CTX genetic element encodes a site-specific recombination system and an intestinal colonization factor.

Abstract

In Vibrio cholerae, the genes encoding cholera toxin (ctxAB) are located on a segment of DNA (termed the "core" region) that is flanked by two or more copies of a repeated sequence called RS1. Together these DNA units comprise the CTX genetic element. Evidence presented here suggests that RS1 sequences encode a site-specific recombination system, which allows integration of a suicide plasmid carrying RS1 into an 18-base-pair sequence (attRSl) located on the chromosome of nontoxigenic V. cholera" strains. Strains of V. cholerae with large deletions removing attRSl and the entire CTX genetic element no longer undergo site-specific recombination with the RS1 sequence. Additionally, these deletion strains show a defect in intestinal colonization. Recombination experiments localize the gene responsible for enhancing colonization to a portion of the core region of the CTX element. The identifled gene encodes a peptide that is highly similar in amino acid sequence to the flexible pilin of Aeromonas hydrophia. These results have important implications in the construction of stable, live attenuated cholera vaccines.The bacterium Vibrio cholerae is the causative agent of cholera, a diarrheal disease that has recently caused an explosive epidemic in Latin America (1). Although this organism has been studied for over 100 years, there still remains a need for an effective cholera vaccine. Recent efforts in developing live attenuated vaccines have focused on the construction of recombinant V. cholerae strains lacking the gene (ctxA) encoding the active subunit of cholera toxin (2-5). Since recombination with wild-type strains could restore toxigenicity to ctxA deletion strains, recA mutations have been examined as a means of stabilizing such live vaccine candidates (6, 7). Naturally occurring strains of V. cholerae that carry duplications of the ctx genes also have duplications of flanking DNA sequences, whereas nontoxigenic strains lack both ctx genes and flanking sequences (2, 8, 9). These observations suggest that the toxin genes are part of a larger genetic element (2,8,9). In V. cholerae strains of the El Tor biotype, this genetic element has been found to consist of a "core" region, which carries the ctxAB operon (10) and the zot gene encoding zona occludens toxin (11), flanked by two or more copies of a 2.7-kb repetitive sequence called RS1. Thus, the ctx genetic element is structurally analogous to compound transposons such as Tn9 (12) with RS1 sequences corresponding to ISJ insertion sequences directly duplicated at the termini. Unlike most transposons, the ctx genetic element appears to be located at the same chromosomal site in all El Tor strains (8), suggesting it may display a high degree of insertion site specificity, as do transposons Tn7 (13) and TnS54 (14).In this paper we show that the RS1 sequences associated with the CTX genetic element apparently mediate recAindependent site-specific recombination between the element and an 18-bp target site on the V. cholerae chromosome called attRS1. In addition...

Replication patterns and organization of replication forks in Vibrio cholerae

“…The bacterial strain used was V. cholerae 2740-80 (CTXQ 2 ) (Pearson et al, 1993). Cells were grown in four different media at 37 uC.…”

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

“…Previously, it has been shown that the genes encoding CT, ctxAB, along with other virulence-related genes reside on a 4?5 kb DNA segment called the core region (Baudry et al, 1992;Pearson et al, 1993;Trucksis et al, 1993). The core region is flanked by one or multiple copies of direct repeat sequences (RSs) that vary in length from 2?4 to 2?7 kb, and this approximately 7 kb DNA segment (RS + core) is called the CTX genetic element (Pearson et al, 1993). However, it has been discovered (Waldor & Mekalanos, 1996) that the CTX genetic element of V. cholerae corresponds to the genome of a filamentous bacteriophage designated CTXf (Fig.…”

CTXφ contains a hybrid genome derived from tandemly integrated elements

“…Unlike the filamentous phages that infect Escherichia coli, CTX can give rise to lysogens, which contain the phage genome integrated into the bacterial chromosome (1,3,4). Integration of CTX DNA into the V. cholerae chromosome is a site-specific process that does not require RecA (5). Both O1 El Tor and O139 strains of V. cholerae, which have caused virtually all recent cholera epidemics, have just one chromosomal locus within which the phage genome is integrated (3,6).…”

“…The core of this integration site (attB) is a 17-bp sequence that is almost identical to an 18-bp sequence within the phage genome. Integration of phage DNA into the genome of an attB ϩ , CTX Ϫ El Tor strain of V. cholerae yields single or tandem prophages flanked by these 17͞18-bp sequences, which are known as end repeats (ERs) (5). If such a lysogen is infected by an additional phage or transformed with the replicative form (RF) (a plasmid) of phage DNA, the new phage DNA will integrate at an ER between tandem prophages or between a 3Ј end and chromosomal DNA; the ER between the 5Ј end of a prophage and adjacent chromosomal DNA is not used (7).…”