described below.
          http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-12-168/MediaObjects/12864_2010_3302_Fig2_HTML.jpg
          Figure 2

          Regions of strain W22703 without counterpart in strain 8081. A) contig 1162: manC, mannose-1-phosphate guanylytransferase; manB, phosphomannomutase; galU/galF, UTP-glucose-1-phosphate uridylyltransferase; gmd, probable GDP-mannose 4,6-dehydratase; per, perosamine synthetase; wbcT/U/V/W, glycosyltransferase. contig 1186: ccp, cytochrome c peroxidase; livF, branched chain amino acid transporter; amdS/fmdA, acetamidase/formamidase; dmsA/B/C, anaerobic DMSO reductase; the gene right of YE0815 encodes a putative nitrilase/cyanide hydratase or apolipoprotein N-acyltransferase; gap ~7,100 bp. contig 1240: the gene right of YE1738 encodes a putative O-acetylhomoserine aminocarboxypropyltransferase; gap ~4,230 bp. contig 1764: the gene right of YE4152 encodes a GCN5-related N-acetyltransferase; gap ~39,960 bp. contig 1785: the gene right of YE3852 encodes a putative acyltransferase; hpmB, hemolysin activator; hlyA, hemolysin A; gap ~20,340 bp. contig 1812: pucG, purine catabolism protein (aminotransferase); glnQ, glutamine ABC transporter ATP-binding protein. contig 1835: mqsR, motility quorum-sensing regulator. contig 1878: gne, uridine diphosphatoacetylglucosamine epimerase; wbcK/P/O/N/M/L/K, glycosyltransferase; wzx, lipopolysaccharide O-unit flippase. contig 1884: gaps ~6,960/21,100 bp. contig 1973: kbaY, tagatose-1,6-bisphosphate aldolase; agaS, tagatose-6-phosphate ketose/aldose isomerase; kbaZ, tagatose 6-phosphate kinase. contig 1976: nrfA-D, formate-dependent nitrate reductase, cytochrome c; dsbE, hypothetical thiol:disulfide interchange protein; ccmE, cytochrome C biogenesis protein. Shaded regions: no counterparts were identified in Y. pseudotuberculosis or Y. pestis genomes. The GC-content of the regions is added to the contig number. Asterisk: low-temperature induced gene as identified by luciferase reportering [60]. B) A novel T3SS (ysa2) of strain W22703 YE0311 putatively encodes a two-component response regulator, the gene left of it a multi-sensor hybrid histidine kinase. Different GC contents were determined for the regions left and right of YE0312.

          Virulence genes or cluster present or absent in W22703 compared with 8081

          While YGI-1, which is responsible for adhesion and includes a T4SS, is completely encoded on contig 1802, the high pathogenicity island (HPI) encoding yersiniabactin [24] is missing in the genome of strain W22703. In contrast, we identified 18 functions probably involved in defence or virulence mechanisms present in W22703, but absent in 8081 (Table 2). Two autotransporter or type V secretion proteins (contig 1177), one of them with homology to an AidA-like adhesin, might play a role in (non-mammalian) host-recognition by W22703. The insecticidal pathogenicity island TC-PAIYe is characteristic for biotype 2-5 strains, but absent in biotype 1A and 1B strains with the exception of WA314 (biotype 1B, serotype O:8) [14, 15]. Another homology group of TC genes was found to be prevalent among clinical biotype 1A strains [25]. Beside a second, non-clustered tccC2 locus [15], no further factors with homology to toxin complex genes could be identified in W22703.
          Table 2

          Predicted functions of genes present in W22703 and absent in 8081

          Transporter

          Contig

          amino acids ABC transporters

          1812, 1658§, 1393, 1802*, 1162*, 1660§, 1186, 1757

          PTS

          1973§, 1884

          major facilitator superfamily

          1950§, 1885

          permeases

          1802, 1240, 1885§, 1996

          sodium/bile acid symporter family

          1785§

          efflux transporter, RND family

          1328

          divalent cation transporter

          1165

          iron complex transport system substrate-binding protein*

          1360

          magnesium transporter

          1165

          Bioenergetic proteins

          Contig

          cytochrome-c protein

          1186, 1976§

          nitrate reductase

          1976§

          Conjugation/replication

          Contig

          conjugation transfer protein, TraD family§

          1170

          MobA/MobL-like protein§

          1170

          type IV prepilin§

          1170§

          chromosome segregation ATPase

          1804

          Metabolism

          Contig

          amidase, hydantoinase/carbamoylase family protein

          1812

          serine-pyruvate transaminase

          1812

          serin protease

          2008

          transferase

          1022

          zinc-dependent protease

          1432

          tagatose-6-phosphate metabolism

          1973

          prolyl endopeptidase

          1967§

          oxidoreduktase

          1963§

          format hydrogenlyase

          1947

          acetamidase/formamidase

          1186

          multisensor hybrid histidine kinase

          1807

          iron-sulfur cluster repair di-iron protein

          1794

          GCN5-like N-acetyltransferases

          1764, 1785§

          L-carnitine-dehydratase*

          1314

          sucrose catabolism§

          1240

          glycogen phosphorylase

          1240

          FAD-dependent pyridine-nucleotide-disulphid-oxidoreductase

          1218

          anaerobic dimethyl-sulfoxide-reductase

          1186

          nitrilase/cyanid-hydratase/apolipoprotein N-acyltransferase

          1186

          thiol-disulfid exchange protein

          1976§

          carbonic-anhydrase§

          1116

          purine catabolism protein

          1812

          NAD-dependent aldehyde-dehydrogenase

          1764§

          Cell wall biogenesis or surface proteins

          Contig

          flagella gene cluster II*

          1803, 1891

          outer membrane protease

          1116

          small integrale membrane protein§

          1246

          perosamine synthetase

          1162

          surface antigen

          1162

          outer membrane protein

          1802, 1177

          pertactin-like protein

          1146

          TonB-dependent heme receptor

          1757

          phosphatidylinositol-diacylglycerol-lyase

          1423

          uridine-diphosphatoacetylglucosamine epimerase

          1878

          pili biogenesis and assembly

          1847, 1393, 1882

          cell wall-associated hydrolases

          1386§, 1192

          integral membrane protein CcmA

          1766

          lipopolysaccharide synthesis

          1162, 1878

          extracellular ligand-binding receptor

          1186

          lipoprotein

          1803

          Defense mechanisms/toxins

          Contig

          T3SS*

          1804, 1807

          bile salt/choloylglycin hydrolase (bsh)

          1088

          toxin-antitoxin system

          1946, 1930

          zonula occludens-toxin

          1920

          Rtx cluster rtxH, rtxC, rtxA

          1867§

          autotransporter cluster

          1177

          insecticidal toxins (TC-PAIYe)

          1579, 1975, 1854, 1855, 1856

          small toxic polypeptide

          1810

          hemolysin; hemolysin activator protein

          1785

          colicin-E2/pyocin S2 immunity protein operon

          1216

          antibiotic acetyltransferase

          1885§, 1921

          Proteins for DNA/RNA regulation and processing

          Contig

          transcriptional regulator

          1088*, 1894, 1885, 1825§, 1835§(2), 1764, 1557§, 1200§, 1171§, 1885(2) §, 1891, 1803, 1802

          RpiR-like transcriptional regulator

          1812

          CRP/FNR family transcriptional regulator*

          1088

          LuxR-like transcriptional regulator

          1921

          negative regulator of β-lactamase expression

          1384

          response regulator receiver protein

          1367, 1186

          methyltransferase

          1835, 1170

          DNA topoisomerase

          1958, 1225

          helix destabilizing protein

          1920

          superfamily I DNA helicase

          1825

          methionyl-tRNA-formyltransferase

          1162

          transcription repressor

          1920

          filamentation induced by cAMP protein Fic

          1088

          sensor protein with MASE1 domain

          1432

          NTP-binding protein

          1116, 1170, 1897, 1882, 1763, 1760, 1837

          Others

          Contig

          plasmid stabilisation system*

          1835

          resolvase

          1404, 1835

          secreted protein

          1211, 1456

          prophage region

          1280, 1796,1920*

          *absent in all Yersinia species sequenced until 12/2010

          §absent in Y. pestis and Y. pseudotuberculosis, but present in other Yersinia species

          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 [26]. 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 plasticity zone of strain 8081 ranges from YE3447 to YE3644 and has a total length of approximately 199 kb with 186 coding sequences (CDS). It was defined by Thomson et al. [18] as the largest region of species-specific genomic variation among Y. enterocolitica biotypes, and it is absent from Y. pestis and Y. pseudotuberculosis. Four contigs revealed to carry PZ genes. We linked contigs 1088/1891 and 1803/1802 due to the presence of truncated hypF and fepG genes, respectively, at their ends. The primer combination 5'-GTTTCTTTATGGGCGCG-3'/5'-TTGGCATGGAGGCCTG-3' hybridizing to the ends of contigs 1891 and 1803 resulted in a PCR product of approximately 1500 bp, thus allowing the linear reconstruction of the W22703-specific PZ (Figure 3). With a total length of ~ 142,000 kb, it is significantly shorter than that of 8081 and exhibits a comparably low density of virulence genes. Many discrete functional units of the 8081 PZ are indeed missing in the W22703 genome as confirmed by BLAST search of any PZ-encoded protein against the translated shotgun sequence. The most prominent ones are (i) the pathogenicity island YAPIYe of 66 CDS including a putative hemolysin, a toxin/antitoxin system ccdA/ccdB, and a type IV pilus operon, (ii) the T3SS ysa important for pathogenicity in an mouse oral infection model [27] and (iii) the T2SS yst1 required for full virulence [28]. Further 8081 PZ genes absent in W22703 are the two putative two component-systems (TCS) YE3561/YE3563 and YE3578/YE3579, a chitinase (YE3576), a putative lipase (YE3614), a putative copper/silver efflux system (YE3626-YE3630), and the arsenic resistance operon. However, it is also worthy of note that PZ loci assumed or known to play a role in pathogenicity towards invertebrates or vertebrates are present in the W22703 genome. Examples are the YGI-1 mentioned above, the hydrogenase 2- (hyb-) locus, fecBCDE encoding an iron transporter, the ferric enterbactin transport system fepBDGC/fes, and proP encoding a betain/proline transporter involved in osmoprotection and osmoregulation.
          http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-12-168/MediaObjects/12864_2010_3302_Fig3_HTML.jpg
          Figure 3

          Plasticity zone of strain W22703. Genes and encoded proteins: speF, ornithine decarboxylase; bsh, chologlycin or bile salt hydrolase; potE, putrescine-ornithine antiporter; mdtN, multidrug resistance protein; map, methionine aminopeptidase; ydfJ, putative metabolite transporter; yjbB, putative Na+/Pi-cotransporter; proP, betain/proline transporter; ydcL, putative lipoprotein; thiM, hydroxyethylthiazole kinase; fic, filamentation induced by cAMP protein; motA/motB, flagellar motor proteins; tnp, transposase; MACPII, methyl-accepting chemotaxis protein; fes, enterochelin esterase; virF, virulence regulon transcriptional activator; rep, IncF plasmid RepFIB replicon; alkB, α-ketoglutarate-dependent dioxygenase; exbB, MotA/TolQ/ExbB proton channel family protein; asterisks: low-temperature induced genes as identified by luciferase reportering [60]. Shaded regions are absent in 8081. See text for more details.

          The recently identified flagellar cluster Flg-2 [29] 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.

          Surface components

          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) [30]; 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 [30].

          Metabolism

          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 [18].

          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

          We identified few enzymes or capabilities that are missing in W22703 in comparison to 8081 (Table 3). Examples are the absence of a chitinase, and of the lipase YE3614 that is probably responsible for the lipase negative reaction of W22703 as a biotype 2 strain [31]. In addition to the arsenic resistance operon on YAPIYe, homologs of a second operon with this function (YE3364-YE3366) are absent in W22703.
          Table 3

          Predicted functions of genes present in 8081 and absent in W22703

          Transporter

          Gene

          copper/silver efflux system

          YE3626-YE3630

          multidrug efflux protein

          YE0443

          ABC transporter

          YE0801-YE0804; YE0818-YE0821

          sugar permease

          YE2605-YE2609

          phosphohistidinoprotein-hexose phosphotransferase component of PTS system (Hpr)

          YE1207

          glucoside specific PTS system

          YE2103-2107

          membrane transport protein

          YE2029, YE3695

          molybdate ABC transporter permease protein

          YE2912

          cation efflux system

          YE3628

          Bioenergetic proteins

          Gene

          sulfite reductase subunit protein

          YE1597

          NADH:flavin oxidoreductase/NADH oxidase family protein

          YE3034

          Conjugation/replication

          Gene

          conjugal transfer protein

          YE1177-YE1178

          type 4 prepilin-like proteins leader peptide processing enzyme

          YE3574

          integrating conjugative element protein

          YE3484, YE3495

          TraG-family protein

          YE3493

          Metabolism

          Gene

          chitinase

          YE3576

          lipase

          YE3614

          zinc metalloprotease

          YE4052

          hydrolase

          YE0306

          dehydrogenase/reductase

          YE0341, YE1369-YE1370

          aldo/keto reductase

          YE3031

          oxidoreductase

          YE0484

          D-serine dehydatase

          YE0800

          glycerol kinase

          YE0824

          UDP-sugar hydrolase/5'-nucleotidase periplasmic precursor

          YE3066

          acetyltransferase

          YE1087, YE2245

          fucose isomerase fucI

          YE0825

          Cell wall biogenesis or surface proteins

          Gene

          adhesin/invasin

          YE0694

          YGI-2 (putative glycolipoprotein)

          YE0894-YE0912

          type-1 fimbrial operon

          YE1111-YE1114

          type-4 pilus

          YE3498-YE3506

          ysaE (invasin)

          YE3549

          yadA

          YE1873

          lipopolysaccharide synthesis

          YE3070-3087

          lipoprotein

          YE0982, YE1234-YE1236, YE1871, YE3483, YE4156

          N-acetylglucosamine-binding protein

          YE3576 (chiY), YE2830, YE3411, YE3494A

          acyl-UDP-N-acetylglucosamine O-acyltransferase, lpxA2

          YE2286

          Defense mechanism/toxins

          Gene

          high pathogenicity island HPI (Yersiniabactin)

          YE2611-YE2622

          HlyA-like hemolysin and transporter (TPS)

          YE2407, YE2408

          hemolysin

          YE3454

          hemophore

          YE0125-YE0126

          heme acquisition system

          YE0123-YE0126

          T3SS effector protein

          YE2447

          autotransporter

          YE1372, YE2049

          ccdA/ccdB, toxin/antitoxin system

          YE3480-YE3481; YE2032-2033

          bifunctional antitoxin/transcriptional repressor RelB

          YE0510, YE0647

          arsenic resistance operon

          YE3364-YE3366; YE3472-YE3478

          metal resistance protein

          YE1299

          ysa, T3SS

          YE3533-YE3561

          yst1, T2SS

          YE3564-YE3575

          iron-sulfur cluster assembly protein

          YE1059

          metallo-β-lactamase superfamily protein

          YE2635

          cytonecrosis factor-like toxin

          YE2091

          actin-ADP-ribosylating toxin

          YE0115

          antibiotic biosynthesis monooxygenase

          YE0340

          Rho-activating domain of cytotoxic necrotizing factor

          YE2091

          Proteins for DNA/RNA regulation and processing

          Gene

          TCS

          YE3561/YE3563; YE3578/YE3579

          LuxR family transcription regulatory protein

          YE0039, YE0343, YE1026, YE1165, YE2050-YE2051, YE3033

          transcriptional regulatory protein

          YE0826, YE4110

          DNA-binding protein

          YE1102, YE1172, YE1825, YE3496A, YE4129

          Cro/CI family transcriptional regulator

          YE1175A

          plasmid-related transcriptional repressor protein

          YE1182

          GntR-family regulatory protein

          YE2670

          DNA helicase

          YE3514

          DEAD-like helicase

          YE2632

          Others

          Gene

          YGI-3, putative integrated plasmid

          YE0975-YE0993

          YGI-4, putative integrated plasmid

          YE1170-YE1183

          YAPIYe

          YE3450-YE3515

          prophage region YE98

          YE0854-YE0888

          prophage region YE185

          YE1667-YE1693

          prophage region YE200

          YE1799-YE1819

          prophage region YE250

          YE2292-YE2363

          zinc-binding protein

          YE0683

          ATP/GTP-binding protein

          YE0986, YE0990, YE3513

          plasmid stabilisation protein

          YE0510A, YE1110

          chromosome partitioning system

          YE3515

          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 [18] 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.

          Discussion

          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 [17].

          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 [32], and confirms the importance of this region for the manifestation of virulence properties of Y. enterocolitica [18].

          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 [33]. Since we used a pYV-free W22703 derivative to demonstrate TC-based insecticidal activity of this strain [13], 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 [34]. 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" [35].

          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 [36]. 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 [37].

          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 [38]. 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 [4143]. 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 [46].

          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 [37]. 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.

          Conclusion

          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.

          Methods

          Bacterial strains and growth conditions

          Y. enterocolitica W22703(pYVe227) is a nalidixic acid-resistant (NalR) restriction mutant (Res-Mod') isolated from strain W227 [47]. 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 [48]. 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 [49], 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; [50]) was used for automatic genome sequence analysis and annotation [16]. Protein coding genes were predicted using the GeneMarkS software program using default settings [51]. Biochemical pathway prediction and reconstruction were performed using the KEGG [52], BRENDA [53], and Microbes online [54] databases. tRNAs were identified using tRNAscanSE [55], rRNA homologs with blastn [56]. 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 [57]. Phylogenetic trees for ROD and T3SS have been automatically calculated using the the software PhyloGenie [58] and the default parameters according to its documentation. We used NCBI nr [59] as reference database and excluded proteins of unclassified taxa.

          Abbreviations

          ABC transporter: 

          ATP-binding cassette transporter

          DMSO: 

          dimethylsulfoxide

          LGT: 

          lateral gene transfer

          PTS: 

          phosphotransferase system

          ROD: 

          region(s) of difference

          TCS: 

          two-partner secretion system

          T3SS: 

          type three secretion system

          T2SS: 

          type two secretion system

          TCS: 

          two component system

          YGI: 

          Yersinia genome island

          Declarations

          Acknowledgements

          We thank Siegfried Scherer for generous support of this study. This work was in part funded by the Deutsche Forschungsgemeinschaft (FU 375/4-1).

          Authors’ Affiliations

          (1)
          Lehrstuhl für Mikrobielle Ökologie, Department Biowissenschaften, Wissenschaftszentrum Weihenstephan, Technische Universität München
          (2)
          Department of Computational Systems Biology, University of Vienna

          References

          1. Sulakvelidze A: Yersiniae other than Y. enterocolitica , Y. pseudotuberculosis , and Y. pestis : the ignored species. Microbes Infect 2000, 2:497–513.PubMedView Article
          2. Sprague LD, Neubauer H: Yersinia aleksiciae sp. nov. Int J Syst Evol Microbiol 2005, 55:831–835.PubMedView Article
          3. Sprague LD, Scholz HC, Amann S, Busse HJ, Neubauer H: Yersinia similis sp. nov. Int J Syst Evol Microbiol 2008, 58:952–958.PubMedView Article
          4. Merhej V, Adekambi T, Pagnier I, Raoult D, Drancourt M: Yersinia massiliensis sp. nov., isolated from fresh water. Int J Syst Evol Microbiol 2008, 58:779–784.PubMedView Article
          5. Hurst MR, Becher SA, Young SD, Nelson TL, Glare TR: Yersinia entomophaga sp. nov. isolated from the New Zealand grass grub Costelytra zealandica . Int J Syst Evol Microbiol 2010.
          6. Murros-Kontiainen AE, Fredriksson-Ahomaa M, Korkeala H, Johansson P, Rahkila R, Bjorkroth J: Yersinia nurmii sp. nov. Int J Syst Evol Microbiol 2010.
          7. Murros-Kontiainen AE, Johansson P, Niskanen T, Fredriksson-Ahomaa M, Korkeala H, Bjorkroth J: Yersinia pekkanenii sp. nov. Int J Syst Evol Microbiol 2010.
          8. Bottone EJ: Yersinia enterocolitica : overview and epidemiologic correlates. Microbes Infect 1999, 1:323–333.PubMedView Article
          9. Wauters G, Kandolo K, Janssens M: Revised biogrouping scheme of Yersinia enterocolitica . Contrib Microbiol Immunol 1987, 9:14–21.PubMed
          10. Tennant SM, Grant TH, Robins-Browne RM: Pathogenicity of Yersinia enterocolitica biotype 1A. FEMS Immunol Med Microbiol 2003, 38:127–137.PubMedView Article
          11. Schubert S, Rakin A, Karch H, Carniel E, Heesemann J: Prevalence of the "high-pathogenicity island" of Yersinia species among Escherichia coli strains that are pathogenic to humans. Infect Immun 1998, 66:480–485.PubMed
          12. Wren BW: The yersiniae - a model genus to study the rapid evolution of bacterial pathogens. Nat Rev Microbiol 2003, 1:55–64.PubMedView Article
          13. Bresolin G, Morgan JA, Ilgen D, Scherer S, Fuchs TM: Low temperature-induced insecticidal activity of Yersinia enterocolitica . Mol Microbiol 2006, 59:503–512.PubMedView Article
          14. Spanier B, Starke M, Higel F, Scherer S, Fuchs TM: Yersinia enterocolitica infection and tcaA -dependent killing of Caenorhabditis elegans . Appl Environ Microbiol 2010, 76:6277–6285.PubMedView Article
          15. Fuchs TM, Bresolin G, Marcinowski L, Schachtner J, Scherer S: Insecticidal genes of Yersinia spp.: taxonomical distribution, contribution to toxicity towards Manduca sexta and Galleria mellonella , and evolution. BMC Microbiol 2008, 8:214.PubMedView Article
          16. Frishman D, Albermann K, Hani J, Heumann K, Metanomski A, Zollner A, Mewes HW: Functional and structural genomics using PEDANT. Bioinformatics 2001, 17:44–57.PubMedView Article
          17. Chen PE, Cook C, Stewart AC, Nagarajan N, Sommer DD, Pop M, Thomason B, Thomason MP, Lentz S, Nolan N, et al.: Genomic characterization of the Yersinia genus. Genome Biol 2010, 11:R1.PubMedView Article
          18. Thomson NR, Howard S, Wren BW, Holden MT, Crossman L, Challis GL, Churcher C, Mungall K, Brooks K, Chillingworth T, et al.: The complete genome sequence and comparative genome analysis of the high pathogenicity Yersinia enterocolitica strain 8081. PLoS Genet 2006, 2:e206.PubMedView Article
          19. Wang X, Li Y, Jing H, Ren Y, Zhou Z, Wang S, Kan B, Xu J, Wang L: Complete genome sequence of a Yersinia enterocolitica "Old World" (3/O:9) strain and comparison with the "New World" (1B/O:8) strain. J Clin Microbiol 2011.
          20. Batzilla J, Hoper D, Antonenka U, Heesemann J, Rakin A: Complete genome sequence of Yersinia enterocolitica subsp. palearctica serogroup O:3. J Bacteriol 2011.
          21. Darling AC, Mau B, Blattner FR, Perna NT: Mauve: multiple alignment of conserved genomic sequence with rearrangements. Genome Res 2004, 14:1394–1403.PubMedView Article
          22. Mulder B, Michiels T, Simonet M, Sory MP, Cornelis G: Identification of additional virulence determinants on the pYV plasmid of Yersinia enterocolitica W227. Infect Immun 1989, 57:2534–2541.PubMed
          23. Hacker J, Blum-Öhler G, Mühldorfer I, Tschape H: Pathogenicity islands of virulent bacteria: structure, function and impact on microbial evolution. Mol Microbiol 1997, 23:1089–1097.PubMedView Article
          24. Carniel E, Guilvout I, Prentice M: Characterization of a large chromosomal "high-pathogenicity island" in biotype 1B Yersinia enterocolitica . J Bacteriol 1996, 178:6743–6751.PubMed
          25. Tennant SM, Skinner NA, Joe A, Robins-Browne RM: Homologues of insecticidal toxin complex genes in Yersinia enterocolitica biotype 1A and their contribution to virulence. Infect Immun 2005, 73:6860–6867.PubMedView Article
          26. Cornelis GR: The Yersinia Ysc-Yop 'type III' weaponry. Nat Rev Mol Cell Biol 2002, 3:742–752.PubMedView Article
          27. Haller JC, Carlson S, Pederson KJ, Pierson DE: A chromosomally encoded type III secretion pathway in Yersinia enterocolitica is important in virulence. Mol Microbiol 2000, 36:1436–1446.PubMedView Article
          28. Iwobi A, Heesemann J, Garcia E, Igwe E, Noelting C, Rakin A: Novel virulence-associated type II secretion system unique to high-pathogenicity Yersinia enterocolitica . Infect Immun 2003, 71:1872–1879.PubMedView Article
          29. Bresolin G, Trcek J, Scherer S, Fuchs TM: Presence of a functional flagellar cluster Flag-2 and low-temperature expression of flagellar genes in Yersinia enterocolitica W22703. Microbiology 2008, 154:196–206.PubMedView Article
          30. Skurnik M, Biedzka-Sarek M, Lubeck PS, Blom T, Bengoechea JA, Perez-Gutierrez C, Ahrens P, Hoorfar J: Characterization and biological role of the O-polysaccharide gene cluster of Yersinia enterocolitica serotype O:9. J Bacteriol 2007, 189:7244–7253.PubMedView Article
          31. Robins-Browne RM: Yersinia enterocolitica. In Food microbiology - Fundamentals and frontiers. Edited by: Doyle MP, Beuchat LR, Montville TJ. Washington D.C., ASM Press; 1997:192–215.
          32. Sory MP, Cornelis G: Yersinia enterocolitica O:9 as a potential live oral carrier for protective antigens. Microb Pathog 1988, 4:431–442.PubMedView Article
          33. Gendlina I, Held KG, Bartra SS, Gallis BM, Doneanu CE, Goodlett DR, Plano GV, Collins CM: Identification and type III-dependent secretion of the Yersinia pestis insecticidal-like proteins. Mol Microbiol 2007, 64:1214–1227.PubMedView Article
          34. ffrench-Constant R, Waterfield N, Daborn P, Joyce S, Bennett H, Au C, Dowling A, Boundy S, Reynolds S, Clarke D: Photorhabdus : towards a functional genomic analysis of a symbiont and pathogen. FEMS Microbiol Rev 2003, 26:433–456.PubMedView Article
          35. Burland V, Shao Y, Perna NT, Plunkett G, Sofia HJ, Blattner FR: The complete DNA sequence and analysis of the large virulence plasmid of Escherichia coli O157:H7. Nucleic Acids Res 1998, 26:4196–4204.PubMedView Article
          36. Thomson NR, Clayton DJ, Windhorst D, Vernikos G, Davidson S, Churcher C, Quail MA, Stevens M, Jones MA, Watson M, et al.: Comparative genome analysis of Salmonella Enteritidis PT4 and Salmonella Gallinarum 287/91 provides insights into evolutionary and host adaptation pathways. Genome Res 2008, 18:1624–1637.PubMedView Article
          37. Heermann R, Fuchs TM: Comparative analysis of the Photorhabdus luminescens and the Yersinia enterocolitica genomes: uncovering candidate genes involved in insect pathogenicity. BMC Genomics 2008, 9:40.PubMedView Article
          38. Winter SE, Thiennimitr P, Winter MG, Butler BP, Huseby DL, Crawford RW, Russell JM, Bevins CL, Adams LG, Tsolis RM, et al.: Gut inflammation provides a respiratory electron acceptor for Salmonella . Nature 2010, 467:426–429.PubMedView Article
          39. Maier RJ, Olczak A, Maier S, Soni S, Gunn J: Respiratory hydrogen use by Salmonella enterica serovar Typhimurium is essential for virulence. Infect Immun 2004, 72:6294–6299.PubMedView Article
          40. Olson JW, Maier RJ: Molecular hydrogen as an energy source for Helicobacter pylori . Science 2002, 298:1788–1790.PubMedView Article
          41. Kunz DA, Wang CS, Chen JL: Alternative routes of enzymic cyanide metabolism in Pseudomonas fluorescens NCIMB 11764. Microbiology 1994, 140:1705–1712.PubMedView Article
          42. Zagrobelny M, Bak S, Ekstrom CT, Olsen CE, Moller BL: The cyanogenic glucoside composition of Zygaena filipendulae (Lepidoptera: Zygaenidae) as effected by feeding on wild-type and transgenic lotus populations with variable cyanogenic glucoside profiles. Insect Biochem Mol Biol 2007, 37:10–18.PubMedView Article
          43. Zagrobelny M, Bak S, Olsen CE, Moller BL: Intimate roles for cyanogenic glucosides in the life cycle of Zygaena filipendulae (Lepidoptera, Zygaenidae). Insect Biochem Mol Biol 2007, 37:1189–1197.PubMedView Article
          44. Bertelsen H, Andersen H, Tvede M: Fermentation of D-tagatose by human intestinal bacteria and diary lactic acid bacteria. Microbial Ecology in Health and Disease 2001, 13:87–95.View Article
          45. Münch A, Stingl L, Jung K, Heermann R: Photorhabdus luminescens genes induced upon insect infection. BMC Genomics 2008, 9:229.PubMedView Article
          46. Jones BV, Begley M, Hill C, Gahan CG, Marchesi JR: Functional and comparative metagenomic analysis of bile salt hydrolase activity in the human gut microbiome. Proc Natl Acad Sci USA 2008, 105:13580–13585.PubMedView Article
          47. Cornelis G, Colson C: Restriction of DNA in Yersinia enterocolitica detected by recipient ability for a derepressed R factor from Escherichia coli . J Gen Microbiol 1975, 87:285–291.PubMed
          48. Sambrook J, Russell DW: Molecular cloning: a laboratory manual. 3rd edition. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y; 2001.
          49. Chain PS, Grafham DV, Fulton RS, Fitzgerald MG, Hostetler J, Muzny D, Ali J, Birren B, Bruce DC, Buhay C, et al.: Genomics. Genome project standards in a new era of sequencing. Science 2009, 326:236–237.PubMedView Article
          50. Walter MC, Rattei T, Arnold R, Guldener U, Munsterkotter M, Nenova K, Kastenmuller G, Tischler P, Wolling A, Volz A, et al.: PEDANT covers all complete RefSeq genomes. Nucleic Acids Res 2009, 37:D408–411.PubMedView Article
          51. Besemer J, Lomsadze A, Borodovsky M: GeneMarkS: a self-training method for prediction of gene starts in microbial genomes. Implications for finding sequence motifs in regulatory regions. Nucleic Acids Res 2001, 29:2607–2618.PubMedView Article
          52. Kanehisa M, Araki M, Goto S, Hattori M, Hirakawa M, Itoh M, Katayama T, Kawashima S, Okuda S, Tokimatsu T, Yamanishi Y: KEGG for linking genomes to life and the environment. Nucleic Acids Res 2008, 36:D480–484.PubMedView Article
          53. Barthelmes J, Ebeling C, Chang A, Schomburg I, Schomburg D: BRENDA, AMENDA and FRENDA: the enzyme information system in 2007. Nucleic Acids Res 2007, 35:D511–514.PubMedView Article
          54. Dehal PS, Joachimiak MP, Price MN, Bates JT, Baumohl JK, Chivian D, Friedland GD, Huang KH, Keller K, Novichkov PS, et al.: MicrobesOnline: an integrated portal for comparative and functional genomics. Nucleic Acids Res 38:D396–400.
          55. Lowe TM, Eddy SR: tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res 1997, 25:955–964.PubMedView Article
          56. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997, 25:3389–3402.PubMedView Article
          57. Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994, 22:4673–4680.PubMedView Article
          58. Frickey T, Lupas AN: PhyloGenie: automated phylome generation and analysis. Nucleic Acids Res 2004, 32:5231–5238.PubMedView Article
          59. Sayers EW, Barrett T, Benson DA, Bolton E, Bryant SH, Canese K, Chetvernin V, Church DM, DiCuccio M, Federhen S, et al.: Database resources of the National Center for Biotechnology Information. Nucleic Acids Res 2011, 39:D38–51.PubMedView Article
          60. Bresolin G, Neuhaus K, Scherer S, Fuchs TM: Transcriptional analysis of long-term adaptation of Yersinia enterocolitica to low-temperature growth. J Bacteriol 2006, 188:2945–2958.PubMedView Article

          Copyright

          © Fuchs et al; licensee BioMed Central Ltd. 2011

          This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

          Advertisement