Cloning and characterization of Photobacterium damselae ssp. piscicida phospholipase: an enzyme that shows haemolytic activity

Abstract

A phospholipase gene of Photobacterium damselae ssp. piscicida (ppp) was cloned from a genomic library and its nucleotide sequence was determined. The open reading frame consisted of 1218 bp encoding a protein of 405 amino acids with a predicted molecular mass of 46 kDa. The PPP had identities (53-55%) with phospholipase and haemolysin of Vibrio spp., while it showed low identities (23-26%) with glycerophospholipid cholesterol acyltransferase of Aeromonas spp. A recombinant PPP (rPPP) with a His tag at the C-terminus expressed in Escherichia coli and purified showed phospholipase activity. The rPPP also showed lecithin-dependent haemolytic activity against mammalian erythrocytes and direct haemolytic activity against fish erythrocytes. The culture supernatant of wild-type P. damselae ssp. piscicida showed phospholipase activity, while that of a PPP gene knockout mutant did not.