Cloning, sequencing, and role in virulence of two phospholipases (A1 and C) from mesophilic Aeromonas sp serogroup O : 34

Two different representative recombinant clones encoding Aeromonas hydrophi la lipases were found upon screening on tributyrin (phospholipase A1) and e gg yolk agar (lecithinase-phospholipase C) plates of a cosmid-based genomic library of Aeromonas hydrophila AH-3 (serogroup O34) introduced into Esche richia coil DH5 alpha. Subcloning, nucleotide sequencing, and in vitro-coup led transcription-translation experiments showed that the phospholipase A1 (pla) and C (plc) genes code for an 83-kDa putative lipoprotein and a 65-kD a protein, respectively. Defined insertion mutants of A. hydrophila AH-3 de fective in either pla or plc genes were defective in phospholipase A1 and C activities, respectively. Lecithinase (phospholipase C) was shown to be cy totoxic but non-hemolytic or poorly hemolytic. A. hydrophila AH-3 plc mutan ts showed a more than 10-fold increase in their 50% lethal dose on fish and mice, and complementation of the plc single gene on these mutants abolishe d this effect, suggesting that Pie protein is a virulence factor in the mes ophilic Aeromonas sp. serogroup O:34 infection process.