CHARACTERIZATION OF THE VIBRIO-CHOLERAE EL-TOR LIPASE OPERON LIPAB AND A PROTEASE GENE DOWNSTREAM OF THE HLY REGION

We have cloned and sequenced a region encoding a lipase operon and a p utative, previously uncharacterized metalloprotease of Vibrio cholerae O1. These lie downstream of hlyA and hlyB, which encode the El Tor he molysin and methyl-accepting chemotactic factor, respectively, Previou s reports identified the hlyC gene downstream of hlyAB, encoding an 18 .3-kDa protein. However, we now show that this open reading frame (ORF ) encodes a 33-kDa protein, and since the amino acid sequence is highl y homologous to the triacylglyceride-specific lipase of Pseudomonas sp p., hlyC has been renamed lipA. LipA contains the highly conserved pen tapeptide and catalytic tried amino acid regions of the catalytic site s of other lipases. The region downstream of lipA has been sequenced a nd has revealed ORFs lipB and prtV. The amino acid sequence of lipB is homologous to those of the accessory lipase proteins (lipase-specific foldase) required by Pseudomonas and various other bacterial species for the production of mature active lipase, and in agreement with this , we show that both lipA and lipB are required to restore a lipase-def icient lipA null mutant of V. cholerae. The intergenic stop codon for lipA overlaps the ribosome-binding site for lipB, and a stem-loop rese mbling a rho-independent terminator is present immediately downstream from lipB, suggesting that lipA and lipB form a lipase operon in V. ch olerae. prtV lies downstream of lipAB but is transcribed in the opposi te direction and is predicted to share the same putative transcription al terminator with lipAB. The zinc-binding and catalytic domains conse rved among many metalloproteases are present in PrtV, which is highly homologous to the immune inhibitor A (InA) metalloprotease of Bacillus thuringiensis. PrtV was visualized as approximately 102 kDa, which is consistent with the coding capacity of the gene. The genetic organiza tion of this region suggests that it is possibly part of a pathogenici ty island, encoding products capable of damaging host cells and/or inv olved in nutrient acquisition by V. cholerae. However, neither lipA no r prtV null mutants were attenuated in the infant mouse model, nor did they exhibit reduced colonization potential compared with wild type i n competition experiments.