Cloning and characterization of the hemolysin determinants from Vibrio cholerae RV79(Hly+), RV79(Hly-), and 569B

Abstract

The Hly region from the chromosome of Vibrio chokrae El Tor strain RV79(Hly-) and the nonhemolytic classical strain 569B were cloned into plasmid vector pBR322. Escherichia coli K-12 transformants possessing these recombinant plasmids were nonhemolytic and were detected with a 32P-labeled hly-specific DNA probe. Restriction endonuclease Sau3AI digestions of the cloned hly loci of two independently obtained RV79(Hly+) convertants, when compared with the digests of cloned RV79(Hly-) loci, revealed that an apparent alteration (10 to 15 base pairs) had occurred. In contrast, an apparent 20-base-pair deletion was present in the cloned hly locus of the classical biotype V. cholerae strain 569B. Maxicell analysis and immunoprecipitation of labeled proteins of E. coli which are encoded by the cloned hly loci of RV79(Hly+) and from nuclease BAL 31-deleted plasmids, as well as immunoprecipitation of [35S]methionine-labeled V. cholerae proteins, suggest that the hemolysin is an 84,000-dalton polypeptide.

Extracellular proteins of Vibrio cholerae: nucleotide sequence of the structural gene (hlyA) for the haemolysin of the haemolytic El Tor strain 017 and characterization of the hlyA mutation in the non‐haemolytic classical strain 569B

“…1). This is in agreement with results obtained by Goldberg and Murphy (1985). We have screened colonies of 569B harbouring pPM2001 for non-reciprocal recombination, such that the chromosomal gene is recombined into the plasmid, resulting in a non-haemolytic phenotype.…”

“…Classical strain 569B contains a small deletion (Goldberg and Murphy, 1985) which we have now confirmed, by sequence analysis, corresponds to an 11 -basepair deletion resulting in a truncated hlyA product which we have designated HlyA'. Analysis of the sequence around the deletion shows a region of inverted repeat homology comprising repeats of 6 base pairs with a 2-base-pair space.…”

“…Nucleotide sequence analysis of the Classical hlyA from pPM2002 revealed only minor changes, which account for the difference in restriction pattern (seen with Sau3A and Taq\) between the El Tor and classical forms of hlyA (Goldberg and Murphy, 1985). The region of difference within the Classical hlyA compared with that of strain 017 is shown in Fig.…”

“…2). Plasmid pPM2002 has identical SauZA and Taq^ restriction patterns to the cloned defective hlyA gene of 569B (Goldberg and Murphy, 1985) and consequently represents the structural defect present in 569B.…”

“…Analysis of non-haemolytic strains by Southern hybridization demonstrates that DNA homologous to the structural gene and surrounding region is present in non-haemolytic strains of both biotypes (Goldberg and Murphy, 1984;Brown and Manning, 1985). Goldberg and Murphy (1985) found a minor change within the vicinity of the structural gene, whereas Brown and Manning (1985) found an alteration upstream of the NyA coding region.…”