Genomic evolution and rearrangement of CTX-Φ prophage elements in Vibrio cholerae during the 2018-2024 cholera outbreaks in eastern Democratic Republic of the Congo.

ABSTRACTBetween 2018 and 2024, we conducted systematic whole-genome sequencing and phylogenomic analysis on 263 V. cholerae O1 isolates from cholera patients across four provinces in the Democratic Republic of Congo (North-Kivu, South-Kivu, Tanganyika, and Kasai Oriental). These isolates were classified into the AFR10d and AFR10e sublineages of AFR10 lineage, originating from the third wave of the seventh El Tor cholera pandemic (7PET). Compared to the strains analysed between 2014 and 2017, both sublineages had few genetic changes in the core genome but recent isolates (2022-2024) had significant CTX prophage rearrangement. AFR10e spread across all four provinces, while AFR10d appeared to be extinct by the end of 2020. Since 2022, most V. cholerae O1 isolates exhibited significant CTX prophage rearrangements, including a tandem repeat of an environmental satellite phage RS1 downstream the ctxB toxin gene of the CTX-Φ-3 prophage on the large chromosome, as well as two or more arrayed copies of an environmental pre-CTX-Φ prophage precursor on the small chromosome. We used Illumina data for mapping and coverage estimation to identify isolates with unique CTX-Φ genomic features. Gene localization was then determined on MinION-derived assemblies, revealing an organization similar to that of non-O1 V. cholerae isolates found in Asia (O139 VC1374, and environmental O4 VCE232), but never described in V. cholerae O1 El Tor from the third wave. In conclusion, while the core genome of AFR10d and AFR10e showed minimal changes, significant alterations in the CTX-Φ and pre-CTX-Φ prophage content and organization were identified in AFR10e from 2022 onwards.

Interactions between the Prophage 919TP and Its Vibrio cholerae Host: Implications of gmd Mutation for Phage Resistance, Cell Auto-Aggregation, and Motility.

Prophage 919TP is widely distributed among Vibrio cholera and is induced to produce free φ919TP phage particles. However, the interactions between prophage φ919TP, the induced phage particle, and its host remain unknown. In particular, phage resistance mechanisms and potential fitness trade-offs, resulting from phage resistance, are unresolved. In this study, we examined a prophage 919TP-deleted variant of V. cholerae and its interaction with a modified lytic variant of the induced prophage (φ919TP cI-). Specifically, the phage-resistant mutant was isolated by challenging a prophage-deleted variant with lytic phage φ919TP cI-. Further, the comparative genomic analysis of wild-type and φ919TP cI--resistant mutant predicted that phage φ919TP cI- selects for phage-resistant mutants harboring a mutation in key steps of lipopolysaccharide (LPS) O-antigen biosynthesis, causing a single-base-pair deletion in gene gmd. Our study showed that the gmd-mediated O-antigen defect can cause pleiotropic phenotypes, e.g., cell autoaggregation and reduced swarming motility, emphasizing the role of phage-driven diversification in V. cholerae. The developed approach assists in the identification of genetic determinants of host specificity and is used to explore the molecular mechanism underlying phage-host interactions. Our findings contribute to the understanding of prophage-facilitated horizontal gene transfer and emphasize the potential for developing new strategies to optimize the use of phages in bacterial pathogen control.

Recent Vibrio cholerae O1 Epidemic Strains Are Unable To Replicate CTXΦ Prophage Genome.

Cholera, an acute diarrheal disease, is caused by pathogenic strains of Vibrio cholerae generated by the lysogenization of the filamentous cholera toxin phage CTXΦ. Although CTXΦ phage in the classical biotype are usually integrated solitarily or with a truncated copy, those in El Tor biotypes are generally found in tandem and/or with related genetic elements. Due to this structural difference in the CTXΦ prophage array, the prophage in the classical biotype strains does not yield extrachromosomal CTXΦ DNA and does not produce virions, whereas the El Tor biotype strains can replicate the CTXΦ genome and secrete infectious CTXΦ phage particles. However, information on the CTXΦ prophage array structure of pathogenic V. cholerae is limited. Therefore, we investigated the complete genomic sequences of five clinical V. cholerae isolates obtained in Kolkata (India) during 2007 to 2011. The analysis revealed that recent isolates possessed an altered CTXΦ prophage array of the prototype El Tor strain. These strains were defective in replicating the CTXΦ genome. All recent isolates possessed identical rstA and intergenic sequence 1 (Ig-1) sequences and comparable rstA expression in the prototype El Tor strain, suggesting that the altered CTXΦ array was responsible for the defective replication of the prophage. Therefore, CTXΦ structures available in the database and literatures can be classified as replicative and nonreplicative. Furthermore, V. cholerae epidemic strains became capable of producing CTXΦ phage particles since the 1970s. However, V. cholerae epidemic strains again lost the capacity for CTXΦ production around the year 2010, suggesting that a significant change in the dissemination pattern of the current cholera pandemic occurred. IMPORTANCE Cholera is an acute diarrheal disease caused by pathogenic strains of V. cholerae generated by lysogenization of the filamentous cholera toxin phage CTXΦ. The analysis revealed that recent isolates possessed altered CTXΦ prophage array of prototype El Tor strain and were defective in replicating the CTXΦ genome. Classification of CTXΦ structures in isolated years suggested that V. cholerae epidemic strains became capable of producing CTXΦ phage particles since the 1970s. However, V. cholerae epidemic strains again lost the capacity for CTXΦ production around the year 2010, suggesting that a critical change had occurred in the dissemination pattern of the current cholera pandemic.

Computational image analysis of the baseplate-tail complex of O1 ElTor vibriophage M4.

We performed an in-depth computational image analysis of the baseplate-tail complex of the M4 vibriophage and identified seven major densities in its baseplate, which notably share structural similarities with baseplate modules of a number of other bacteriophages belonging to different species. Employing computational analysis, we explained the helical organization of the sheath protein, wrapping the tail tube. Based on the results obtained in this work along with the proteomics information published previously, we are able to decipher the plausible roles assigned to the different components of the M4 baseplate during infection of the host.

[Genomic recombination of the vibrio cholerae serogroup O1 El Tor pandemic strains].

Objective: To analyze the genomic recombination of the vibrio cholerae serogroup O1 El Tor pandemic strains. Methods: A total of 292 complete or draft genome sequences of Vibrio cholerae O1 serogroup El Tor strains isolated from 1937 to 2015 were selected from National Biotechnology Information Center database. The genome alignment of strains was computed by snippy software by using N16961 as reference sequence. Then ClonalFrameML software was used to do the recombinant analysis. The wilcox.test function in agricolae package was used to compare the number recombinant segments and the total length of recombinant regions between small and large chromosomes. The kruskal function was used to compare the number recombinant segments and the total length of recombinant regions among different isolation continents. The KOBAS tool was used to do the gene ontology enrichment analysis of recombinant hotspot genes. Results: Of all 292 strains of Vibrio cholerae, 163 strains (55.8%) were recombined. The median of normalized recombinant segment number of small chromosome was 4.7×10(-6) (9.3×10(-7), 2.0×10(-5)), which was significantly larger than that of large chromosome [2.4×10(-6) (3.4×10(-7), 5.7×10(-6))] (P<0.001). The median (P(25),P(75)) of recombinant segment number of strains isolated from Africa, Asia, Europe, North America and South America were 23(1.0,33.0), 1.0(0.0,34.0), 6.0(2.0,13.0), 0.0(0.0,1.0) and 29.5(6.8,56.8), respectively, and the difference was statistically significant (P<0.001). The median (P(25),P(75)) of total length of recombinant regions of strains isolated from Africa, Asia, Europe, North America and South America were 233.0(4.0, 461.0), 11.0(0.0, 695.5), 56.0(4.0,111.0), 0.0(0.0,9.0) and 347.5(132.8,1 323.5) bp, respectively, and the difference was statistically significant (P<0.001). Gene ontology Enrichment analysis showed that the functions of 62 recombinant hotspot genes were mainly enrichment in chemotaxis, taxis, response to external stimulus, receptor activity and molecular transducer activity. Conclustion: In this study, we found that there were significant differences in the number of recombinant fragments and the length of recombinant regions between large and small chromosomes of Vibrio cholerae El Tor. We also found significant differences in the number of recombinant fragments and the total length of recombinant regions among different continents.

A novel pre-CTX prophage in the Vibrio cholerae serogroup O139 strain.

In Vibrio cholerae, the lysogenic bacteriophage CTXΦ carries the cholera toxin genes ctxAB, which can be transferred from toxigenic strains to nontoxigenic strains through infection and lysogenic conversion of CTXΦ. This phage also has the precursor genome which does not harbor ctxAB, named pre-CTXΦ. Based on the sequences of the transcriptional regulator-encoding gene rstR alleles in CTXΦ/pre-CTXΦ, multiple types of these prophages have been classified and identified in toxigenic and nontoxigenic V. cholerae strains. In this study, by combining the short-read and long-read sequencing approaches of next generation sequencing, we obtained the complete genome sequence of the studied V. cholerae toxigenic serogroup O139 strain and identified the CTXΦ and a pre-CTXΦ genome type encoding a novel rstR allele, pre-CTXZHJΦ. This pre-CTX prophage integrates into the small chromosome of the V. cholerae host strain and coexists with a typical CTXETΦ prophage present in the large chromosome, which is commonly present in the seventh pandemic serogroup O1 and toxigenic serogroup O139 strains. RstRZHJ could bind to the ig-2 region in the RstAB promotor in the pre-CTXZHJΦ genome, and could repress the expression of its own rstAB genes but could not repress rstAB expression in CTXETΦ and CTXclassΦ, suggesting that the V. cholerae strains carrying the pre-CTXZHJΦ prophage cannot prevent the infection of these epidemic CTXΦs, hence have the potentiality to become toxigenic by acquiring and lysogenic conversion of CTXΦs. Our study identified a novel pre-CTXΦ type, and presents the new evidence for the complexity and diversity of the CTXΦ/pre-CTXΦ family in V. cholerae.

Analysis of 19 Highly Conserved Vibrio cholerae Bacteriophages Isolated from Environmental and Patient Sources Over a Twelve-Year Period.

The Vibrio cholerae biotype "El Tor" is responsible for all of the current epidemic and endemic cholera outbreaks worldwide. These outbreaks are clonal, and it is hypothesized that they originate from the coastal areas near the Bay of Bengal, where the lytic bacteriophage ICP1 (International Centre for Diarrhoeal Disease Research, Bangladesh cholera phage 1) specifically preys upon these pathogenic outbreak strains. ICP1 has also been the dominant bacteriophage found in cholera patient stools since 2001. However, little is known about the genomic differences between the ICP1 strains that have been collected over time. Here, we elucidate the pan-genome and the phylogeny of the ICP1 strains by aligning, annotating, and analyzing the genomes of 19 distinct isolates that were collected between 2001 and 2012. Our results reveal that the ICP1 isolates are highly conserved and possess a large core-genome as well as a smaller, somewhat flexible accessory-genome. Despite its overall conservation, ICP1 strains have managed to acquire a number of unknown genes, as well as a CRISPR-Cas system which is known to be critical for its ongoing struggle for co-evolutionary dominance over its host. This study describes a foundation on which to construct future molecular and bioinformatic studies of these V. cholerae-associated bacteriophages.

Visualizing a Vibrio cholerae O1 El Tor typing bacteriophage belonging to the Myoviridae group and the packaging of its genomic ends inside the phage capsid.

Phage D10, an O1 El Tor tying vibriophage, has been successfully employed to tract the outspread of cholera epidemic. Using Transmission Electron Microscopy and computational image analysis, we have determined the structures of the capsid, head-to-tail connector, the contractile helical tail, the baseplate and combined them to form the complete three-dimensional (3D) D10 phage structure. Using partial denaturation experiments on the genome and using the computed 3D structure of the phage, we have established the packing of the genome ends inside the capsid together with the release styles during the phage infection, respectively. Finally, using the 3D density maps of the different components of the D10 phage, we have presented a simplified picture of morphogenesis of the D10 vibriophage. Using the complete assembled structure of the D10 phage, we have traced the path of the phage genome during the infection process, all the way from the phage head down the tail tube of the tail to the top of the baseplate. To the best of our knowledge, this is first structural study for a long-tailed vibriophage. We have tabulated the structural features of the different components of the phages belonging to the Myoviridae and Siphoviridae. The comparative study suggested the possibility of a common origin of the bacteriophages, irrespective of belonging to different groups and species.

The role of CTX and RS1 satellite phages genomic arrangement in Vibrio cholera toxin production in two recent cholera outbreaks (2012 and 2013) in IR Iran.

The objective of the present study was to investigate the genomic arrangement of CTX/RS1 prophages in 30 Vibrio cholerae strains obtained from 2 consecutive years of cholera outbreak and to compare the role of different CTX/RS1 arrangements in cholera toxin expression among the El Tor strains. Profile A with TLC-RS1-CTX-RTX arrangement was observed in 46.7% of the isolates with RS1 phage locating adjacent to TLC element. About 50% of the isolates showed Profile B with TLC-CTX-RS1-RTX arrangement and one single isolate (3.3%) revealed TLC-CTX-RS1-RS1-RTX arrangement (Profile C). No RS1 element was detected to be adjacent to TLC element in B and C profiles. No truncated CTX phage genome was detected among the isolates of 2 years. Different CTX-RS1 arrangement profiles (A, B, and C) with different RS1 copy numbers and locations uniformly showed low level of cholera toxin production in El Tor strains with no significant difference, revealing that different RS1 copy numbers and locations have no effect on cholera toxin production level (p-value >0.05). However, increased cholera toxin expression was observed for control V. cholerae classical biotype strain. In conclusion, variations in RS1 prophage did not affect CT expression level in related El Tor V. cholerae strains. CTX genotyping establishes a more valuable database for epidemiologic, pathogenesis, and source tracking purposes.

Environmental bacteriophages active on biofilms and planktonic forms of toxigenic Vibrio cholerae: Potential relevance in cholera epidemiology.

METHODS: Phages isolated from environmental waters in Bangladesh were tested for their host specificity towards V. cholerae O1 and O139, and the ability to disperse V. cholerae biofilms formed in the laboratory. Representative phages were further characterized by electron microscopy and whole genome sequencing. Selected phages were then introduced in various combinations to biofilms of toxigenic V. cholerae added to samples of river water, and the dispersion of biofilms as well as the growth kinetics of V. cholerae and the phages were monitored. RESULTS: A phage cocktail composed of three different phages isolated from surface waters in Bangladesh and designated as JSF7, JSF4, and JSF3 could significantly influence the distribution and concentration of the active planktonic form and biofilm associated form of toxigenic V. cholerae in water. While JSF7 showed a biofilm degrading activity and dispersed cells from both V. cholerae O1 and O139 derived biofilms thus increasing the concentration of planktonic V. cholerae in water, JSF4 and JSF3 showed strong bactericidal activity against V. cholerae O1 and O139 respectively. A mixture of all three phages could effectively reduce both biofilm-associated and planktonic V. cholerae in river water microcosms. SIGNIFICANCE: Besides potential applicability in phage-mediated control of cholera, our results have relevance in appreciating possible intricate role of diverse environmental phages in the epidemiology of the disease, since both biofilms and phages influence the prevalence and infectivity of V. cholerae in a variety of ways.

Replication of Vibrio cholerae classical CTX phage.

The toxigenic classical and El Tor biotype Vibrio cholerae serogroup O1 strains are generated by lysogenization of host-type-specific cholera toxin phages (CTX phages). Experimental evidence of the replication and transmission of an El Tor biotype-specific CTX phage, CTX-1, has explained the evolution of V. cholerae El Tor biotype strains. The generation of classical biotype strains has not been demonstrated in the laboratory, and the classical biotype-specific CTX phage, CTX-cla, is considered to be defective with regard to replication. However, the identification of atypical El Tor strains that contain CTX-cla-like phage, CTX-2, indicates that CTX-cla and CTX-2 replicate and can be transmitted to V. cholerae strains. The replication of CTX-cla and CTX-2 phages and the transduction of El Tor biotype strains by various CTX phages under laboratory conditions are demonstrated in this report. We have established a plasmid-based CTX phage replication system that supports the replication of CTX-1, CTX-cla, CTX-2, and CTX-O139. The replication of CTX-2 from the tandem repeat of lysogenic CTX-2 in Wave 2 El Tor strains is also presented. El Tor biotype strains can be transduced by CTX phages in vitro by introducing a point mutation in toxT, the transcriptional activator of the tcp (toxin coregulated pilus) gene cluster and the cholera toxin gene. This mutation also increases the expression of cholera toxin in El Tor strains in a sample single-phase culture. Our results thus constitute experimental evidence of the genetic mechanism of the evolution of V. cholerae.

Genetic characterization of ØVC8 lytic phage for Vibrio cholerae O1.

BACKGROUND: Epidemics and pandemics of cholera, a diarrheal disease, are attributed to Vibrio cholera serogroups O1 and O139. In recent years, specific lytic phages of V. cholera have been proposed to be important factors in the cyclic occurrence of cholera in endemic areas. However, the role and potential participation of lytic phages during long interepidemic periods of cholera in non-endemic regions have not yet been described. The purpose of this study was to isolate and characterize specific lytic phages of V. cholera O1 strains. METHODS: Sixteen phages were isolated from wastewater samples collected at the Endhó Dam in Hidalgo State, Mexico, concentrated with PEG/NaCl, and purified by density gradient. The lytic activity of the purified phages was tested using different V. cholerae O1 and O139 strains. Phage morphology was visualized by transmission electron microscopy (TEM), and phage genome sequencing was performed using the Genome Analyzer IIx System. Genome assembly and bioinformatics analysis were performed using a set of high-throughput programs. Phage structural proteins were analyzed by mass spectrometry. RESULTS: Sixteen phages with lytic and lysogenic activity were isolated; only phage ØVC8 showed specific lytic activity against V. cholerae O1 strains. TEM images of ØVC8 revealed a phage with a short tail and an isometric head. The ØVC8 genome comprises linear double-stranded DNA of 39,422 bp with 50.8 % G + C. Of the 48 annotated ORFs, 16 exhibit homology with sequences of known function and several conserved domains. Bioinformatics analysis showed multiple conserved domains, including an Ig domain, suggesting that ØVC8 might adhere to different mucus substrates such as the human intestinal epithelium. The results suggest that ØVC8 genome utilize the "single-stranded cohesive ends" packaging strategy of the lambda-like group. The two structural proteins sequenced and analyzed are proteins of known function. CONCLUSIONS: ØVC8 is a lytic phage with specific activity against V. cholerae O1 strains and is grouped as a member of the VP2-like phage subfamily. The encoding of an Ig domain by ØVC8 makes this phage a good candidate for use in phage therapy and an alternative tool for monitoring V. cholerae populations.

The Hybrid Pre-CTXΦ-RS1 Prophage Genome and Its Regulatory Function in Environmental Vibrio cholerae O1 Strains.

The cholera toxin genes of Vibrio cholerae are encoded by CTXΦ, a lysogenic bacteriophage. Infection with this phage plays a determinant role in toxigenicity conversion and the emergence of new clones of pathogenic V. cholerae. Multiple phage alleles, defined by sequence types of the repressor gene rstR, have been found, showing the divergence of phage genomes. Pre-CTXΦ, which is characterized by the absence of toxin genes, is predicted to be the precursor of CTXΦ. We have found a new pre-CTXΦ prophage genome (named pre-CTXZJΦ for its novel rstR allele) in nontoxigenic V. cholerae O1 isolates that were obtained during surveillance of the estuary water of the Zhujiang River. A novel hybrid genome of the helper phage RS1 was identified in an environmental strain carrying pre-CTXZJΦ in this study. The chromosomal integration and genomic arrangement of pre-CTXZJΦ and RS1 were determined. The RS2 of pre-CTXZJΦ was shown to have a function in replication, but it seemed to have lost its ability to integrate. The RstR of pre-CTXZJΦ exerted the highest repression of its own rstA promoter compared to other RstRs, suggesting rstR-specific phage superinfection immunity and potential coinfection with other pre-CTXΦ/CTXΦ alleles. The environmental strain carrying pre-CTXZJΦ could still be infected by CTXETΦ, the most common phage allele in the strains of the seventh cholera pandemic, suggesting that this nontoxigenic clone could potentially undergo toxigenicity conversion by CTXΦ infection and become a new toxigenic clone despite already containing the pre-CTXΦ prophage.

Molecular insights into the evolutionary pathway of Vibrio cholerae O1 atypical El Tor variants.

Pandemic V. cholerae strains in the O1 serogroup have 2 biotypes: classical and El Tor. The classical biotype strains of the sixth pandemic, which encode the classical type cholera toxin (CT), have been replaced by El Tor biotype strains of the seventh pandemic. The prototype El Tor strains that produce biotype-specific cholera toxin are being replaced by atypical El Tor variants that harbor classical cholera toxin. Atypical El Tor strains are categorized into 2 groups, Wave 2 and Wave 3 strains, based on genomic variations and the CTX phage that they harbor. Whole-genome analysis of V. cholerae strains in the seventh cholera pandemic has demonstrated gradual changes in the genome of prototype and atypical El Tor strains, indicating that atypical strains arose from the prototype strains by replacing the CTX phages. We examined the molecular mechanisms that effected the emergence of El Tor strains with classical cholera toxin-carrying phage. We isolated an intermediary V. cholerae strain that carried two different CTX phages that encode El Tor and classical cholera toxin, respectively. We show here that the intermediary strain can be converted into various Wave 2 strains and can act as the source of the novel mosaic CTX phages. These results imply that the Wave 2 and Wave 3 strains may have been generated from such intermediary strains in nature. Prototype El Tor strains can become Wave 3 strains by excision of CTX-1 and re-equipping with the new CTX phages. Our data suggest that inter-chromosomal recombination between 2 types of CTX phages is possible when a host bacterial cell is infected by multiple CTX phages. Our study also provides molecular insights into population changes in V. cholerae in the absence of significant changes to the genome but by replacement of the CTX prophage that they harbor.

High prevalence and diversity of pre-CTXΦ alleles in the environmental Vibrio cholerae†O1 and O139 strains in the Zhujiang River estuary.

Toxigenic conversion of environmental Vibrio cholerae strains through lysogenic infection by the phage CTXΦ is an important step in the emergence of new pathogenic clones. The precursor form of the CTXΦ phage, pre-CTXΦ, does not carry the cholera toxin gene. During our investigation, we frequently found pre-CTXΦ prophages in non-toxigenic isolates in the serogroups of O1 and O139 strains in the Zhujiang estuary. We observed high amounts of sequence variation of rstR and gIII(CTX) in the pre-CTXΦ alleles as well as in the tcpA sequences within the strains. In addition, a new pre-CTXΦ allele, with a novel rstR sequence type and hybrid RS2, was identified. Our findings show that active, complicated gene recombination and horizontal transfer of pre-CTXΦs occurs within V. cholerae environmental strains, which creates a complex intermediate pool for the generation of toxigenic clones in the estuarine environment.

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.

CTX prophages in Vibrio cholerae O1 strains.

The classical biotype strains of the Vibrio cholerae O1 serogroup harbor the biotype-specific cholera-toxin encoding phage (CTX) CTX(cla), and the El Tor biotype strains contain CTX-1. Although the classical biotype strains have become extinct, a remnant of classical CTX phage is transferred to the El Tor biotype strains. The prototype El Tor strains, which produce the biotype-specific cholera toxin, are now being replaced by atypical El Tor variant strains producing classical biotype cholera toxin. The genome sequences of the CTX phages in atypical El Tor strains indicate that the CTX phages in atypical El Tor strains are a mosaic of CTXcla and CTX-1. Before the emergence of atypical El Tor stains in the early 1990s, unusual pre-seventh pandemic strains were isolated in the US Gulf Coast between 1973 and 1986. These strains have characteristics of atypical El Tor strains since they are El Tor biotype strains containing CTX(cla), yet the genome sequence of this CTX phage indicates that it is different from CTXcla and is therefore classified separately as CTX(US Gulf).

Isolation and characterization of lytic vibriophage against Vibrio cholerae O1 from environmental water samples in Kelantan, Malaysia.

Water samples from a variety of sources in Kelantan, Malaysia (lakes, ponds, rivers, ditches, fish farms, and sewage) were screened for the presence of bacteriophages infecting Vibrio cholerae. Ten strains of V. cholerae that appeared to be free of inducible prophages were used as the host strains. Eleven bacteriophage isolates were obtained by plaque assay, three of which were lytic and further characterized. The morphologies of the three lytic phages were similar with each having an icosahedral head (ca. 50-60 nm in diameter), a neck, and a sheathed tail (ca. 90-100 nm in length) characteristic of the family Myoviridae. The genomes of the lytic phages were indistinguishable in length (ca. 33.5 kb), nuclease sensitivity (digestible with DNase I, but not RNase A or S1 nuclease), and restriction enzyme sensitivity (identical banding patterns with HindIII, no digestion with seven other enzymes). Testing for infection against 46 strains of V. cholerae and 16 other species of enteric bacteria revealed that all three isolates had a narrow host range and were only capable of infecting V. cholerae O1 El Tor Inaba. The similar morphologies, indistinguishable genome characteristics, and identical host ranges of these lytic isolates suggests that they represent one phage, or several very closely related phages, present in different water sources. These isolates are good candidates for further bio-phage-control studies.

Phase variable O antigen biosynthetic genes control expression of the major protective antigen and bacteriophage receptor in Vibrio cholerae O1.

The Vibrio cholerae lipopolysaccharide O1 antigen is a major target of bacteriophages and the human immune system and is of critical importance for vaccine design. We used an O1-specific lytic bacteriophage as a tool to probe the capacity of V. cholerae to alter its O1 antigen and identified a novel mechanism by which this organism can modulate O antigen expression and exhibit intra-strain heterogeneity. We identified two phase variable genes required for O1 antigen biosynthesis, manA and wbeL. manA resides outside of the previously recognized O1 antigen biosynthetic locus, and encodes for a phosphomannose isomerase critical for the initial step in O1 antigen biosynthesis. We determined that manA and wbeL phase variants are attenuated for virulence, providing functional evidence to further support the critical role of the O1 antigen for infectivity. We provide the first report of phase variation modulating O1 antigen expression in V. cholerae, and show that the maintenance of these phase variable loci is an important means by which this facultative pathogen can generate the diverse subpopulations of cells needed for infecting the host intestinal tract and for escaping predation by an O1-specific phage.

Vibrio cholerae typing phage N4: genome sequence and its relatedness to T7 viral supergroup.

OBJECTIVES: In countries where cholera is endemic, Vibrio cholerae O1 bacteriophages have been detected in sewage water. These have been used to serve not only as strain markers, but also for the typing of V. cholerae strains. Vibriophage N4 (ATCC 51352-B1) occupies a unique position in the new phage-typing scheme and can infect a larger number of V. cholerae O1 biotype El Tor strains. Here we characterized the complete genome sequence of this typing vibriophage. METHODS: The complete DNA sequence of the N4 genome was determined by using a shotgun sequencing approach. RESULTS: Complete genome sequence explored that phage N4 is comprised of one circular, double-stranded chromosome of 38,497 bp with an overall GC content of 42.8%. A total of 47 open reading frames were identified and functions could be assigned to 30 of them. Further, a close relationship with another vibriophage, VP4, and the enterobacteriophage T7 could be established. DNA-DNA hybridization among V. cholerae O1 and O139 phages revealed homology among O1 vibriophages at their genomic level. CONCLUSIONS: This study indicates two evolutionary distinctive branches of the possible phylogenetic origin of O1 and O139 vibriophages and provides an unveiled collection of information on viral gene products of typing vibriophages.