INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Vibrio cholerae is the etiologic agent of the diarrheal disease cholera. Humans become infected with V. cholerae after ingestion of contaminated food or water. Of the nearly 200 recognized serogroups of V. cholerae, only the O1 and O139 serogroups are associated with epidemics of cholera (27). The V. cholerae O1 serogroup is further divided into the classical and El Tor biotypes on the basis of several phenotypic differences. Since 1817, seven cholera pandemics have been described. The classical biotype is believed to have given rise to the first six cholera pandemics (2). The ongoing seventh pandemic of cholera, which began in 1961, is caused by the El Tor biotype. In 1992, a newly recognized serogroup, O139, emerged and resulted in cholera epidemics in Southeast Asia (14). The emergence of this novel V. cholerae serogroup, along with the re-emergence in 1991 of cholera in South America after a nearly 100-year absence, has renewed interest in the origins and evolution of this pathogen.

Pathogenic V. cholerae isolates colonize the small intestine and secrete cholera toxin (CT), an A-B-type toxin, to cause the profuse secretory diarrhea characteristic of cholera (44). CT is encoded by ctxA and ctxB, which are not integral components of the V. cholerae genome but, instead, reside in the genome of CTX, a filamentous bacteriophage that infects V. cholerae, as well as its close relative V. mimicus (8, 18, 48). CTX utilizes the V. cholerae type IV pilus TCP, an essential intestinal colonization factor (46), as its receptor (48). In contrast to the well-characterized filamentous bacteriophages derived from Escherichia coli, such as f1, the CTX genome integrates into the genome of V. cholerae to form a prophage (48). Integration of CTX is site specific (39, 48). However, following infection of classical strains or El Tor strains lacking a CTX integration site, the El Tor-derived CTX remains extrachromosomal, replicating as a plasmid (48, 49). This plasmid form of CTX was designated the phage replicative form (RF), since cells harboring this plasmid produce relatively large amounts of viral particles (48, 49).

The 6.9-kb CTX genome has a modular structure composed of two functionally distinct domains, the core and RS2 regions (Fig. 1) (48). The core region encodes CT and the genes involved in phage morphogenesis, including genes that are thought to encode the major and minor phage coat proteins (Psh, Cep, OrfU, and Ace) and a protein required for CTX assembly (Zot) (48). The RS2 region encodes genes required for replication (rstA), integration (rstB), and regulation (rstR) of CTX (49). RS2 also contains two intergenic regions, ig-1 and ig-2 (Fig. 1). The RS2 region genes only show sequence similarity to two recently described Vibrio sp.-derived filamentous phages (13, 23). In contrast, the genes of the core region (with the exceptions of ctxA and ctxB) show sequence similarity to the morphogenesis genes of filamentous phages from a range of bacterial species (48). Interestingly, the percent GC contents of the ctxA and ctxB genes, 38 and 33%, respectively (Fig. 1), are significantly different from those of the rest of the core region genes. The distinct GC content of ctxAB, compared to the remainder of the CTX genome, suggests that these genes evolved separately from the remainder of the phage genome and that they were acquired after the emergence of a precursor form of CTX that lacked ctxAB. In fact, there have been a number of reports in the literature of zot⁺ V. cholerae isolates lacking ctxAB (15, 31).

View larger version (17K): [in this window] [in a new window]   FIG. 1.   (Top) Schematic representation of the organization of the El Tor-derived CTX genome. Open arrows represent CTX ORFs and the direction of transcription of each gene. Numbers within the arrows indicate the genes' percent GC contents. The horizontal bar below the CTX map indicates the position of the core probe used in DNA hybridization analysis. The two arrows below the CTX map indicate the positions of the PCR primers used to amplify the region between zot and ig-1 from pre-CTX RF DNA. (Bottom) Regional variation in the mean proportion of GC content based on a sliding window of 100 nucleotides.

The diversity among CTX genomes has only been examined with reference to the RS2 region genes. DNA sequence analysis has revealed that rstA and rstB from CTX^ET CTX^class, and CTX^calc are highly similar (99% amino acid identity) (16, 29). In contrast, comparisons of rstR sequences among CTX^ET, CTX^class, and CTX^calc revealed that they were extremely diverse, with less than 30% amino acid sequence similarity (16, 29). Furthermore, the three known rstR genes have a percent GC content (34 to 37%) that is distinct from those of most of the other CTX genes (Fig. 1). The hypervariability of rstR and its distinct GC content suggest that CTX variants obtained diverse rstR cassettes via horizontal gene transfer, followed by recombination. A similar mechanism is believed to account for the diversity of repressors in lambdoid phages (12). Since recombination, rather than mutation, probably accounts for the distinct CTX rstR genes, the diversity of rstR sequences does not provide an insight into the relatedness of distinct CTXs.

We set out to study the evolution and relatedness of CTX variants and to examine whether distinct CTX genotypes are associated with each of the V. cholerae O1 biotypes, with different V. cholerae serogroups, or with V. mimicus. During the course of this work, three strains harboring CTX-like prophages that lacked the ctxA and ctxB genes were identified. We hypothesize that these CTX-like prophages lacking ctxAB represent derivatives of the ancestral precursor of CTX. Comparative nucleotide sequence analyses of two CTX core region genes, orfU and zot, from 13 V. cholerae strains revealed that there are distinct phage lineages in classical and El Tor V. cholerae isolates. These analyses suggest that acquisition of CTX by Vibrio spp. has occurred multiple times and has involved several CTX genotypes.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Bacterial strains. All of the bacterial strains used were cultured in Luria-Bertani (34) broth at 37°C. These strains included 13 V. cholerae and 4 V. mimicus isolates that encompassed seven serogroups (Table 1). These Vibrio sp. isolates were derived from both human and environmental sources with a wide geographic distribution between 1930 and 1993 (Table 1).

Molecular analyses. V. cholerae DNA was extracted and purified using the G-nome DNA isolation kit from Bio 101, Inc., Vista, Calif. To assay for the presence of the CTX genome among Vibrio isolates, two pairs of primers were used for PCR assays, orfU1 plus zot2 and ctxA1 plus ctxB2, which were designed from published DNA sequences as previously described (8). For Southern hybridization analyses, a CTX core region DNA probe spanning the region between psh and zot was used (Fig. 1). Southern hybridization was carried out using horseradish peroxidase-labeled DNA probes, which were prepared and hybridized using the ECL direct labeling and detection system (Amersham Pharmacia, Little Chalfont, Buckinghamshire, England) in accordance with the manufacturer's instructions.

Nucleotide sequencing. Sequencing was performed with an Applied Biosystems 373A automated DNA sequencing system using the DyeDeoxy terminator cycle sequencing kit at the Tufts Medical School sequencing facility. For all of the genes analyzed, both DNA strands were sequenced. The BLAST programs (1) were used to compare sequences to those in the GenBank databases. A 715-bp region of orfU and a 708-bp segment of zot were amplified by PCR and sequenced. PCR primers to amplify the chromosomal mdh locus were designed from the mdh sequence of V. cholerae strain N16961. With the forward (5' atgaaagtcgctgttatt 3') and reverse (5' gtatctaacatgccatcc 3') primers, an 892-bp region of the 939-bp mdh sequence was amplified. PCR products were purified using the Qiaquick (Qiagen) PCR purification kit and subsequently sequenced on both strands. To generate sequencing templates from the three strains that were found to contain CTX-like prophage genomes that lacked ctxAB, the DNA sequences between zot and ig-1 in the RF were amplified with primers zot5 and rig1 (Fig. 1) using plasmid DNA derived from these strains as templates. The forward primer zot5 (5' gcagtagcctttgactgag 3') lies within the zot gene, and the reverse primer rig1 (5' cacgctacgtcgcttatgt 3') is located within the conserved part of the first intergenic region of CTX. The PCR products were cloned into pCR2.1 (Invitrogen, Carlsbad, Calif.), and the resulting plasmids were subsequently used as templates for sequencing.

Phylogenetic analyses. Regional variation in GC content over the entire length of the CTX genome was calculated using a sliding window of 100 nucleotides. The GC content of each CTX gene was also calculated using the MacVector program. DNA sequence data were assembled and edited with Eyeball Sequence Editor (11). Gene trees were constructed with MEGA (30). Rates (per site) of synonymous (k_S) and nonsynonymous (k_N) substitutions were calculated by the methods of Nei and Gojobori (36) and Nei and Jin (37). The proportions of synonymous (p_S) and nonsynonymous (p_N) substitutions in the orfU and zot genes between pairs of strains were tabulated in a sliding-window analysis of 30 codons along each gene by the program PSWIN (T. S. Whittam, Pennsylvania State University).

Nucleotide sequence accession numbers. The nucleotide sequences obtained during this study for orfU, zot, and mdh have been deposited in GenBank under accession numbers AF238329 to AF238373.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Identification and analysis of potential CTX precursors. To begin to assess the diversity of CTX, we examined our laboratory collection of natural V. cholerae isolates for the presence of CTX-related sequences using PCR tests for the presence of orfU, ace, zot, and ctxAB. We identified two isolates, 151 (38) and 208 (38), that contained the CTX sequences orfU, ace, and zot but did not encode ctxAB. In addition to these two isolates, we obtained a third V. cholerae strain, C325, that was previously reported to contain CTX core region sequences without ctxAB (31). V. cholerae strains 151, 208, and C325 belonged to three serogroups, O11, O37, and O1, respectively, and were isolated in Mexico, Thailand, and India, respectively. Southern blots revealed that the phage structural genes were present within the chromosomal DNAs of these strains (data not shown). We hypothesized that the phage genes might be components of a prophage that could represent a derivative of a precursor of CTX (pre-CTX) that lacked ctxAB. The distinct GC content of ctxAB relative to the remainder of the CTX genome strongly suggests that the toxin genes were acquired subsequent to the development of a pre-CTX. To test whether the phage genes are part of functional prophages in these strains, we first examined whether these three strains contained extrachromosomal DNA that corresponded to the RF of the pre-CTX DNA. Southern blot analyses of plasmid DNAs prepared from these strains showed the presence of extrachromosomal DNA that hybridized with a core region probe in all three cases (Fig. 2 and data not shown). For C325 and 151, this plasmid was ~6.0 kb, a size consistent with a CTX-like RF that is missing ctxAB. In strain 208, this plasmid was ~7.0 kb. Furthermore, PCR analyses strongly suggested that the gene content and gene order of core region and RS2 region genes in these three plasmids are identical to those of CTX, with the exception of the missing ctxAB genes (Fig. 3). Finally, supernatants derived from all three strains could transmit these pre-CTX genomes to a classical recipient strain (Fig. 4 and data not shown). These results indicate that these putative pre-CTX prophages give rise to infectious particles. These experiments demonstrate that these three CTX-like prophages lacking ctxAB are functional and are consistent with the hypothesis that CTX evolved from a pre-CTX lacking ctxAB.

View larger version (81K): [in this window] [in a new window]   FIG. 2.   Detection of an extrachromosomal form of the pre-CTX prophage from ctxAB strain C325. Plasmid DNA was prepared from C325, classical strain O395 (a negative control), El Tor strain E7946 (a positive control), and Bah-2(pCTX-Kn). Southern hybridization with a core probe was then used to detect either SphI-digested or undigested plasmid DNA.

View larger version (59K): [in this window] [in a new window]   FIG. 3.   PCR analyses of the genomic organizations of the plasmid form of CTX from V. cholerae strain N16961 (El Tor) or from putative pre-CTXs from V. cholerae strains 151, 208, and C325. The primer pairs used are indicated below the lanes. The left- and rightmost lanes contained molecular size markers.

View larger version (85K): [in this window] [in a new window]   FIG. 4.   Detection of an infectious form of pre-CTX derived from strain C325. Cell-free supernatants from C325, O395 (a negative control), and E7946 (a positive control) were mixed with agglutinated O395. Twenty-four hours later, plasmid DNA was prepared from these three cultures and the presence of CTX core genes was determined by Southern blot analysis.

View larger version (49K): [in this window] [in a new window]   FIG. 5.   Alignment of the nucleotide sequences of zot to ig-1 from V. cholerae strain N16961 (El Tor) and from V. cholerae strains 151, 208, and C325. The latter three sequences were determined from PCR-amplified pre-CTX RF DNAs derived from these strains. The arrow above the sequences depicts the 3' end of the zot ORFs. Dots indicate nucleotide identity, and dashes represent gaps introduced to allow alignment of the CTX sequences with the pre-CTX sequences. Boxed and italicized nucleotides indicate the repeat sequences that play a role in ToxR binding. The boxed nucleotides designated ER represent the end repeat sequence thought to constitute the core of the CTX attachment site. The ctxAB genes of strain N16961 are not shown. M represents the ctxA start codon, and the asterisk designates the stop codons of ctxB and zot. The line above the N16961 sequence beginning after the ctxB stop codon represents the El Tor ig-1 sequence.

Diversity of CTX core region sequences. Since our understanding of the diversity of CTXs was limited and rested entirely on RS2 region sequences, we compared the sequences of core region genes from a set of Vibrio sp. isolates that we found to harbor CTX. This set included 13 V. cholerae strains and 4 V. mimicus strains (Table 1). These strains were chosen to represent the diversity of CTXs in our laboratory collection based on the dates and sites of their isolation. The sample of V. cholerae included seven O1 serogroup strains (three classical and four El Tor biotype strains). Although classical and El Tor strains are thought to be essentially clonal (3, 10, 28), the similarity of the CTXs in these strains has not been assessed. The six non-O1 serogroup strains included four serotypes (O139, O37, O141, and O11) and two strains that contained the putative pre-CTX derivatives (151 and 208).

View this table: [in this window] [in a new window]   TABLE 2.   Nucleotide sequence variation in the orfU and zot genes of CTXs derived from V. cholerae and V. mimicus strains

Spatial distribution of polymorphic sites. The OrfU protein is thought to be functionally equivalent to pIII of filamentous phages derived from E. coli (22, 48). pIII is a phage coat protein that mediates phage attachment to a host cell. We hypothesized that the significant differences in the OrfU sequences between classical strain- and El Tor strain-derived CTXs reflect the functional constraints that these two OrfU proteins face in binding to the CTX receptor, TCP. The sequence of TcpA, the major subunit of TCP, is known to vary considerably between the two V. cholerae O1 biotypes (24, 42). To begin to address this possibility, we analyzed whether the synonymous and nonsynonymous substitutions within orfU are clustered in the central domain of the genes, which encodes the putative TCP-binding domain of OrfU (21). The results of this analysis, shown in Fig. 6, revealed a striking clustering of synonymous and nonsynonymous site variation in the central portion of orfU. Two distinct peaks of nonsynonymous site variation were present in the central region. We predict that these two orfU hypervariable regions constitute parts of OrfU that are important for OrfU-TcpA interaction. In contrast, similar analysis of the zot sequences did not reveal any clustering of the polymorphic sites.

View larger version (18K): [in this window] [in a new window]   FIG. 6.   Regional variation in the mean proportion of synonymous (pS) differences between pairs of strains and the mean proportion of nonsynonymous (pN) differences between pairs of strains based on a sliding window of 90 nucleotides in orfU. Squares represent pN values, and diamonds indicate pS values.

Evolutionary relationships among CTXs and their Vibrio sp. host strains. To compare the evolutionary relationships between CTXs and their Vibrio sp. host strains, the 17 zot, orfU, and mdh sequences were used to construct three phylogenetic trees. These three trees, shown in Fig. 7, were constructed by the neighbor-joining method from a matrix of pairwise genetic distances based on all polymorphic nucleotide sites, with correction for multiple substitutions by the Jukes-Cantor method (25, 43). The most notable feature of this analysis is the divergent clustering of CTX genes from classical and El Tor biotype V. cholerae isolates (with the exception of RV79). The orfU and zot sequences derived from strains of the same biotype invariably clustered together (Fig. 7). For the most part, orfU sequences derived from nonepidemic V. cholerae isolates clustered with El Tor orfU sequences. The relationships among the V. mimicus strains based on CTX gene trees are very different. V. mimicus strain PT5 clusters with the El Tor strains on both the orfU and zot gene trees. However, V. mimicus strains PT48, 523-80, and 9583 grouped with the classical strains in the orfU tree but with the El Tor strains in the zot gene tree (Fig. 7). These discrepancies are probably due to the low number of polymorphic sites analyzed at the zot locus, as indicated by the low bootstrap values obtained for these nodes in the zot gene tree. Similarly, the limited number of polymorphic sites among the zot sequences probably explains the discrepancy between the orfU and zot gene trees for non-O1 serogroup strains V52, 208, and V46. Another notable feature of Fig. 7 is the lack of congruence of the phage gene trees with the gene tree derived from mdh sequences. The classical and El Tor epidemic V. cholerae isolates are all identical based on mdh sequence analysis, yet they have very divergent CTX sequences. This lack of similarity between the phage and chromosomal gene trees is indicative of horizontal transmission of CTX, as expected for a mobile genetic element like CTX. The clustering of V. mimicus strain PT5-derived CTX sequences with El Tor V. cholerae CTX sequences and the V. mimicus strain PT48-derived orfU sequence with the classical orfU sequence (Fig. 7) provides additional evidence for the proposition (8) that horizontal transfer of CTX between V. cholerae and V. mimicus occurred relatively recently. Similarly, these gene trees suggest that there was a relatively recent transfer of CTX between O1 and non-O1 strains of V. cholerae.

View larger version (32K): [in this window] [in a new window]   FIG. 7.   Evolutionary relationships based on synonymous site variation in the orfU, zot, and mdh genes. The neighbor-joining method was used to construct the trees. The V. cholerae El Tor strains are in red, classical strains are in blue, non-O1 serogroup strains are in black with the particular serogroup in the superscript, and V. mimicus strains are in green. Bootstrap values based on 1,000 computer-generated trees are indicated at the nodes, and only values greater than 50 are shown.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

CTX evolution. A number of toxins and other virulence factors are encoded within the genomes of bacteriophages (4, 47). In most, if not all, cases, phage-encoded virulence genes are not thought to directly influence the biological properties of the phage. Rather, these genes are believed to be accessories to the bacteriophage genome that can affect the properties of the host cell and thereby potentially indirectly influence the viability of the phage genome. Since neither CT nor its structural genes seem to affect CTX functions and since the GC content of ctxAB differs from that of most of the other CTX genes, we hypothesized that ctxAB was acquired after the evolution of a pre-CTX that lacked these genes.

Diverse CTXs in the evolution of toxigenic Vibrio spp. Comparative sequence analyses of the chromosomal mdh locus in CTX host strains yields an inferred phylogeny that differs significantly from the inferred phylogeny based on either zot or orfU sequences (compare Fig. 7A with B or C). It was recently reported (10) that epidemic isolates of V. cholerae are very closely related, and our comparisons of mdh sequences confirm this finding. All of the mdh sequences for V. cholerae serogroup O1 epidemic isolates that we analyzed were identical and thus cluster together on the mdh gene tree. However, this was not the case for the two CTX gene trees. The phage sequences derived from classical and El Tor strains formed two divergent branches on both the orfU and zot gene trees. This finding of distinct CTX lineages within essentially identical V. cholerae chromosomal backgrounds is not consistent with a simple evolutionary scenario of clonal descent, in which a single CTX progenitor infected an ancestral V. cholerae isolate and evolved within V. cholerae to the present level of diversity. Rather, it suggests the hypothesis that distinct CTXs independently infected ctxAB progenitors of the classical and El Tor V. cholerae pandemic strains (Fig. 8).

View larger version (15K): [in this window] [in a new window]   FIG. 8.   Model for acquisition of ctxAB by the two V. cholerae O1 biotypes. An ancestral V. cholerae isolate gave rise to the classical and El Tor biotypes, which were subsequently independently infected with divergent CTXs. CTX probably arose from a precursor CTX that acquired the CT genes by imprecise excision from a unknown donor strain.