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.The hemolysin (HlyA) produced by many El Tor and non-O1 strains of Vibrio cholerae has been shown to be a pore-forming toxin and has been implicated as a virulence determinant (4,25,28,29,63). The production of HlyA is controlled by levels of available iron, and thus, it has been suggested that HlyA may be involved in iron scavenging (54). HlyA expression is also upregulated by the DNA-binding protein HlyU, and studies of a hlyU null mutant suggest that HlyU may also regulate the expression of additional virulence determinants (63).The hlyA locus is closely linked to a number of other potential virulence determinants. A lecithinase-phospholipase gene, lecA, is transcribed divergently from hlyA (37), and downstream of hlyA lies hlyB, whose gene product is highly homologous to methyl-acceptin...

Identification and Characterization of a Hemolysin Gene Cluster in Vibrio anguillarum

“…The gene order of the hemolysin region was found to be highly conserved in various Vibrio species (8,20). As illustrated in Fig.…”

“…No homologue to hlyB is found in the hemolysin region of V. anguillarum. It has been proposed that the genetic organization of this region of V. cholerae is part of a pathogenicity island, encoding products capable of damaging host cells and/or involved in nutrient acquisition (20). The same may be the case in V. anguillarum as well.…”

“…2, the hemolysin region in V. cholerae contains genes (lec, hlyA, hlyB, lipA, and lipB) that, with the exception of hlyB, are homologous to plp, vah1, llpA, and llpB of V. anguillarum, respectively. The hlyB gene of V. cholerae apparently encodes a methyl-accepting chemotactic protein (20). No homologue to hlyB is found in the hemolysin region of V. anguillarum.…”

Identification of genes involved in the activation of the Bacillus thuringiensis inhA metalloprotease gene at the onset of sporulation The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AF287346.

“…It is also very similar to the protease PrtV of Vibrio cholerae (Ogierman et al, 1997). Its amino acid sequence contains the zinc-binding and catalytic active site residues common to these various metalloproteases.…”

A low-M r lipase activation factor cooperating with lipase modulator protein LimL in Pseudomonas sp. strain 109

“…The co-existence of a downstream limL gene, either on the same plasmid or on a separate plasmid, was found to be essential for the functional expression of LipL (Ihara et al, 1992). A similar requirement of a secondary gene has been observed for several lipases (Frenken et al, 1993a ;Iizumi et al, 1991 ;Jørgensen et al, 1991 ;Kok et al, 1995 ;Ogierman et al, 1997 ;Siomi et al, 1992 ;Wohlfarth & Winkler, 1992), and studies of Burkholderia cepacia (LimA ; Hobson et al, 1993), Pseudomonas glumae (LipB ; Frenken et al, 1993b), Pseudomonas aeruginosa (LipB ; Hirayama et al, 1993) and Pseudomonas sp. strain KWI-56 (Act ;Iizumi & Fukase, 1994) suggested that the secondary gene product is required for correct lipase folding.…”