A poly-proline II helix in YadA from Yersinia enterocolitica serotype O:9 facilitates heparin binding through electrostatic interactions.

Poly-proline II helices are secondary structure motifs frequently found in ligand-binding sites. They exhibit increased flexibility and solvent exposure compared to the strongly hydrogen-bonded α-helices or ß-strands and can therefore easily be misinterpreted as completely unstructured regions with an extremely high rotational freedom. Here, we show that the adhesin YadA of Yersinia enterocolitica serotype O:9 contains a poly-proline II helix interaction motif in the N-terminal region. The motif is involved in the interaction of YadAO:9 with heparin, a host glycosaminoglycan. We show that the basic residues within the N-terminal motif of YadA are required for electrostatic interactions with the sulfate groups of heparin. Biophysical methods including CD spectroscopy, solution-state NMR and SAXS all independently support the presence of a poly-proline helix allowing YadAO:9 binding to the rigid heparin. Lastly, we show that host cells deficient in sulfation of heparin and heparan sulfate are not targeted by YadAO:9 -mediated adhesion. We speculate that the YadAO:9 -heparin interaction plays an important and highly strain-specific role in the pathogenicity of Yersinia enterocolitica serotype O:9.

Structure of the Yersinia injectisome in intracellular host cell phagosomes revealed by cryo FIB electron tomography.

Many pathogenic bacteria use the type III secretion system (T3SS), or injectisome, to secrete toxins into host cells. These protruding systems are primary targets for drug and vaccine development. Upon contact between injectisomes and host membranes, toxin secretion is triggered. How this works structurally and functionally is yet unknown. Using cryo-focused ion beam milling and cryo-electron tomography, we visualized injectisomes of Yersinia enterocolitica inside the phagosomes of infected human myeloid cells in a close-to-native state. We observed that a minimum needle length is required for injectisomes to contact the host membrane and bending of host membranes by some injectisomes that contact the host. Through subtomogram averaging, the structure of the entire injectisome was determined, which revealed structural differences in the cytosolic sorting platform compared to other bacteria. These findings contribute to understanding how injectisomes secrete toxins into host cells and provides the indispensable native context. The application of these cryo-electron microscopy techniques paves the way for the study of the 3D structure of infection-relevant protein complexes in host-pathogen interactions.

Growth of Yersinia pseudotuberculosis Strains at Different Temperatures, pH Values, and NaCl and Ethanol Concentrations.

Maximum growth temperature and growth limits in Luria-Bertani broth at different pH values and NaCl and ethanol concentrations were determined for 49 Yersinia pseudotuberculosis strains representing serotypes O:1, O:2, O:3, O:4, and O:5. In addition, the ability of the strains to grow at 0°C and the growth parameters at 1°C were determined. The maximum growth temperatures measured by Gradiplate temperature incubator varied between 42.2 and 43.7°C. All strains were able to grow at 0°C in Luria-Bertani broth within 17 days of incubation. At 1°C, differences were observed among strains in the maximum growth rates and area under the curve values based on optical density data, which suggests that some Y. pseudotuberculosis strains adapt faster to colder conditions. The mean maximum growth rates and area under the curve values at 1°C, as well as the mean maximum growth temperatures, were statistically significantly higher among serotype O:1 strains compared with O:3 strains and among biotype 1 compared with biotype 2 strains. All strains grew at pH 4.5, whereas none of the strains were able to grow at pH 4.2. The highest pH at which growth was observed varied between 9.0 and 9.3. For 14 strains the maximum NaCl concentration at which growth was observed was 4.8%, whereas 35 of the strains were able to grow at 5.0% NaCl. None of the strains showed growth at 5.2% NaCl. All strains were able to grow at 4.5% ethanol concentration (v/v), whereas 5.0% ethanol concentration was completely inhibitory to all strains. The observed limited physiological diversity among various Y. pseudotuberculosis strains may stem from the genetic homogeneity of the species.

Bacterial LPX motif-harboring virulence factors constitute a species-spanning family of cell-penetrating effectors.

Effector proteins are key virulence factors of pathogenic bacteria that target and subvert the functions of essential host defense mechanisms. Typically, these proteins are delivered into infected host cells via the type III secretion system (T3SS). Recently, however, several effector proteins have been found to enter host cells in a T3SS-independent manner thereby widening the potential range of these virulence factors. Prototypes of such bacteria-derived cell-penetrating effectors (CPEs) are the Yersinia enterocolitica-derived YopM as well as the Salmonella typhimurium effector SspH1. Here, we investigated specifically the group of bacterial LPX effector proteins comprising the Shigella IpaH proteins, which constitute a subtype of the leucine-rich repeat protein family and share significant homologies in sequence and structure. With particular emphasis on the Shigella-effector IpaH9.8, uptake into eukaryotic cell lines was shown. Recombinant IpaH9.8 (rIpaH9.8) is internalized via endocytic mechanisms and follows the endo-lysosomal pathway before escaping into the cytosol. The N-terminal alpha-helical domain of IpaH9.8 was identified as the protein transduction domain required for its CPE ability as well as for being able to deliver other proteinaceous cargo. rIpaH9.8 is functional as an ubiquitin E3 ligase and targets NEMO for poly-ubiquitination upon cell penetration. Strikingly, we could also detect other recombinant LPX effector proteins from Shigella and Salmonella intracellularly when applied to eukaryotic cells. In this study, we provide further evidence for the general concept of T3SS-independent translocation by identifying novel cell-penetrating features of these LPX effectors revealing an abundant species-spanning family of CPE.

Structural Insight into Inhibition of CsrA-RNA Interaction Revealed by Docking, Molecular Dynamics and Free Energy Calculations.

The carbon storage regulator A (CsrA) and its homologs play an important role in coordinating the expression of bacterial virulence factors required for successful host infection. In addition, bacterial pathogens with deficiency of CsrA are typically attenuated for virulence. In 2016, the first series of small-molecule inhibitors of CsrA-RNA interaction were identified, which were found to achieve the CsrA-RNA inhibition by binding to the CsrA, without interfering with the RNA. However, the binding mechanism of these inhibitors of CsrA is not known. Herein, we applied molecular docking, molecular dynamics and binding free energy calculations to investigate the binding mode of inhibitors to CsrA. We found that the G11(RNA)-binding site is the most important binding site for CsrA inhibitors. An inhibitor with the proper size range can bind to that site and form a stable complex. We also found that inhibitors with larger size ranges bind to the entire CsrA-RNA interface, but have loose binding. However, this loose binding still resulted in inhibitory activity. The calculated binding free energy from MM/GBSA has a good correlation with the derived experimental binding energy, which might be used as a tool to further select CsrA inhibitors after a first-round of high-throughput virtual screening.

X-ray crystal structures of the pheromone-binding domains of two quorum-hindered transcription factors, YenR of Yersinia enterocolitica and CepR2 of Burkholderia cenocepacia.

The ability of LuxR-type proteins to regulate transcription is controlled by bacterial pheromones, N-acylhomoserine lactones (AHLs). Most LuxR-family proteins require their cognate AHLs for activity, and some of them require AHLs for folding and stability, and for protease-resistance. However, a few members of this family are able to fold, dimerize, bind DNA, and regulate transcription in the absence of AHLs; moreover, these proteins are antagonized by their cognate AHLs. One such protein is YenR of Yersinia enterocolitica, which is antagonized by N-3-oxohexanoyl-l-homoserine lactone (OHHL). This pheromone is produced by the OHHL synthase, a product of the adjacent yenI gene. Another example is CepR2 of Burkholderia cenocepacia, which is antagonized by N-octanoyl-l-homoserine lactone (OHL), whose synthesis is directed by the cepI gene of the same bacterium. Here, we describe the high-resolution crystal structures of the AHL binding domains of YenR and CepR2. YenR was crystallized in the presence and absence of OHHL. While this ligand does not cause large scale changes in the YenR structure, it does alter the orientation of several highly conserved YenR residues within and near the pheromone-binding pocket, which in turn caused a significant movement of a surface-exposed loop.

Identifying components required for OMP biogenesis as novel targets for antiinfective drugs.

The emergence of multiresistant Gram-negative bacteria requires new therapies for combating bacterial infections. Targeting the biogenesis of virulence factors could be an alternative strategy instead of killing bacteria with antibiotics. The outer membrane (OM) of Gram-negative bacteria acts as a physical barrier. At the same time it facilitates the exchange of molecules and harbors a multitude of proteins associated with virulence. In order to insert proteins into the OM, an essential oligomeric membrane-associated protein complex, the ß-barrel assembly machinery (BAM) is required. Being essential for the biogenesis of outer membrane proteins (OMPs) the BAM and also periplasmic chaperones may serve as attractive targets to develop novel antiinfective agents. Herein, we aimed to elucidate which proteins belonging to the OMP biogenesis machinery have the most important function in granting bacterial fitness, OM barrier function, facilitating biogenesis of dedicated virulence factors and determination of overall virulence. To this end we used the enteropathogen Yersinia enterocolitica as a model system. We individually knocked out all non-essential components of the BAM (BamB, C and E) as well as the periplasmic chaperones DegP, SurA and Skp. In summary, we found that the most profound phenotypes were produced by the loss of BamB or SurA with both knockouts resulting in significant attenuation or even avirulence of Ye in a mouse infection model. Thus, we assume that both BamB and SurA are promising targets for the development of new antiinfective drugs in the future.

Interactions between the Cytoplasmic Domains of PspB and PspC Silence the Yersinia enterocolitica Phage Shock Protein Response.

The phage shock protein (Psp) system is a widely conserved cell envelope stress response that is essential for the virulence of some bacteria, including Yersinia enterocolitica Recruitment of PspA by the inner membrane PspB-PspC complex characterizes the activated state of this response. The PspB-PspC complex has been proposed to be a stress-responsive switch, changing from an OFF to an ON state in response to an inducing stimulus. In the OFF state, PspA cannot access its binding site in the C-terminal cytoplasmic domain of PspC (PspCCT), because this site is bound to PspB. PspC has another cytoplasmic domain at its N-terminal end (PspCNT), which has been thought to play a role in maintaining the OFF state, because its removal causes constitutive activation. However, until now, this role has proved recalcitrant to experimental investigation. Here, we developed a combination of approaches to investigate the role of PspCNT in Y. enterocolitica Pulldown assays provided evidence that PspCNT mediates the interaction of PspC with the C-terminal cytoplasmic domain of PspB (PspBCT) in vitro Furthermore, site-specific oxidative cross-linking suggested that a PspCNT-PspBCT interaction occurs only under noninducing conditions in vivo Additional experiments indicated that mutations in pspC might cause constitutive activation by compromising this PspCNT binding site or by causing a conformational disturbance that repositions PspCNT in vivo These findings have provided the first insight into the regulatory function of the N-terminal cytoplasmic domain of PspC, revealing that its ability to participate in an inhibitory complex is essential to silencing the Psp response. IMPORTANCE: The phage shock protein (Psp) response has generated widespread interest because it is linked to important phenotypes, including antibiotic resistance, biofilm formation, and virulence in a diverse group of bacteria. Therefore, achieving a comprehensive understanding of how this response is controlled at the molecular level has obvious significance. An integral inner membrane protein complex is believed to be a critical regulatory component that acts as a stress-responsive switch, but some essential characteristics of the switch states are poorly understood. This study provides an important advance by uncovering a new protein interaction domain within this membrane protein complex that is essential to silencing the Psp response in the absence of an inducing stimulus.

The YsrS Paralog DygS Has the Capacity To Activate Expression of the Yersinia enterocolitica Ysa Type III Secretion System.

UNLABELLED: The Yersinia enterocolitica Ysa type III secretion system (T3SS) is associated with intracellular survival, and, like other characterized T3SSs, it is tightly controlled. Expression of the ysa genes is only detected following growth at low temperatures (26°C) and in high concentrations of sodium chloride (290 mM) in the medium. The YsrSTR phosphorelay (PR) system is required for ysa expression and likely responds to NaCl. During our investigations into the Ysr PR system, we discovered that genes YE3578 and YE3579 are remarkably similar to ysrR and ysrS, respectively, and are probably a consequence of a gene duplication event. The amino acid differences between YE3578 and ysrR are primarily clustered into two short regions. The differences between YE3579 and ysrS are nearly all located in the periplasmic sensing domain; the cytoplasmic domains are 98% identical. We investigated whether these paralogs were capable of activating ysa gene expression. We found that the sensor paralog, named DygS, is capable of compensating for loss of ysrS, but the response regulator paralog, DygR, cannot complement a ysrR gene deletion. In addition, YsrR, but not DygR, interacts with the histidine phosphorelay protein YsrT. Thus, DygS likely activates ysa gene expression in response to a signal other than NaCl and provides an example of a phosphorelay system in which two sensor kinases feed into the same regulatory pathway. IMPORTANCE: All organisms need mechanisms to promote survival in changing environments. Prokaryotic phosphorelay systems are minimally comprised of a histidine kinase (HK) that senses an extracellular stimulus and a response regulator (RR) but can contain three or more proteins. Through gene duplication, a unique hybrid HK was created. We show that, while the hybrid appears to retain all of the phosphorelay functions, it responds to a different signal than the original. Both HKs transmit the signal to the same RR, which activates a promoter that transcribes a set of genes encoding a type III secretion system (T3SS) whose function is not yet evident. The significance of this work lies in finding that two HKs regulate this T3SS, highlighting its importance.

Solid-state NMR Study of the YadA Membrane-Anchor Domain in the Bacterial Outer Membrane.

MAS-NMR was used to study the structure and dynamics at ambient temperatures of the membrane-anchor domain of YadA (YadA-M) in a pellet of the outer membrane of E. coli in which it was expressed. YadA is an adhesin from the pathogen Yersinia enterocolitica that is involved in interactions with the host cell, and it is a model protein for studying the autotransport process. Existing assignments were sucessfully transferred to a large part of the YadA-M protein in the E. coli lipid environment by using (13) C-(13) C DARR and PDSD spectra at different mixing times. The chemical shifts in most regions of YadA-M are unchanged relative to those in microcrystalline YadA-M preparations from which a structure has previously been solved, including the ASSA region that is proposed to be involved in transition-state hairpin formation for transport of the soluble domain. Comparisons of the dynamics between the microcrystalline and membrane-embedded samples indicate greater flexibility of the ASSA region in the outer-membrane preparation at physiological temperatures. This study will pave the way towards MAS-NMR structure determination of membrane proteins, and a better understanding of functionally important dynamic residues in native membrane environments.

Conserved structure and domain organization among bacterial Slc26 transporters.

The Slc26 proteins are a ubiquitous superfamily of anion transporters conserved from bacteria to humans, among which four have been identified as human disease genes. Our functional knowledge of this protein family has increased but limited structural information is available. These proteins contain a transmembrane (TM) domain and a C-terminal cytoplasmic sulfate transporter and anti-sigma factor (STAS) domain. In a fundamental step towards understanding the structure/function relationships within the family we have used small-angle neutron scattering (SANS) on two distantly related bacterial homologues to show that there is a common, dimeric and structural architecture among Slc26A transporters. Pulsed electron-electron double resonance (PELDOR) spectroscopy supports the dimeric SANS-derived model. Using chimaeric/truncated proteins we have determined the domain organization: the STAS domains project away from the TM core and are essential for protein stability. We use the SANS-generated envelopes to assess a homology model of the TM core.

Bioinformatics analysis of a non-specific nuclease from Yersinia enterocolitica subsp. palearctica.

In this paper, the physical and chemical characteristics, biological structure and function of a non-specific nuclease from Yersinia enterocolitica subsp. palearctica (Y. NSN) found in our group were studied using multiple bioinformatics approaches. The results showed that Y. NSN had 283 amino acids, a weight of 30,692.5 ku and a certain hydrophilic property. Y. NSN had a signal peptide, no transmembrane domains and disulphide bonds. Cleavage site in Y. NSN was between pos. 23 and 24. The prediction result of the secondary structure showed Y. NSN was a coil structure-based protein. The ratio of α-helix, ß-folded and random coil were 18.73%, 16.96% and 64.31%, respectively. Active sites were pos. 124, 125, 127, 157, 165 and 169. Mg(2+) binding site was pos. 157. Substrate binding sites were pos. 124, 125 and 169. The analysis of multisequencing alignment and phylogenetic tree indicated that Y. NSN shared high similarity with the nuclease from Y. enterocolitica subsp. enterocolitica 8081. The enzyme activity results showed that Y. NSN was a nuclease with good thermostability.

Rapid subtyping of Yersinia enterocolitica by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for diagnostics and surveillance.

In this study, an alternative to the current traditional bioserotyping techniques was developed for subtyping Y. enterocolitica using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The most common pathogenic bioserotypes could easily be distinguished using only a few bioserotype-specific biomarkers. However, biochemical methods should still be used to distinguish biotype 1A from 1B.

OmpC-like porin from outer membrane of Yersinia enterocolitica: molecular structure and functional activity.

OmpC-like porin was isolated from the outer membrane (OM) of Yersinia enterocolitica cultured at 37°C (the "warm" variant) and its physicochemical and functional properties were studied. The amino acid sequence of OmpC porin was established, and the primary structure and transmembrane topology of this protein were analyzed in comparison with the OmpF porin isolated from Y. enterocolitica cultured at 6°C (the "cold" variant). Both porins of Y. enterocolitica had a high homology degree (65%) between themselves and with OmpC and OmpF porins from OM of Escherichia coli (58 and 76% homology, respectively). The secondary structure of OmpC and OmpF porins from OM of Y. enterocolitica consists of 16 ß-strands connected by short "periplasmic" and longer "extracellular" loops with disordered structure, according to the topological model developed for porins of E. coli. The molecular structures of OmpC and OmpF porins of Y. enterocolitica have significant differences in the structure of the "extracellular" loops and in the position of one of three tryptophan residues. Using the bilayer lipid membrane (BLM) technique, pores formed by OmpC porin of Y. enterocolitica were shown to differ in electrophysiological characteristics from channels of OmpF protein of this microorganism. The isolated OmpC porin reconstructed into BLM displayed functional plasticity similarly to OmpF protein and nonspecific porins of other enterobacteria. The conductivity level of the channels formed by this protein in the BLM was regulated by value of the applied potential.

Enterobacterial common antigen and O-specific polysaccharide coexist in the lipopolysaccharide of Yersinia enterocolitica serotype O:3.

Yersinia enterocolitica serotype O : 3 produces two types of lipopolysaccharide (LPS) molecules to its surface. In both types the lipid A (LA) structure is substituted by inner core (IC) octasaccharide to which either outer core (OC) hexasaccharide or homopolymeric O-polysaccharide (OPS) is linked. In addition, enterobacterial common antigen (ECA) can be covalently linked to LPS, however, via an unknown linkage. To elucidate the relationship between ECA and LPS in Y. enterocolitica O : 3 and the effect of temperature on their expression, LPS was isolated from bacteria grown at 22 °C and 37 °C by consequent hot phenol/water and phenol-chloroform-light petroleum extractions to obtain LPS preparations free of ECA linked to glycerophospholipid. In immunoblotting, monoclonal antibodies TomA6 and 898, specific for OPS and ECA, respectively, reacted both with ladder-like bands and with a slower-migrating smear suggesting that the ECA and OPS epitopes coexist on the same molecules. These results were supported by immunoblotting with a monovalent Y. enterocolitica O : 3 ECA-specific rabbit antiserum. Also, two or three 898-positive (and monovalent-positive) TomA6-negative bands migrated at the level of the LA-IC band in LPS samples from certain OC mutants, most likely representing LA-IC molecules carrying 1-3 ECA repeat units but no OPS. These bands were also present in Y. enterocolitica O : 9 OC mutants; however, coexistence of ECA and OPS in the same molecules could not be detected. Finally, the LA-IC-ECA bands were missing from LPS of bacteria grown at 37 °C and also the general reduction in wild-type bacteria of ECA-specific monovalent-reactive material at 37 °C suggested that temperature regulates the expression of ECA. Indeed, RNA-sequencing analysis showed significant downregulation of the ECA biosynthetic gene cluster at 37 °C.

Recombinant production of Yersinia enterocolitica pyruvate kinase isoenzymes PykA and PykF.

The glycolytic enzyme pyruvate kinase (PK) generates ATP from ADP through substrate-level phosphorylation powered by the conversion of phosphoenolpyruvate to pyruvate. In contrast to other bacteria, Enterobacteriaceae, such as pathogenic yersiniae, harbour two pyruvate kinases encoded by pykA and pykF. The individual roles of these isoenzymes are poorly understood. In an attempt to make the Yersinia enterocolitica pyruvate kinases PykA and PykF amenable to structural and functional characterisation, we produced them untagged in Escherichia coli and purified them to near homogeneity through a combination of ion exchange and size exclusion chromatography, yielding more than 180 mg per litre of batch culture. The solution structure of PykA and PykF was analysed through small angle X-ray scattering which revealed the formation of PykA and PykF tetramers and confirmed the binding of the allosteric effector fructose-1,6-bisphosphate (FBP) to PykF but not to PykA.

Characteristics of Yersinia enterocolitica biotype 1A strains isolated from patients and asymptomatic carriers.

Yersinia enterocolitica biotype 1A strains are frequently isolated from the environment, foods, and animals, and also from humans with yersiniosis. There are controversial reports on the pathogenicity of biotype 1A strains. In this study, 811 fecal samples from asymptomatic humans from Switzerland were studied for the presence of Y. enterocolitica. Nine (1.1%) of the 811 samples were positive for Y. enterocolitica 1A. These strains were compared with 12 Y. enterocolitica 1A strains from Swiss patients with diarrhea isolated in the same year. Almost all (20/21) Y. enterocolitica 1A strains carried the ystB gene, seven strains carried the hreP gene, and none carried the ail, ystA, myfA, yadA, or virF genes. Most (17/21) Y. enterocolitica 1A strains belonged to two major clusters, A and B, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Strains of cluster B were only isolated from humans with diarrhea; however, ystB and hreP genes were detected in strains from both clinical and non-clinical samples and from strains of clusters A and B. Using ribotyping, six restriction patterns among biotype 1A strains were obtained with HindIII enzyme. The most common ribotype (RT I) was found in strains isolated from humans with and without diarrhea. All biotype 1A strains had a unique NotI profile by pulsed-field gel electrophoresis (PFGE), showing a very high genetic diversity. In this study, Y. enterocolitica 1A strains from clinical and non-clinical samples could not be clearly differentiated from each other. More research is needed in order to prove that biotype 1A strains are a primary cause for human yersiniosis and not only a secondary finding.

Yersinia enterocolitica in diagnostic fecal samples from European dogs and cats: identification by fourier transform infrared spectroscopy and matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Yersinia enterocolitica is the main cause of yersiniosis in Europe, one of the five main bacterial gastrointestinal diseases of humans. Beside pigs, companion animals, especially dogs and cats, were repeatedly discussed in the past as a possible source of pathogenic Y. enterocolitica. To investigate the presence and types of Y. enterocolitica in companion animals, a total of 4,325 diagnostic fecal samples from dogs and 2,624 samples from cats were tested. The isolates obtained were differentiated by using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and Fourier transform infrared spectroscopy (FT-IR). Isolated Y. enterocolitica strains were bioserotyped. The detection of the ail gene by PCR and confirmation by FT-IR were used as a pathogenicity marker. Y. enterocolitica strains were isolated from 198 (4.6%) of the dog and 8 (0.3%) of the cat fecal samples investigated. One hundred seventy-nine isolates from dogs were analyzed in detail. The virulence factor Ail was detected in 91.6% of isolates. Isolates of biotype 4 (54.7%) and, to a lesser extent, biotypes 2 (23.5%), 3 (11.2%), and 5 (2.2%) were detected. The remaining 8.4% of strains belonged to the ail-negative biotype 1A. All 7 isolates from cats that were investigated in detail were ail positive. These results indicate that companion animals could be a relevant reservoir for a broad range of presumptively human-pathogenic Y. enterocolitica types. MALDI-TOF MS and FT-IR proved to be valuable methods for the rapid identification of Y. enterocolitica, especially in regard to the large number of samples that were investigated in a short time frame.

Enhanced mid-infrared chemical imaging (IRCI) detection of DNA microarrays.

We report on the optimization of a recently proposed mid-infrared chemical imaging (IRCI) detection method for the analysis of DNA microarrays. The improved protocol allowed for a ten-fold reduction in the time needed to generate a mosaic image of an entire microarray and the production of IR images with high contrast that would facilitate data analysis and interpretation. Advantages of using this protocol were evaluated by applying it to the analysis of four virulence genes in the genomes of 19 strains of the food bacterial pathogen Yersinia enterocolitica.