Use of poly-beta-oxybutyric acid by Yersinia pseudotuberculosis and Listeria monocytegenes bacteria at various temperatures

A comparative investigation of the intracellular content of poly-beta-hydroxybutyric acid showed that Yersinia pseudotuberculosis strains accumulated, on the average, lower amounts of this reserve substance than Listeria monocytogenes strains. The intracellular pool of poly-beta-hydroxybutyric acid was responsible for the growth of the bacteria at low temperatures (4-6 degrees C) in the absence of any exogenous carbon and energy source.     

Requirement for RNA helicase CsdA for growth of Yersinia pseudotuberculosis IP32953 at low temperatures

The expression of csdA, encoding an RNA helicase, was induced at 3°C in Yersinia pseudotuberculosis. The role of CsdA in Y. pseudotuberculosis under cold conditions was confirmed by impaired growth of insertional csdA mutants at 3°C. The results suggest that CsdA is crucial for Y. pseudotuberculosis survival in the chilled food chain.     

The biochemical mechanisms of energy support in Yersinia pseudotuberculosis cells at a low cultivation temperature

The object of the study was the first stage of biological oxidation: the transfer of hydrogen electrons to the components of the respiratory chain of Y. pseudotuberculosis cells by NAD and NADF, coenzymes of pyridine-dependent dehydrogenases, having labile redox properties. The study revealed that in the low-temperature cultivation of Y. pseudotuberculosis an increase in the content of NAD and NADF was 1.5- to 2.0-fold greater than that observed at 37 degrees C, which was indicative of the fact that at low environmental temperature pyridine-dependent dehydrogenases played a more important role than at high temperature (37 degrees C). This, in combination with other mechanisms, made it possible for bacterial cells to maintain the level of energy metabolism, necessary for their survival, in the environment with low and constantly changing temperature.     

Expression of membrane beta-barrel protein in E. coli at low temperatures: Structure of Yersinia pseudotuberculosis OmpF porin inclusion bodies

The recombinant OmpF porin of Yersinia pseudotuberculosis as a model of transmembrane protein of the β-barrel structural family was used to study low growth temperature effect on the structure of the produced inclusion bodies (IBs). This porin showed a very low expression level in E. coli at a growth temperature below optimal 37 °C. The introduction of a N-terminal hexahistidine tag into the mature porin molecule significantly increased the biosynthesis of the protein at low cultivation temperatures. The recombinant His-tagged porin (rOmpF-His) was expressed in E. coli at 30 and 18 °C as inclusion bodies (IB-30 and IB-18). The properties and structural organization of IBs, as well as the structure of rOmpF-His solubilized from the IBs with urea and SDS, were studied using turbidimetry, electron microscopy, dynamic light scattering, optical spectroscopy, and amyloid-specific dyes. IB-18, in comparison with IB-30, has a higher solubility in denaturants, suggesting a difference between IBs in the conformation of the associated polypeptide chains. The spectroscopic analysis revealed that rOmpF-His IBs have a high content of secondary structure with a tertiary-structure elements, including a native-like conformation, the proportion of which in IB-18 is higher than in IB-30. Solubilization of the porin from IBs is accompanied by a modification of its secondary structure. The studied IBs also contain amyloid-like structures. The results obtained in this study expand our knowledge of the structural organization of IBs formed by proteins of different structural classes and also have a contribution into the new approaches development of producing functionally active recombinant membrane proteins.     

A cloned chromosomal DNA fragment which differentiates Yersinia pestis from Yersinia pseudotuberculosis and Yersinia enterocolitica

Chromosomal DNA from reference Yersinia strains was digested individually with 9 restriction endonucleases. DNA fragments were separated and analyzed by electrophoresis through agarose gels. The clearest fragment patterns were obtained when EcoRI was employed. The Y. pestis fragment pattern obtained after the use of this enzyme showed the presence of a unique DNA fragment with molecular mass 1400 bp. This DNA fragment was cloned, purified, labeled with 32P and then used to probe EcoRI digests of all three Yersinia species. A strong hybridization signal was obtained with Y. pestis strain. No such signal was found with Y. pseudotuberculosis or Y. enterocolitica. These results indicate that the DNA fragment is species specific and could be used as a diagnostic DNA probe for Y. pestis.     

Isolation and characterization of the immunoglobulin-binding protein from Yersinia pseudotuberculosis

A high molecular weight immunoglobulin-binding protein localized on the surface of bacterial cells has been isolated from the protein fraction of the outer membrane of Yersinia pseudotuberculosis, and its properties are described. The immunoglobulin-binding protein is a trypsin-resistant and temperature-sensitive -structured protein. As shown by MALDI-TOF mass spectrometry, after heating at 100°C the molecular weight of the protein constituted 37.5 kD. The native protein is capable of interacting with human and rabbit IgG but looses the ability to bind the immunoglobulins after the temperature denaturation. The immunoglobulin-binding protein binds to the Fc-fragments of the immunoglobulins and binding depends on the presence of calcium ions.     

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.     

Effect of cultivation temperature on nucleic acid level in bacteria Listeria monocytogenes and Yersinia pseudotuberculosis

The relationship between the multiplication of bacteria, the content of nucleic acid and the specific rate of their growth during their batch cultivation in nutrient broth and mineral medium at temperatures of 37 degrees C and 4-6 degrees C was studied in the causative agents of saprozoonotic infections with L. monocytogenes and Y. pseudotuberculosis used as typical representatives of such bacteria. The content of DNA was shown to remain practically unchanged after the alteration of cultivation temperature and the conditions of nutrition. The linear relationship between the content of RNA and specific growth rate was registered both at 37 degrees C and 4-6 degrees C. However a higher content of RNA at low temperatures was found to correspond to one and the same specific growth rate, which was linked with the additional synthesis of this nucleic acid.     

The biosynthesis and biological role of 6-deoxyheptose in the lipopolysaccharide O-antigen of Yersinia pseudotuberculosis

Yersinia pseudotuberculosis O:2a harbours 6-deoxy- d - manno -heptose in its O-antigen. The biological function of 6-deoxyheptose and its role in virulence is unknown and its biosynthetic pathway has not been demonstrated experimentally. Here, we show that dmhA and dmhB are necessary for 6-deoxyheptose biosynthesis in Y. pseudotuberculosis . Their disruption resulted in the lack of 6-deoxyheptose in the O-unit and its replacement by d - glycero - d - manno -heptose, thus indicating relaxed specificity of the glycosyltransferases, polymerase and ligase involved in lipopolysaccharide synthesis. The dmhB mutant exhibited a lower content in ketooctonic acid (Ko)-containing core molecules and reduced ligation and polymerization of the O-unit. We also show that Tyr128 is essential for activity of DmhB, and that DmhB functions as an oligomer, based on the dominant negative effect of overexpression of DmhB Y128F in dmhA . Moreover, we demonstrate that 6-deoxyheptose is important for virulence-related functions of the outer membrane and its appendages in vitro , such as barrier function against bile salts, polymyxin and novobiocin, and flagella-mediated motility. Although both mutants colonized the mouse ceacum as well as the wild type, the dmhB mutant was impaired for colonization of the liver, suggesting that DmhB represents a potential therapeutic target.     

Isolation and characterization of a low-molecular-weight immunoglobulin-binding protein from Yersinia pseudotuberculosis

A low-molecular-weight immunoglobulin-binding protein (IBP) bound with the cell envelope has been isolated from Yersinia pseudotuberculosis cells and partially characterized. This IBP is a hydrophilic protein with a high polarity index of 55.3%. The molecular weight of the protein has been determined by MALDI-TOF mass spectrometry as 14.3 kD. CD spectroscopy showed that the IBP has high contents of the beta-structure and random coil structure. The IBP contains glycine as the N-terminal amino acid. The protein can be stored for a long time at acidic pH values but aggregates and loses activity at alkaline and neutral pH. The IBP binds rabbit IgG with optimum at pH of 6.0-7.5. The IBP interacts with IgG molecule in the Fc-fragment region. The protein retains activity after heating at 100 degrees C in the presence of SDS.     

DNA probe for detecting Yersinia pestis and serovariant I of Yersinia pseudotuberculosis by detecting specific DNA repeating sequences]

In order to construct a DNA probe for the plague pathogen detection, we have obtained the recombinant plasmid pRD100 carrying an EcoRI-flanked 140 bp fragment from the genetically silent region of Yersinia pestis species-specific plasmid pYP1. When used as a DNA probe for hybridization of DNA from various strains of 25 bacterial species, this DNA fragment was shown to have the complementary sequences in all investigated Yersinia pestis strains (200), including the plasmid pYP1 lacking ones, and in all the studied Yersinia pseudotuberculosis serotype I strains (80). The search for the probe target in these species has led us to conclusion that it is a specific repeated DNA sequence present in more copies in Yersinia pestis than in Yersinia pseudotuberculosis serotype I. The hybridization of these sequences with the radioactive probe and 24 hours autography makes possible the detection of 1.3 x 10(5) cells of Yersinia pestis and 3 x 10(6) cells of Yersinia pseudotuberculosis serotype I immobilized on the nitrocellulose membranes. Use of the probe for analysis of the nitrocellulose membrane fixed spleen smears from animals that died of experimental plague made possible the detection of Yersinia pestis cells within 48 h.     

Genomic comparison of Yersinia pestis and Yersinia pseudotuberculosis by combination of suppression subtractive hybridization and DNA microarray

In order to further figure out the genetic differences between Yersinia pestis and Yersinia pseudotuberculosis, and to provide novel insights into the evolution of Y. pestis, we compared the genomes of Y. pseudotuberculosis serogroup I strain ATCC29833 and Y. pestis Antiqua strain 49006 using a combination of suppression subtractive hybridization (SSH) and comparative genomic hybridization with DNAs from a diverse panel of Y. pestis and Y. pseudotuberculosis strains. SSH followed by BLAST analysis revealed 112 SSH fragments specific to strain ATCC29833, compared to the genomic sequence data of Y. pestis strains CO92, KIM and 91001. We identified 17 SSH fragments that appeared to be newly determined genetic contents of Y. pseudotuberculosis. The combination of SSH and microarray analysis showed that the parallel loss of genes contributed greatly not only to the significant genomic divergence between Y. pestis and Y. pseudotuberculosis but also to the intra-species microevolution of both of species. The results confirmed our earlier hypothesis that Y. pestis Antiqua isolates from the natural plague focus B in China represented the most ancestral strains in China, hence phylogenetically the closest isolates to Y. pseudotuberculosis.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .Xiaoyi Wang and Dongsheng Zhou contributed equally to this work.