Cloning, expression, and nucleotide sequence of the glycerophospholipid : cholesterol acyltransferase gene from Aeromonas hydrophila
Date
1988
Authors
Thornton, Julian C.
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Abstract
The purpose of this study was to examine the extracellular glycerophospholipid:cholesterol acyltransferase (GCAT) from & hydrophila. The primary structure of GCAT was determined by sequencing the GCAT gene using the chain termination method of Sanger (Sanger et al. 1979). This gene, gcatA, was subsequently cloned into a wide-host-range expression vector (pMMB66; Furst et al. 1986), and the ability of both Escherichia coli and Aeromonas salmonicida to secrete the protein was studied.
It was revealed that gcatA from & hydrophila encoded a 31 kD enzyme capable of carrying out acyltransfer between glycerophospholipids and cholesterol.· This enzyme, which is normally extracellular in cultures of & hydrophila (MacIntyre and Buckley 1978), was secreted extracellularly by the related bacteria & salmonicida when the & hydrophila GCAT gene was present on a wide-host-range plasmid. In contrast, !L.. coli clones bearing the same recombinant plasmid only secreted GCAT to their periplasmic space, thus suggesting a lack of the necessary GCAT secretory machinery in the !L._ coli outer membrane.
A typical !L._ coli consensus-like promoter was not seen in the nucleotide sequence upstream of the GCAT gene. Although a region sharing some similarities with the CAP binding-site consensus sequence of !L._ coli (de Crombrugghe et al. 1984) was identified approximately 130-150 nucleotides upstream of the start codon of the GCAT gene.
The sequence of the structural gene, gcatA, contained two regions that share sequence simtlarities with other lipases. These regions are believed to be the active site and the interfacial lipid-binding site of the other lipases (Maragnore and Henrickson 1986}.
Polyclonal antibodies directed against GCAT from both hydrophila and salmonicida cross reacted with both proteins in ELISA and in Western immunoblots. This suggests that although the molecular weights and amino acid compositions of the two related proteins are quite different, some of the antigenic determinants of GCAT are conserved in these two proteins.