Cloning of the Bacillus firmus OF4 cls gene and characterization of its gene product

Abstract

The gene that codes for cardiolipin (CL) synthase and an adjacent gene that codes for a MecA homolog in the alkaliphilic bacteria Bacillus firmus OF4 have been cloned and sequenced (GenBank accession number U88888). The cls gene contains 1509 nucleotides, corresponding to a polypeptide of 57.9 kDa. The predicted amino acid sequence has 129 identities and 100 similarities with the Escherichia coli CL synthase. Homologies were also noted with polypeptide sequences from putative cls genes from Bacillus subtilis and Psuedomonas putida. Conserved histidine, tyrosine, and serine residues may be part of the active site and participate in phosphatidyl group transfer. The B. firmus OF4 cls gene product was inserted into plasmid pET3 to form a recombinant plasmid pDG2, which overproduces CL synthase in E. coli. A membrane fraction containing the overproduced enzyme converts phosphatidylglycerol to CL and glycerol. The B. firmus enzyme is stimulated by potassium phosphate, inhibited by CL and phosphatidate, and has a slightly higher pH optimum than the E. coli enzyme.