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The review contains some brief information on cholera epidemics in Africa. Based on the results of the whole genome sequencing of 30 clinical strains isolated in Africa in different periods of the 7th cholera pandemic (1985-2012), extensive genetic diversity has been revealed. It is demonstrated that at present cholera epidemics in Africa are caused by new variants of the agent, which emerged in South- Eastern Asia in consequence of not only new genes acquisition, but also genome alterations of pandemicity and pathogenicity islands. SNP analysis of 53 strains circulating at different times in the territory of the continent, as well as isolated in South-Eastern Asia, has been carried out. Phylogenetic relations between the majority of the African and Asian strains have been established. In addition, strains were shown to exist that are, apparently, endemic to the African region. Identified genetic diversity of the strains with varying virulence and drug resistance points out the necessity of continuous molecular monitoring of the cholera agent in Africa.

Experimental modeling of origination of the virulent Vibrio cholerae El Tor genovariants is presented. It was demonstrated that the genovariants obtained did not differ from the natural genetically modified strains emerged in a natural population of the agent, either in phenotypical or genotypic properties. Using the PCR assay and sequencing techniques it was proved that the constructed genovariants carried a CTX(Class phi) prophage genome region with ctxBl gene of the V. cholerae classical biovar in the chromosome. It is shown that the prophage structure alterations lead to the increase in the toxigenicity and virulence in the genovariants compared to the typical strain-recipient. Moreover, as regards proteomics, changes in the expression of 26 proteins that perform various functions in the cell, such as metabolism, energy exchange, transportation, etc., were demonstrated. The data are indicative of the impact that a new DNA region in the genome of the genovariants has on the expression level of different house-keeping genes. The results obtained testify to the fact that one of the mechanisms of the genovariant emergence in the natural populations of the agent can be horizontal gene transfer.

A key pathogenicity factor of the cholera etiologic agent is cholera toxin (CT) whose synthesis is encoded by the ctxAB operon forming apart of the CTXphi ptophage. Alterations in the virulent properties of the cholera vibrios are based on the variability of the CTXphi prophage containing the genes for ctxAB, zot, ace, cep, orfU, and psh in its core region. At the same time, the mechanism of the porophage genome reorganization needs further and more profound analysis. The goal of this work was to demonstrate that transposon Tn5-Mob (Kmr), when introduced into the chromosome of the V. cholera model strain MAK757 El Tor biovar containing two copies of the CTXphi prophage provoked a reorganization in the CTXphi prophage consisting in the deletion of zot, ace, cep, orfU genes. The level of the CT biosynthesis in the insertion mutants MAK757 chr::Tn5-Mob still retaining only the ctxAB operon, increased more than 2000 times as compared to that of the original strain. The enhanced CT production was shown to be associated with the altered structure of the chromosomal DNA region containing one copy of the ctxAB operon encoding this protein biosynthesis. The mutation in the CTXphi genome induced by Tn5-Mob was unstable. Among 600 isolated colonies obtained after dissemination of the MAK757 chr::Tn5-Mob transposant capable of CT overproduction in the full medium with no antibiotics, 5.8% gave clones that in parallel to the loss of Kmr marker, appeared to be deprived of the ctxAB operon thus becoming non-toxinogenic. The observed formation of the V. cholerae insertion mutants both capable of CT overproduction and non-toxinogenic ones, may be indicative of an important role played in the evolution of the cholera pathogen by the CTXphi genome variability induced by Tn elements. The plasmidless V. cholerae El Tor strain characterized by type II CT hyperproduction thus obtained in our experiments could be used for the production of this protein routinely applied to construct efficient cholera diagnostic and prophylactic preparations.

The conjugative recombinant plasmid pIEM3 (KmR TcR) was constructed in order to introduce the cloned ctxB gene encoding the cholera toxin B subunit into the Vibrio cholerae cells. The plasmid was obtained as a result of co-integration of two plasmids: a conjugative plasmid, pIEM1(KmR), carrying mini-kan transposon and IS1 element, as well as the pCTdelta27(TcR) plasmid that is a derivative of the pBR322 which carries the cloned ctxB gene. The avirulent Vibrio cholerae strain eltor biovar deprived (according to the PCR analysis) of the key structural and regulatory pathogenicity genes and carrying a mutation in a single gene of the O1 antigen was chosen as the pIEM3 plasmid carrier strain. The cointegrate uncoupling was shown to take place in 5% the cholera vibrio cells followed by retention of only the multi-copy pCTdelta7 plasmid. This event leads to the formation of the TcRKmS clones characterized by high levels of the cholera toxin secreted B subunit production (10 to 14 microg/ml), one of these (KM93) being selected as a strain-producer of the protein. Molecular-genetic and biochemical assays were used to elucidate peculiar features of inheritance and expression of the cloned ctxAB gene within the KM93 cells. The expression of the cloned ctxB gene was shown to be independent of the presence of the toxR, tcpP, tcpH, toxT regulatory genes suggesting the existence of some other mechanisms that might exert their control over the transcriptional activity of the cholera toxin B subunit gene. Effective production of the cholera toxin B subunit would be also observed if the constructed producer strain was cultured under the conditions of industrial process. This indicates a possibility of its employment as a source of this protein involved in manufacturing cholera immunodiagnostic and prophylactic preparations.

The evolution of the genome of the pathogenic agent of the seventh cholera pandemia Vibrio cholerae eltor biovariant was thought to occur by acquiring not only structural genes of virulence but also regulatory systems as a result of horizontal transfer events. The polymerase chain reaction revealed the presence of the following regulatory genes that control the virulence gene expression in the chromosome of pre-pandemic and pandemic strains of cholera vibrios eltor: toxR, toxT, tcpP, tcpH, luxS, luxO, crp, vicH, pepA. The avirulent V. cholerae strain ATCC14033 isolated in 1910 (hypothetical predecessor of the cholera eltor agent) was shown to be lacking the regulatory genes toxT, tcpP, tcpHlocalized in the pathogenicity island VPI-1, and to be capable of realizing positive control over the expression of the virulence genes involved in the ToxR regulon. The virulent strains isolated from cholera patients during the local cholera outbreak in Indonesia in 1937 did not differ from the strains that caused cholera eltor pandemic in 1961. The strains had identical content of the regulatory genes tested. Only one strain of the four isolates studied contained no tcpPgene. Two key regulatory genes, toxR and toxT, were sequenced in all the isolates. The toxR nucleotide sequence of three pre-pandemic strains was shown to be indistinguishable from that of the pandemic isolates. On the other hand, the clinical strain MAK757 isolated prior to the emergence of the epidemic demonstrated an altered nucleotide sequence in its toxR gene. Experiments with the intra-intestinal challenge of suckling rabbits were indicative of similar virulence levels for the pre-pandemic and pandemic clinical strains. These results may serve as the evidence of the in vivo activity of the pre-pandemic strains of the toxT, tcpH, and tcpP positive regulatory genes that acquired in V. cholerae during the evolutionary process.

Cholera, plague, and anthrax, the diseases that have accounted for millions of human victims, still endanger the entire mankind by possible development of epidemic outbreaks due to their spread or application as bioterrorist agents. Generalized results of research into the genomic features of the Vibrio cholerae, Yersinia pestis, and Bacillus anthracis are discussed. Despite different frequencies of evolutional transformations occurring in their genomes, that are likely to be associated with diverse life cycles of the pathogens, clones with altered diagnostic, and virulence characteristics were shown to have a fair probability of formation. Also presented in the review, are literature data concerning the main evolutional stages for any of these pathogens, determination of new genetic variants, consideration of the mechanisms facilitating maintenance of the microbial agents during the interepidemic periods.

A comparative analysis of the genome of V. cholerae O139 strains isolated in Russia's territory from patients with cholera and from the environment showed essential differences in their structures. The genome of clinical strains possessed all tested genes associated with virulence (ctxAB, zot, ace, rstC, rtxA, hap, toxR and toxT) and the at-tRS site for the CTXp phage DNA integration. As for the O139 V. cholerae chromosome strains isolated from water, 70% of the studied genes (ctxAB, zot, ace, rstC, tcpA, and toxT) and the attRS sequence were not detected in them. A lack of the key virulence genes in O139-serogroup "water" vibrios, including genes of toxin-coregulated adhesion pili. (that are receptors for the CTXp phage), and of the attachment site of the above phage are indicative of that the O139 V. cholerae strains isolated from open water sources located in different Russia's regions are epidemically negligible.

The paper contains a survey of published data about the use of DNA-diagnostics in indicating and identifying the causative agents of highly dangerous infections like plague, cholera and anthrax. A discussion of data about the genetic relationship between strains of the mentioned causative agents isolated from different sources by using the molecular-typing methods as well as about the evolution ties between strains of different origins is in the focus of attention. Results of comparative studies of nucleotide sequences of genomes or of individual genomes in different Yersinia pestis, Vibrio cholerae and Bacillus anthracis strains, which are indicative of the evolution of their pathogenicity, are also under discussion.

Infection of V. cholerae 01 (classical and eltor biovars) cells with the temperate cholera phage 139 derived from V. cholerae serogroup 0139 followed by integration of the phage genome into the bacterial chromosome significantly increased the production of cholera toxin, the main virulence factor. The level of toxin biosynthesis in the lysogenic V. cholerae classical strain increased 3-fold and that in V. eltor thirty times in comparison with the parental strains. Increased production of cholera toxin was not associated with an increase in the number of copies of genes involved in its biosynthesis but seemed to be due to changes in toxinogenesis regulation.

Polymerase chain reaction (PCR) detected the presence of various genes associated with virulence in genome of strains V. cholerae eltor isolated in Turkmenistan territory during epidemic and epidemic-free perios. It was found that a complete set of virulence genes (ctxA+, tcpA+ and toxR+) contained strains isolated from patients, carriers and environment only in cholera epidemics. Strains isolated from the environment in the period free of epidemics did not contain ctxA and tcpA in 78.2% of cases, but 5.2% of the strains carried a complete set of virulence genes. There were also nontoxigenic strains containing genes tcpA and toxR. Such strains were isolated from the environment (16.6%) and vibrion carriers (42.9%). Isolated were also strains V.cholerae eltor carrying bacteriophage CTX phi with incomplete set of virulence genes and having genotype ctxA-, ace+ and zot+. Almost all the strains ctxA-, tcpA+ carry attRS1-site in genome. This shows that such strains may transform into toxigenic as a result of infection with bacteriophage CTX phi.

The analysis of modern data concerning new genes of Vibrio cholerae of O139 serogroup and genetic control of their biosynthesis is represented. The special attention is paid to description of molecular genetic events induced the derivation the cholera causative agent of new serogroup. The structure of the moderate phages of V. cholerae O139 and their role in virulence of the cholera causative agent of the new serogroup and variability of its genome are discussed.

Specific oligonucleotide primers were chosen for identifying the fragments of the four major virulence genes of V. cholerae eltor (ctxA, tcpA, toxR, and hap) using the polymerase chain reaction (PCR). In order to estimate the efficiency of complex PCR testing of V. cholerae for evaluation of their epidemiological significance, a collection of 80 V. cholerae eltor strains with known virulence was selected, whose most important specific features had been studied previously. The hap was appropriate species-specific gene making it possible to detect V. cholerae strains regardless of their virulence. The most complete and objective data for evaluating the epidemic significance can be obtained by detecting the presence of three virulence genes (ctxA, tcpA, and toxR) in their chromosome. The prevalence of the above four genes in various V. cholerae strains isolated from the environment during epidemic and non-epidemic periods was studied.

Molecular genetic features of Vibrio cholerae classical strains which caused an epidemic of Asian cholera in Russia in 1942 have been studied for the first time. These strains had a high level of choleric toxin production and toxin-coregulated adhesion piles, the main virulence factors; all the strains were auxotrophs and needed purine and/or amino acids for growth in minimal medium. Moreover, having hapA structural gene in the chromosome (according to polymerase chain reaction), they did not produce soluble hemagglutinin protease promoting propagation of vibrios in the environment. These features of natural V. cholerae classical strains are apparently responsible for the peculiar infectious and epidemic processes in the cholera epidemic.

Restriction analysis of temperate cholera phage 139 isolated from Vibrio cholerae P16064, serogroup 0139, showed its DNA to be double-stranded linear with cohesive terminals. DNA-DNA hybridization on nylon membranes revealed that many V. cholerae strains of serogroup 0139 isolated in different regions contained a temperate cholera phage 139 in their genomes. Southern blot hybridization of chromosomal DNA PST-fragments with phage probe showed that the temperate phage 139 was identical to the temperate phage of serogroup II V. eltor. The phage integrated in the chromosome near genes encoding motility (mot) and production of the capsule (cap) and purine (pur). Phage genome is apparently responsible for instability of cap, pur, and mot genes whose products are important for the development of an infectious process in cholera.

Reviews modern data on the genetic control of the key factors of Vibrio cholerae pathogenicity: cholera toxin and toxin-coregulated adhesion pili. Pays special attention to the temperate filamentous CTX bacteriophage, whose genome contains structural genes of cholera toxin, and the "pathogenicity island" carrying tcp genes responsible for the most important factor of the human small intestine colonization with V. cholerae. Discusses the mechanism of coordinated regulation of the activity of the main genes of V. cholerae pathogenicity genes.

A conjugational gene transfer system consisting of donor and recipient strains has been developed for genetic analysis of Vibrio cholerae 0139 serogroup, a new cholera agent. Donor strains constructed using the Tn5-Mob carrying the origin of transfer (ori T) of plasmid RP4 and helper plasmid pRP4-4 were able to perform a directed transfer of chromosomal markers. Recipient strains carried mutations in auxotrophic genes as well as in virulence genes. Based on this gene transfer system, a genetic map of V. cholerae chromosome has been created showing the order of 17 gene markers. Relationship between the genetic structure of V. cholerae 0139 and 01 is discussed.

Conjugational-mating experiments showed mrh genes coding for the synthesis of mannose and fucose resistant hemagglutinin/adhesin and vibrio motility gene mot to be localized in the chromosome of V. cholerae Dacca strain 35 close to tox-2 mutation providing a high level of toxin production and rfb locus which controls 01 antigen synthesis. The results indicate the presence of a block of virulence genes within ura-pur chromosomal region of V. cholerae.

A clinical isolate Vibrio cholerae P16064 serogroup 0139 was shown to produce two different kinds of colonies: opaque encapsulated and translucent devoid of capsule. Low virulence of translucent colonies seems to be due to not only loss of capsule which determines serum resistance, but also to decreased expression of genes controlling the motility, and production of protease and mannose-sensitive adhesion pili. Analysis of the lysogenic properties of the two types of colonies permitted us to propose that simultaneous spontaneous alteration of capsule production and the above-mentioned virulence factors in translucent colonies may be caused by a temperate phage in turbid clones of strain P16064.

A new mutation tox-2 defining the increased level of cholera exotoxin production by the strain Vibrio cholerae Dakka 35 isolated from nature has been mapped by conjugational crosses of donor and recipient strains differing by toxin production and serovar. The mutation has been localized on the chromosomal fragment containing the ilv, pur, ura, rfb genes adjacent to ura-94 locus. The linkage of the tox-2 mutation with the rfb locus coding for the synthesis of somatic Ol-antigen has been also established.

The nonagglutinating vibrions having Tn-elements inserted into the chromosome were obtained as a result of conjugal transfer of vector plasmids carrying the different transposons (Tn9, Tn10, Tn601, Tn5-Mob) and of the consequent isolation of plasmid-free clones of Vibrio cholerae non OI. Identification of auxotrophic mutations induced by the transposons inserted into the bacterial genome made possible the construction of the primary chromosomal map of Vibrio cholerae non OI. The efficient donor strains of Vibrio cholerae non OI were constructed by introducing the transposon Tn5-Mob and the helper plasmid RP-4. The donors are capable of oriented conjugal transfer of chromosome.

The mutations oagM and oagR affecting the synthesis of somatic O-antigen have been localized on the chromosome of Vibrio cholerae El Jor and classic biotypes by conjugational crosses between different donor and recipient strains. The mutations are localized in the vicinity of the arg marker in both classic and El Tor biotypes of Vibrio cholerae.