The YompC Protein of Yersinia enterocolitica

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including invasin Inv and adhesins Ail, Myf and YadA, are the OM proteins of chromosomal (Inv, Ail, Myf) or plasmid origin (YadA) (Bottone 1997). The Inv protein ...
Folia Microbiol. 52 (1), 73–80 (2007)

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The YompC Protein of Yersinia enterocolitica: Molecular and Physiological Characterization K. BRZOSTEK, A. RACZKOWSKA Department of Applied Microbiology, Institute of Microbiology, Warsaw University, 02-096 Warsaw, Poland e-mail [email protected] Received 7 December 2005 Revised version 18 April 2006

ABSTRACT. The structural gene coding for YompC has been identified in the genome of a pathogenic strain of Yersinia enterocolitica O:9, and was subsequently cloned and sequenced. Detailed alignment of the deduced amino acid sequence showed that YompC is a member of the OmpC porin family with the highest degree of homology to Klebsiella pneumoniae. The mutant lacking YompC porin was constructed by insertional inactivation of the yompC gene which resulted from the integration of suicide vector at the yompC locus. In intact cells of Y. enterocolitica, loss of the YompC protein reduced the outer membrane permeability for β-lactam antibiotics and tetracycline and resulted in a 2–5-fold increase in resistance to these compounds, depending on their chemical properties. Mutation in the ompR regulatory gene resulted in the loss of both YompC and YompF porins, which led to a greater increase of resistance to antibiotics, as compared with the YompC mutant strain. Moreover, the binding assay with HEp-2 cells suggests that YompC may play a role in the adhesion properties of Y. enterocolitica strains.

Abbreviations Chl Kan Nal NB

chloramphenicol kanamycin nalidixic acid nutrient broth

OM PAGE PBS Y.ent.

outer membrane(s) polyacrylamide gel electrophoresis phosphate-buffered saline Yersinia enterocolitica

Y.ent. is a gastrointestinal pathogen of humans causing mesenteric lymphadenitis, diarrhea and enteritis (Brubaker 1991; Sonnevend et al. 2005). Most studies of Y.ent. pathogenicity have focused on the expression of Yop proteins, main virulence markers that are responsible for the resistance to nonspecific host defenses and are coded by the 70-kb virulence plasmid pYV (Cornelis et al. 1998). Other virulence markers, including invasin Inv and adhesins Ail, Myf and YadA, are the OM proteins of chromosomal (Inv, Ail, Myf) or plasmid origin (YadA) (Bottone 1997). The Inv protein promotes both attachment to and invasion into eukaryotic cells by yersiniae (Pepe and Miller 1993). It plays the main role in the “zipper mechanism” by which Y.ent. can invade epithelial cells. YadA as the “multifaceted protein” is involved in interactions with mammalian cells at different stages of infection (El Tahir and Skurnik 2001). The role of Ail (Miller and Falkow 1988) and Myf (Iriarte et al. 1993), the membrane exposed proteins, in the process of pathogenesis of Y.ent. has also been proven. The porins of the OM of G– bacteria are proteins that function as pores for nonspecific diffusion of small hydrophilic molecules (Benz and Bauer 1988; Nikaido 1992; Mallea et al. 1995; Hernandez-Alles et al. 1999; Nitzan et al. 1999; Simonet et al. 2003). Many of these proteins traverse the OM as homooligomers tightly associated with the peptidoglycan layer. Due to the cell surface exposure some of them serve also as receptor sites for binding of phages and bacteriocins. The classical porins are the major OM proteins (≈105 per cell) and are encoded by 1 (ompC – Escherichia coli B), 2 (ompC, ompF – E. coli K-12) or 3 structural genes (ompC, ompF, ompD – Salmonella enterica sv. Typhimurium). Porins were identified also in other members of the Enterobacteriaceae and some of them, functioning in pathogenic strains, are involved in their interactions with eukaryotic cells and with host defence mechanisms. The homologous OmpC protein of Shigella flexneri mediates resistance to host killing and extracellular spreading of these bacteria between epithelial cells (Bernardini et al. 1993). Porins from Salmonella spp. have been shown to activate the complement system, to induce the secretion of several cytokines (Galdiero et al. 1995) and to stimulate bactericidal activity by macrophages (Tabaraie et al. 1994; Blanco et al. 1997). The major OM porin OmpC of S. enterica sv. Typhimurium has been recognized as a protein that mediates adherence to macrophages (Negm and Pistole 1999). Not all porin proteins are involved in bacterial interaction with mammalian cells. PhoE (porin induced by phosphorus starvation) of S. enterica sv. Typhimurium has no effect on the ability to

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invade or survive in J774 or HEp-2 cells. Contrary to OmpC, the OmpF porin of S. flexneri does not appear to be involved in the virulence of this pathogen (Bernardini et al. 1993). Virulence associated with the expression of porins of Y.ent. O:9 has never been studied. Based on the osmoregulating feature of E. coli porins we have previously identified homologs of OmpC and OmpF in the OM of Y.ent. O:5 (nonpathogenic 1A biotype) and pore-forming activity of Y.ent. YompC has been demonstrated in the black lipid bilayer experiments (Brzostek et al. 1989). Here we have focused on the identification and characterization of the homolog of OmpC protein in the pathogenic strain Y.ent. O:9 (biotype 2). Using isogenic strains (with or without the YompC protein), two aspects of the physiology of the YompC porin have been studied; (1) ompC mutant was used to determine the biological function of this porin in terms of adhesion properties of Y.ent. O:9, (2) we tried to find out if the lack of the nonspecific diffusion channel in the OM influences the susceptibility of bacterial cells to antibiotics. The results reported here support our suggestion about the involvement of YompC in the OM permeability and in interactions of this porin with mammalian cells.

MATERIALS AND METHODS Bacterial strains, plasmids and media. For strains and plasmids see Table I. Y.ent. O:9 strains were routinely grown in Luria–Bertani medium (LB) at 23 or 37 °C for E. coli strains. In osmolarity experiments NB without or with NaCl (300 mmol/L) was used. When necessary, media were supplemented with (in mg/L): kanamycin 50, chloramphenicol 25, and nalidixic acid 30. Table I. Bacterial strains and plasmids Strains or plasmids

Description

Reference

wild type strain (serotype O:9), pYVO9+ Ye9, yompC mutant, ChlR OP3, pBBRC4, ChlR, KanR Ye9, ompR mutant, KanR

State Institute of Hygiene, Warsaw this study this study Brzostek et al. 2003

F– φ80lacZ ΔM15 (lacZYA-orgF) U169 deoR recA1 endA1 hsdR17 phoA supE44 λ– thi-1 gyrA96 relA1 F'{lacIqTn10(TpR)}mcrAΔ(mrr-hsdRMSmcrBC) φ80lacZ ΔM15 ΔlacX74 deoR recA1 araD139 Δ(ara-leu)7697 galU galK rpsL (SmR) endA1 nupG TpR SmR recA thi pro hsdR–M– [RP4-2-Tc::Mu:KanR Tn7] (λpir)

Hanahan 1983

cloning vector, KanR pDrive, carrying the entire yompC coding sequence with rbs site (1179 bp) pDrive, carrying the yompC gene fragment (755 bp) oriR6K, mobRP4, ChlR pEP185.2, carrying KpnI/XbaI fragment from pDC12 (yompC gene fragment), ChlR mob, KanR pBBR1MCS-2, carrying SmaI/XbaI fragment from pDC11 (entire coding sequence of yompC) helper plasmid, KanR

Qiagen this study

Y. enterocolitica strains Ye9 OP3 OP5 AR4 E. coli strains DH5α Top10F´

S17-1 λpir

Invitrogen

De Lorenzo and Timmis 1994

Plasmids pDrive pDC11 pDC12 pEP185.2 pEPC2 pBBR1MCS-2 pBBRC4 pRK2013

this study Kinder et al. 1993 this study Kovach et al. 1995 this study Figurski and Helinski 1979

The MIC values (the lowest concentration of antibiotics that brought about complete inhibition of visible growth) were determined by serial 2-fold dilutions of the antibiotics (at different range) prepared in LB broth and distributed in glass tubes. Each tube contained 1 mL of an antibiotic dilution and 1 mL of Y.ent. inoculum. Tubes were incubated at 23 °C for 1 d. OM protein extraction and SDS-PAGE. Y.ent. strains grown at 26 °C to late exponential phase were centifuged (6000 g, 10 min). Pellets were resuspended in PBS (pH 7.2) containing 1 mmol/L phenylme-

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thanesulfonyl fluoride (Sigma). The cell suspensions were sonicated, broken debris was removed by centrifugation and membranes fractions were collected after centrifugation (40 000 g, 1 h). Proteins of the inner membrane were solubilized with 10 mmol/L Tris buffer (pH 7.4) with 0.1 % Triton X-100 per 30 min, then removed by centrifugation. OM proteins were extracted from the membrane fraction with electrophoretic sample buffer and boiled for 10 min. Sample extracts were analyzed by SDS-PAGE (10–12 % system). Proteins were visualized by Coomassie blue staining and destaining procedure. DNA manipulation and analysis. Basic recombinant DNA techniques (Sambrook et al. 1997) were employed. Southern blots were performed with the digoxigenin-labeled PCR probe by a random prime kit (Roche). PCR amplification of yompC DNA was performed in an automated thermal cycler (MJ Research) with DNA polymerase kit (Promega). Cycling parameters included a 5-min denaturation at 94 °C, followed by 30 cycles of denaturation (94 °C, 1 min), annealing (56 °C, 1 min) and extension (72 °C, 2 min), ending in a final extension period (72 °C, 10 min). The primers used to amplify internal (755 bp) fragment of the yompC gene, i.e. YompC1 (5´-GGT TAT GGT CAG TGG GAA TAT CAG-3´) and YompC2 (5´-GTC GCA CCA ATA TCA ACA TA-3´) were designed. The nucleotide sequence of ORF (1179 kb) with the 5´-untranslated region upstream of the yompC containing rbs region was amplified with a pair of primers YompC3Sma (5´-CCC CGG GGA TCT AAT TAG AGG ATA ATA ACG ATG-3´) and YompC4Xba (5´-TGC TCT AGA TTA GAA CTG GTA CAC CAA ACC AAC-3´) (underlined – restriction sites for SmaI and XbaI). Sequencing of yompC of Y. enterocolitica Ye9. To determine the nucleotide sequence of yompC, the PCR product containing the entire yompC coding sequence was ligated to the pDrive vector (Qiagen). The generating plasmid pDC11 was used for sequencing with universal M13 forward and reverse primers. The nucleotide sequence of the yompC gene was determined using the ABI Prism BigDye terminator Cycle Sequencing System (Perkin-Elmer). This was subsequently read on an ABI Prism 377 DNA Sequencer (DNA Sequencing and Oligonucleotide Synthesis Laboratory, Institute of Biochemistry and Biophysics, Polish Academy of Science, Warsaw). Alignment procedures and phylogeny. Using EMBLE software we aligned homologous sequences of OmpC derived from protein databanks (Swissprot, Swisspir) and our data obtained for YompC Y.ent. Ten amino acid sequences of OmpC porin homologs from protein databanks and amino acid sequence of Y.ent. Ye9 predicted from nucleotide sequence of yompC were compared. The ClustalW program was used to determine the sequence-specific amino acid homology and phylogeny between the aligned sequences. Construction of yompC mutant of Y. enterocolitica Ye9. (1) Amplified DNA of the yompC gene fragment (755 bp) was ligated to 3.85 kb pDrive cloning vector (Qiagen) following the manufacturer’s protocol and the resulting plasmid pDC12 was transformed into the E. coli TOP10F' strain; (2) the 840-bp KpnI/XbaI DNA fragment from pDC12, which contained a part of the yompC gene, was cloned into the KpnI and XbaI sites of the mobilizable suicide vector pEP185.2 (4.6 kb). The obtained plasmid pEPC2 was transformed into E. coli S17-1 λpir strain and then was conjugated into Y.ent. Ye9 NalR, using the transfer function provided by the host. Exconjugants were selected on LB plates as ChlR NalR strains. Transcomplementation of YompC mutation. The pBBR1MCS-2 plasmid was used in the yompC complementation experiments. The 1179-kb PCR product containing the entire yompC coding sequence with 5´ untranslated upstream region including the rbs region was ligated to the pDrive cloning vector. The generating plasmid pDC11 was transformed into the E. coli TOP10F´ strain. Plasmid pDC11 isolated from KanR transformants was digested with SmaI/XbaI to obtain the appropriate yompC insert. This fragment was then cloned into the SmaI and XbaI sites of the 5.1-kb mobilizable pBBR1MCS-2 vector, yielding pBBRC4 construct. This plasmid was next transformed into the E. coli TOP10F´ strain. Using E. coli DH5α strain containing helper plasmid pRK2013, which provides tra and mob genes, transfer of pBBRC4 plasmid from donor strain E. coli TOP10F´ into recipient strain mutant OP3 (ChlR) in triparental mating was performed. The transconjugants were selected on LB plates with Chl and Kan. HEp-2 cell adhesion assay. The HEp-2 cell line was maintained at 37 °C with 5 % CO2 in supplemented DMEM medium (Sigma) with 10 % heat-inactivated fetal bovine serum. HEp-2 cells were cultured overnight in a 24 mm-diameter plastic wells (5 × 105 cells per well). Nonadherent cells were removed by washing the wells with PBS prior to infection. Bacterial suspensions of Ye9 wild-type strain, OP3 mutant and OP5 strain all grown in the osmolarity of the LB medium, corresponding to a multiplicity of infection of 10, were added to each monolayer and incubated at 37 °C with 5 % CO2 for 1 h. Subsequently, the monolayers were washed 3× with PBS. The total number of surface-bound (adherent) bacteria was determined by

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cell lysis using 0.1 % Triton and plating on LB agar plates. The number of bacteria recovered after a 1-h infection was compared with the number of bacteria initially added to the monolayer of HEp-2 cells.

RESULTS AND DISCUSSION SDS-PAGE of OM proteins from Y.ent. Ye9 revealed the presence of 2 proteins with apparent molar mass of ≈38–39 kDa associated with a peptidoglycan. In this property they resembled the OM porin proteins of E. coli. Optimal resolution of these two OM proteins isolated from Ye9 cells grown in NB broth (low osmolarity) has not been achieved using standard PAGE system usually applied for separation of porins of E. coli. These two proteins were seen as a single, unresolved band at a position corresponding to apparent molar mass of ≈38 kDa (data not shown). Application of the high osmolarity for growth of Y.ent. Ye9 strain slightly improved this resolution due to different expression of both proteins in response to these conditions (Fig. 1, lane 1). The expression of the first of the porins, YompF (upper band on the gel), was reduced under high osmolarity (300 mmol/L NaCl) and the second, YompC (lower band), was induced. The elevated level of YompC and simultaneously reduced of YompF improved the resolution of these proteins and two bands were easier to distinguish. This shift in protein balance suggests that the YompC, YompF proteins are probably the equivalents of OmpC, OmpF porins of E. coli K-12.

Fig. 1. Electrophoretic separation (SDS-PAGE) of OM proteins from Y. enterocolitica O:9; strains were grown in NB medium with (lanes 1, 3, 5, 7) or without (2, 4, 6) 300 mmol/L NaCl; 1 – Ye9 wild-type strain, 2, 3 – mutant AR4 (YompC–, YompF–), 4, 5 – OP3 (YompC–, YompF+), 6, 7 – OP3/pBBRC4; arrows – the positions of YompC and YompF.

To investigate the role of YompC in the physiology of Y.ent., an isogenic yompC mutant of Y.ent. Ye9 strain was constructed. The internal fragment of yompC gene (755 bp) generated by PCR with primers YompC1 and YompC2 was cloned into the pDrive vector and subcloned into the mobilizable suicide vector pEP185.2 (Fig. 2). The resulting pEPC2 plasmid was subsequently transferred into Y.ent. Ye9 by conjugation. Transconjugants were selected for resistance to Chl (25 mg/L). Insertional inactivation of yompC resulted from homologous recombination between nucleotide sequences of yompC avaliable on the suicide vector and the bacterial chromosome at the yompC locus. Integration of pEPC2 into the chromosome resulted in duplicate, nonidentical, truncated copies of the yompC gene separated by suicide vector. The obtained yompC mutant (OP3 strain) was verified by Southern hybridization (data not shown). To confirm the mutagenic inactivation of yompC in the OP3 strain, the profile of porins in the OM was analyzed by SDS-PAGE (Fig. 1). OM fractions were prepared from the mutant OP3 strain grown under different osmolarity conditions. In addition, the OM proteins from the Y.ent. strain AR4 devoid of the osmotic regulator OmpR protein (Brzostek et al. 2003) were prepared. Electrophoretic profiles of OM proteins revealed that mutant strain OP3 did not produce the YompC protein, as the lower band corresponding to this porin was not visible, especially in the OM sample prepared from cells growing at high osmolarity (Fig. 1, lane 5). Moreover, the ompR mutant (AR4 strain) lacked both YompC, YompF porins. When a wild copy of the yompC gene (pBBRC4) was introduced into the OP3 strain, a thick band, most probably containing two proteins corresponding to YompF and YompC, was present on the gel (Fig. 1, lanes 6, 7). This result indicates that the YompC protein appeared in the OM of this mutated strain. Since the wild copy of the yompC gene was introduced on a plasmid, the number of YompC molecules in the OM sample, prepared from cells grown at high osmolarity, far exceeded the amount of YompF (reduced under these conditions) and resulted in the lower position of the thick band compared to samples at low osmolarity (Fig. 1, lane 7).

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Fig. 2. Sequence alignment of OmpC homologs. Comparative alignment of deduced YompC amino acid sequence from Y. enterocolitica Ye9 with OmpC homologs from E. coli and Y. pseudotuberculosis. Protein sequences were aligned using the ClustalW program (EMBL); asterisks – amino acids in that column are identical in all aligned sequences, colons – the conserved substitutions, dots – the semi-conserved substitutions.

The search for the yompC gene in the genome of Y.ent. O:9 (Ye9 strain) was based on the Y.ent. O:8 genome sequence which appeared in the Sanger Database and its comparison with the known ompC sequence of E. coli K-12 (EMBL). The amplified PCR product of yompC gene was obtained with primers YompC3Sma and YompC4Xba and subsequently cloned and sequenced. The open reading frame yompC of Y.ent. Ye9 contains 1161 bases. This corresponds to a protein with 387 amino acids. The deduced amino acid sequence of YompC was compared with certain amino acid sequences of known OmpC porin family found in the protein databases SwissProt and Protein Information Resource (PIR). The derived amino-acid sequence of the Y.ent. Ye9 YompC showed 85 % similarity to OmpC Y. pseudotuberculosis and 73 % to OmpC E. coli K-12 (Fig. 3). The sequence alignment of ten OmpC homologs was the basis of the phylogram in Fig. 4 indicating the relationships within the OmpC family. The sequences of porins belonging to OmpC family were aligned and analyzed by the ClustalW program. The tree diagram shows that YompC is a member of the OmpC porin family with the highest degree of homology to Klebsiella pneumoniae. It has been reported that deficiency or loss of porin expression in E. coli, either one (OmpF or OmpC) or both is accompanied by an increased resistance to β-lactam antibiotics and tetracycline (Nikaido and Vaara 1985). Similarly, reduced OM permeability due to different expression of porin proteins was observed for the pathogenic strains of E. coli O157:H7, K. pneumoniae, Enterobacter aerogenes, S. enterica sv. Wien (Martinez et al. 2001; Martinez-Martinez et al. 1996; Chevalier et al. 1999; Armand-Lefevre et al.

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2003). Y.ent. strain lacking the YompC protein was tested for its sensitivity to different β-lactam antibiotics and tetracycline (Table II). The MIC values were compared for different Y.ent. strains, wild type and mutants lacking one or two porins. Susceptibility testing confirmed high resistance of wild type Ye9 strain to

Fig. 3. Possible phylogenetic tree derived from similarity of OmpC porin sequences (analyzed using the ClustalW program). Each branching point shows the level of maximum similarity between the analyzed proteins. All amino acid sequence data except YompC Y. enterocolitica Ye9 come from the EMBL.

numerous β-lactams due to the activity of two chromosomally encoded β-lactamases (Seoane and GarciaLobo 1991; Seoane et al. 2003). Meanwhile, we observed a variation in the susceptibility to cephalosporins between the tested strains which was closely related to the loss of YompC, YompF porins. The lack of YompC in the OM has been associated with more than a 2-fold increase in resistance to cephaloridine and 4–5-fold increase to other cephalosporins tested. The loss of both porins resulted in a further increase in resistance to the antibiotics used. These data support the role of Y.ent. porins in the OM permeability towards tested Table II. MIC (mg/La) of antimicrobial agents against Y. enterocolitica O:9 strains with different patterns of porin expression Antibiotic Benzylpenicillin Cefazoline Cephalotin Cefalexin Cephaloridine Cephalosporin C Cefoxitin Tetracycline

Wild type (Ye9) >500 >500 >500 82 68 32 20 1.5

YompC– (OP3)

YompC–, YompF– (AR4)

>500 >500 >500 340 150 136 120 4.1

>500 >500 >500 600 460 340 340 4.8

aDetermined in LB broth at 23 °C; means of 3 independent determinations.

antibiotics. Although the significant level of resistance appearing with the loss of one or two major porins of Y.ent. may be the result of a combined effect of decreased permeability of the OM with enzymic inactivation by β-lactamases. Mutants lacking one or two porins showed almost Table III. Ability of Y. enterocolitica the same pattern of resistance to tetracycline which suggests a direct strains to adhere to HEp-2 cellsa (relative association of the YompC porin with the resistance phenotype and adhesion, %) OM permeability to this antibiotic. To determine whether the YompC porin plays a role in microStrain % bial attachment to eukaryotic cells, we tested the ability of the yompC mutant (OP3 strain) to adhere to HEp-2 cells (Table III). Y.ent. wildYe9 (wild type) 7.2 type strain was used for positive control, OP5 (OP3/pBBRC4) strain OP3 (yompC mutant) 2.8 was enclosed to study the effect of the complementation of yompC OP5 (OP3/pBBRC4) 5.3 mutation. aCell binding determined after 1 h of incuMutant OP3 exhibited more than a 2-fold decrease in the abibation at 37 °C by plating the bacteria lity to adhere to HEp-2 cells than the Ye9 strain. Introduction of after separation from HEp-2 cells; perpBBRC4 with wild copy of yompC into OP3 strain partially restored formed 3×, data represent a typical expethe wild-type adhesion level. It would suggest that the YompC proriment. tein partially determines the adhesion properties of Y.ent. Up-to-date, we cannot rule out the possibility that the loss of YompC exerts its

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effect by elimination of the protein itself, because we cannot eliminate the possible changes in intermolecular interactions with lipolysaccharide, murein, or other OM proteins. However, on the other hand, YompC

Fig. 4. Construction of the pEPC2 plasmid. The internal fragment of yompC gene (755 bp) generated by PCR with specific primers was cloned into the pDrive vector, subcloned into the mobilizable suicide vector pEP185.2 and subsequently transferred into Y. enterocolitica Ye9 by conjugation. Insertional inactivation of yompC resulted from homologous recombination between two nucleotide sequences: fragment of yompC available on the suicide vector and the bacterial chromosome at the yompC locus.

presented on the surface of ingested bacteria in the intestinal tract could influence the first stages of adherence, enhanced later by Inv and other known Y.ent. adhesins. The high expression of YompC in the high osmolarity of the ileum could be advantageous for this kind of interaction.

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REFERENCES ARMAND-LEFEVRE L., LEFLON-GUIBOUT V., BREDIN J., BARGUELLIL F., AMOR A., PEGES J.M., NICOLAS-CHANOINE M.H.: Imipenem resistance in Salmonella enterica serovar Wien related to porin loss and CMY-4 β-lactamase production. Antimicrob.Agents Chemother. 47, 1165–1168 (2003). BENZ R., BAUER K.: Permeation of hydrophilic molecules through the outer membrane of Gram-negative bacteria. Eur.J.Biochem. 176, 1–19 (1988). BERNARDINI M.L., SANNA M.G., FONTAINE A., SANSONETTI P.J.: OmpC is involved in invasion of epithelial cells by Shigella flexneri. Infect.Immun. 61, 3625–3635 (1993). BLANCO L.P., TORO C.S., ROMERO J.M., SANTIVIAGO C.A., MORA G.C.: Salmonella typhi Ty2 OmpC porin induces bactericidal activity on U937 monocytes. Microbiol.Immunol. 41, 999–1003 (1997). BOTTONE E.J.: Yersinia enterocolitica: the charisma continues. Clin.Microbiol.Rev. 10, 257–276 (1997). BRUBAKER R.R.: Factors promoting acute and chronic diseases caused by yersiniae. Clin.Microbiol.Rev. 4, 309–324 (1991). BRZOSTEK K., HREBENDA J., BENZ R., BOOS W.: The OmpC protein of Yersinia enterocolitica: purification and properties. Res.Microbiol. 140, 599–614 (1989). BRZOSTEK K., RACZKOWSKA A., ZASADA A.: The osmotic regulator OmpR is involved in the response of Yersinia enterocolitica O:9 to environmental stresses and survival within macrophages. FEMS Microbiol.Lett. 228, 265–271 (2003). CHEVALIER J., PAGES J.M., MALLEA M.: In vivo modification of porin activity conferring antibiotic resistance to Enterobacter aerogenes. Biochem.Biophys.Res.Commun. 266, 248–251 (1999). CORNELIS G.R., BOLAND A., BOYD A.P., GEUIJEN C., IRIARTE M., NEYT C., SORY M.P., STAINIER I.: The virulence plasmid of Yersinia, an antihost genome. Microbiol.Mol.Biol.Rev. 62, 1315–1352 (1998). DE LORENZO V., TIMMIS K.N.: Analysis and construction of stable phenotypes in Gram-negative bacteria with Tn5- and Tn10-derived minitransposon. Meth.Enzymol. 235, 386–405 (1994). EL TAHIR Y., SKURNIK M.: YadA, the multifaceted Yersinia adhesion. Internat.J.Med.Microbiol. 291, 209–218 (2001). FIGURSKI D.H., HELINSKI D.R.: Replication of an origin containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc.Nat.Acad.Sci.USA 75, 1648–1652 (1979). GALDIERO F., TUFANO M.A., SOMMESE L., FOLGORE A., TEDESCO F.: Activation of complement system by porins extracted from Salmonella typhimurium. Infect.Immun. 46, 559–563 (1984). GALDIERO F., SOMMESE L., SCRAFOGLIERO P., GALDIERO M.: Biological activities – lethality, Shwartzman reaction and pyrogenicity of Salmonella typhimurium porins. Microbiol.Pathog. 16, 111–119 (1995). HANAHAN D.: Studies on transformation of Escherichia coli with plasmids. J.Mol.Biol. 166, 557–580 (1983). HERNANDEZ-ALLES S., ALBERTI S., ALVARES D., DOMENECH-SAMNCHES A., MARTINEZ-MARTINEZ L., TOMAS J.M., BENEDI V.J.: Porin expression in clinical isolates of Klebsiella pneumoniae. Microbiology 145, 673–679 (1999). IRIARTE M., VANOOTEGHEN J.C., DELOR I., DIAZ R., KNUTTON S., CORNELIS G.R.: The Myf fibrillae Yersinia enterocolitica. Mol. Microbiol. 9, 507–520 (1993). KINDER S.A., BADGER J.L., BRYANT G.O., PEPE J.C., MILLER V.L.: Cloning of the YenI restriction endonuclease and methyltransferase from Yersinia enterocolitica serotype O8 and construction of a transformable R-M+ mutant. Gene 136, 271–275 (1993). KOVACH M.E., ELZER P.H., HILL D.S., ROBERTSON G.T., FARRIS M.A., ROOP II R.M., PETERSON K.M.: Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166, 175–176 (1995). MALLEA M., SIMONET V., LEE E.H., GERVIER R., COLLATZ E., GUTMANN L., PAGES J.M.: Biological and immunological comparisons of Enterobacter cloacae and Escherichia coli porins. FEMS Microbiol.Lett. 129, 273–280 (1995). MARTINEZ M.B., FICKINGER M., HIGGINS L., KRICK T., NELSESTUEN G.L.: Reduced outer membrane permeability of Escherichia coli O157:H7: suggested role of modified outer membrane porins and theoretical function in resistance to antimicrobial agents. Biochemistry 40, 11965–11974 (2001). MARTINEZ-MARTINEZ L., HERNANDEZ-ALLES S., ALBERTI S., TOMAS J.M., BENEDI V.J., JACOBY G.A.: In vivo election of porindeficient mutants of Klebsiella pneumoniae with increased resistance to cefoxitin and expanded-spectrum cephalosporins. Antimicrob.Agents Chemother. 40, 342–348 (1996). MILLER V.L., FALKOW S.: Evidence for two genetic loci in Yersinia enterocolitica that can promote invasion of epithelial cells. Infect. Immun. 56, 1242–1248 (1988). NEGM R.S., PISTOLE T.G.: The porin OmpC of Salmonella typhimurium mediates adherence to macrophages. Can.J.Microbiol. 45, 658–669 (1999). NIKAIDO H.: Porins and specific channels of bacterial outer membranes. Mol.Microbiol. 6, 435–442 (1992). NIKAIDO H., VAARA M.: Molecular basis of bacterial outer membrane permeability. Microbiol.Rev. 49, 1–32 (1985). NITZAN Y., ORLOVSKY K., PECHATNIKOV I.: Characterization of porins isolated from the outer membrane of Serratia liquefaciens. Curr.Microbiol. 38, 71–79 (1999). PEPE J., MILLER V.L.: Yersinia enterocolitica invasin: a primary role in the initiation of infection. Proc.Nat.Acad.Sci.USA 90, 6473– 6477 (1993). SAMBROOK J.E.F., FRITSCH E.F., MANIATIS T.: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor (NY) 1997. SEOANE A., GARCIA-LOBO J.M.: Cloning of chromosomal β-lactamase genes from Yersinia enterocolitica. J.Gen.Microbiol. 137, 141– 146 (1991). SEOANE A., SANCHEZ E., GARCIA-LOBO J.M.: Tandem amplification of a 28-kilobase region from Yersinia enterocolitica chromosome containing the blaA gene. Animicrob.Agents Chemother. 47, 682–688 (2003). SIMONET V.C., BASLE A., KLOSE K.E., DELCOUR A.H.: The Vibrio cholerae porins OmpU and OmpT have distinct channel properties. J.Biol.Chem. 278, 17539–17545 (2003). SONNEVEND Á., CZIRÓK É., PÁL T.: Yersinia Yop-specific IgA antibodies in Hungarian blood donors. Folia Microbiol. 50, 269–272 (2005). TABARAIE B., SHARMA B.K., SHARMA P.R., SEHGAL R., GANGULY N.K.: Stimulation of macrophage oxygen free radical producion and lymphocyte blastogenic response by immunization with porins. Microbiol.Immunol. 38, 561–565 (1994).