scholarly journals Comparative Genomic Analyses of the Vibrio Pathogenicity Island and Cholera Toxin Prophage Regions in Nonepidemic Serogroup Strains of Vibrio cholerae

2003 ◽  
Vol 69 (3) ◽  
pp. 1728-1738 ◽  
Author(s):  
Manrong Li ◽  
Mamuka Kotetishvili ◽  
Yuansha Chen ◽  
Shanmuga Sozhamannan
Keyword(s):  
Cholera Toxin ◽  
Vibrio Cholerae ◽  
Fragment Length Polymorphism ◽  
Pathogenicity Island ◽  
Filamentous Phage ◽  
Comparative Genomic ◽  
Genomic Analyses ◽  
Ctxφ Prophage ◽  
Horizontal Acquisition ◽  
Restriction Fragment Length

ABSTRACT Two major virulence factors are associated with epidemic strains (O1 and O139 serogroups) of Vibrio cholerae: cholera toxin encoded by the ctxAB genes and toxin-coregulated pilus encoded by the tcpA gene. The ctx genes reside in the genome of a filamentous phage (CTXφ), and the tcpA gene resides in a vibrio pathogenicity island (VPI) which has also been proposed to be a filamentous phage designated VPIφ. In order to determine the prevalence of horizontal transfer of VPI and CTXφ among nonepidemic (non-O1 and non-O139 serogroups) V. cholerae, 300 strains of both clinical and environmental origin were screened for the presence of tcpA and ctxAB. In this paper, we present the comparative genetic analyses of 11 nonepidemic serogroup strains which carry the VPI cluster. Seven of the 11 VPI+ strains have also acquired the CTXφ. Multilocus sequence typing and restriction fragment length polymorphism analyses of the VPI and CTXφ prophage regions revealed that the non-O1 and non-O139 strains were genetically diverse and clustered in lineages distinct from that of the epidemic strains. The left end of the VPI in the non-O1 and non-O139 strains exhibited extensive DNA rearrangements. In addition, several CTXφ prophage types characterized by novel repressor (rstR) and ctxAB genes and VPIs with novel tcpA genes were found in these strains. These data suggest that the potentially pathogenic, nonepidemic, non-O1 and non-O139 strains identified in our study most likely evolved by sequential horizontal acquisition of the VPI and CTXφ independently rather than by exchange of O-antigen biosynthesis regions in an existing epidemic strain.

scholarly journals Examination of Diverse Toxin-Coregulated Pilus-Positive Vibrio cholerae Strains Fails To Demonstrate Evidence for Vibrio Pathogenicity Island Phage

2003 ◽  
Vol 71 (6) ◽  
pp. 2993-2999 ◽  
Author(s):  
Shah M. Faruque ◽  
Jun Zhu ◽  
Asadulghani ◽  
M. Kamruzzaman ◽  
John J. Mekalanos
Keyword(s):  
Vibrio Cholerae ◽  
Virulence Factors ◽  
Genetic Variants ◽  
Horizontal Transfer ◽  
Pathogenicity Island ◽  
Culture Conditions ◽  
Filamentous Phage ◽  
Colonization Factor ◽  
Toxin Coregulated Pilus ◽  
El Tor

ABSTRACT The major virulence factors of toxigenic Vibrio cholerae are cholera toxin, which is encoded by a lysogenic filamentous bacteriophage (CTXΦ), and toxin-coregulated pilus (TCP), an essential colonization factor that is also the receptor for CTXΦ. The genes involved in the biosynthesis of TCP reside in a pathogenicity island, which has been reported to correspond to the genome of another filamentous phage (designated VPIΦ) and to encode functions necessary for the production of infectious VPIΦ particles. We examined 46 V. cholerae strains having diverse origins and carrying different genetic variants of the TCP island for the production of the VPIΦ and CTXΦ in different culture conditions, including induction of prophages with mitomycin C and UV irradiation. Although 9 of 10 V. cholerae O139 strains and 12 of 15 toxigenic El Tor strains tested produced extracellular CTXΦ, none of the 46 TCP-positive strains produced detectable VPIΦ in repeated assays, which detected as few as 10 particles of a control CTX phage per ml. These results contradict the previous report regarding VPIΦ-mediated horizontal transfer of the TCP genes and suggest that the TCP island is unable to support the production of phage particles. Further studies are necessary to understand the mechanism of horizontal transfer of the TCP island.

scholarly journals Development of a PCR-restriction fragment length polymorphism assay for detection and subtyping of cholix toxin variant genes of Vibrio cholerae

2014 ◽  
Vol 63 (5) ◽  
pp. 667-673 ◽  
Author(s):  
Sharda Prasad Awasthi ◽  
Masahiro Asakura ◽  
Sucharit Basu Neogi ◽  
Atsushi Hinenoya ◽  
T. Ramamurthy ◽  
...  
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Vibrio Cholerae ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Pcr Rflp ◽  
Cholix Toxin ◽  
Rflp Assay ◽  
Restriction Fragment Length

Cholix toxin (ChxA) is an exotoxin reported in Vibrio cholerae non-O1/non-O139. Apart from its prototype (ChxA I) we have recently identified two novel variants of this toxin, ChxA II and ChxA III. Our previous investigations indicated that the first two variants may instigate extra-intestinal infections and ChxA II can be more lethal than ChxA I in mice. However, all three cholix toxins (ChxA I to III) failed to show any enterotoxicity in rabbit ileal loops. In this study we developed a PCR-restriction fragment length polymorphism (RFLP) assay to differentiate all three chxA variants to further understand the importance of each subtype. By using 53 V. cholerae non-O1/non-O139 strains harbouring chxA genes, which were previously categorized by sequencing, and various other strains as negative controls, the PCR-RFLP assay showed 100 % typability and specificity. Furthermore, when applied to differentiate additional V. cholerae strains, which were also screened for the chxA gene by colony hybridization, this assay identified chxA I and chxA II genes among 18.5 % and 4.5 % of non-O1/non-O139 strains (n = 178), respectively. One non-O1/non-O139 strain was untypable due to the insertion of an IS911-like element. Interestingly, the chxA I gene was detected in 10 out of 137 cholera toxin gene-negative V. cholerae O1 strains. These results suggest that the PCR-RFLP assay developed in this study can be a rapid and simple method to differentiate the chxA subtypes.

scholarly journals Effect of LexA on Chromosomal Integration of CTXϕ in Vibrio cholerae

2015 ◽  
Vol 198 (2) ◽  
pp. 268-275 ◽  
Author(s):  
Archana Pant ◽  
D Anbumani ◽  
Satyabrata Bag ◽  
Ojasvi Mehta ◽  
Pawan Kumar ◽  
...  
Keyword(s):  
Cholera Toxin ◽  
Vibrio Cholerae ◽  
Fine Tuning ◽  
Filamentous Phage ◽  
Chromosomal Integration ◽  
Content Type ◽  
Ssdna Binding ◽  
Tyrosine Recombinases ◽  
Reporter Strains ◽  
In Cells

ABSTRACTThe genesis of toxigenicVibrio choleraeinvolves 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, ofV. choleraebecause of its known role in controlling levels of Ssb proteins in other bacteria. CTXϕ integration decreased in cells with a ΔlexAmutation 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.IMPORTANCECholera 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 choleraehas 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 theV. choleraegenome and created several reporter strains carrying ΔlexAorlexA(Ind−) alleles. Using the reporter strains, the importance of LexA control of Ssb expression in the integration efficiency of CTXϕ was demonstrated.

scholarly journals Vibrio cholerae LexA Coordinates CTX Prophage Gene Expression

2009 ◽  
Vol 191 (22) ◽  
pp. 6788-6795 ◽  
Author(s):  
Harvey H. Kimsey ◽  
Matthew K. Waldor
Keyword(s):  
Gene Expression ◽  
Cholera Toxin ◽  
Vibrio Cholerae ◽  
Single Site ◽  
Virion Production ◽  
Regulatory Circuit ◽  
Ctxφ Prophage ◽  
Gene Regulatory ◽  
Ctx Prophage ◽  
Operator Site

ABSTRACT The filamentous bacteriophage CTXΦ transmits the cholera toxin genes by infecting and lysogenizing its host, Vibrio cholerae. CTXΦ genes required for virion production initiate transcription from the strong P A promoter, which is dually repressed in lysogens by the phage-encoded repressor RstR and the host-encoded SOS repressor LexA. Here we identify the neighboring divergent rstR promoter, P R, and show that RstR both positively and negatively autoregulates its own expression from this promoter. LexA is absolutely required for RstR-mediated activation of P R transcription. RstR autoactivation occurs when RstR is bound to an operator site centered 60 bp upstream of the start of transcription, and the coactivator LexA is bound to a 16-bp SOS box centered at position −23.5, within the P R spacer region. Our results indicate that LexA, when bound to its single site in the CTXΦ prophage, both represses transcription from P A and coactivates transcription from the divergent P R. We propose that LexA coordinates P A and P R prophage transcription in a gene regulatory circuit. This circuit is predicted to display transient switch behavior upon induction of CTXΦ lysogens.

scholarly journals Comparative genomic analysis of the secondary flagellar (flag-2) system in the order Enterobacterales

2019 ◽  
Author(s):  
Pieter De Maayer ◽  
Talia Pillay ◽  
Teresa A Coutinho
Keyword(s):  
Evolutionary History ◽  
Potential Role ◽  
Genomic Analysis ◽  
Comparative Genomic ◽  
Flagellar Biosynthesis ◽  
Genomic Analyses ◽  
Variable Feature ◽  
Locomotory Organ ◽  
Protein Number ◽  
Horizontal Acquisition

Abstract Background The order Enterobacterales encompasses a broad range of metabolically and ecologically versatile bacterial taxa, most of which are motile by means of peritrichous flagella. Flagellar biosynthesis has been linked to a primary flagella locus, flag -1, encompassing ~ 50 genes. A discrete locus, flag -2, encoding a distinct flagellar system, has been observed in a limited number of enterobacterial taxa, but its function remains largely uncharacterized.Results and Discussion Comparative genomic analyses showed that orthologous flag -2 loci are present in 592/4,028 taxa belonging to 5/8 and 31/76 families and genera, respectively, in the order Enterobacterales. Furthermore, the presence of the outermost flag- 2 genes only in many taxa suggest that this locus was far more prevalent and has subsequently been lost through gene deletion events. The flag -2 loci range in size from ~3.4 to 81.1 kilobases and code for between five and 102 distinct proteins. The discrepancy in size and protein number can be attributed to the presence of cargo gene islands within the loci. Evolutionary analyses revealed a complex evolutionary history for the flag -2 loci, representing ancestral elements in some taxa, while showing evidence of recent horizontal acquisition in other enterobacteria.Conclusions The flag -2 flagellar system is a relatively common, but highly variable feature among members of the Enterobacterales. Given the energetic burden of flagellar biosynthesis and functioning, the prevalence of a second flagellar system suggests it plays important biological roles in the enterobacteria and we postulate on its potential role as locomotory organ or as secretion system.

scholarly journals Emergence of Vibrio cholerae O1 Biotype El Tor Serotype Inaba from the Prevailing O1 Ogawa Serotype Strains in India

2000 ◽  
Vol 38 (11) ◽  
pp. 4249-4253 ◽  
Author(s):  
Pallavi Garg ◽  
Ranjan K. Nandy ◽  
Papiya Chaudhury ◽  
Nandini Roy Chowdhury ◽  
Keya De ◽  
...  
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Vibrio Cholerae ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Gel Electrophoresis ◽  
Fragment Length Polymorphism ◽  
Pulsed Field Gel Electrophoresis ◽  
Vibrio Cholerae O1 ◽  
El Tor ◽  
Restriction Fragment Length

The toxigenic Inaba serotype of Vibrio cholerae O1 biotype El Tor reappeared in India in 1998 and 1999, almost 10 years after its last dominance in Calcutta in 1989. Extensive molecular characterization by ribotyping, restriction fragment length polymorphism, and pulsed-field gel electrophoresis indicated that recent Inaba strains are remarkably different from the earlier Inaba strains but are very similar to the prevailing V. choleraeO1 Ogawa El Tor biotype strains. The antibiograms of the Inaba strains were also similar to those of the recent V. cholerae Ogawa strains. These V. cholerae O1 Inaba strains appear to have evolved from the currently prevailing Ogawa strains and are likely to dominate in the coming years.

scholarly journals Comparative genomic analysis of the secondary flagellar (flag-2) system in the order Enterobacterales

2020 ◽  
Author(s):  
Pieter De Maayer ◽  
Talia Pillay ◽  
Teresa A Coutinho
Keyword(s):  
Evolutionary History ◽  
Potential Role ◽  
Genomic Analysis ◽  
Comparative Genomic ◽  
Flagellar Biosynthesis ◽  
Genomic Analyses ◽  
Variable Feature ◽  
Locomotory Organ ◽  
Protein Number ◽  
Horizontal Acquisition

Abstract Background The order Enterobacterales encompasses a broad range of metabolically and ecologically versatile bacterial taxa, most of which are motile by means of peritrichous flagella. Flagellar biosynthesis has been linked to a primary flagella locus, flag -1, encompassing ~ 50 genes. A discrete locus, flag -2, encoding a distinct flagellar system, has been observed in a limited number of enterobacterial taxa, but its function remains largely uncharacterized. Results and Discussion Comparative genomic analyses showed that orthologous flag -2 loci are present in 592/4,028 taxa belonging to 5/8 and 31/76 families and genera, respectively, in the order Enterobacterales. Furthermore, the presence of only the outermost flag- 2 genes in many taxa suggests that this locus was far more prevalent and has subsequently been lost through gene deletion events. The flag -2 loci range in size from ~3.4 to 81.1 kilobases and code for between five and 102 distinct proteins. The discrepancy in size and protein number can be attributed to the presence of cargo gene islands within the loci. Evolutionary analyses revealed a complex evolutionary history for the flag -2 loci, representing ancestral elements in some taxa, while showing evidence of recent horizontal acquisition in other enterobacteria. Conclusions The flag -2 flagellar system is a fairly common, but highly variable feature among members of the Enterobacterales. Given the energetic burden of flagellar biosynthesis and functioning, the prevalence of a second flagellar system suggests it plays important biological roles in the enterobacteria and we postulate on its potential role as locomotory organ or as secretion system.

scholarly journals Vibrio Pathogenicity Island and Cholera Toxin Genetic Element-Associated Virulence Genes and Their Expression in Non-O1 Non-O139 Strains of Vibrio cholerae

2002 ◽  
Vol 70 (8) ◽  
pp. 4735-4742 ◽  
Author(s):  
Amit Sarkar ◽  
Ranjan K. Nandy ◽  
G. Balakrish Nair ◽  
Asoke C. Ghose
Keyword(s):  
Cholera Toxin ◽  
Vibrio Cholerae ◽  
Recombinant Strain ◽  
Virulence Genes ◽  
Pathogenicity Island ◽  
Homologous Region ◽  
Genetic Element ◽  
Rt Pcr ◽  
Pathogenic Potential ◽  
Regulated Expression

ABSTRACT A non-O1 non-O139 Vibrio cholerae strain, 10259, belonging to the serogroup O53 was shown to harbor genes related to the vibrio pathogenicity island (VPI) and a cholera toxin (CT) genetic element called CTX. While the nucleotide sequence of the strain 10259 tcpA gene differed significantly (26 and 28%) from those of O1 classical and El Tor biotype strains, respectively, partial sequence analysis data of certain other VPI-associated genes (aldA, tagA, tcpP/H, toxT, acfB/C, and int) and intergenic regions (tcpF to toxT and tcpH to tcpA) of the strain showed only minor variations (0.4 to 4.8%) from corresponding sequences in O1 strains. Strain 10259 also contained CTX element-associated toxin genes with sequences almost identical to those of O1 strains. Growth of the organism in Luria broth (LB) under ToxR inducing conditions (30°C and pH 6.5) led to transcriptional activation of tcpP/H, toxR, toxT, and tcpA genes, but not of ctxA, as determined by reverse transcription-PCR (RT-PCR). Subsequent analysis revealed that strain 10259 possessed only two copies (instead of three or more copies found in epidemic-causing O1 or O139 strains) of the heptanucleotide (TTTTGAT) repeats in the intergenic region upstream of ctxAB. Therefore, a strain 10259 mutant was generated by replacement of this region with a homologous region (1.4 kb) derived from a V. cholerae O1 classical biotype strain (O395) that contained seven such repeats. The resultant recombinant strain (10259R) was found to be capable of coordinately regulated expression of toxT, ctxA, and tcpA when grown under the ToxR inducing conditions. Serological studies also demonstrated that the recombinant strain produced TcpA and a significantly (∼1,000-fold) higher level of CT in vitro compared to that of the parent strain. Virulence gene expression in two other non-O1 non-O139 strains (serogroup O37) containing VPI and the CTX element was studied by RT-PCR and serological assay. One strain (S7, which was involved in an epidemic in Sudan in 1968) showed coordinately regulated expression of virulence genes leading to the production of both CT and TcpA in LB medium. However, the other strain, V2, produced RT-PCR-detectable transcripts of toxT, ctxA, or tcpA genes in the early phase (6 h), but not in the late phase (16 h) of growth in LB medium. These results are consistent with the low levels of production of CT and TcpA by the strain that were serologically detectable. The significance of these results is discussed in relation to the role of virulence genes and their expression to the pathogenic potential of V. cholerae strains belonging to non-O1 serogroups.

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