[Genetic basis of the variability of nitrate reduction trait in Yersinia pestis strains].

The genetic basis of the varying ability to reduce nitrate in strains belonging to different biovars and subspecies of plague-causing microbe has been investigated and the inability to reduce nitrate observed in different intraspecies groups of Yersinia pestis has been shown to stem from mutations in different genes involved in the expression of this trait. The absence of denitrifying activity in strains of altaica and hissarica subspecies was not due to a mutation at position 613 of the periplasmic reductase napA observed in the strains of the biovar medievalis of the main subspecies, but rather was due to a mutation in the sequence encoding the nitrate-binding domain of the ABC transporter protein SsuA; a thymine insertion (+T) was detected at position 302 from the start of the ssuA gene. Five strains of biovar antiqua isolated at different times in Mongolia, China, and Africa were shown to lack the ability to reduce nitrate. A PCR test targeting two chromosomal regions containing deletions of 19 and 24 bp in size has been developed for the identification of strains of the biovar medievalis. This test can be combined with the test for the marker mutation in the napA gene for a more reliable detection of Y. pestis strains belonging to this biovar.

[Standard algorithm of molecular typing of Yersinia pestis strains].

AIM: Development of the standard algorithm of molecular typing of Yersinia pestis that ensures establishing of subspecies, biovar and focus membership of the studied isolate. Determination of the characteristic strain genotypes of plague infectious agent of main and nonmain subspecies from various natural foci of plague of the Russian Federation and the near abroad. MATERIALS AND METHODS: Genotyping of 192 natural Y. pestis strains of main and nonmain subspecies was performed by using PCR methods, multilocus sequencing and multilocus analysis of variable tandem repeat number. RESULTS: A standard algorithm of molecular typing of plague infectious agent including several stages of Yersinia pestis differentiation by membership: in main and nonmain subspecies, various biovars of the main subspecies, specific subspecies; natural foci and geographic territories was developed. The algorithm is based on 3 typing methods--PCR, multilocus sequence typing and multilocus analysis of variable tandem repeat number using standard DNA targets--life support genes (terC, ilvN, inv, glpD, napA, rhaS and araC) and 7 loci of variable tandem repeats (ms01, ms04, ms06, ms07, ms46, ms62, ms70). The effectiveness of the developed algorithm is shown on the large number of natural Y. pestis strains. Characteristic sequence types of Y. pestis strains of various subspecies and biovars as well as MLVA7 genotypes of strains from natural foci of plague of the Russian Federation and the near abroad were established. CONCLUSION: The application of the developed algorithm will increase the effectiveness of epidemiologic monitoring of plague infectious agent, and analysis of epidemics and outbreaks of plague with establishing the source of origin of the strain and routes of introduction of the infection.

[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.

[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.

[The biofilm formation in Yersinia pestis strains isolated in Astrakhan region].

AIM: To study biofilm formation in strains of Yersinia pestis isolated in 2009 in Astrakhan region. MATERIALS AND METHODS: Study of biofilm formation was performed on abiotic surfaces as well as on cuticle of nematode Caenorhabditis elegans. Detection of genes was performed by PCR with specific primers. RESULTS: Study of phenotypic (fermentation of sugars and alcohols) as well as genetic (presence of plasmids, genes of chromosome region of pigmentation) characteristics of Y. pestis strains showed that they are typical for strains isolated in Astrakhan region. All isolated in 2009 strains formed well developed biofilm on abiotic surfaces and cuticle of C. elegans nematode. They contained genes of hms operon and regulatory genes hmsT and hmsP, which are necessary for formation of pigmented colonies on Congo red medium as well as biofilm on abiotic and biotic surfaces. CONCLUSION: Strains of Y. pestis isolated in 2009 in Astrakhan region formed well developed biofilm on different types of surfaces, which could facilitate their survival in complex parasitic biocenosis of plague natural focus.

[Comparative genetic characteristic of vaccine strain of Yersinia pestis EV and its putative "virulent derivatives"].

AIM: To perform a comparison of genetic characteristics of vaccine strain EV and its putative "virulent derivatives", obtained after passages through highly susceptible animals, in order to identify the strains-"revertants" and establish their possible origin. MATERIALS AND METHODS: Yersinia pestis EV strains and its putative "virulent derivatives" were used in the study. Polymerase chain reaction and DNA-DNA hybridization were used for genetic analysis. RESULTS: Comparison of several genetic characteristics of vaccine strain EV and its putative "virulent derivatives" allowed to establish that virulent "revertants" are not derivatives of vaccine strain EV because they do not belong to East biovar, do not have ribotype characteristic for EV strain and contain pigmentation area, which is absent in vaccine strain. CONCLUSION: Obtained results evidence against possibility of reversion of vaccine EV strain to virulent forms in organisms of highly susceptible animals and confirm its safety for vaccination.

[Comparative analysis of distribution of pseudogenes in the genome of strains of basic and supplementary species of the plague infection agent].

Comparative analysis of distribution of pseudogenes (YPO1582, YPO1728, YPO1967, and YPO4008) of strains of basic and supplementary species of the plague infection agent and pseudotuberculosis infection agent was performed. The genome of basic subspecies of plague infection agent species strain contains 3 different variants: intact genes, genes with IS-element inserts, or individual fragments. The pseudogene profile can be used as genetic marker of the Y. Pestis strains of basic subspecies from natural foci of plague. Strains of supplementary subspecies of Y. Pestis and Y. pseudotuberculosis contain these genes as the wild-type gene alleles. In addition to other factors, this fact can be regarded as evidence of ancient origin of plague infection agent supplementary subspecies and their similarity to pseudotuberculosis infection agent.

[Study of ability to form biofilms in main and non-main subspecies of Yersinia pestis strains].

AIM: To compare biofilm formation in main and non-main subspecies of Yersinia pestis strains as well as in Yersinia pseudotuberculosis strains and to study influence of different genes on expression of this characteristic in different subspecies of Y. pestis. MATERIALS AND METHODS: Study of biofilm formation was performed bygrowing cultures on LB broth in polystyrene Petri dishes with subsequent staining of biofilms formed on the dishes' bottom with crystal violet as well as by electron microscopy. Pigment-sorption sign was detected on differential medium with Congo red. RESULTS: It was shown that the majority of Y. pestis strains and all strains of Y. pseudotuberculosis form well-expressed biofilms on abiotic surface. Formation of biofilms by Y. pestis strains is clearly correlateswith their ability to form pigmented colonies on solid medium with dyestuff. Genes which according to literature data are necessary for biofilm formation by Y. pestis and Y. pseudotuberculosis were found in genome of non-main species. CONCLUSION: Ability of Y. pestis strains belonging to main and non-main subspecies to form biofilm on abiotic surface was revealed.

[Prevalence of type III secretion system genes in cholera vibrios from different serogroups].

Prevalence of vcs genes coding the type III secretion system (T3SS) in cholera vibrios of different serogroups isolated in Russia and neighboring countries was studied for the first time. Virulent strains of O1 and O139 serogroups as well as toxigenic Vibrio cholerae strains of other serogroups contained no T3SS genes. Unlike mentioned strains, 29.2% of atoxigenic non O1/non O139 cholera vibrios isolated from patients in Russia and neighboring countries contained the T3SS genes cluster, which might contribute to the pathogenic properties of these strains.

[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.

[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.

[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 analysis and simulation of the virulence of Yersinia pestis]. / Geneticheskii analiz i modelirovanie virulentnosti 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.