Acquisition of classical CTX prophage from Vibrio cholerae O141 by El Tor strains aided by lytic phages and chitin-induced competence

Abstract

The El Tor biotype of Vibrio cholerae O1, causing the current seventh pandemic of cholera, has replaced the classical biotype, which caused the sixth pandemic. The CTX prophages encoding cholera toxin in the two biotypes have distinct repressor (rstR) genes. Recently, new variants of El Tor strains that carry the classical type (CTX(class)) prophage have emerged. These "hybrid" strains apparently originate through lateral gene transfer and recombination events. To explore possible donors of the CTX(class) prophage and its mode of transfer, we tested environmental V. cholerae isolates for the presence of CTX(class) prophage and mobility of the phage genome. Of the 272 environmental V. cholerae isolates tested, 6 were found to carry the CTX(class) prophage; all of these belonged to the O141 serogroup. These O141 strains were unable to produce infectious CTX(class) phage or to transmit the prophage to recipient strains in the mouse model of infection; however, the CTX(class) prophage was acquired by El Tor strains when cultured with the O141 strains in microcosms composed of filtered environmental water, a chitin substrate, and a V. cholerae O141-specific bacteriophage. The CTX(class) prophage either coexisted with or replaced the resident CTX(ET) prophage, resulting in El Tor strains with CTX genotypes similar to those of the naturally occurring hybrid strains. Our results support a model involving phages and natural chitin substrate in the emergence of new variants of pathogenic V. cholerae. Furthermore, the O141 strains apparently represent an alternative reservoir of the CTX(class) phage genome, because the classical V. cholerae O1 strains are possibly extinct.

Fig. 1.

Analysis of genetically marked V. cholerae O141 strains carrying a Km^R marker in the resident CTX^class prophage. These strains were produced by recombination with a PCR amplicon derived from strain O395NT using the chitin-induced transformation protocol (see text for details). Lane 1 shows molecular size markers corresponding to 1-kb DNA Ladder Plus (Invitrogen); lane 2, the PCR amplicon spanning zot and ctxB genes derived from O395NT, comprising the Km^R marker flanked by CTX genes; lane 3, O141 strain V51 (native); lane 6, O141 strain 2615 (native); lane 7, a derivative of strain 2615 carrying the Km^R marker; and lane 8, O141 strain 2634 (native). Lanes 4 and 5 show derivatives of V51 carrying the Km^R marker in the CTX prophage, and lanes 9 and 10 show derivatives of strain 2634 carrying the Km^R marker.

Fig. 2.

PCR analysis of the ctxAB genes (Left) and the rstR genes (Right) of CTX prophages carried by different derivatives of toxigenic V. cholerae O1 El Tor biotype strains that were subjected to transformation in microcosms in the presence of a genetically marked toxigenic O141 strain carrying a Km^R marker in the resident CTX^class prophage, a lytic phage for O141 (JSF141Bφ), and pieces of sterile shrimp shell as a source of chitin. Lane 1 shows donor O141 strain 2615-K_m; lane 2, wild-type V11–2615; and lane 8, strain C6706ΔhapA before transformation. Lanes 3–7 show different transformants of strain V11–2615 (toxigenic El Tor strain), and lanes 9–13 show different transformants of strain C6706ΔhapA. The markers in lanes marked “M” correspond to the 1-kb DNA Ladder Plus (Invitrogen). These findings demonstrate the origination of strains with diverse CTX genotype from the same parent strain. Although some of these retain the original CTX phage, others replace part or whole of the original prophage with the CTX^class phage genome.

Fig. 3.

Role of lytic phages in chitin-mediated uptake of foreign DNA by V. cholerae in the aquatic environment. Lytic phages acting on V. cholerae cells release DNA from the donor cells. The free DNA fragments are taken up by recipient strains, aided by chitin-induced competence. Possible recombination between the foreign DNA and the host chromosome can cause genetic changes in the recipient cell.