Molecular analyses of a putative CTXphi precursor and evidence for independent acquisition of distinct CTX(phi)s by toxigenic Vibrio cholerae

Abstract

The genes encoding cholera toxin (ctxA and ctxB) are encoded in the genome of CTXphi, a filamentous phage that infects Vibrio cholerae. To study the evolutionary history of CTXphi, we examined genome diversity in CTX(phi)s derived from a variety of epidemic and nonepidemic Vibrio sp. natural isolates. Among these were three V. cholerae strains that contained CTX prophage sequences but not the ctxA and ctxB genes. These prophages each gave rise to a plasmid form whose genomic organization was very similar to that of the CTXphi replicative form, with the exception of missing ctxAB. Sequence analysis of these three plasmids revealed that they lacked the upstream control region normally found 5' of ctxA, as well as the ctxAB promoter region and coding sequences. These findings are consistent with the hypothesis that a CTXphi precursor that lacked ctxAB simultaneously acquired the toxin genes and their regulatory sequences. To assess the evolutionary relationships among additional CTX(phi)s, two CTXphi-encoded genes, orfU and zot, were sequenced from 13 V. cholerae and 4 V. mimicus isolates. Comparative nucleotide sequence analyses revealed that the CTX(phi)s derived from classical and El Tor V. cholerae isolates comprise two distinct lineages within otherwise nearly identical chromosomal backgrounds (based on mdh sequences). These findings suggest that nontoxigenic precursors of the two V. cholerae O1 biotypes independently acquired distinct CTX(phi)s.

FIG. 1

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

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.

FIG. 3

PCR analyses of the genomic organizations of the plasmid form of CTXφ from V. cholerae strain N16961 (El Tor) or from putative pre-CTXφs 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.

FIG. 4

Detection of an infectious form of pre-CTXφ 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 CTXφ core genes was determined by Southern blot analysis.

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-CTXφ 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 CTXφ sequences with the pre-CTXφ 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 CTXφ 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.

FIG. 6

Regional variation in the mean proportion of synonymous (p_S) differences between pairs of strains and the mean proportion of nonsynonymous (p_N) differences between pairs of strains based on a sliding window of 90 nucleotides in orfU. Squares represent p_N values, and diamonds indicate p_S values.

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.

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.

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 CTXφs. CTXφ probably arose from a precursor CTXφ that acquired the CT genes by imprecise excision from a unknown donor strain.