Molecular cloning of the hemolysin determinant from Vibrio cholerae El Tor

Vibrio cholerae El Tor RV79 is phenotypically nonhemolytic; however, strongly hemolytic convertants are occasionally observed on blood agar plates. We have cloned DNA sequences corresponding to the hemolysin determinant from RV79 (Hly+) in the lambda L47.1 and pBR322 vectors. A 2.3-kilobase fragment of V. cholerae DNA was found to be necessary for hemolytic activity. This cloned DNA sequence was used as a probe in Southern blot hybridization analysis of chromosomal restriction digests of a variety of El Tor and classical biotype V. cholerae strains. In all cases, DNA fragments with the same electrophoretic mobilities hybridized to the Hly probe. The results presented demonstrate that the cloned hemolysin determinant is the hly locus. By using mutator vibriophage VcA-3 insertion to promote high-frequency transfer, the hly locus was mapped between arg and ilv on the V. cholerae RV79 chromosome.