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The ctxAB operon, encoding cholera toxin (CT) in Vibrio cholerae, is carried by the genome of a filamentous phage, CTX[PHI]. Usually, specific CTX[PHI] infect each of the two important biotypes, classical and El Tor, of epidemic V. cholerae strains belonging to serogroup O1, and are called [CTX.sup.class][PHI] and [CTX.sup.ET][PHI], respectively. However, an unusual hybrid El Tor strain carrying [CTX.sup.class][PHI] caused the cholera epidemic in Mozambique in 2004. To understand the evolution of that strain, we have further analysed some representative hybrid El Tor strains isolated in Kolkata, India, in 1992, and the results indicate that both the Mozambique and the Indian strains are infected with a unique [CTX.sup.class][PHI] having only four copies of the tandem heptamer repeat sequence 5'-TTTTGAT-3' present in the ctxAB promoter ([P.sub.ctxAB]) region, like in [CTX.sup.ET][PHI]. Usually, the [P.sub.ctxAB] of the classical biotype contains seven to eight copies of such sequences. However, sequence analyses of the [P.sub.ctxAB] regions of several classical strains indicated that the copy number of heptamer repeat sequences might vary from four to eight copies, which was previously unknown. Since the hybrid strains analysed in this study carry four copies of the heptamer sequences, it may thus serve as a marker to trace the strain in future. Interestingly, while the Mozambique strain is devoid of an El Tor-specific free RS1 element or pre-CTX prophage, the Indian hybrid strains carry such elements. The free RS1 has been mapped, cloned and sequenced. As in pre-CTX and CTX prophages, multiple copies of free RS1 elements were found to be integrated in tandem in the large chromosomal dif site. Since Indian hybrid El Tor strains carry either free RS1 or pre-CTX prophage in their large chromosomes, it is possible that the Mozambique hybrid El Tor strain has evolved from these progenitor strains by step-wise deletion of CTX genetic elements from their large chromosomes. DOI 10.1099/mic.0.032458-0

The cgtA gene, coding for the conserved G protein CgtA, is essential in bacteria. In contrast to a previous report, here we show by using genetic analysis that cgtA is essential in Vibrio cholerae even in a [DELTA]relA background. Depletion of CgtA affected the growth of V. cholerae and rendered the cells highly sensitive to the replication inhibitor hydroxyurea. Overexpression of V. cholerae CgtA caused distinct elongation of Escherichia coli cells. Deletion analysis indicated that the C-terminal end of CgtA plays a critical role in its proper function.

The cholera toxin (CT) is a critical determinant of the virulence of epidemic Vibrio cholerae strains. The ctxAB operon encoding CT is part of the genome of a filamentous bacteriophage CTX[PHI], which may integrate as a single copy or as multiple copies in the genome of V. cholerae. The CTX[PHI] genome is composed of RS2 (2.4 kb) and core (4.5 kb) regions. In the present study extensive genetic mapping analyses indicated that two copies of tandemly arrayed CTX prophages are integrated in the small chromosome of an environmental V. cholerae strain, VCE232, belonging to serogroup 04. Further mapping revealed that the integration of prophages has occurred in the same genetic locus of the small chromosome of VCE232 as that of V. cholerae O1 biotype El Tor strains. Interestingly, a new type of RS2-like element 3.5 kb in size was found in the CTX prophage genome in the small chromosome of VCE232. Cloning followed by sequencing of the new RS2-like element of VCE232 revealed the presence of three ORFs, which probably encode highly divergent types of phage regulatory proteins. Furthermore, the strain VCE232 also harbours two copies of a tandemly arranged CTX prophage devoid of the ctxAB genes, called pre-CTX prophage, in its large chromosome. The presence of multiple copies of diverse CTX prophages in both the chromosomes of VCE232 suggests that toxigenic environmental V. cholerae non-O1, non-O139 strains could play a role in the emergence of new epidemic clones. DOI: 10.1099/mic.0.2006/000117-0

The relA gene product determines the level of (p)ppGpp, the effector nucleotides of the bacterial stringent response that are also involved in the regulation of other functions, like antibiotic production and quorum sensing. In order to explore the possible involvement of relA in the regulation of virulence of Vibrio cholerae, a relA homolog from the organism (rel[A.sub.VCH]) was cloned and sequenced. The rel[A.sub.VCH gene encodes a 738-aminoacid protein having functions similar to those of other gram-negative bacteria, including Escherichia coli. A [DELTA]relA::kan allele was generated by replacing ~31% of the open reading frame of wild-type relA of V. cholerae El Tor strain C6709 with a kanamycin resistance gene. The V. cholerae relA mutant strain thus generated, SHK17, failed to accumulate (p)ppGpp upon amino acid deprivation. Interestingly, compared to the wild type, C6709, the mutant strain SHK17 exhibited significantly reduced in vitro production of two principal virulence factors, cholera toxin (CT) and toxin-coregulated pilus (TCP), under virulence gene-inducing conditions. In vivo experiments carried out in rabbit ileal loop and suckling mouse models also confirmed our in vitro results. The data suggest that (p)ppGpp is essential for maximal expression of CT and TCP during in vitro growth, as well as during intestinal infection by virulent V. cholerae. Northern blot and reverse transcriptase PCR analyses indicated significant reduction in the transcript levels of both virulence factors in the relA mutant strain SHK17. Such marked alteration of virulence phenotypes in SHK17 appears most likely to be due to down regulation of transcript levels of toxR and toxT, the two most important virulence regulatory genes of V. cholerae. In SHKI7, the altered expression of the two outer membrane porin proteins, OmpU and OmpT, indicated that the relA mutation most likely affects the ToxR-dependent virulence regulatory pathway, because it had been shown earlier that ToxR directly regulates their expression independently of ToxT.

Research has been conducted on Vibrio cholerae O1 El Tor strains. Results demonstrate that the copies of CTX prophage integrate in the chromosome of some Vibrio cholerae O1 El Tor strains.

A macrorestriction map of the genome of strain SG24 of Vibrio cholerae O139 Bengal was constructed using the enzymes NotI, SfiI and CeuI. More than 80 homologous and heterologous genes, including a number of operons, have been positioned on the physical map. When the map of the SG24 genome was compared with that of classical O1 sstrain 569B, considerable diversity in DNA restriction sites was noted and hypervariable regions were identified. A number of genetic markers are positioned differently in the SG24 and 569B genomes.

A physical map of the genome of the classical O1 hypertoxinogenic strain 569B of Vibrio cholerae has been constructed. It consisted of 22 homologous and heterologous genes and seven rrn operons that were generated via partial restriction fragment digestion. This physical restriction map is suggested to provide a novel approach for the analysis of genetic heterogeneity among V. cholerae strains.

The strain of the Noti- and Sfil-digested genome of Vibrio Cholerae 0139 isolated from different epidemic regions of India reveals clonal origin and the genome size about 2.2 Mb. Examination of the electrophoretic profiles of the genome of these strains, the RFLP of the cholera toxin gene and Southern blot hybridization of Noti-linking clone of classical strain 569B suggest that these strains have high resemble to V. cholerae 01 biotype EI Tor but differ from the classical 01 vibrios. Genetic heterogeneity in the population is demonstrated by the organization of the virulence gene cassettes in different 0139 strains.

Vibrio cholerae belonging to serovars O1 and non-O1, which are biochemically and gentically indistinguishable, differ in cell surface fragility, with O1 being unable to survive environmental fluctuations. An investigation was conducted to determinethe cell surface architecture of non-O1 vibrios. The results showed that non-O1vibrios are more resistant to hydrophobic compounds, detergents and lysis in hypotonic solutions, as compared to O1 isolates. In addition, non-O1 vibrios donot have phospholipids in the outer leaflet of the outer membrane.

Bacterial species are hosts to horizontally acquired mobile genetic elements (MGEs), which encode virulence, toxin, antimicrobial resistance, and other metabolic functions. The bipartite genome of Vibrio cholerae harbors sporadic and conserved MGEs that contribute in the disease development and survival of the pathogens. For a comprehensive understanding of dynamics of MGEs in the bacterial genome, we engineered the genome of V. cholerae and examined in vitro and in vivo stability of genomic islands (GIs), integrative conjugative elements (ICEs), and prophages. Recombinant vectors carrying the integration module of these GIs, ICE and CTXΦ, helped us to understand the efficiency of integrations of MGEs in the V. cholerae chromosome. We have deleted more than 250 acquired genes from 6 different loci in the V. cholerae chromosome and showed contribution of CTX prophage in the essentiality of SOS response master regulator LexA, which is otherwise not essential for viability in other bacteria, including Escherichia coli. In addition, we observed that the core genome-encoded RecA helps CTXΦ to bypass V. cholerae immunity and allow it to replicate in the host bacterium in the presence of similar prophage in the chromosome. Finally, our proteomics analysis reveals the importance of MGEs in modulating the levels of cellular proteome. This study engineered the genome of V. cholerae to remove all of the GIs, ICEs, and prophages and revealed important interactions between core and acquired genomes. [ABSTRACT FROM AUTHOR]

Vibrio cholerae cells shifted from 37 degrees Celsius to various low temperatures when it was exponentially grown, revealing that the organism could adapt and grow at temperatures down o 15 degrees Celsius, below which the growth was completely arrested. Analyses of proteins of cold-exposed cells revealed significant induction of two major cold shock proteins (Csps), whose molecular masses were 7.7 kDa and 7.5 kDa and six other Csps, most of which were much larger.

The Bay of Bengal is known as the epicenter for seeding several devastating cholera outbreaks across the globe. Vibrio cholerae, the etiological agent of cholera, has extraordinary competency to acquire exogenous DNA by horizontal gene transfer (HGT) and adapt them into its genome for structuring metabolic processes, developing drug resistance, and colonizing the human intestine. Antimicrobial resistance (AMR) in V. cholerae has become a global concern. However, little is known about the identity of the resistance traits, source of AMR genes, acquisition process, and stability of the genetic elements linked with resistance genes in V. cholerae. Here we present details of AMR profiles of 443 V. cholerae strains isolated from the stool samples of diarrheal patients from two regions of India. We sequenced the whole genome of multidrug-resistant (MDR) and extensively drug-resistant (XDR) V. cholerae to identify AMR genes and genomic elements that harbor the resistance traits. Our genomic findings were further confirmed by proteome analysis. We also engineered the genome of V. cholerae to monitor the importance of the autonomously replicating plasmid and core genome in the resistance profile. Our findings provided insights into the genomes of recent cholera isolates and identified several acquired traits including plasmids, transposons, integrative conjugative elements (ICEs), pathogenicity islands (PIs), prophages, and gene cassettes that confer fitness to the pathogen. The knowledge generated from this study would help in better understanding of V. cholerae evolution and management of cholera disease by providing clinical guidance on preferred treatment regimens. [ABSTRACT FROM AUTHOR]

Background: Chronic arsenic exposure via contaminated drinking water is a global environmental health problem associated with hematological, hepatic and many serious systemic disorders. This study on adult male rats evaluated the protective effects of vitamin E (VE) and vitamin C (VC) against arsenic-mediated hematological and hepatic toxicities. Methods: Arsenic was administered orally as arsenic trioxide (3 mg/kg body weight/day), as a single dose for 30 consecutive days or along with VC/ascorbic acid (200 mg/kg body weight/day dissolved in water) and VE/α-tocopherol (400 mg/kg body weight/day dissolved in olive oil) as supplements. Multiple hematological and hepatic parameters were assessed. Results: Arsenic exposure caused significant reduction of erythrocyte counts (p < 0.05), leukocyte counts (p < 0.01) and hemoglobin (Hb) levels (p < 0.01). Arsenic exposure also led to marked echinocytic transformation of erythrocytes resulting in increased morphological index (p < 0.001). Altered serum oxidative balance was observed with a higher oxidative stress index (p < 0.001). The results also showed a significant increase of serum cholesterol (p < 0.05), low-density lipoprotein (p < 0.001) and triglycerides (p < 0.01), and decreased high-density lipoprotein (p < 0.01) along with total protein (p < 0.01). A marked elevation of hepatic thiobarbituric acid reactive substance (p < 0.05) along with decreased reduced glutathione (p < 0.001) levels were also observed. Interestingly, co-administration of VC and VE significantly prevented all the arsenic-induced alterations (p < 0.05) except Hb content and serum protein. Conclusions: The present investigation offers strong evidence regarding the protective efficacy of co-administration of VC and VE against hematotoxicity and hepatotoxicity in adult male rats caused by chronic arsenic exposure. [ABSTRACT FROM AUTHOR]