Molecular cloning of the hemolysin determinant from Vibrio cholerae El Tor

Vibrio cholerae El Tor RV79 is phenotypically nonhemolytic; however, strongly hemolytic convertants are occasionally observed on blood agar plates. We have cloned DNA sequences corresponding to the hemolysin determinant from RV79 (Hly+) in the lambda L47.1 and pBR322 vectors. A 2.3-kilobase fragment of V. cholerae DNA was found to be necessary for hemolytic activity. This cloned DNA sequence was used as a probe in Southern blot hybridization analysis of chromosomal restriction digests of a variety of El Tor and classical biotype V. cholerae strains. In all cases, DNA fragments with the same electrophoretic mobilities hybridized to the Hly probe. The results presented demonstrate that the cloned hemolysin determinant is the hly locus. By using mutator vibriophage VcA-3 insertion to promote high-frequency transfer, the hly locus was mapped between arg and ilv on the V. cholerae RV79 chromosome.     

Comparison of chitin-induced natural transformation in pandemic Vibrio cholerae O1 El Tor strains

The human pathogen Vibrio cholerae serves as a model organism for many important processes ranging from pathogenesis to natural transformation, which has been extensively studied in this bacterium. Previous work has deciphered important regulatory circuits involved in natural competence induction as well as mechanistic details related to its DNA acquisition and uptake potential. However, since competence was first reported for V. cholerae in 2005, many researchers have struggled with reproducibility in certain strains. In this study, we therefore compare prominent seventh pandemic V. cholerae isolates, namely strains A1552, N16961, C6706, C6709, E7946, P27459, and the close relative MO10, for their natural transformability and decipher underlying defects that mask the high degree of competence conservation. Through a combination of experimental approaches and comparative genomics based on new whole-genome sequences and de novo assemblies, we identify several strain-specific defects, mostly in genes that encode key players in quorum sensing. Moreover, we provide evidence that most of these deficiencies might have recently occurred through laboratory domestication events or through the acquisition of mobile genetic elements. Lastly, we highlight that differing experimental approaches between research groups might explain more of the variations than strain-specific alterations.     

Culture conditions for stimulating cholera toxin production by Vibrio cholerae O1 El Tor

A method that stimulates cholera toxin (CT) production by Vibrio cholerae O1 biotype El Tor (El Tor vibrios) to the level of several micrograms per ml in the culture fluid was established. Such a large amount of CT was obtained by the following method: El Tor vibrios were cultured in AKI medium (1.5% Bacto peptone, 0.4% yeast extract-Difco, 0.5% NaCl, 0.3% NaHCO3) at 37 C for 4 hr in a stationary test tube and then for 16 hr in a shaken flask, with inoculum sizes of 10(5) to 10(7)/ml. With this method, 35 strains out of 60 examined produced 2 to 16 micrograms/ml of CT as determined by the reversed passive latex agglutination test (RPLA). Thirty-three randomly selected strains out of the 60 produced reasonable amounts of rabbit skin vascular permeability factor, reflecting the amount of CT titrated with RPLA.     

A proteome reference map for Vibrio cholerae El Tor

A proteome reference map has been constructed for Vibrio cholerae El Tor, in the pI range of 4.0 to 7.0. The map is based on two-dimensional gels (2-D) and the identification, by peptide mass fingerprint, of proteins in 94 spots, corresponding to 80 abundant proteins. Two strains are compared, strain N16961 and a Latin American El Tor strain C3294. The consensus map contains 340 spots consistently seen with both strains grown in Luria-Bertani broth (LB) or minimal M9 medium. The results were obtained from nine gels run with 18 cm immobilized pH gradient strips and precast gels. The 2-D gels were anchored to real N16961 proteins identified by mass spectrometry. Various energy metabolism components and periplasmic ATP-binding cassette (ABC) transporter proteins were identified among the abundant proteins. Two isoforms of OmpU were found. Five operons are proposed and seven hypothetical proteins were experimentally confirmed. Comparisons are made with protein 2-D gels for a classical strain and to microarray analysis available for the N16961 El Tor strain. New results were obtained from the proteome analysis, indicating an abundance of periplasmic ABC transporter proteins not found in microarray studies.     

Steps in the development of a Vibrio cholerae El Tor biofilm

We report that, in a simple, static culture system, wild-type Vibrio cholerae El Tor forms a three-dimensional biofilm with characteristic water channels and pillars of bacteria. Furthermore, we have isolated and characterized transposon insertion mutants of V. cholerae that are defective in biofilm development. The transposons were localized to genes involved in (i) the biosynthesis and secretion of the mannose-sensitive haemagglutinin type IV pilus (MSHA); (ii) the synthesis of exopolysaccharide; and (iii) flagellar motility. The phenotypes of these three groups suggest that the type IV pilus and flagellum accelerate attachment to the abiotic surface, the flagellum mediates spread along the abiotic surface, and exopolysaccharide is involved in the formation of three-dimensional biofilm architecture.     

Association of Vibrio cholerae O1 El Tor and O139 Bengal with the Copepods Acartia tonsa and Eurytemora affinis

The association of Vibrio cholerae with zooplankton has been suggested as an important factor in transmission of human epidemic cholera, and the ability to colonize zooplankton surfaces may play a role in the temporal variation and predominance of the two different serogroups (V. cholerae O1 El Tor and O139) in the aquatic environment. To date, interactions between specific serogroups and species of plankton remain poorly understood. Laboratory microcosm experiments were carried out to compare quantitatively the colonization of two copepod species, Acartia tonsa and Eurytemora affinis, by each of the epidemic serogroups. V. cholerae O1 consistently achieved higher abundances than V. cholerae O139 in colonizing adults of each copepod species as well as the multiple life stages of E. affinis. This difference in colonization may be significant in the general predominance of V. cholerae O1 in cholera epidemics in rural Bangladesh where water supplies are taken directly from the environment.     

霍乱弧菌N16961超级整合子重复序列及基因盒分析

目的:确定O1群El Tor型霍乱弧菌N16961超级整合子(SI)中霍乱弧菌重复序列(VCR)的序列特点,以及VCR和基因盒的数量及位置。方法:用局部序列比对软件BLAST将VCR参考序列与霍乱弧菌N16961的Ⅱ号染色体进行比对,用Artemis Comparison Tool查看比对结果获得比对区域的位置信息,并采用perl语言脚本获得霍乱弧菌N16961的Ⅱ号染色体VCR相应区域的序列;用全局比对软件Clustal W将上一步获得的所有VCR序列进行多序列比对,采用perl语言脚本处理比对结果获得一致性序列;用MEGA4.0软件查看多序列比对结果,并采用perl语言脚本计算各位置变异频率,据此分析霍乱弧菌N16961的Ⅱ号染色体上VCR和基因盒的特点。结果:在N16961的超级整合子中有158个VCR,其核苷酸长度为117～124 bp;其一致性序列有126个核苷酸,其中37个为保守核苷酸位点,89个为可变核苷酸位点;139个VCR与相邻的VCR之间至少有1个基因,19个VCR相互之间没有任何基因;N16961的SI中共存在146个基因盒,基因盒大小为390～5924 bp不等,每个基因盒中整合的基因数目为1～9个不等。结论:建立了SI中VCR和基因盒的分析流程,分析了SI中VCR的保守及变异位点,明确了霍乱弧菌N16961的SI中VCR和基因盒的信息,为霍乱弧菌和其他细菌中SI的研究提供了分析基础。     

Variation in epitopes of the B subunit of El Tor and classical biotype Vibrio cholerae O1 cholera toxin

Variation in epitopes of the B subunit of cholera toxin (CT-B) produced by strains of El Tor and classical biotype Vibrio cholerae O1 was examined using monoclonal antibodies prepared to V. cholerae 569B CT. CT-B epitopes were markedly conserved for V. cholerae classical biotypes. In contrast, epitope variation was observed for El Tor biotypes, which produced both a classical-like CT-B and a unique CT-B lacking at least one epitope common to 569B CT-B. The missing epitope was located outside the GM1 ganglioside-binding site. From results of the study reported here, genetic divergence is exhibited in the El Tor biotype CT-B versus classical CT-B. Furthermore, at least five unique epitopes of V. cholerae 569B CT-B can be defined.     

Molecular characterization of Vibrio cholerae outbreak strains with altered El Tor biotype from southern India

Forty-four Vibrio cholerae isolates collected over a 7-month period in Chennai, India in 2004 were characterized for gene traits, antimicrobial susceptibility and genomic fingerprints. All 44 isolates were identified as O1 El Tor Ogawa, positive for various toxigenic and pathogenic genes viz. ace, ctxB, hlyA, ompU, ompW, rfbO1, rtx, tcpA, toxR and zot. Nucleotide sequencing revealed the presence of cholera toxin B of classical biotype in all the El Tor isolates, suggesting infection of isolates by classical CTXΦ. Antibiogram analysis showed a broad-spectrum antibiotic resistance that was also confirmed by the presence of resistant genes in the genomes. All isolates contained a class 1 integron and an SXT constin. However, isolates were sensitive to chloramphenicol and tested negative for the chloramphenicol resistant gene suggesting a deletion in SXT constin. Fingerprinting analysis of isolates by ERIC- and Box PCR revealed similar DNA patterns indicating the clonal dissemination of a single predominant V. cholerae O1 strain throughout the 2004 outbreak in Chennai.     

Cryptic Appearance of a New Clone of Vibrio cholerae Serogroup O1 Biotype El Tor in Calcutta,India

In this study, pulsed-field gel electrophoresis (PFGE) was applied to determine if the Vibrio cholerae O1 strains which reappeared after being temporarily displaced in Calcutta by the O139 serogroup were different from those isolated before the advent of the O139 serogroup. NotI digestion generated a total of 11 different patterns among the 24 strains of V. cholerae randomly selected to represent different time frames. Among the V. cholerae O1 strains isolated after July 1993, 4 PFGE banding patterns designated as H through K were observed with pattern H dominating. Pattern H was distinctly different from all other patterns encountered in this study including patterns A, B and C of V. cholerae O1 El Tor, which dominated before November 1992, and pattern F, which was the dominant V. cholerae O139 pattern. Further, pattern H was also different from the NotI banding patterns of the representative strains of the 4 toxigenic clonal groups of V. cholerae O1 El Tor currently prevailing in different parts of the world. NotI fragments of the new clone of V. cholerae O1 did not hybridize with an O139 specific DNA probe, indicating that there was no O139 genetic material in the new clone of V. cholerae O1. Hybridization data with an O1-specific DNA probe again differentiated between the clones of V. cholerae O1 existing before the genesis of the O139 serogroup and the O1 strains currently prevalent.     

Cellular localization and export of the soluble haemolysin of Vibrio cholerae El Tor

Summary The cellular location of the haemolysin of Vibrio cholerae El Tor strain 017 has been analyzed. This protein is found both in the periplasmic space and the extracellular medium in Vibrio cholerae. However, when the cloned gene, present on plasmid pPM431, is introduced into E. coli K-12 this protein remains localized predominantly in the periplasmic space with no activity detected in the extracellular medium. Mutants of E. coli K-12 (tolA and tolB) which leak periplasmic proteins mimic excretion and release the haemolysin into the growth medium. Secretion of haemolysin into the periplasm is independent of perA (envZ) and in fact, mutants in perA (envZ) harbouring pPM431 show hyperproduction of periplasmic haemolysin. These results in conjunction with those for other V. cholerae extracellular proteins suggest that although E. coli K-12 can secrete these proteins into the periplasm, it lacks a specific excretion mechanism, present in V. cholerae, for the release of soluble proteins into the growth medium.     

El Tor霍乱弧菌双精氨酸转运系统功能单位的分析研究

本文旨在分析El Tor霍乱弧菌双精氨酸转运(Tat)系统的基因簇构成,对其功能进行分析,确定其最小功能单位.通过与大肠埃希菌Tat系统基因簇的基因同源性比较,推测霍乱国际测序标准株N16961的Tat系统基因簇构成;分别对霍乱弧菌Tat系统的基因簇进行单基因缺失、双基因缺失和全基因缺失,建立缺失株和回补株,并与野生株...     

Detection and molecular characterization of Vibrio cholerae O1 Inaba biotype El Tor strain in Kerala, S. India

The resurgence of enteric pathogen Vibrio cholerae, the causative organism of epidemic cholera, remains a major health problem in many developing countries. The outbreaks of cholera follow a seasonal pattern in regions of endemicity. The southern Indian state of Kerala is endemic to cholera. A V. cholerae strain isolated from the stool sample of a patient in Piravam, Kerala, South India, was analysed. However, this case occurred at a time not associated with cholera outbreaks, leading to concern among the State health officials. We compared the virulence potential of the isolate with that of the standard or reference strains, that have been widely used as positive control. The isolate was identified as V. cholerae O1 biotype El Tor serotype Inaba. The resistance pattern of the isolate to common antibiotics was examined and it was found to be multi-drug resistant in nature. The strain was analysed for the presence of the CTX genetic element, which encodes genes for cholera toxin and other important regulatory genes. It was found to be positive for all the genes tested. In Kerala, most of the cholera outbreaks have been reported to be caused by V. cholerae O1 El Tor belonging to Ogawa serotype. Interestingly, the V. cholerae strain isolated from this case has been found to be of Inaba serotype, which is rarely reported.     

Outer Membrane Protein OmpW Is the Receptor for Typing Phage VP5 in the Vibrio cholerae O1 El Tor Biotype

Phage typing is used for the subtyping of clones of epidemic bacteria. In this study, we identified the outer membrane protein OmpW as the receptor for phage VP5, one of the typing phages for the Vibrio cholerae O1 El Tor biotype. A characteristic 11-bp deletion in ompW was observed in all epidemic strains resistant to VP5, suggesting that this mutation event can be used as a tracing marker in cholera surveillance.     

Genetic determination of the vibriocinogenicity trait in Vibrio cholerae of the El Tor biotype

In two different strains of cholera vibrios two recA-dependent plasmids, pVib I (1.9-2.2 Md) and pVib II (5.2-5.8 Md), have been detected. These plasmids determine the synthesis of vibriocin, coagulase and fibrinolysin, which has been established by the cotransformation of the DNA of plasmids pVib I and pBR322 and by the transfer mobilization with the use of plasmid RP4.     

Molecular Characterization of Vibrio cholerae O1 Reveals Continuous Evolution of Its New Variants in India

Vibrio cholerae, the causing agent of cholera is still a major health challenge in most of the developing countries. In this study, V. cholerae strains collected from different cholera outbreaks in India over a period of past 7 years were found to have various toxigenic, pathogenic and regulatory genes viz. ctxAB, zot, tcp, hlyA, ace, ompU, ompW, rfbO1, toxT and toxR. The biotype specific genes rstR and rtxC revealed the El Tor biotype in majority of the isolates. However, variants among the isolates were found having genotype of both the biotypes. Sequencing of ctxB gene revealed the presence of altered ctxB of classical biotype with additional variations in isolates of 2007. Mismatch amplification mutation assay PCR also confirmed the isolates belonging to classical biotype. Antibiogram of the isolates revealed resistance for nalidixic acid, co-trimoxazole, streptomycin, and polymyxin B and susceptibility for tetracycline among most of the isolates from India. However, V. cholerae isolates from a recent outbreak in Eastern India were resistant to tetracycline. The study corroborated the continuous emergence and wide-spread of multidrug resistant El Tor variant strains in the Indian subcontinent.     

A Role for the Mannose-Sensitive Hemagglutinin in Biofilm Formation by Vibrio cholerae El Tor

While much has been learned regarding the genetic basis of host-pathogen interactions, less is known about the molecular basis of a pathogen's survival in the environment. Biofilm formation on abiotic surfaces represents a survival strategy utilized by many microbes. Here it is shown that Vibrio cholerae El Tor does not use the virulence-associated toxin-coregulated pilus to form biofilms on borosilicate but rather uses the mannose-sensitive hemagglutinin (MSHA) pilus, which plays no role in pathogenicity. In contrast, attachment of V. cholerae to chitin is shown to be independent of the MSHA pilus, suggesting divergent pathways for biofilm formation on nutritive and nonnutritive abiotic surfaces.