title: Characterization of the Vibrio cholerae El Tor lipase operon lipAB and a protease gene downstream of the hly region.
abstract: We have cloned and sequenced a region encoding a lipase operon and a putative, previously uncharacterized metalloprotease of Vibrio cholerae O1. These lie downstream of hlyA and hlyB, which encode the El Tor hemolysin and methyl-accepting chemotactic factor, respectively. Previous 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 highly homologous to the triacylglyceride-specific lipase of Pseudomonas spp., hlyC has been renamed lipA. LipA contains the highly conserved pentapeptide and catalytic triad amino acid regions of the catalytic sites of other lipases. The region downstream of lipA has been sequenced and 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-deficient lipA null mutant of V. cholerae. The intergenic stop codon for lipA overlaps the ribosome-binding site for lipB, and a stem-loop resembling a rho-independent terminator is present immediately downstream from lipB, suggesting that lipA and lipB form a lipase operon in V. cholerae. prtV lies downstream of lipAB but is transcribed in the opposite direction and is predicted to share the same putative transcriptional terminator with lipAB. The zinc-binding and catalytic domains conserved 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 organization of this region suggests that it is possibly part of a pathogenicity island, encoding products capable of damaging host cells and/or involved in nutrient acquisition by V. cholerae. However, neither lipA nor prtV null mutants were attenuated in the infant mouse model, nor did they exhibit reduced colonization potential compared with wild type in competition experiments.
authors.affiliation: Department of Microbiology and Immunology, University of Adelaide, South Australia.
authors.forename: M A
authors.initials: MA
authors.lastname: Ogierman
authors.forename: A
authors.initials: A
authors.lastname: Fallarino
authors.forename: T
authors.initials: T
authors.lastname: Riess
authors.forename: S G
authors.initials: SG
authors.lastname: Williams
authors.forename: S R
authors.initials: SR
authors.lastname: Attridge
authors.forename: P A
authors.initials: PA
authors.lastname: Manning
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D002771:Cholera / Q000235:genetics; D002874:Chromosome Mapping; D002876:Chromosomes, Bacterial / Q000235:genetics; D003001:Cloning, Molecular; D018388:Codon, Terminator; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006461:Hemolysis; D008049:Lipase / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D008666:Metalloendopeptidases / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D051379:Mice; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D009876:Operon; D010453:Peptide Synthases / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011549:Pseudomonas / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013604:T-Phages / Q000235:genetics; D014158:Transcription, Genetic; D014734:Vibrio cholerae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D014774:Virulence / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D018388:Codon, Terminator; D004269:DNA, Bacterial; C079999:LipA protein, Bacteria; C094673:HlyC lipase, Vibrio cholerae; D008049:Lipase; D008666:Metalloendopeptidases; C073584:Prt protein, Streptomyces; D010453:Peptide Synthases; C072322:lipoate-protein ligase