Molecular evidence favouring step-wise evolution of Mozambique Vibrio cholerae O1 El Tor hybrid strain

The ctxAB operon, encoding cholera toxin (CT) in Vibrio cholerae, is carried by the genome of a filamentous phage, CTXPhi. Usually, specific CTXPhi infect each of the two important biotypes, classical and El Tor, of epidemic V. cholerae strains belonging to serogroup O1, and are called CTX(class)Phi and CTX(ET)Phi, respectively. However, an unusual hybrid El Tor strain carrying CTX(class)Phi caused the cholera epidemic in Mozambique in 2004. To understand the evolution of that strain, we have further analysed some representative hybrid El Tor strains isolated in Kolkata, India, in 1992, and the results indicate that both the Mozambique and the Indian strains are infected with a unique CTX(class)Phi having only four copies of the tandem heptamer repeat sequence 5'-TTTTGAT-3' present in the ctxAB promoter (P(ctxAB)) region, like in CTX(ET)Phi. Usually, the P(ctxAB) of the classical biotype contains seven to eight copies of such sequences. However, sequence analyses of the P(ctxAB) regions of several classical strains indicated that the copy number of heptamer repeat sequences might vary from four to eight copies, which was previously unknown. Since the hybrid strains analysed in this study carry four copies of the heptamer sequences, it may thus serve as a marker to trace the strain in future. Interestingly, while the Mozambique strain is devoid of an El Tor-specific free RS1 element or pre-CTX prophage, the Indian hybrid strains carry such elements. The free RS1 has been mapped, cloned and sequenced. As in pre-CTX and CTX prophages, multiple copies of free RS1 elements were found to be integrated in tandem in the large chromosomal dif site. Since Indian hybrid El Tor strains carry either free RS1 or pre-CTX prophage in their large chromosomes, it is possible that the Mozambique hybrid El Tor strain has evolved from these progenitor strains by step-wise deletion of CTX genetic elements from their large chromosomes.