Cloning, characterization, and chromosomal mapping of a phospholipase (lecithinase) produced by Vibrio cholerae.

ABSTRACT

Phospholipases are associated with virulence in bacterial diseases. Vibrio cholerae produces a phospholipase (lecithinase), with enzyme production visualized as a zone of clearing around colonies plated on egg yolk agar. The role of phospholipase in gut colonization or disease pathogenesis is unknown. We used the egg yolk agar assay to clone and characterize a gene encoding a phospholipase from V. cholerae El Tor strain E7946. Sequence analysis revealed a 1,254-bp open reading frame (lec) encoding a 418-amino-acid protein with a predicted molecular weight of 47,600. The predicted sequence exhibits DNA homology to other Vibrionaceae phospholipases. A potential signal sequence exists in the predicted amino acid sequence, as does a lipid binding motif found in prokaryotic and eukaryotic phospholipases and lipases. Polyacrylamide gel electrophoresis combined with an egg yolk agarose overlay demonstrated phospholipase activity migrating at a relative molecular weight of 45,000 in preparations of V. cholerae and the Escherichia coli clone. Restriction mapping and Southern blot analysis revealed that lec, hlyA (hemolysin), and hlyC (lipase) are adjacent on the V. cholerae chromosome, and chromosomal digests of several El Tor, classical, and O139 (Bengal) strains demonstrated conservation of this gene arrangement. An in-frame internal deletion of the lec gene was constructed and recombined into the chromosome of attenuated V. cholerae El Tor strain CVD 110. The resulting mutant lacked lecithinase activity on egg yolk agar but had undiminished reactivity in rabbit ligated ileal loop assays.