Molecular-epidemiological characteristic and possible origin of Vibrio cholerae non O1/non O139 with complete and limited set of virulence genes

Study of molecular-epidemiological characteristics of Vibrio cholerae non O1/non O139 serogroup with complete and limited set of virulence genes was performed. Differences of their genes composition as compared to these of O1 serogroup (classic and El Tor biovars) were revealed, which points to their origin from avirulent environmental cholera vibrios.     

Genetic analysis and simulation of the virulence of Yersinia pestis

The genetic analysis of Y. pestis virulence factors accomplished in the 358 strain isogenic system allowed us to determine a minimal set of known factors providing pathogenicity. The combination of chromosomal marker Pgm+ and calcium dependence plasmid (pCad) is shown to be sufficient for preserving the virulence of Y. pestis. Experimental modelling of virulence in this microorganism by the genetic exchange methods was carried out. The reduced virulence of the strains Pgm+ and pCad+ for guinea pigs was detected.     

Determination of genetic bases of auxotrophy in Yersinia pestis ssp. caucasica strains

Based on the results of computer analysis of nucleotide sequences in strains Yersinia pestis and Y. pseudotuberculosis recorded in the files of NCBI GenBank database, differences between genes argA, aroG, aroF, thiH, and thiG of strain Pestoides F (subspecies caucasica) were found, compared to other strains of plaque agent and pseudotuberculosis microbe. Using PCR with calculated primers and the method of sequence analysis, the structure of variable regions of these genes was studied in 96 natural Y. pestis and Y. pseudotuberculosis strains. It was shown that all examined strains of subspecies caucasica, unlike strains of plague-causing agent of other subspecies and pseudotubercolosis microbe, had identical mutations in genes argA (integration of the insertion sequence IS100), aroG (insertion of ten nucleotides), aroF (inserion of IS100), thiH (insertion of nucleotide T), and thiG (deletion of 13 nucleotides). These mutations are the reason for the absence in strains belonging to this subspecies of the ability to synthesize arginine, phenylalanine, tyrosine, and vitamin B1 (thiamine), and cause their auxotrophy for these growth factors.     

A study of the nucleotide sequence variability of rha locus genes of Yersinia pestis main and non-main subspecies

A study of the structural and regulatory genes, determining rhamnose fermentation, that are located in the rha locus of the chromosome of Yersinia pestis main and non-main subspecies and of Yersinia pseudotuberculosis of serogroups I-III was performed. The nucleotide sequence of Y. pestis main subspecies differs substantially from those of non-main subspecies and Y. pseudotuberculosis by the presence of a nucleotide substitution in 671 bp location of rhaS gene resulting presumably in the Y. pestis non-main subsp inability to utilize rhamnose. This results in incapability of Y. pestis non-main subspecies to utilize rhamnose. Other nucleotide substitutions found in Y. pestis non-main subspecies strains influence only upon expression efficiency of this diagnostic criterion.     

Genotyping of Yersinia pestis strains on the basis of variation of ribosomal rRNA biosynthesis genes

Analysis of restriction fragment length polymorphism of rRNA genes of Yersinia pestis and Y. pseudotuberculosis strains, circulating in Russian Federation and abroad revealed the effectiveness of ribotyping for differentiation between these microorganisms, as well as for differentiation between different Y. pestis biovars and main and nonmain subspecies of this agent. Use of this method was shown to be promising as a component for the complex molecular typing system of Y. pestis. Variant ribotypes of main and non-main subspecies of Y. pestis strains are presented.     

Genetic bases of methionine dependence in <Emphasis Type="Italic">Yersinia pestis</Emphasis> strains of main and non-main subspecies

Structural and functional organization of genes responsible for biosynthesis of amino acid methionine, which plays a leading role in cellular metabolism of bacteria, was studied in 24 natural Yersinia pestis strains of the major and non-main subspecies from various natural plague foci located in the territory of Russian Federation and neighbouring foreign countries, and also in Y. pestis and Y. pseudotuberculosis strains recorded in the files of NCBI GenBank database. Conservatism of genes metA, metC, metE, and metH as well as regulatory genes metR and metJ involved in biosynthesis of this amino acid was established. Sequencing of the variable locus of gene metB in natural Y. pestis strains of major and non-main subspecies revealed that the reason for the methionine dependence of strains belonging to the main subspecies is a deletion of a single nucleotide (−G) in the 988 position from the beginning of the gene, whereas this dependence in strains belonging to subspecies hissarica results from the appearance of a single nucleotide (+G) insertion in the 989 position of gene metB. These mutations are absent in strains of the caucasica, altaica, and ulegeica subspecies of the plague agent and in strains of pseudotuberculosis microbe, which correlates with their capacity for methionine biosynthesis.     

Structural analysis of genes participating in melibiose fermentation and isocitrate lyase production in <Emphasis Type="Italic">Yersinia pestis</Emphasis> strains of the main and nonmain subspecies

Comparative analysis of nucleotide sequences of genes participating in melibiose fermentation and isocitrate lyase production was conducted in 90 natural Yersinia pestis strains of main and nonmain subspecies. It was ascertained that the lack of the ability to utilize disaccharide melibiose in strains of the main subspecies is caused by integration of the insertion sequence IS285 at 73 bp from the beginning of the structural gene melB that encodes the transport protein galactoside permease. In contrast, strains of nonmain subspecies (caucasica, altaica, hissarica and ulegeica) contain the intact gene melB and are capable of fermenting melibiose. Differences in the manifestation of the other differential trait, production of isocitrate lyase, are connected with the presence in strains of the main species of mutation (insertion of two nucleotides +CC) in the regulatory gene iclR encoding repressor protein of the acetate operon, which is the reason for constitutive synthesis of this enzyme. Strains of nonmain subspecies do not contain mutations in gene iclR, and this correlates in these strains with their capacity for inducible synthesis of isocitrate lyase.     

Genetic bases of methionine dependence in Yersinia pestis strains of major and non-major subspecies

Structural and functional organization of genes responsible for biosynthesis of amino acid methionine, which plays a leading role in cellular metabolism of bacteria, was studied in 24 natural Yersinia pestis strains of the major and minor subspecies from various natural plague foci located in the territory of Russian Federation and neighbouring foreign countries, and also in Y. pestis and Y. pseudotuberculosis strains recorded in the files of NCBI GenBank database. Conservatism of genes metA, metB, metC, metE, and metH as well as regulatory genes metR and metJ involved in biosynthesis of this amino acid was established. Sequencing of the variable locus of gene metB in natural Y. pestis strains of major and minor subspecies revealed that the reason for the methionine dependence of strains belonging to the major subspecies is a deletion of a single nucleotide (-G) in the 988 position from the beginning of the gene, whereas this dependence in strains belonging to subspecies hissarica results from the appearance of a single nucleotide (+G) insertion in the 989 position of gene metB. These mutations are absent in strains of the caucasica, altaica, and ulegeica subspecies of the plague agent and in strains of pseudotuberculosis microbe, which correlates with their capacity for methionine biosynthesis.     

Structural analysis of genes participating in melibiose fermentation and isocitrate lyase production in Yersinia pestis strains of main and non main subspecies

Comparative analysis of nucleotide sequences of genes participating in melibiose fermentation and isocitrate lyase production was conducted in 90 natural Yersinia pestis strains of main and non main subspecies. It was ascertained that the lack of the ability to utilize disaccharide melibiose in strains of the main subspecies is caused by integration of the insertion sequence IS285 at 73 bp from the beginning of the structural gene melB that encodes the transport protein galactoside permease. In contrast, strains of non main subspecies (caucasica, altaica, and ulegeica) contain the intact gene melB and are capable of fermenting melibiose. Differences in the manifestation of the other differential trait, production of isocitrate lyase, are connected with the presence of mutation (insertion of two nucleotides +CC) in the regulatory gene iclR encoding repressor protein of the acetate operon, which is the reason for constitutive synthesis of this enzyme. Strains of non main subspecies do not contain mutations in gene iclR, and this correlates in these strains with their capacity for inducible synthesis of isocitrate lyase.