Genesis of variants of Vibrio cholerae O1 biotype El Tor: role of the CTX  array and its position in the genome

Abstract

The gene encoding cholera toxin, the principal virulence factor of Vibrio cholerae, is encoded by a filamentous, lysogenic bacteriophage known as CTXf. The genome of V. cholerae, the host for CTXf, consists of two chromosomes, one large and one small. Here, it is shown that localization and array of CTX prophage DNA in either the large or small chromosome of V. cholerae is likely to be one of the reasons for the emergence of O1 biotype El Tor variants isolated just before and after the V. cholerae O139 cholera outbreak in 1992. Analyses of the organization of the CTX region of the genome of pre-O139 El Tor strains revealed that these strains carry two distinct CTX prophages integrated in the small chromosome in tandem: CTX ET , the prophage having a conserved NotI site in its repeat sequence segment which seems to be specific for the El Tor strains so far examined, followed by CTX calc -like genome, the prophage found in recent O139 clinical isolates from Calcutta. In sharp contrast, in post-O139 El Tor strains only one copy of the CTX ET prophage was found to be integrated in the large chromosome. To the authors' knowledge, the presence of CTX prophage in the small chromosome of O1 El Tor strains has not been reported previously. It is also shown that the difference in the CTX copy number and the position of the bacteriophage on the genomes of pre-and post-O139 El Tor strains have an effect on cholera toxin production. While a pre-O139 strain produced maximum cholera toxin in yeast extract/peptone medium at 30˚C, a post-O139 El Tor strain showed maximal yield at 37˚C, indicating differential regulation of cholera toxin between the strains. It appears from this study that the variation in the integration site of the CTX prophage, its copy number and the presence of diverse phage genomes in V. cholerae O1 biotype El Tor may be strategically important for generating variants with subtle phenotypic modulations of virulence factor production in this longest-ruling seventh pandemic strain.

Genetic organization of pre-CTX and CTX prophages in the genome of an environmental Vibrio cholerae non-O1, non-O139 strain

“…The genes of the RS2 element encode functions required for regulation (rstR gene product), replication (rstA gene product) and integration (rstB gene product) of CTXW in the genome of V. cholerae (Waldor et al, 1997). Several distinct CTXW variants have been described, namely CTX ET W derived from V. cholerae O1 El Tor, CTX class W derived from classical V. cholerae, CTX Calc W derived from O139 V. cholerae (Davis et al, 1999), CTX Env W derived from non-O1, non-O139 V. cholerae (Mukhopadhyay et al, 2001), and CTX var W present in pre-O139 V. cholerae El Tor isolates (Nandi et al, 2003). Apart from these genes, some RS2 elements may contain an additional gene, rstC (Fig.…”

“…V. cholerae possesses two circular chromosomes, one small and one large (Heidelberg et al, 2000;Trucksis et al, 1998). In V. cholerae El Tor/O139 genomes the CTX prophage may be present either as a single copy or as multiple copies arranged in tandem (Bhadra et al, 1995;Mekalanos, 1983;Nandi et al, 2003). In sharp contrast, in V. cholerae classical strains, the prophage is usually present in two copies, one copy in each chromosome (Nandi et al, 2003;Trucksis et al, 1998).…”

“…In V. cholerae El Tor/O139 genomes the CTX prophage may be present either as a single copy or as multiple copies arranged in tandem (Bhadra et al, 1995;Mekalanos, 1983;Nandi et al, 2003). In sharp contrast, in V. cholerae classical strains, the prophage is usually present in two copies, one copy in each chromosome (Nandi et al, 2003;Trucksis et al, 1998). The whole-genome sequencing of the V. cholerae El Tor strain N16961 has revealed the integration of only one copy of the CTX prophage flanked by the RS1 element in the large chromosome (Heidelberg et al, 2000).…”

Isolation of Vibrio cholerae O1 strains similar to pre-seventh pandemic El Tor strains during an outbreak of gastrointestinal disease in an island resort in Fiji

“…The identity of these strains was first reconfirmed by V. cholerae-species-specific ompW PCR developed by Nandi et al (2003). The serogroup of these strains was subsequently reconfirmed using polyvalent O1 and monovalent Inaba and Ogawa antisera and by a multiplex PCR targeted to identify genes encoding O1 (rfbO1) and O139 (rfbO139)-specific O biosynthetic genes and cholera toxin gene (ctxA) as described previously (Hoshino et al, 1998).…”

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

“…CTX⌽/pre-CTX⌽ integrates between the toxin-linked cryptic (TLC) element and RTX toxin loci in the large chromosome of epidemic strains or between the traF (VCA0570) and yciH (VCA0568) loci in the small chromosome of classical strains and in certain clinical El Tor strains (20,(42)(43)(44)(45). To identify the integration site of pre-CTX ZJ ⌽ in strain VC06-18, PCR and sequencing were performed using primers InI/InJ and inA/inB (see Table S1 in the supplemental material), which span the attB sites in the small and large chromosomes of V. cholerae, respectively.…”