RESUMEN
BACKGROUND: In recent years, non-O1, O139 serogroups of Vibrio cholerae have become a major source of pathogenic infection. However, the origin and acquisition of their virulence properties remain under explored. In this regard bacteriophages of Vibrio cholerae are well known to be the carriers of pathogenic traits across various strains. So, any possible association of vibriophages and non-O1, O139 serogroups would provide a deeper insight of their pathogenic threats. MATERIAL/METHODS: Ten non-O1, O139 clinical isolates of Vibrio cholerae were induced by mitomycin C. Virulence profiles of those isolates were determined by multiplex PCR. BglII, KpnI and HaeII were used to generate the restriction profile of isolated bacteriophage. Two of the phage harboring strains was ribotyped by Southern hybridization. RESULTS: In the present study, ten non-O1, O139 diarrheal isolates of Vibrio cholerae were examined for their ability to produce infectious phage particles out of which two strains, PG128 and PG130 were found to be positive. The host range and restriction profile of phage particles were identical to a biotype converting temperate vibriophage PS166. Both PG128 and PG130 carried unique ribotype pattern and lacked the major virulence determinants. But PG128 was found to carry hlyA, mshA, rtxC and toxR, a set of accessory virulence determinants. CONCLUSIONS: The evidences present here provide definite clues for a possible phage mediated emergence of newer Vibrio choleare pathogens.
Asunto(s)
Bacteriófagos/aislamiento & purificación , Lisogenia/fisiología , Vibrio cholerae/virología , Bacteriófagos/clasificación , Bacteriófagos/patogenicidad , Interacciones Huésped-Patógeno/fisiología , Reacción en Cadena de la Polimerasa , Mapeo Restrictivo , Ribotipificación , Vibrio cholerae/fisiología , Virulencia , Activación Viral/fisiologíaRESUMEN
Alkaline phosphatase (APase) of Vibrio cholerae is the first monomeric alkaline phosphatase reported [Roy, N.K., Ghosh, R.K., Das, J., 1982a. Monomeric alkaline phosphatase of V. cholerae. J. Bacteriol. 150, 1033-1039.]. The gene (phoA(VC)) encoding this enzyme is not identified in the published genome sequence of the V. cholerae serogroup O1 El Tor strain N16961 [Heidelberg et al., 2000, DNA sequence of both the chromosome of cholera pathogen V. cholerae. Nature 406, 477-484.]. However two genes (phoB(VC) and phoR(VC)) regulating the synthesis of alkaline phosphatase in this organism, equivalent to phoB and phoR of Escherichia coli, are located in tandem on chromosome I of V. cholerae. We have identified the phoA(VC) gene on the N16961 genome sequence by amino acid sequence analysis of the purified alkaline phosphatase of V. cholerae classical strain 569B followed by BLAST search. The gene was found to be located on the hypothetical protein locus VCA0033 of chromosome II. The identity of the gene was confirmed by expressing the cloned VCA0033 locus in phoA mutant E. coli E15 and JC9223 cells and isolating V. cholerae monomeric alkaline phosphatase. Insertional inactivation of the gene also resulted in complete loss of the phenotype. Unlike in E. coli where phoB, phoR and phoA are closely linked, phoA(VC) is not linked to phoB(VC) and phoR(VC).
Asunto(s)
Fosfatasa Alcalina/genética , Genes Bacterianos/genética , Vibrio cholerae/genética , Fosfatasa Alcalina/química , Fosfatasa Alcalina/metabolismo , Secuencia de Aminoácidos , Clonación Molecular , ADN Bacteriano/química , ADN Bacteriano/genética , Escherichia coli/genética , Genoma Bacteriano , Datos de Secuencia Molecular , Mutagénesis Insercional , Mutación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ADN , Análisis de Secuencia de Proteína , Vibrio cholerae/enzimologíaRESUMEN
We have cloned, sequenced and analysed all the five classes of the intergenic (16S-23S rRNA) spacer region (ISR) associated with the eight rrn operons (rrna-rrnh) of Vibrio cholerae serogroup O1 El Tor strains isolated before, during and after the O 139 outbreak. ISR classes 'a' and 'g' were found to be invariant, ISR-B (ISRb and ISRe) exhibited very little variation, whereas ISR-C (ISRc, ISRd, and ISRf) and ISRh showed the maximum variation. Phylogenetic analysis conducted with all three ISR classes (ISR-B, ISR-C and ISRh) showed that the pre-O 139 serogroup and post-O 139 serogroup O1 El Tor strains arose out of two independent clones, which was congruent with the observation made by earlier workers suggesting that analyses of ISR-C and ISR-h, instead of all five ISR classes, could be successfully used to study phylogeny in this organism.
Asunto(s)
ADN Intergénico/genética , Filogenia , Vibrio cholerae O1/clasificación , Vibrio cholerae O1/genética , Clonación Molecular/métodos , ADN Espaciador Ribosómico/genética , Electroforesis en Gel de Agar , Heterogeneidad Genética , Genoma Bacteriano/genética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Ribosómico 16S/genética , ARN Ribosómico 23S/genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , Vibrio cholerae O1/aislamiento & purificación , Vibrio cholerae O139/clasificación , Vibrio cholerae O139/genéticaRESUMEN
Nine major transfer RNA (tRNA) gene clusters were analysed in various Vibrio cholerae strains. Of these, only the tRNA operon I was found to differ significantly in V. cholerae classical (sixth pandemic) and El Tor (seventh pandemic) strains. Amongst the sixteen tRNA genes contained in this operon, genes for tRNA Gln3 (CAA) and tRNA Leu6 (CUA) were absent in classical strains as compared to El Tor strains. The observation strongly supported the view that the above two pandemic strains constitute two different clones.
Asunto(s)
Operón/genética , ARN de Transferencia/química , Vibrio cholerae/genética , ARN de Transferencia/genética , Vibrio cholerae/clasificación , Operón de ARNr/genéticaRESUMEN
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.