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 β-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.     

The protective properties of a porin from the outer membrane of Yersinia pseudotuberculosis

The protective properties of the species-specific pore-forming polypeptide of Y. pseudotuberculosis outer membrane (porin) were studied. The study showed that 80-90% of mice immunized with porin survived after challenge with 10-30 LD50 of Y. pseudotuberculosis, serovars 1 and 3. The LD50 of the preparation exceeded its ED50 more than 100-fold. Immunization made in two injections was more effective than immunization in one injection. The immunization of the animals with porin led to an increase in the activity of peritoneal exudate macrophages with respect to Y. pseudotuberculosis, serovars 1 and 3.     

Isolation and physico-chemical properties of a protein, included in an endotoxin from Yersinia pseudotuberculosis

A major protein of the endotoxin from Yersinia pseudotuberculosis was isolated from the complex lipid A--protein by treatment with SDS and triton X-100 followed by gel-chromatography on Sephacryl S-300. Protein has apparent molecular mass 40 kDa and alanine as N-terminal amino acid residue. CD and IR spectroscopy conformational changes of the protein molecule in the process of its isolation. The thermal and pH stabilities of the protein were investigated by the methods of intrinsic fluorescence and differential scanning microcalorimetry. The isolated protein revealed two thermal transitions (at 30-35 and 50-55 degrees C), which depend on Ca2+ concentration.     

Molecular cloning, isolation, and properties of chaperone Skp from Yersinia pseudotuberculosis

The skp gene of Yersinia pseudotuberculosis was expressed without its signal sequence in Escherichia coli BL21(DE3) cells. The recombinant protein Skp accumulated in soluble form in the cytoplasm of the producer strain. The protein was isolated and characterized: the molecular weight, isoelectric point, N-terminal amino acid sequence (20 amino acid residues), and the content of the secondary structure elements were determined. Using cross-linking stabilization and high-mass MALDI-TOF mass spectrometric analysis, it was found that rSkp forms a stable homotrimer in solution and interacts with human IgG. Three-dimensional models of the Skp trimer and its complexes with Fc- and Fab-fragments of human IgG1 were constructed by computer modeling.     

Biological and physico-chemical properties of Yersinia pseudotuberculosis bacterial culture having the fra-operon Yersinia pestis

The biological and physico-chemical properties of cultures of two isogenous recombinant variants of Yersinia pseudotuberculosis were studied. The cell genomes of the cultures are distinguished from one another only by the presence or by the absence of the fra-operon, which is a determined attribute of the plague microbe capsule-forming process. The expression of the attribute is amplified by rising the microbial biomass cultivation temperature and stimulates the decrease in the viability of the bacteria and adaptation potential in vitro. In the warm-blooded owner organism the microbes of the capsule-forming recombinant variant are characterized by the greater residual pathogenicity and immunogenic ability to the experimental plague of the laboratory animals as compared to the reference-variant cells. These specific features could be explained by more expressed colonizing ability of the capsule-forming microbes provided by owner cells' stability to the phagocyte process.     

A novel OmpY porin from Yersinia pseudotuberculosis: structure, channel-forming activity and trimer thermal stability

A novel OmpY porin was predicted based on the Yersinia pseudotuberculosis genome analysis. Whereas it has the different genomic annotation such as "outer membrane protein N" (ABS46310.1) in str. IP 31758 or "outer membrane protein C2, porin" (YP_070481.1) in str. IP32953, it might be warranted to rename the OmpN/OmpC2 to OmpY, "outer membrane protein Y", where letter "Y" pertained to Yersinia. Both phylogenetic analysis and genomic localization clearly support that the OmpY porin belongs to a new group of general bacterial porins. The recombinant OmpY protein with its signal sequence was overexpressed in porin-deficient Escherichia coli strain. The mature rOmpY was shown to insert into outer membrane as a trimer. The OmpY porin, isolated from the outer membrane, was studied employing spectroscopic, electrophoretic and bilayer lipid membranes techniques. The far UV CD spectrum of rOmpY was essentially identical to that of Y. pseudotuberculosis OmpF. The near UV CD spectrum of rOmpY was weaker and smoother than that of OmpF. The rOmpY single-channel conductance was 180 ± 20 pS in 0.1 M NaCl and was lower than that of the OmpF porin. As was shown by electrophoretic and bilayer lipid membrane experiments, the rOmpY trimers were less thermostable than the OmpF trimers. The porins differed in the trimer-monomer transition temperature by about 20°C. The three-dimensional structural models of the Y. pseudotuberculosis OmpY and OmpF trimers were generated and the intra- and intermonomeric interactions stabilizing the porins were investigated. The difference in the thermal stability of OmpY and OmpF trimers was established to correlate with the difference in intermonomeric polar contacts.     

Isolation and characterization of recombinant OmpF-like porin from the Yersinia pseudotuberculosis outer membrane

The encoding sequence of the pore-forming OmpF-like protein from the Yersinia pseudotuberculosis outer membrane was cloned and expressed in Escherichia coli cells. Conditions for isolation and refolding of recombinant monomer and porin trimer were selected. Their spatial structures were characterized by the intrinsic protein fluorescence and CD spectroscopy. It was shown that recombinant porins are similar in the composition of secondary structure elements to isolated porins, but have a considerably less compact tertiary structure. The pore-forming activities of the recombinant proteins are similar to those of Y. pseudotuberculosis native porins.     

Isolation and characterization of recombinant OmpF-like porin from the Yersinia pseudotuberculosis outer membrane

The encoding sequence of the pore-forming OmpF-like protein from the Yersinia pseudotuberculosis outer membrane was cloned and expressed in Escherichia coli cells. Conditions were selected for isolation and refolding of recombinant monomer and porin trimer. Their spatial structures were characterized by the intrinsic protein fluorescence and CD spectroscopy. It was shown that the recombinant porins are similar in the composition of secondary structure elements to the isolated porins, but have a considerably less compact tertiary structure. The pore-forming activities of the recombinant proteins are similar to those of Y. pseudotuberculosis native porins. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http://www.maik.ru.     

Study of effect of substitution of the penultimate amino acid residue on expression,structure, and functional properties of Yersinia pseudotuberculosis OmpY porin

The purpose of the study was to compare the expression of two Yersinia pseudotuberculosis proteins, wild-type porin OmpY and the mutant porin OmpY designated as OmpY-Q having the uncharged amino acid residue Gln instead of positively charged Arg at the penultimate position in the same heterologous host. According to the literature, a similar substitution (Lys to Gln) of the penultimate amino acid residue in Neisseria meningitidis porin PorA drastically improved the assembly of the protein in the E. coli outer membrane in vivo. Site-directed mutagenesis was used to replace Arg by Gln (R338Q) in OmpY, and the conditions for optimal expression and maturation of OmpY-Q were selected. It was found that the growth rates of E. coli strains producing OmpY and OmpY-Q and the expression levels of the porins were approximately equal. Comparative analysis of recombinant OmpY and OmpY-Q did not show significant differences in structure, antigenic, and functional properties of the porins, or any noticeable effect of the R338Q substitution in OmpY on its assembly in the E. coli outer membrane in vivo. The probable causes of discrepancies between our results and the previous data on porin PorA are discussed considering the known mechanisms of biogenesis of porins at the periplasmic stage.     

Sedimentation properties of a lipopolysaccharide-protein complex from Yersinia pseudotuberculosis

It was shown that in water media lipopolysaccharide-protein complex (LPSPC-) forms compact units with molecular weight 1.29 X 10(6) inclined to self-association. Disaggregation of LPSPC to subunits with molecular weight 7 X 10(4) under the action of the detergent and EDTA reveals the substantial role of hydrophobic interaction of ions of divalent metals in the formation of high molecular weight aggregates.     

Effects of pH on structural and functional properties of porin from the outer membrane of Yersinia pseudotuberculosis. II. Characterization of pH-induced conformational intermediates of yersinin

pH-Induced intermediates of Omp F-like porin from the outer membrane of Yersinia pseudotuberculosis (yersinin) were characterized by fluorescence and fluorescent probe spectroscopy and circular dichroism. The most dramatic changes in the intrinsic fluorescence of the protein induced by pH titration correlated with different conformational states of the porin molecule. pH-induced conformational transitions of yersinin can be described in terms of a three-state model: (1) disordering of porin associates and formation of porin trimers structurally similar to the native protein; (2) unfolding of individual porin domains followed by cooperative dissociation of trimers into monomers; (3) formation of two loosely structured forms of monomer intermediates. It is assumed that one of these monomeric forms (at pH 3.0) corresponds to the molten-globule state of porin with native secondary structure, while the other one (at 2.0) represents a partly denatured (misfolded) monomer, which retains no more than 50% of the regular secondary structure. The putative mechanism of low pH-induced β-barrel unfolding is discussed in terms of a theoretical model of yersinin spatial structure.     

Direct isolation of Yersinia enterocolitica and Yersinia pseudotuberculosis from meat

Studies were done to determine the usefulness of dilute alkali (KOH) treatment of meat samples for direct isolation of Yersinia enterocolitica and Yersinia pseudotuberculosis, without enrichment. Virulent Y. enterocolitica and Y. pseudotuberculosis in pork contaminated with 10(2), 10(3), and 10(4) cells per g survived the direct KOH treatment and were never recovered by using KOH postenrichment treatment. From 6 (4.8%) of 125 samples of retail ground pork, four biotype 4 serotype O3 and one biotype 3B serotype O3 strains of Y. enterocolitica and one Y. pseudotuberculosis serotype 4b strain were recovered by using direct KOH treatment without enrichment. As these isolations were attained without using enrichment cultural procedures, they represent an important time-saving alternative to simplify and speed isolation of Yersinia spp. from meat.     

Molecular cloning and sequencing of a pectate lyase gene from Yersinia pseudotuberculosis.

A pectate lyase gene (pelY) from Yersinia pseudotuberculosis was cloned in Escherichia coli DH-5 alpha. The gene was expressed in either orientation in pUC plasmids, indicating that the insert DNA carried a Y. pseudotuberculosis promoter which functioned in E. coli. However, when cloned in the orientation which placed the coding region downstream of the vector lac promoter, expression of pelY was nine times higher than it was in the opposite orientation and the growth of E. coli cells was inhibited. Nucleotide sequence analysis of the pelY gene disclosed an open reading frame of 1,623 base pairs (PLY). The peptide sequence at the amino-terminal end of the protein contains a typical signal peptide sequence, consistent with the observation that the mature PLY protein accumulated largely in the periplasmic space of E. coli. The pI of PLY produced in E. coli cells was 4.5, and its activity was inhibited 90% or more by EDTA. The enzyme macerated cucumber tissue about 1,000 times less efficiently than did PLe from Erwinia chrysanthemi EC16. The pelY gene has no sequence similarity to the pel genes thus far sequenced from Erwinia spp.     

Structural-functional characteristics of Yersinia pseudotuberculosis plasmid pVM82

The restriction map of Yersinia pseudotuberculosis plasmid pVM82 was established using the "chromosome walking" method. According to transpositional mutagenesis, the plasmid pVM82 appeared to be conjugative and was able to be transmitted from Y. pseudotuberculosis to the E. coli K-12 cells.     

Interaction of porin from Yersinia pseudotuberculosis with lipopolysaccharides. Effect of ionic strength, pH, and divalent cations on the binding parameters

Interaction of the pore-forming protein (porin) from Yersinia pseudotuberculosis with S- and R-forms of the endogenous lipopolysaccharide (LPS) was studied at various ionic strengths (20-600 mM NaCl), concentrations of divalent cations (5-100 mM CaCl2, MgCl2), and pH values from 3.0 to 9.0. The interaction of the R-LPS with porin has been shown in all experimental conditions to be in consensus with the model suggesting binding at independent sites of two types. S-LPS binds to interacting sites of relatively high affinity and to independent sites of low affinity at all pH values examined and at low NaCl concentration. The cooperative interaction of the S-LPS and porin is not observed at high ionic strength and in divalent cation-free medium. The number of binding sites of porin and association constants (Ka) for both LPS forms decrease significantly on increasing the solution ionic strength. The Ka values for the R- and S-LPS change oppositely on changing the pH: the Ka value for the R-LPS is maximal (Ka = 6.7 x 10(5) M-1), but that for S-LPS is minimal (Ka = 0.4 x 10(5) M(-1) at pH 5.0-5.5. The number of high-affinity and low-affinity binding sites for both LPS forms is maximal at pH 5.0-5.5. In this case, the numbers of high- and low-affinity sites for R-LPS are 3 and 10, respectively, and those for the S-LPS are 7 and 20, respectively. These data suggest an important role of electrostatic interactions on binding of LPS to porin. The contribution of conformational changes of the ligand and protein and hydrophobic interactions are discussed.     

Molecular genetic monitoring of Yersinia pseudotuberculosis using PCR O-genotyping

Irkutsk PCR O-genotyping of 117 Yersinia pseudotuberculosis serotype O:1-O:4 strains isolated in Siberia and Far East was performed. These methods allowed both O-genotype and its variants (a, b, c) to be detected. It was demonstrated that three Y. pseudotuberculosis O-genotypes were circulated in Siberia (O:1a, O:1b and 0:3) and six genotypes (O:1a, O:1b, O:1c, O:2a, O:3 and O:4b) were circulated at Far East. Genotype O:1b dominates at both regions (87.8%). PCR-algorithm for identification of Y. pseudotuberculosis O-genotypes having epidemic significance was developed. The identification of the etiological agent O-genotype without bacteriological isolation of the stimulus is possible by PCR analysis of the clinical material.     

Direct isolation of Yersinia enterocolitica and Yersinia pseudotuberculosis from meat.

Studies were done to determine the usefulness of dilute alkali (KOH) treatment of meat samples for direct isolation of Yersinia enterocolitica and Yersinia pseudotuberculosis, without enrichment. Virulent Y. enterocolitica and Y. pseudotuberculosis in pork contaminated with 10(2), 10(3), and 10(4) cells per g survived the direct KOH treatment and were never recovered by using KOH postenrichment treatment. From 6 (4.8%) of 125 samples of retail ground pork, four biotype 4 serotype O3 and one biotype 3B serotype O3 strains of Y. enterocolitica and one Y. pseudotuberculosis serotype 4b strain were recovered by using direct KOH treatment without enrichment. As these isolations were attained without using enrichment cultural procedures, they represent an important time-saving alternative to simplify and speed isolation of Yersinia spp. from meat.