Cholerae Genome Research Articles

CRISPR-Cas systems are present predominantly on mobile genetic elements in Vibrio species

BackgroundBacteria are prey for many viruses that hijack the bacterial cell in order to propagate, which can result in bacterial cell lysis and death. Bacteria have developed diverse strategies to counteract virus predation, one of which is the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR associated (Cas) proteins immune defense system. Species within the bacterial family Vibrionaceae are marine organisms that encounter large numbers of phages. Our goal was to determine the significance of CRISPR-Cas systems as a mechanism of defense in this group by investigating their prevalence, phylogenetic distribution, and genome context.ResultsHerein, we describe all the CRISPR-Cas system types and their distribution within the family Vibrionaceae. In Vibrio cholerae genomes, we identified multiple variant type I-F systems, which were also present in 41 additional species. In a large number of Vibrio species, we identified a mini type I-F system comprised of tniQcas5cas7cas6f, which was always associated with Tn7-like transposons. The Tn7-like elements, in addition to the CRISPR-Cas system, also contained additional cargo genes such as restriction modification systems and type three secretion systems. A putative hybrid CRISPR-Cas system was identified containing type III-B genes followed by a type I-F cas6f and a type I-F CRISPR that was associated with a prophage in V. cholerae and V. metoecus strains. Our analysis identified CRISPR-Cas types I-C, I-E, I-F, II-B, III-A, III-B, III-D, and the rare type IV systems as well as cas loci architectural variants among 70 species. All systems described contained a CRISPR array that ranged in size from 3 to 179 spacers. The systems identified were present predominantly within mobile genetic elements (MGEs) such as genomic islands, plasmids, and transposon-like elements. Phylogenetic analysis of Cas proteins indicated that the CRISPR-Cas systems were acquired by horizontal gene transfer.ConclusionsOur data show that CRISPR-Cas systems are phylogenetically widespread but sporadic in occurrence, actively evolving, and present on MGEs within Vibrionaceae.

Comparative genomics for non-O1/O139 Vibrio cholerae isolates recovered from the Yangtze River Estuary versus V. cholerae representative isolates from serogroup O1

Vibriocholerae, which is autochthonous to estuaries worldwide, can cause human cholera that is still pandemic in developing countries. A number of V. cholerae isolates of clinical and environmental origin worldwide have been subjected to genome sequencing to address their phylogenesis and bacterial pathogenesis, however, little genome information is available for V. cholerae isolates derived from estuaries, particularly in China. In this study, we determined the complete genome sequence of V. cholerae CHN108B (non-O1/O139 serogroup) isolated from the Yangtze River Estuary, China and performed comparative genome analysis between CHN108B and other eight representative V. cholerae isolates. The 4,168,545-bp V. cholerae CHN108B genome (47.2% G+C) consists of two circular chromosomes with 3,691 predicted protein-encoding genes. It has 110 strain-specific genes, the highest number among the eight representative V. cholerae whole genomes from serogroup O1: there are seven clinical isolates linked to cholera pandemics (1937–2010) and one environmental isolate from Brazil. Various mobile genetic elements (such as insertion sequences, prophages, integrative and conjugative elements, and super-integrons) were identified in the nine V. cholerae genomes of clinical and environmental origin, indicating that the bacterium undergoes extensive genetic recombination via lateral gene transfer. Comparative genomics also revealed different virulence and antimicrobial resistance gene patterns among the V. cholerae isolates, suggesting some potential virulence factors and the rising development of resistance among pathogenic V. cholerae. Additionally, draft genome sequences of multiple V. cholerae isolates recovered from the Yangtze River Estuary were also determined, and comparative genomics revealed many genes involved in specific metabolism pathways, which are likely shaped by the unique estuary environment. These results provide additional evidence of V. cholerae genome plasticity and will facilitate better understanding of the genome evolution and pathogenesis of this severe water-borne pathogen worldwide.

Novel Genetic Tool to Study the Stability of Genomic Islands

Genomic islands (GIs) are discrete segments of mobile DNA with defined boundaries according to recent patents, acquired in the bacterial genome from another organism by horizontal gene transfer during the course of evolution. GIs contribute significantly to virulence, disease development, antimicrobial resistance and metabolic process. The present study focuses on the development of a vector based genetic tool carrying selectable and counter-selectable markers, in order to flag the GIs in the bacterial chromosome and monitor their stability under in vitro and in vivo conditions. We engineered suicide vectors, pSB40 and pSB41, carrying single or tandem copies of chloramphenicol acetyltransferase (cat) and levansucrase (sacB) alleles, respectively. The sacB-cat allele in both the vectors is flanked by several restriction sites. To test the suitability of sacB-cat allele for monitoring GI loss, we introduced the allele in the Vibrio Pathogenicity Island-1 (VPI-1) in Vibrio cholerae genome. The V. cholerae strain carrying sacB-cat allele in VPI-1 element showed resistance to chloramphenicol and sensitivity to sucrose at optimal growth conditions. Loss of VPI-1 element from the V. cholerae genome was simply monitored by growing the cells on selection agar plates supplemented with sucrose. Our results showed that the genetic tool we developed is suitable for monitoring GI stability in the bacterial genome. The present study indicates that pSB40 and pSB41are efficient and sensitive genetic tool that can be used for reverse genetics experiments and monitoring stability of mobile genetic elements in the bacterial genome.

CAMP Receptor Protein Controls Vibrio cholerae Gene Expression in Response to Host Colonization.

ABSTRACTThe bacterium Vibrio cholerae is native to aquatic environments and can switch lifestyles to cause disease in humans. Lifestyle switching requires modulation of genetic systems for quorum sensing, intestinal colonization, and toxin production. Much of this regulation occurs at the level of gene expression and is controlled by transcription factors. In this work, we have mapped the binding of cAMP receptor protein (CRP) and RNA polymerase across the V. cholerae genome. We show that CRP is an integral component of the regulatory network that controls lifestyle switching. Focusing on a locus necessary for toxin transport, we demonstrate CRP-dependent regulation of gene expression in response to host colonization. Examination of further CRP-targeted genes reveals that this behavior is commonplace. Hence, CRP is a key regulator of many V. cholerae genes in response to lifestyle changes.

Biology of Vibrio cholera. Editorial overview.

In this monographic issue, we have the pleasure to present contributions from six of the leading laboratories at the forefront of Vibrio cholerae genetics, ecology and evolution, together with a brief tribute by Diego Romero to Doctor Jaime Ferrán y Clua, a pioneering Spanish bacteriologist who developed the first vaccine against this pathogen. V. cholerae is a free-living aquatic bacterium that interacts with and infects a variety of organisms. In humans it causes cholera, the deadly diarrhoea that was responsible for millions of deaths during seven pandemics since 1817, and still thousands every year. The Boucher lab presents a study of the ecology, evolution and dispersal of pandemic V. cholerae biotypes in relation to environmental reservoirs. They show how both species-specific and lineage-specific genetic determinants play a role in the ability of V. cholerae strains to cause pandemics, having evolved gradually over centuries. One of the key aspects that makes a particularly successful pathogen is its genomic plasticity. The V. cholerae genome contains a superintegron (SI) that is involved in development and dissemination of antibiotic resistance genes among diverse bacterial species, permitting population expansion in challenging conditions. Escudero and Mazel review the SI as a true hotspot of V. cholerae's genomic diversity and low-cost memory of adaptive functions in its complex lifestyle and ecology. Another remarkable aspect of V. cholerae 's genetics is the presence of two chromosomes. Segregation and division in multi-chromosomal becteria is relatively complex, and V. cholerae remains the paradigm. Espinosa and colleagues review the cell cycle of V. cholerae , comparing and contrasting with that of E. coli . In addition to genome plasticity, V. cholerae uses a variety of attack/defence strategies to compete and thrive in different niches, through interaction with bacteriophages, bacteria and eukaryotes. The role of phages in the life cycle of V. cholerae has been increasingly recognized and investigated over the past decade. Andrew Camilli and colleagues take us through the exciting evolutionary arms race between V. cholerae and virulent bacteriophages, based both on mechanisms of phage resistance in the bacterium and a unique phage-encoded CRISPR-Cas system used to counteract this resistance. Finally, the authors discuss the impact of these predator-prey dynamics in the context of infection, and their use as a strategy to limit cholera transmission within a community. In regards to its ability to coexist with other microbes, V. cholerae can produce effectors that are either released to the extracellular media, or delivered via intimate cell-to-cell contact such as those injected via the type VI secretion system (T6SS). The Pukatzki lab reviews the versatility of the T6SS to produce different combinations of such effectors, which establishes the strains of V. cholerae that can co-exist in the environment. After killing a cell, its DNA is released and incorporated by natural competence into other living cells, thereby being a potential source of diversification for V. cholerae's T6SS effectors. Finally, I revisisit the discovery of non-canonical D-amino acids, recently identified effectors secreted by V. cholerae which have increasingly been shown to be important in enhancing the ability of the bacterium to colonize and persist in a particular niche. I focus on recent observations that suggest different D-amino acids influence distinct cellular processes in bacteria, and dis cuss their role in modulating environmental bacterial biodiversity.

Systematic genetic dissection of chitin degradation and uptake in Vibrio cholerae.

Vibrio cholerae is a natural resident of the aquatic environment, where a common nutrient is the chitinous exoskeletons of microscopic crustaceans. Chitin utilization requires chitinases, which degrade this insoluble polymer into soluble chitin oligosaccharides. These oligosaccharides also serve as an inducing cue for natural transformation in Vibrio species. There are 7 predicted endochitinase-like genes in the V. cholerae genome. Here, we systematically dissect the contribution of each gene to growth on chitin as well as induction of natural transformation. Specifically, we created a strain that lacks all 7 putative chitinases and from this strain, generated a panel of strains where each expresses a single chitinase. We also generated expression plasmids to ectopically express all 7 chitinases in our chitinase deficient strain. Through this analysis, we found that low levels of chitinase activity are sufficient for natural transformation, while growth on insoluble chitin as a sole carbon source requires more robust and concerted chitinase activity. We also assessed the role that the three uptake systems for the chitin degradation products GlcNAc, (GlcNAc)2 and (GlcN)2 , play in chitin utilization and competence induction. Cumulatively, this study provides mechanistic details for how this pathogen utilizes chitin to thrive and evolve in its environmental reservoir.

Sequential displacement of Type VI Secretion System effector genes leads to evolution of diverse immunity gene arrays in Vibrio cholerae

Type VI secretion systems (T6SS) enable bacteria to engage neighboring cells in contact-dependent competition. In Vibrio cholerae, three chromosomal clusters each encode a pair of effector and immunity genes downstream of those encoding the T6SS structural machinery for effector delivery. Different combinations of effector-immunity proteins lead to competition between strains of V. cholerae, which are thought to be protected only from the toxicity of their own effectors. Screening of all publically available V. cholerae genomes showed that numerous strains possess long arrays of orphan immunity genes encoded in the 3′ region of their T6SS clusters. Phylogenetic analysis reveals that these genes are highly similar to those found in the effector-immunity pairs of other strains, indicating acquisition by horizontal gene transfer. Extensive genomic comparisons also suggest that successive addition of effector-immunity gene pairs replaces ancestral effectors, yet retains the cognate immunity genes. The retention of old immunity genes perhaps provides protection against nearby kin bacteria in which the old effector was not replaced. This mechanism, combined with frequent homologous recombination, is likely responsible for the high diversity of T6SS effector-immunity gene profiles observed for V. cholerae and closely related species.

Exception to the Rule: Genomic Characterization of Naturally Occurring Unusual Vibrio cholerae Strains with a Single Chromosome.

The genetic make-up of most bacteria is encoded in a single chromosome while about 10% have more than one chromosome. Among these, Vibrio cholerae, with two chromosomes, has served as a model system to study various aspects of chromosome maintenance, mainly replication, and faithful partitioning of multipartite genomes. Here, we describe the genomic characterization of strains that are an exception to the two chromosome rules: naturally occurring single-chromosome V. cholerae. Whole genome sequence analyses of NSCV1 and NSCV2 (natural single-chromosome vibrio) revealed that the Chr1 and Chr2 fusion junctions contain prophages, IS elements, and direct repeats, in addition to large-scale chromosomal rearrangements such as inversions, insertions, and long tandem repeats elsewhere in the chromosome compared to prototypical two chromosome V. cholerae genomes. Many of the known cholera virulence factors are absent. The two origins of replication and associated genes are generally intact with synonymous mutations in some genes, as are recA and mismatch repair (MMR) genes dam, mutH, and mutL; MutS function is probably impaired in NSCV2. These strains are ideal tools for studying mechanistic aspects of maintenance of chromosomes with multiple origins and other rearrangements and the biological, functional, and evolutionary significance of multipartite genome architecture in general.

A genomic island in Vibrio cholerae with VPI-1 site-specific recombination characteristics contains CRISPR-Cas and type VI secretion modules.

Cholera is a devastating diarrhoeal disease caused by certain strains of serogroup O1/O139 Vibrio cholerae. Mobile genetic elements such as genomic islands (GIs) have been pivotal in the evolution of O1/O139 V. cholerae. Perhaps the most important GI involved in cholera disease is the V. cholerae pathogenicity island 1 (VPI-1). This GI contains the toxin-coregulated pilus (TCP) gene cluster that is necessary for colonization of the human intestine as well as being the receptor for infection by the cholera-toxin bearing CTX phage. In this study, we report a GI (designated GIVchS12) from a non-O1/O139 strain of V. cholerae that is present in the same chromosomal location as VPI-1, contains an integrase gene with 94% nucleotide and 100% protein identity to the VPI-1 integrase, and attachment (att) sites 100% identical to those found in VPI-1. However, instead of TCP and the other accessory genes present in VPI-1, GIVchS12 contains a CRISPR-Cas element and a type VI secretion system (T6SS). GIs similar to GIVchS12 were identified in other V. cholerae genomes, also containing CRISPR-Cas elements and/or T6SS’s. This study highlights the diversity of GIs circulating in natural V. cholerae populations and identifies GIs with VPI-1 recombination characteristics as a propagator of CRISPR-Cas and T6SS modules.

The Nucleoid Binding Protein H-NS Biases Genome-Wide Transposon Insertion Landscapes.

ABSTRACTTransposon insertion sequencing (TIS; also known as TnSeq) is a potent approach commonly used to comprehensively define the genetic loci that contribute to bacterial fitness in diverse environments. A key presumption underlying analyses of TIS datasets is that loci with a low frequency of transposon insertions contribute to fitness. However, it is not known whether factors such as nucleoid binding proteins can alter the frequency of transposon insertion and thus whether TIS output may systematically reflect factors that are independent of the role of the loci in fitness. Here, we investigated whether the histone-like nucleoid structuring (H-NS) protein, which preferentially associates with AT-rich sequences, modulates the frequency of Mariner transposon insertion in the Vibrio cholerae genome, using comparative analysis of TIS results from wild-type (wt) and Δhns V. cholerae strains. These analyses were overlaid on gene classification based on GC content as well as on extant genome-wide identification of H-NS binding loci. Our analyses revealed a significant dearth of insertions within AT-rich loci in wt V. cholerae that was not apparent in the Δhns insertion library. Additionally, we observed a striking correlation between genetic loci that are overrepresented in the Δhns insertion library relative to their insertion frequency in wt V. cholerae and loci previously found to physically interact with H-NS. Collectively, our findings reveal that factors other than genetic fitness can systematically modulate the frequency of transposon insertions in TIS studies and add a cautionary note to interpretation of TIS data, particularly for AT-rich sequences.

From cholera to corals: Viruses as drivers of virulence in a major coral bacterial pathogen.

Disease is an increasing threat to reef-building corals. One of the few identified pathogens of coral disease is the bacterium Vibrio coralliilyticus. In Vibrio cholerae, infection by a bacterial virus (bacteriophage) results in the conversion of non-pathogenic strains to pathogenic strains and this can lead to cholera pandemics. Pathogenicity islands encoded in the V. cholerae genome play an important role in pathogenesis. Here we analyse five whole genome sequences of V. coralliilyticus to examine whether virulence is similarly driven by horizontally acquired elements. We demonstrate that bacteriophage genomes encoding toxin genes with homology to those found in pathogenic V. cholerae are integrated in V. coralliilyticus genomes. Virulence factors located on chromosomal pathogenicity islands also exist in some strains of V. coralliilyticus. The presence of these genetic signatures indicates virulence in V. coralliilyticus is driven by prophages and other horizontally acquired elements. Screening for pathogens of coral disease should target conserved regions in these elements.

A Cytosine Methyltransferase Modulates the Cell Envelope Stress Response in the Cholera Pathogen [corrected

DNA methylation is a key epigenetic regulator in all domains of life, yet the effects of most bacterial DNA methyltransferases on cellular processes are largely undefined. Here, we used diverse techniques, including bisulfite sequencing, transcriptomics, and transposon insertion site sequencing to extensively characterize a 5-methylcytosine (5mC) methyltransferase, VchM, in the cholera pathogen, Vibrio cholerae. We have comprehensively defined VchM’s DNA targets, its genetic interactions and the gene networks that it regulates. Although VchM is a relatively new component of the V. cholerae genome, it is required for optimal V. cholerae growth in vitro and during infection. Unexpectedly, the usually essential σE cell envelope stress pathway is dispensable in ∆vchM V. cholerae, likely due to its lower activation in this mutant and the capacity for VchM methylation to limit expression of some cell envelope modifying genes. Our work illuminates how an acquired DNA methyltransferase can become integrated within complex cell circuits to control critical housekeeping processes.

Effect of LexA on Chromosomal Integration of CTXϕ in Vibrio cholerae.

The genesis of toxigenic Vibrio cholerae involves acquisition of CTXϕ, a single-stranded DNA (ssDNA) filamentous phage that encodes cholera toxin (CT). The phage exploits host-encoded tyrosine recombinases (XerC and XerD) for chromosomal integration and lysogenic conversion. The replicative genome of CTXϕ produces ssDNA by rolling-circle replication, which may be used either for virion production or for integration into host chromosome. Fine-tuning of different ssDNA binding protein (Ssb) levels in the host cell is crucial for cellular functioning and important for CTXϕ integration. In this study, we mutated the master regulator gene of SOS induction, lexA, of V. cholerae because of its known role in controlling levels of Ssb proteins in other bacteria. CTXϕ integration decreased in cells with a ΔlexA mutation and increased in cells with an SOS-noninducing mutation, lexA (Ind(-)). We also observed that overexpression of host-encoded Ssb (VC0397) decreased integration of CTXϕ. We propose that LexA helps CTXϕ integration, possibly by fine-tuning levels of host- and phage-encoded Ssbs. Cholera toxin is the principal virulence factor responsible for the acute diarrheal disease cholera. CT is encoded in the genome of a lysogenic filamentous phage, CTXϕ. Vibrio cholerae has a bipartite genome and harbors single or multiple copies of CTXϕ prophage in one or both chromosomes. Two host-encoded tyrosine recombinases (XerC and XerD) recognize the folded ssDNA genome of CTXϕ and catalyze its integration at the dimer resolution site of either one or both chromosomes. Fine-tuning of ssDNA binding proteins in host cells is crucial for CTXϕ integration. We engineered the V. cholerae genome and created several reporter strains carrying ΔlexA or lexA (Ind(-)) alleles. Using the reporter strains, the importance of LexA control of Ssb expression in the integration efficiency of CTXϕ was demonstrated.

Vibrio cholerae Response Regulator VxrB Controls Colonization and Regulates the Type VI Secretion System.

Two-component signal transduction systems (TCS) are used by bacteria to sense and respond to their environment. TCS are typically composed of a sensor histidine kinase (HK) and a response regulator (RR). The Vibrio cholerae genome encodes 52 RR, but the role of these RRs in V. cholerae pathogenesis is largely unknown. To identify RRs that control V. cholerae colonization, in-frame deletions of each RR were generated and the resulting mutants analyzed using an infant mouse intestine colonization assay. We found that 12 of the 52 RR were involved in intestinal colonization. Mutants lacking one previously uncharacterized RR, VCA0566 (renamed VxrB), displayed a significant colonization defect. Further experiments showed that VxrB phosphorylation state on the predicted conserved aspartate contributes to intestine colonization. The VxrB regulon was determined using whole genome expression analysis. It consists of several genes, including those genes that create the type VI secretion system (T6SS). We determined that VxrB is required for T6SS expression using several in vitro assays and bacterial killing assays, and furthermore that the T6SS is required for intestinal colonization. vxrB is encoded in a four gene operon and the other vxr operon members also modulate intestinal colonization. Lastly, though ΔvxrB exhibited a defect in single-strain intestinal colonization, the ΔvxrB strain did not show any in vitro growth defect. Overall, our work revealed that a small set of RRs is required for intestinal colonization and one of these regulators, VxrB affects colonization at least in part through its regulation of T6SS genes.

Worldwide occurrence of integrative conjugative element encoding multidrug resistance determinants in epidemic Vibrio cholerae O1.

In the last decades, there has been an increase of cholera epidemics caused by multidrug resistant strains. Particularly, the integrative and conjugative element (ICE) seems to play a major role in the emergence of multidrug resistant Vibrio cholerae. This study fully characterized, by whole genome sequencing, new ICEs carried by multidrug resistant V. cholerae O1 strains from Nigeria (2010) (ICEVchNig1) and Nepal (1994) (ICEVchNep1). The gene content and gene order of these two ICEs are the same, and identical to ICEVchInd5, ICEVchBan5 and ICEVchHai1 previously identified in multidrug resistant V. cholerae O1. This ICE is characterized by dfrA1, sul2, strAB and floR antimicrobial resistance genes, and by unique gene content in HS4 and HS5 ICE regions. Screening for ICEs, in publicly available V. cholerae genomes, revealed the occurrence and widespread distribution of this ICE among V. cholerae O1. Metagenomic analysis found segments of this ICE in marine environments far from the direct influence of the cholera epidemic. Therefore, this study revealed the epidemiology of a spatio-temporal prevalent ICE in V. cholerae O1. Its occurrence and dispersion in V. cholerae O1 strains from different continents throughout more than two decades can be indicative of its role in the fitness of the current pandemic lineage.

The two Cis-acting sites, parS1 and oriC1, contribute to the longitudinal organisation of Vibrio cholerae chromosome I.

The segregation of bacterial chromosomes follows a precise choreography of spatial organisation. It is initiated by the bipolar migration of the sister copies of the replication origin (ori). Most bacterial chromosomes contain a partition system (Par) with parS sites in close proximity to ori that contribute to the active mobilisation of the ori region towards the old pole. This is thought to result in a longitudinal chromosomal arrangement within the cell. In this study, we followed the duplication frequency and the cellular position of 19 Vibrio cholerae genome loci as a function of cell length. The genome of V. cholerae is divided between two chromosomes, chromosome I and II, which both contain a Par system. The ori region of chromosome I (oriI) is tethered to the old pole, whereas the ori region of chromosome II is found at midcell. Nevertheless, we found that both chromosomes adopted a longitudinal organisation. Chromosome I extended over the entire cell while chromosome II extended over the younger cell half. We further demonstrate that displacing parS sites away from the oriI region rotates the bulk of chromosome I. The only exception was the region where replication terminates, which still localised to the septum. However, the longitudinal arrangement of chromosome I persisted in Par mutants and, as was reported earlier, the ori region still localised towards the old pole. Finally, we show that the Par-independent longitudinal organisation and oriI polarity were perturbed by the introduction of a second origin. Taken together, these results suggest that the Par system is the major contributor to the longitudinal organisation of chromosome I but that the replication program also influences the arrangement of bacterial chromosomes.

Second-Pandemic Strain of Vibrio cholerae from the Philadelphia Cholera Outbreak of 1849

In the 19th century, there were several major cholera pandemics in the Indian subcontinent, Europe, and North America. The causes of these outbreaks and the genomic strain identities remain a mystery. We used targeted high-throughput sequencing to reconstruct the Vibrio cholerae genome from the preserved intestine of a victim of the 1849 cholera outbreak in Philadelphia, part of the second cholera pandemic. This O1 biotype strain has 95 to 97% similarity with the classical O395 genome, differing by 203 single-nucleotide polymorphisms (SNPs), lacking three genomic islands, and probably having one or more tandem cholera toxin prophage (CTX) arrays, which potentially affected its virulence. This result highlights archived medical remains as a potential resource for investigations into the genomic origins of past pandemics.

Comparative genome analysis of non-toxigenic non-O1 versus toxigenic O1 Vibrio cholerae.

Pathogenic strains of Vibrio cholerae are responsible for endemic and pandemic outbreaks of the disease cholera. The complete toxigenic mechanisms underlying virulence in Vibrio strains are poorly understood. The hypothesis of this work was that virulent versus non-virulent strains of V. cholerae harbor distinctive genomic elements that encode virulence. The purpose of this study was to elucidate genomic differences between the O1 serotypes and non-O1 V. cholerae PS15, a non-toxigenic strain, in order to identify novel genes potentially responsible for virulence. In this study, we compared the whole genome of the non-O1 PS15 strain to the whole genomes of toxigenic serotypes at the phylogenetic level, and found that the PS15 genome was distantly related to those of toxigenic V. cholerae. Thus we focused on a detailed gene comparison between PS15 and the distantly related O1 V. cholerae N16961. Based on sequence alignment we tentatively assigned chromosome numbers 1 and 2 to elements within the genome of non-O1 V. cholerae PS15. Further, we found that PS15 and O1 V. cholerae N16961 shared 98% identity and 766 genes, but of the genes present in N16961 that were missing in the non-O1 V. cholerae PS15 genome, 56 were predicted to encode not only for virulence–related genes (colonization, antimicrobial resistance, and regulation of persister cells) but also genes involved in the metabolic biosynthesis of lipids, nucleosides and sulfur compounds. Additionally, we found 113 genes unique to PS15 that were predicted to encode other properties related to virulence, disease, defense, membrane transport, and DNA metabolism. Here, we identified distinctive and novel genomic elements between O1 and non-O1 V. cholerae genomes as potential virulence factors and, thus, targets for future therapeutics. Modulation of such novel targets may eventually enhance eradication efforts of endemic and pandemic disease cholera in afflicted nations.

Architecture of the superintegron in Vibrio cholerae: identification of core and unique genes

Background: Vibrio cholerae , the etiologic agent of cholera, is indigenous to aquatic environments. The V. cholerae genome consists of two chromosomes; the smallest of these harbors a large gene capture and excision system called the superintegron (SI), of ~120 kbp. The flexible nature of the SI that results from gene cassette capture, deletion and rearrangement is thought to make it a hotspot of V. cholerae diversity, but beyond the basic structure it is not clear if there is a core genome in the SI and if so how it is structured. The aim of this study was to explore the core genome structure and the differences in gene content among strains of V. cholerae . Methods: From the complete genomes of seven V. cholerae and one Vibrio mimicus representative strains, we recovered the SI sequences based on the locations of the structural gene IntI4 and the V. cholerae repeats. Analysis of the pangenome, including cluster analysis of functional genes, pangenome profile analysis, genetic variation analysis of functional genes, strain evolution analysis and function enrichment analysis of gene clusters, was performed using a pangenome analysis pipeline in addition to the R scripts, splitsTree4 and genoPlotR. Results and conclusions: Here, we reveal the genetic architecture of the V. cholerae SI. It contains eight core genes when V. mimicus is included and 21 core genes when only V. cholerae strains are considered; many of them are present in several copies. The V. cholerae SI has an open pangenome, which means that V. cholerae may be able to import new gene cassettes to SI. The set of dispensable SI genes is influenced by the niche and type species. The core genes are distributed along the SI, apparently without a position effect.

Architecture of the superintegron in Vibrio cholerae: identification of core and unique genes.

Background: Vibrio cholerae, the etiologic agent of cholera, is indigenous to aquatic environments. The V. cholerae genome consists of two chromosomes; the smallest of these harbors a large gene capture and excision system called the superintegron (SI), of ~120 kbp. The flexible nature of the SI that results from gene cassette capture, deletion and rearrangement is thought to make it a hotspot of V. cholerae diversity, but beyond the basic structure it is not clear if there is a core genome in the SI and if so how it is structured. The aim of this study was to explore the core genome structure and the differences in gene content among strains of V. cholerae. Methods: From the complete genomes of seven V. cholerae and one Vibrio mimicus representative strains, we recovered the SI sequences based on the locations of the structural gene IntI4 and the V. cholerae repeats. Analysis of the pangenome, including cluster analysis of functional genes, pangenome profile analysis, genetic variation analysis of functional genes, strain evolution analysis and function enrichment analysis of gene clusters, was performed using a pangenome analysis pipeline in addition to the R scripts, splitsTree4 and genoPlotR. Results and conclusions: Here, we reveal the genetic architecture of the V. cholerae SI. It contains eight core genes when V. mimicus is included and 21 core genes when only V. cholerae strains are considered; many of them are present in several copies. The V. cholerae SI has an open pangenome, which means that V. cholerae may be able to import new gene cassettes to SI. The set of dispensable SI genes is influenced by the niche and type species. The core genes are distributed along the SI, apparently without a position effect.