Molecular analysis of the rstR and orfU genes of the CTX prophages integrated in the small chromosomes of environmental Vibrio cholerae non-O1, non-O139 strains.

The ctxAB genes encoding cholera toxin, reside in the genome of a filamentous bacteriophage CTXphi. The presence of CTX prophage in non-epidemic environmental Vibrio cholerae strains is rare. The CTX prophage, the lysogenic form of CTXphi in V. cholerae, is comprised of the 'RS2' and the 'Core'. Analysis of the rstR gene present in the RS2 region of the CTX prophage revealed the presence of new alleles of the prophages in four environmental non-O1, non-O139 strains VCE22 (O36), VCE228 (O27), VCE232 (O4) and VCE233 (O27), and the CTX prophages are located in the small chromosomes. Phylogenetic analysis based on the nucleotide sequences of the rstR and orfU (present in the core) genes of these prophages placed them in a single unique cluster, which is distally located compared with that of epidemic V. cholerae O1 strains. Further analysis indicated that the genome of the prophage present in the strain VCE22 is devoid of the ctxAB genes, called pre-CTX prophage and the strain also possess the toxin-coregulated pilus protein coding gene tcpA of classical type, another important pathogenicity determining locus of the epidemic V. cholerae strains. Comparative analysis of the nucleotide sequences of the rstR and orfU genes indicated that the pre-CTX prophage of VCE22 might be the progenitor of new alleles of the CTX prophages present in these environmental strains.

Isolation and characterization of unserotypable lysogens of Vibrio cholerae phage PS166.

BACKGROUND: Temperate phages of Vibrio cholerae are the major force in the emergence of newer pathogenic clones. PS166, a temperate vibriophage, has the ability to convert pathogenic biotypes of the V. Cholerae O1 serogroup. During the conversion process, PS166 also tends to produce a group of lysogens neither reported nor characterized so far. Characterization of those lysogens may provide insight into the emergence of newer pathogenic clones of V. Cholerae. MATERIAL/METHODS: The V. cholerae eltor strain MAK757 was used to generate PS166 lysogens which were serotyped by using antisera specific to V. Cholerae O1 and O139 strains. Multiplex PCR was used to compare the status of various pathogenic markers of those lysogens. The outer membrane proteins (OMP) of the lysogens were determined by SDS-PAGE. RESULTS: Temperate phage PS166 infection of V. cholerae eltor MAK757 gave rise to a new kind of lysogens termed unserotypable due to their lack of reactivity to both O1 and O139 antisera. Moreover, these lysogens displayed a new phage sensitivity profile different from both classical and parental eltor biotypes. The genetic profiles of the CTX element and VPI region of these lysogens were found to be unaltered, together with the biotype-specific markers mshA and hlyA. Though the genetic loci of major outer membrane proteins did not change after lysogenization, the outer membrane protein profile of these lysogens showed the absence of a 40-kDa band corresponding to the toxR-controlled porin, ompT. CONCLUSIONS: We provide evidence for the vibriophage PS166-mediated lysogenic conversion of pathogenic cholera strains.

[The genome polymorphism of Vibrio cholerae ctxAB(-) strains, containing the proximal part of the CTX element]. / Izuchenie genomnogo polimorphizma ctxAB(-)-shtammov kholernykh vibrionov, soderzhashchikh proksimal'nuiu chast' CTX-elementa.

The comparative analysis of the hybridization patterns of DNA restricts for 20 V. cholerae, groups 01 and non-01 (non-0139), containing the incomplete CTX element (ctxAB-) was carried out with the use of probes, complementary to the genes of the proximal part of the virulence cassettle and flanking its RS1 sequences. This group was found to be heterogeneous both in the number of copies of "truncated" CTX prophage and their localizations in the genome, as well as in the position of the sites of restriction endonucleases HindlII and BglII. Among 17 clinically noncholerigenic isolates, 5 etiologically dangerous clones were found, each of them characterized by the definite time and place of isolation. At least one of them proved to be the causative agent of the local outbreak of diarrheal diseases in Uzbekistan.