Genetic analysis of the chromosomal region encoding lysophospholipase L(2) of Vibrio cholerae O1

From the Tn5-inserted mutant library of Vibrio cholerae O1, we found a mutant, NF404, which lost the production of both hemolysin and cholera toxin (CT) even though the Tn5-insertion site was out side from the structural genes for hemolysin and CT. Cloning and sequencing analysis of the homologous region from the wild-type strain, revealed that the sequence spanning the coding region of an ORF1 nominated as lypA, encoding a 39.5 kDa protein. Deduced amino acid sequence of the lypA gene had 37.6% identity to the lysophospholipase L(2) (EC 3.1.1.5) of Escherichia coli. In the downstream of lypA, a second open reading frame (ORF2) encoding an unknown protein with molecular weight of 19.9 kDa was found. Assaying the lysophospholipase L(2) activity in the cell extract of E. coli harboring lypA in an expression vector showed clear increase in the enzymatic activity.