Isolation of enterotoxin structural gene deletion mutations in Vibrio cholerae induced by two mutagenic vibriophages.

Abstract

Phenotypically nontoxinogenic mutants of V. cholerae were isolated after infection with either of 2 mutagenic vibriophages, VcA1 and VcA2cts1. DNA isolated from these mutants was analyzed for toxin gene sequences by the Southern blotting method with 32P-labeled probes derived from the cloned A and B subunit genes for the heat-labile enterotoxin of Escherichia coli, designated LT. Several of the mutant isolates lost all sequences hybridizing to the LT probes, indicating that these clones contain deletion mutations that removed the structural gene(s) for cholera toxin. The mutants were prototrophic and grew normally, in vitro, demonstrating that the toxin is not essential for the growth and viability of V. cholerae. The toxin gene deletion mutants multiplied well in vivo in ligated rabbit intestine. Because of these growth properties and the stability of deletion mutations, these strains are promising candidates for testing as live oral vaccine strains for protection against cholera in humans.