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.     

Effects of growth temperature and pVM82 plasmid on fatty acids of lipid A from Yersinia pseudotuberculosis

Effects of cultivation temperature (8 or 37 degrees C) and plasmid profile on the lipid A fatty acids of three isogenic Yersinia pseudotuberculosis strains (plasmidless (82-) and strains containing pVM82 (82+) or p57 (57+) plasmids) obtained by alkaline hydrolysis of the whole bacterial cells and differentiated from fatty acids of other membrane lipids were investigated. On the basis of the analysis, it is concluded that lipids A of all studied samples contain 3-hydroxytetradecanoic and dodecanoic acids, a part of which exists as the 3-dodecanoyloxytetradecanoic derivative. The effect of temperature appears in the higher contents of ester- and amide-linked 3-acyloxyalkanoic residues in lipid A from the "cold" variants of the bacteria and is determined by chromosomal genes. The plasmid effect is seen as various responses of the isogenic derivatives to change of growth temperature: in cells of strains 82+ and 82- grown in the cold, the share of lipid A fatty acids in the total population of cellular fatty acids is reduced, while in strains with plasmid p57 it is increased. The temperature variants of the 57+ strain differ by the low contents of amide-linked 3-acyloxyalkanoic acids. Finally, lack of plasmid pVM82 in the "warm" variants of the bacteria results in accumulation of glycolipid molecules deprived of dodecanoic acid. Correlation between growth temperature and plasmid profiles, on one hand, and lipid A fatty acid composition and potential pathogenic properties of the Y. pseudotuberculosis, on the other hand, and also possible mechanisms of thermal adaptation of this organism are discussed.     

A new pathogenic trait encoded by Yersinia pseudotuberculosis pVM82 plasmid

The strains of Yersinia pseudotuberculosis isolated from patients in the course of outbreaks of infection (epidemic strains) were found to possess at least two plasmids with molecular masses of 45 and 82 MD. In contrast, the strains obtained in sporadic cases harbored different sets of plasmids, but never the 82 MD plasmids. These plasmids designated pVM82 and isolated from strains of different geographic regions of the country were identical. pVM82 have no homology with Y. pestis plasmids of the similar size coding for the FraI antigen. The pVM82 DNA was found to be composed of the 57 MD plasmid DNA and the 25 MD fragment of Y. pseudotuberculosis DNA. Using Western blot hybridization technique it was shown that the presence of pVM82 suppressed formation of antibody against some major antigenic determinants of Y. pseudotuberculosis. Immunosuppression took place when the animals were infected with bacteria grown below 20 but not at 37 degrees C. The 57 MD plasmid failed to produce immunosuppression. It was concluded that the 25 MD fragment of pFN82 encoded a novel pathogenic factor responsible for immunosuppression.     

Alternative localization of the determinant of pathogenicity coded by the Yersinia pseudotuberculosis pVM82 plasmid]

The chromosomal DNA regions in Yersinia pseudotuberculosis strains occur that are homologous to 25 Md DNA segment of the plasmid pVM82 encoding the bacterial capability of immunosuppression. The character of the chromosomal DNA regions dispersion reacting with the 25 Md segment probes is different in epidemiologically hazardous and nonvirulent strains of Yersinia pseudotuberculosis. The specific DNA regions occur as well as identical ones. The suppression of antibody formation to a number of main Yersinia pseudotuberculosis antigens by epidemiologically hazardous strain is demonstrated. The suppression is analogous to the one previously described for Yersinia pseudotuberculosis strains harbouring the plasmid pVM82.     

Effect of a culturing method and growth phase on composition of lipopolysaccharides in Yersinia pseudotuberculosis

The effects of the culturing method (suspension cultures in a liquid nutrient broth or colonies on a solid agarized medium) and the growth phase on the lypopolysaccharide (LPS) composition of Yersinia pseudotuberculosis (O:Ib serovar, strain KS 3058) grown in cold (5 degrees C) were studied. The amount the LPS synthesized by cells depended on the bacteria growth phase for both media. The LPS acylation degree was constant, whereas the length of the O-specific polysaccharide chain varied with the culture age and achieved maximum in the stationary growth phase for both media. The bacteria culturing on the nutrient agar stimulated more intensive synthesis of LPS, which were extracted more easily, had longer polysaccharide O-chains, and were more toxic than LPS of the bacteria cultured in the liquid medium. It was proposed that the culturing of Yersinia pseudotuberculosis in cold as colonies on the agar surface causes an increase in the bacterial virulence.     

Structure of O-specific side chains of lipopolysaccharides from Yersinia pseudotuberculosis

Lipopolysaccharide prepared from cells of Yersinia (Pasteurella) pseudotuberculosis of serogroups I, II, III, IV, and V is known to contain the 3,6-dideoxyhexose (DDH) paratose, abequose, paratose, tyvelose, and ascarylose in its respective O-specific side chains. Lipopolysaccharides or lipid-free polysaccharides of all of the 10 known serogroups and subgroups were subjected to methylation analysis and determined as alditol acetates by gas-liquid chromatography and mass spectrometry. The results indicated that the O-specific side chains of nine serotypes are composed of oligosaccharide repeating units in the form of four alternative general structures in which a terminal DDH may vary. These structures are DDH [Formula: see text] 6-deoxy-d-manno-heptose [Formula: see text] d-galactose (serogroups IA, IIA, and IVB), DDH [Formula: see text] d-mannose [Formula: see text] l-fucose (serogroups IB and IIB), and two configurations similar to the latter except that the 4-position of l-fucose was either linked to the d-mannose residue (serogroups VA and VB) or to the DDH residue (serogroups III and IVA). In contrast, O-groups in lipopolysaccharide of the newly discovered serogroup VI contained the DDH colitose and 2-acetamido-2-deoxy-d-galactose. Accordingly, all five known types of DDH have now been detected in lipopolysaccharides of Y. pseudotuberculosis. The sugar 6-deoxy-d-manno-heptose, present in O-specific side chains of serogroups IA, IIA, and IVB, has not yet been reported to occur elsewhere in nature.     

Heterogeneity of lipopolysaccharides from Yersinia pseudotuberculosis: chemical characterization of various molecular types

Prior studies have shown some unusual changes in the lipopolysaccharides (LPSs) from Yersinia pseudotuberculosis that occur when the microbe is grown at low temperature; the specific features of these LPSs in comparison with the LPSs from other enteropathogens may be due to unusual thermal adaptation mechanisms. To gain insight into this question, the chemical composition of Y. pseudotuberculosis LPS has been determined. The data indicate that two different S-form LPS species are produced in "cold"-grown bacteria. These have an identical set of bands after SDS-PAGE, similar elution profiles during gel-filtration on a Sephadex G-200 column in the presence of sodium deoxycholate, identical monosaccharide and fatty acid compositions, and similar polymerization degrees, but they have different acylation degree. On the whole, the macromolecularly different LPS populations, varying not only in their smooth or rough nature and hydrophobicity, but also in their localization in the outer membrane and, probably, their interactions with other cell components, are synthesized in "cold"-grown Y. pseudotuberculosis. The biological sense of the heterogeneity and its connection with psychrophilic and pathogenic properties of pseudotuberculosis organisms are discussed.     

Analysis of the plasmid composition of Yersinia pseudotuberculosis strains and its use for typing pseudotuberculosis pathogens

Electrophoresis in agarose gel has been used to study the plasmid spectra of 854 Yersinia pseudotuberculosis strains isolated from different sources. The plasmids found in the microbial strains are represented by the elements with molecular masses 82; 57; 45; 5.5; 4.4; 3.5; 2.7; 2.4; 2.3 Md. The variable spectra of plasmids is peculiar only for serovar I of Yersinia pseudotuberculosis. Plasmids p45 and p82 are classified as the main, while other plasmids as auxiliary ones. In accord with the classification all plasmid containing strains are divided into 8 plasmid strains. Using the proposed method for intraspecific typing of Yersinia pseudotuberculosis permits one to perfect the epidemiological analysis of pseudotuberculosis infection and make concrete the direction of prophylactic and antiepidemic measures.     

The effect of temperature on the interaction of Yersinia pseudotuberculosis lipopolysaccharide with chitosan

The mechanism of binding of lipopolysaccharide (LPS) from Yersinia pseudotuberculosis to low-molecular-weight chitosan was investigated using sedimentation analysis, centrifugation in glycerol and percoll density gradients, and isopicnic centrifugation in cesium chloride. The LPS interaction with chitosan was shown to be a multistage process that depended on time and reaction temperature. A stable LPS-chitosan complex could be formed only after preliminary incubation of the initial components at an elevated temperature (37 degrees C). This temperature caused the LPS dissociation and promoted its binding to chitosan. The LPS binding to chitosan results in further dissociation of the endotoxin and formation of the complex with a molecular weight that is tens of times less than the initial molecular weight of LPS. The obtained complex remained stable in solutions of high ionic strength.     

The effect of the trophic and temperature conditions of cultivation on lipid synthesis by Yersinia pseudotuberculosis

The comparative study of the synthesis lipids in Y. pseudotuberculosis, depending on the conditions of their cultivation (at different temperatures in mineral media and in media, containing organic compounds), has been carried out. As demonstrated in this study, temperature in the main inducing factor, affecting the synthesis of lipids of definite classes and fatty acids, incorporated into these lipids. During the cultivation of Y. pseudotuberculosis in mineral and organic media under the conditions of low temperature their lipid composition remains unchanged, but at 6 degrees C the synthesis of unsaturated fatty acids prevails, while at 37 degrees C saturated fatty acids are mainly synthesized. On mineral media at 37 degrees C bacteria synthesize mostly nonpolar lipids in the form of reserve substances, represented by triglycerides and free fatty acids.     

The effect of Yersinia pestis EV76 6 MD plasmid on the composition of outer membrane proteins of Yersinia pseudotuberculosis YPIII]

On the basis of Yersinia pseudotuberculosis strain YPIII the isogenic variants containing the different combinations of 47 Md plasmids from Yersinia pestis or Yersinia pseudotuberculosis cells with the 6 Md pYP plasmid from Yersinia pestis EV (intact or having impaired the pla gene determining the synthesis of plasmocoagulase). The degradation of the secreted proteins encoded by the 47 Md plasmids of Yersinia pestis and Yersinia pseudotuberculosis in the cells harbouring the 6Md pYP plasmid has been registered. Yersinia pseudotuberculosis strain YPIII carrying its own 47Md and pYP plasmids also contained no YOP1 protein, in contract to the parent strain. The damage of the pla gene eliminated the destructive effect on the outer membrane proteins. Imposition of the 47Md and 6Md plasmids from Yersinia pestis in Yersinia pseudotuberculosis cells may be used for obtaining and study of the physiological role of low molecular mass proteins resulting from proteolysis of proteins encoded by the 47Md virulence plasmid of Yersinia.     

Fatty-acid composition of cells and lipopolysaccharides in different Yersinia species under the conditions of growth at low temperature

Y. pestis, Y. pseudotuberculosis, Y. enterocolitica, Y. frederiksenii, Y. intermedia, Y. kristensenii and Y. ruckeri grown at 4 degrees C were characterized by fatty acid composition with a high content of C16:1 and C18:1, as well as the proportion of saturated to nonsaturated fatty acids equal to, on the average, 2.0. In Yersinia lipopolysaccharides a relatively high level of C16:1 and C12:0 was observed with the prevalence of 3-OH-C14:0. In the fatty-acid spectra of both cells and lipopolysaccharides no essential difference was noted. Thus, during growth at low temperature differences, earlier detected in the studied Yersinia species grown at 37 degrees C and making it possible to divide 7 Yersinia species into 2 groupes, were completely leveled. These results confirmed the close phylogenetic relationship between the Yersinia species under study and were indicative of more pronounced biological community of Yersinia under the conditions of growth at low temperature.     

Yersinia pseudotuberculosis plasmids and their significance in the realization of the epidemic process in pseudotuberculosis

The results obtained in the electrophoretic study of the plasmid spectra of 190 Y. pseudotuberculosis strains, isolated from different sources, in 0.6% agarose gel are presented. 11 types of plasmids differing in their molecular weight have been detected. Plasmids with a molecular weight of 45 MD determine Ca2+ dependence, bacterial virulence for white mice and autoagglutination. The presence of differences in Y. pseudotuberculosis strains of serovars I, III and IV has been established, which is manifested by their differing plasmid spectra. The relationship between the presence of plasmids with a molecular weight of 75 and 45 DM in the strains and the character of pseudotuberculosis morbidity in the population has been demonstrated. The epidemic course of infection correlates with the presence of both these plasmids and the sporadic course of infection, with the presence of the plasmid with a molecular weight of 45 MD only.     

Action of sodium chloride on Yersinia pseudotuberculosis and Yersinia enterocolitica

The bacteriostatic and bactericidal action of sodium chloride on 60 Y. pseudotuberculosis strains, 75 Y. enterocolitica strains and 158 urine-fermenting strains has been studied. A new specific feature of Y. pseudotuberculosis has been revealed: high sensitivity to sodium chloride. The suitability of the sodium chloride test has been shown for the identification of Yersinia and the differentiation of Y. pseudotuberculosis and Y. enterocolitica.     

Various characteristics of Yersinia pseudotuberculosis determined by plasmid 45 Md associated with calcium dependence

The clones of two types (T+ and T-) have been identified among the strains of Y. pseudotuberculosis. The difference in the morphology of the clones is based on the ability of T+ clones to agglutinate in the process of growth. The identified clones are different in calcium dependence, in the ability to agglutinate and in their virulence for the laboratory animals. The differences have been proved to be connected with the presence of a 45 Md plasmid in T+ cells. Replication of this plasmid is suppressed by the acridine orange dye in concentrations of 12.5 or 25.0 mkg X ml-1. The plasmid is spontaneously lost from the strains during their continuous storage. The microscopy of colonies permits the selection of clones with the parental phenotype from the populations having lost the 45 Md plasmid.     

The isolation of Yersinia intermedia strains with a plasmid of 82-megadalton molecular weight in natural populations of Yersinia

For the first time Y. intermedia strains containing plasmids with a molecular weight of 82 MD have been detected in natural populations of urease-positive Yersinia. Such populations have been isolated from two species of birds and from the soil in the area where they have been killed (the Maritime Territory), as well as from washings from the surface of onions in a vegetable store (Chita Province). The strains, administered orally to white mice, proved to be nonpathogenic. Plasmids with a molecular weight of 82 MD are supposed to occur in natural populations of other Yersinia species.