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The gene encoding cholera toxin, the principal virulence factor of Vibrio cholerae, is encoded by a filamentous, lysogenic bacteriophage known as CTXφ. The genome of V. cholerae, the host for CTXφ, 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[sup ET], the prophage having a conserved Notl site in its repeat sequence segment which seems to be specific for the El Tor strains so far examined, followed by CYX[sub calc]-like genome, the prophage found in recent O139 clinical isolates from Calcutta. In sharp contrast, in post-C139 El Tor strains only one copy of the CTX[sup 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 Tot 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-C139 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... [ABSTRACT FROM AUTHOR]