Alternative mechanism of cholera toxin acquisition by Vibrio cholerae: generalized transduction of CTXPhi by bacteriophage CP-T1

Abstract

Horizontal transfer of genes encoding virulence factors has played a central role in the evolution of many pathogenic bacteria. The unexpected discovery that the genes encoding cholera toxin (ctxAB), the main cause of the profuse secretory diarrhea characteristic of cholera, are encoded on a novel filamentous phage named CTXPhi, has resulted in a renewed interest in the potential mechanisms of transfer of virulence genes among Vibrio cholerae. We describe here an alternative mechanism of cholera toxin gene transfer into nontoxigenic V. cholerae isolates, including strains that lack both the CTXPhi receptor, the toxin coregulated pilus (TCP), and attRS, the chromosomal attachment site for CTXPhi integration. A temperature-sensitive mutant of the V. cholerae generalized transducing bacteriophage CP-T1 (CP-T1ts) was used to transfer a genetically marked derivative of the CTX prophage into four nontoxigenic V. cholerae strains, including two V. cholerae vaccine strains. We demonstrate that CTXPhi transduced by CP-T1ts can replicate and integrate into these nontoxigenic V. cholerae strains with high efficiency. In fact, CP-T1ts transduces the CTX prophage preferentially when compared with other chromosomal markers. These results reveal a potential mechanism by which CTXPhi(+) V. cholerae strains that lack the TCP receptor may have arisen. Finally, these findings indicate an additional pathway for reversion of live-attenuated V. cholerae vaccine strains.

FIG. 1

Chromosomal arrangement of TLC, RS1, CTXΦ, and RTX in the V. cholerae chromosome. The open reading frames are shown as arrows. The black triangles represent attRS sequences. Vertical lines indicate restriction enzyme sites. Horizontal bars show the five DNA probes used in this study.

FIG. 2

Southern blot analysis of plasmid DNAs derived from transductants 468-83Kn, 2740-80Kn, Bah-2Kn, Bah-3Kn, and O395 (pCTX-Kn). Plasmid DNAs from the indicated strains were digested with SphI, which cuts once in CTXΦ, separated on an agarose gel, transferred to nitrocellulose, and then probed with a CTXΦ core region fragment.

FIG. 3

Southern blot analysis of the integrated copies of CTXΦ in strains SM115, 468-83Kn, 2740-80Kn, and Bah-2-Kn. Equal amounts of chromosomal DNAs from the indicated strains were digested with XbaI, electrophoresed, transferred to nitrocellulose, and probed with a CTXΦ core region fragment. J, left junction fragment of integrated CTXΦ. The ∼10- and ∼6-kb bands represent CTX prophages with or without an intervening RS1 sequence, respectively.

FIG. 4

Southern blot analysis of the integrated copies of CTXΦ in strains SM115, 468-83Kn, 2740-80Kn, and Bah-2Kn. BglII-digested chromosomal DNA was separated on an agarose gel, transferred to nylon membranes, and hybridized with the RS1-specific probe rstC. The ∼4.0-kb bands represent the right chromosomal junction of an integrated RS1, and the ∼2.7-kb bands represent RS1 sequences 5′ of the CTX prophage.

FIG. 5

Chromosomal arrangement of CTXΦ, RS1, TLC, and rtxA in the original donor strain SM115 and in the transductants 468-83Kn, 2740-80Kn, and Bah-2Kn as determined by Southern blot and PCR analysis. Vertical lines indicate restriction enzyme sites; XbaI cuts twice in the Kn^r gene in SM115. The flanking regions of the integrated CTXΦ were determined by PCR analyses with core CTXΦ, TLC, and rtxA primers (Table 2). Open boxes indicate the TLC sequence and the rtxA sequence. Stippled boxes represent the RS1 sequence, and solid boxes represent the CTXΦ.