Genetic Behavior of R Factors in Vibrio cholerae

The fi⁺ and fi⁻ R factors are unstable in Vibrio cholerae even on selective media containing antibiotics, although a temperature-sensitive kanamycin resistance factor and its recombinants are stably maintained in V. cholerae at low temperatures.     

Transcriptomic profiling of the oyster pathogen Vibrio splendidus opens a window on the evolutionary dynamics of the small RNA repertoire in the Vibrio genus

Work in recent years has led to the recognition of the importance of small regulatory RNAs (sRNAs) in bacterial regulation networks. New high-throughput sequencing technologies are paving the way to the exploration of an expanding sRNA world in nonmodel bacteria. In the Vibrio genus, compared to the enterobacteriaceae, still a limited number of sRNAs have been characterized, mostly in Vibrio cholerae, where they have been shown to be important for virulence, as well as in Vibrio harveyi. In addition, genome-wide approaches in V. cholerae have led to the discovery of hundreds of potential new sRNAs. Vibrio splendidus is an oyster pathogen that has been recently associated with massive mortality episodes in the French oyster growing industry. Here, we report the first RNA-seq study in a Vibrio outside of the V. cholerae species. We have uncovered hundreds of candidate regulatory RNAs, be it cis-regulatory elements, antisense RNAs, and trans-encoded sRNAs. Conservation studies showed the majority of them to be specific to V. splendidus. However, several novel sRNAs, previously unidentified, are also present in V. cholerae. Finally, we identified 28 trans sRNAs that are conserved in all the Vibrio genus species for which a complete genome sequence is available, possibly forming a Vibrio “sRNA core.”     

Influence of domain architecture and codon usage pattern on the evolution of virulence factors of Vibrio cholerae

Cholera remains a heavy burden to human health in some developing countries including India where sanitation is poor and health care is limited. After the publication of the complete genome sequence of Vibrio cholerae, the etiological agent of cholera, extensive possibilities, earlier unavailable, have opened up to understand the genetic organization of V. cholerae. In the present study, we analyzed all the pathogenic non-horizontally transferred genes of V. cholerae to know the ancestral relationship and how the pathogenic genes have been evolved in V. cholerae genome. We observed that protein domain has important role in developing pathogenicity, and codon usage pattern of the pathogenic protein domain is also subject to selection. The present study unambiguously depict that the patterns of synonymous codon usage within a protein domain can change dramatically during the course of evolution to give rise to pathogenicity.     

gbpA as a Novel qPCR Target for the Species-Specific Detection of Vibrio cholerae O1, O139, Non-O1/Non-O139 in Environmental,Stool, and Historical Continuous Plankton Recorder Samples

The Vibrio cholerae N-acetyl glucosamine-binding protein A (GbpA) is a chitin-binding protein involved in V. cholerae attachment to environmental chitin surfaces and human intestinal cells. We previously investigated the distribution and genetic variations of gbpA in a large collection of V. cholerae strains and found that the gene is consistently present and highly conserved in this species. Primers and probe were designed from the gbpA sequence of V. cholerae and a new Taq-based qPCR protocol was developed for diagnostic detection and quantification of the bacterium in environmental and stool samples. In addition, the positions of primers targeting the gbpA gene region were selected to obtain a short amplified fragment of 206 bp and the protocol was optimized for the analysis of formalin-fixed samples, such as historical Continuous Plankton Recorder (CPR) samples. Overall, the method is sensitive (50 gene copies), highly specific for V. cholerae and failed to amplify strains of the closely-related species Vibrio mimicus. The sensitivity of the assay applied to environmental and stool samples spiked with V. cholerae ATCC 39315 was comparable to that of pure cultures and was of 10² genomic units/l for drinking and seawater samples, 10¹ genomic units/g for sediment and 10² genomic units/g for bivalve and stool samples. The method also performs well when tested on artificially formalin-fixed and degraded genomic samples and was able to amplify V. cholerae DNA in historical CPR samples, the earliest of which date back to August 1966. The detection of V. cholerae in CPR samples collected in cholera endemic areas such as the Benguela Current Large Marine Ecosystem (BCLME) is of particular significance and represents a proof of concept for the possible use of the CPR technology and the developed qPCR assay in cholera studies.     

The Transmission and Antibiotic Resistance Variation in a Multiple Drug Resistance Clade of Vibrio cholerae Circulating in Multiple Countries in Asia

Vibrio cholerae has caused massive outbreaks and even trans-continental epidemics. In 2008 and 2010, at least 3 remarkable cholera outbreaks occurred in Hainan, Anhui and Jiangsu provinces of China. To address the possible transmissions and the relationships to the 7^th pandemic strains of those 3 outbreaks, we sequenced the whole genomes of the outbreak isolates and compared with the global isolates from the 7^th pandemic. The three outbreaks in this study were caused by a cluster of V. cholerae in clade 3.B which is parallel to the clade 3.C that was transmitted from Nepal to Haiti and caused an outbreak in 2010. Pan-genome analysis provided additional evolution information on the mobile element and acquired multiple antibiotic resistance genes. We suggested that clade 3.B should be monitored because the multiple antibiotic resistant characteristics of this clade and the ‘amplifier’ function of China in the global transmission of current Cholera pandemic. We also show that dedicated whole genome sequencing analysis provided more information than the previous techniques and should be applied in the disease surveillance networks.     

Rapid and Sensitive Quantification of Vibrio cholerae and Vibrio mimicus Cells in Water Samples by Use of Catalyzed Reporter Deposition Fluorescence In Situ Hybridization Combined with Solid-Phase Cytometry

A new protocol for rapid, specific, and sensitive cell-based quantification of Vibrio cholerae/Vibrio mimicus in water samples was developed. The protocol is based on catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) in combination with solid-phase cytometry. For pure cultures, we were able to quantify down to 6 V. cholerae cells on one membrane with a relative precision of 39% and down to 12 cells with a relative precision of 17% after hybridization with the horseradish peroxidase (HRP)-labeled probe Vchomim1276 (specific for V. cholerae and V. mimicus) and signal amplification. The corresponding position of the probe on the 16S rRNA is highly accessible even when labeled with HRP. For the first time, we were also able to successfully quantify V. cholerae/V. mimicus via solid-phase cytometry in extremely turbid environmental water samples collected in Austria. Cell numbers ranged from 4.5 × 10¹ cells ml⁻¹ in the large saline lake Neusiedler See to 5.6 × 10⁴ cells ml⁻¹ in an extremely turbid shallow soda lake situated nearby. We therefore suggest CARD-FISH in combination with solid-phase cytometry as a powerful tool to quantify V. cholerae/V. mimicus in ecological studies as well as for risk assessment and monitoring programs.     

Lessons to be learned from studying Vibrio cholerae in model systems

A response to The complete genome sequence of Vibrio cholerae: a tale of two chromosomes and of two lifestyles, by Gary K Schoolnik and Fitnat H Yildiz, Genome Biology 2000 1:reviews1016.1-1016.3.     

PVv3, a New Shuttle Vector for Gene Expression in Vibrio vulnificus

An efficient electroporation procedure for Vibrio vulnificus was designed using the new cloning vector pVv3 (3,107 bp). Transformation efficiencies up to 2 × 10⁶ transformants per μg DNA were achieved. The vector stably replicated in both V. vulnificus and Escherichia coli and was also successfully introduced into Vibrio parahaemolyticus and Vibrio cholerae. To demonstrate the suitability of the vector for molecular cloning, the green fluorescent protein (GFP) gene and the vvhBA hemolysin operon were inserted into the vector and functionally expressed in Vibrio and E. coli.     

Ribotyping and TCP gene cluster analysis of environmental and clinical Vibrio cholerae strains isolated in Iran

The aim of this study was to investigate the presence of TCP gene clusters among clinical and environmental Vibrio cholerae isolates and to explore the genetic relatedness of isolates using ribotyping technique. A total of 50 V. cholerae strains (30 clinical and 20 environmental) were included in this study. Three clinical isolates were negative for TCP cluster genes while the cluster was absent in all of the environmental strains. Ribotyping of rRNA genes with BglI produced 18 different ribotype patterns, three of which belonged to clinical O1 serotype isolates. The remaining 15 ribotypes belonged to clinical non-O1, non-O139 serogroups (two patterns) and environmental non-O1, non-O139 serogroups (13 patterns). Clinical V. cholerae O1 strains from 2004 through 2006 and several environmental non-O1, non-O139 V. cholerae strains from 2006 showed 67.3 % similarity and fell within one single gene cluster. Ribotyping analysis made it possible to further comprehend the close originality of clinical isolates as very little changes have been occurred within rRNA genes of different genotypes of V. cholerae strains through years. In conclusion, ribotyping analysis of environmental V. cholerae isolates showed a substantial genomic diversity supporting the fact that genetic changes within bacterial genome occurs during years in the environment, while only little changes may arise within the genome of clinical isolates.     

Occurrence and distribution of plankton-associated and free-living toxigenic Vibrio cholerae in a tropical estuary of a cholera endemic zone

Cholera epidemics are thought to be influenced by changes in populations of estuarine Vibrio cholerae. We investigated the abundance and distribution of this bacterium, as ??free-living?? (<20???m fraction) and associated with microphytoplankton (>20???m) or zooplankton (>60???m), in the Karnaphuli estuary of Bangladesh during pre- and post-monsoon seasons. Cultivable Vibrio populations were ~10²?C10⁴ colony forming units (CFU) ml^?1 in the high saline zone (19?C23 practical salinity unit, PSU) and declined in freshwater (<10¹?CFU?ml^?1). Culture independent detection of toxigenic V. cholerae O1 and O139 serogroups revealed a higher abundance of ??free-living?? (10⁴?C10⁵ cells?l^?1) than those attached to plankton (10¹?C10³ cells?l^?1). However, ??free-living?? O1 and O139 cells were sometimes absent in the medium saline and freshwater areas (0.0?C11 practical salinity unit [PSU]). In contrast, plankton samples always harbored these serogroups despite changes in salinity and other physico-chemical properties. Microphytoplankton and zooplankton were dominated by diatoms and blue-green algae, and copepods and rotifers, respectively. Toxigenic V. cholerae abundance did not correlate with plankton abundance or species but had a positive correlation with chitin in the <20???m fraction, where suspended particulate matter (SPM), V. cholerae and chitin concentrations were highest. C:N ratios indicated that organic matter in SPM originated predominantly from plankton. The differential occurrence of ??free-living?? and attached V. cholerae suggests a pivotal function of plankton in V. cholerae spreading into freshwater areas. The probable association of this pathogen with organisms and particles in the nanoplankton (<20???m) fraction requires validation of the concept of the ??free living?? state of V. cholerae in aquatic habitats.     

Excess SeqA leads to replication arrest and a cell division defect in Vibrio cholerae

Although most bacteria contain a single circular chromosome, some have complex genomes, and all Vibrio species studied so far contain both a large and a small chromosome. In recent years, the divided genome of Vibrio cholerae has proven to be an interesting model system with both parallels to and novel features compared with the genome of Escherichia coli. While factors influencing the replication and segregation of both chromosomes have begun to be elucidated, much remains to be learned about the maintenance of this genome and of complex bacterial genomes generally. An important aspect of replicating any genome is the correct timing of initiation, without which organisms risk aneuploidy. During DNA replication in E. coli, newly replicated origins cannot immediately reinitiate because they undergo sequestration by the SeqA protein, which binds hemimethylated origin DNA. This DNA is already methylated by Dam on the template strand and later becomes fully methylated; aberrant amounts of Dam or the deletion of seqA leads to asynchronous replication. In our study, hemimethylated DNA was detected at both origins of V. cholerae, suggesting that these origins are also subject to sequestration. The overproduction of SeqA led to a loss of viability, the condensation of DNA, and a filamentous morphology. Cells with abnormal DNA content arose in the population, and replication was inhibited as determined by a reduced ratio of origin to terminus DNA in SeqA-overexpressing cells. Thus, excessive SeqA negatively affects replication in V. cholerae and prevents correct progression to downstream cell cycle events such as segregation and cell division.     

Ordered Cloned DNA Map of the Genome of Vibrio cholerae 569B and Localization of Genetic Markers

By using a low-resolution macrorestriction map as the foundation (R. Majumder et al., J. Bacteriol. 176:1105–1112, 1996), an ordered cloned DNA map of the 3.2-Mb chromosome of the hypertoxinogenic strain 569B of Vibrio cholerae has been constructed. A cosmid library the size of about 4,000 clones containing more than 120 Mb of V. cholerae genomic DNA (40-genome equivalent) was generated. By combining landmark analysis and chromosome walking, the cosmid clones were assembled into 13 contigs covering about 90% of the V. cholerae genome. A total of 92 cosmid clones were assigned to the genome and to regions defined by NotI, SfiI, and CeuI macrorestriction maps. Twenty-seven cloned genes, 9 rrn operons, and 10 copies of a repetitive DNA sequence (IS1004) have been positioned on the ordered cloned DNA map.     

Caenorhabditis elegans Recognizes a Bacterial Quorum-sensing Signal Molecule through the AWCON Neuron

In a process known as quorum sensing, bacteria use chemicals called autoinducers for cell-cell communication. Population-wide detection of autoinducers enables bacteria to orchestrate collective behaviors. In the animal kingdom detection of chemicals is vital for success in locating food, finding hosts, and avoiding predators. This behavior, termed chemotaxis, is especially well studied in the nematode Caenorhabditis elegans. Here we demonstrate that the Vibrio cholerae autoinducer (S)-3-hydroxytridecan-4-one, termed CAI-1, influences chemotaxis in C. elegans. C. elegans prefers V. cholerae that produces CAI-1 over a V. cholerae mutant defective for CAI-1 production. The position of the CAI-1 ketone moiety is the key feature driving CAI-1-directed nematode behavior. CAI-1 is detected by the C. elegans amphid sensory neuron AWC^ON. Laser ablation of the AWC^ON cell, but not other amphid sensory neurons, abolished chemoattraction to CAI-1. These analyses define the structural features of a bacterial-produced signal and the nematode chemosensory neuron that permit cross-kingdom interaction.     

Genomic and Phenotypic Diversity of Coastal Vibrio cholerae Strains Is Linked to Environmental Factors

Studies of Vibrio cholerae diversity have focused primarily on pathogenic isolates of the O1 and O139 serotypes. However, autochthonous environmental isolates of this species routinely display more extensive genetic diversity than the primarily clonal pathogenic strains. In this study, genomic and metabolic profiles of 41 non-O1/O139 environmental isolates from central California coastal waters and four clinical strains are used to characterize the core genome and metabolome of V. cholerae. Comparative genome hybridization using microarrays constructed from the fully sequenced V. cholerae O1 El Tor N16961 genome identified 2,787 core genes that approximated the projected species core genome within 1.6%. Core genes are almost universally present in strains with widely different niches, suggesting that these genes are essential for persistence in diverse aquatic environments. In contrast, the dispensable genes and phenotypic traits identified in this study should provide increased fitness for certain niche environments. Environmental parameters, measured in situ during sample collection, are correlated to the presence of specific dispensable genes and metabolic capabilities, including utilization of mannose, sialic acid, citrate, and chitosan oligosaccharides. These results identify gene content and metabolic pathways that are likely selected for in certain coastal environments and may influence V. cholerae population structure in aquatic environments.     

Growth of Vibrio cholerae O1 in Red Tide Waters off California

Vibrio cholerae serotype O1 is autochthonous to estuarine and coastal waters. However, its population dynamics in such environments are not well understood. We tested the proliferation of V. cholerae N16961 during a Lingulodinium polyedrum bloom, as well as other seawater conditions. Microcosms containing 100-kDa-filtered seawater were inoculated with V. cholerae or the 0.6-μm-pore-size filterable fraction of seawater assemblages. These cultures were diluted 10-fold with fresh 100-kDa-filtered seawater every 48 h for four cycles. Growth rates ranged from 0.3 to 14.3 day⁻¹ (4.2 day⁻¹ ± 3.9) for V. cholerae and 0.1 to 9.7 day⁻¹ (2.2 ± 2.8 day⁻¹) for bacterial assemblage. Our results suggest that dissolved organic matter during intense phytoplankton blooms has the potential to support explosive growth of V. cholerae in seawater. Under the conditions tested, free-living V. cholerae was able to reach concentrations per milliliter that were up to 3 orders of magnitude higher than the known minimum infectious dose (10⁴ cell ml⁻¹) and remained viable under many conditions. If applicable to the complex conditions in marine ecosystems, our results suggest an important role of the growth of free-living V. cholerae in disease propagation and prevention during phytoplankton blooms.     

Response of Pathogenic Vibrio Species to High Hydrostatic Pressure

Vibrio parahaemolyticus ATCC 17802, Vibrio vulnificus ATCC 27562, Vibrio cholerae O:1 ATCC 14035, Vibrio cholerae non-O:1 ATCC 14547, Vibrio hollisae ATCC 33564, and Vibrio mimicus ATCC 33653 were treated with 200 to 300 MPa for 5 to 15 min at 25°C. High hydrostatic pressure inactivated all strains of pathogenic Vibrio without triggering a viable but nonculturable (VBNC) state; however, cells already existing in a VBNC state appeared to possess greater pressure resistance.     

The extracellular nuclease Dns and its role in natural transformation of Vibrio cholerae

Free extracellular DNA is abundant in many aquatic environments. While much of this DNA will be degraded by nucleases secreted by the surrounding microbial community, some is available as transforming material that can be taken up by naturally competent bacteria. One such species is Vibrio cholerae, an autochthonous member of estuarine, riverine, and marine habitats and the causative agent of cholera, whose competence program is induced after colonization of chitin surfaces. In this study, we investigate how Vibrio cholerae's two extracellular nucleases, Xds and Dns, influence its natural transformability. We show that in the absence of Dns, transformation frequencies are significantly higher than in its presence. During growth on a chitin surface, an increase in transformation efficiency was found to correspond in time with increasing cell density and the repression of dns expression by the quorum-sensing regulator HapR. In contrast, at low cell density, the absence of HapR relieves dns repression, leading to the degradation of free DNA and to the abrogation of the transformation phenotype. Thus, as cell density increases, Vibrio cholerae undergoes a switch from nuclease-mediated degradation of extracellular DNA to the uptake of DNA by bacteria induced to a state of competence by chitin. Taken together, these results suggest the following model: nuclease production by low-density populations of V. cholerae might foster rapid growth by providing a source of nucleotides for the repletion of nucleotide pools. In contrast, the termination of nuclease production by static, high-density populations allows the uptake of intact DNA and coincides with a phase of potential genome diversification.     

Genetic lesions within the 3a gene of SARS-CoV

Phage N5 is one of the phages of Vibrio cholerae serovar O1 biotype El Tor (Ghosh, A. N., Ansari, M. Q., and Dutta, G. C. Isolation and morphological characterization of El Tor cholera phages. J. Gen. Virol. 70: 2241–2243, 1989). In the present communication the growth curve, molecular weight and confirmation of the genome, partial denaturation map and restriction endonuclease digestion pattern have been determined. Partial denaturation map indicates that the genome has non-permuted / invariant sequence. Presence of cohesive ends has also been documented.