Cloning of genes involved in membrane lipid synthesis: effects of amplification of phosphatidylglycerophosphate synthase in Escherichia coli.

Abstract

The structural gene (pgsA) for the CDP-diacylglycerol:sn-glycero-3-phosphate phosphatidyltransferase (EC 2.7.8.5, phosphatidylglycerophosphate synthase) from Escherichia coli has been cloned, using pSC101 as the vector. The resulting hybrid plasmids not only correct the lack of in vitro synthase activity in pgsA strains but also cause an amplification (6- to 40-fold over wild-type levels) in enzymatic activity in direct proportion to the copy number of the plasmids found in vivo. The cloned gene also corrects the abnormally low level of polyglycerophosphatides found in pgsA strains and actually increases the level of phosphatidylglycerol to above that normally found in E. coli. The degree of alteration in phospholipid composition brought about by these hybrid plasmids is not of the order expected if fluctuations in enzyme levels in vivo were an important regulatory mechanism in phospholipid metabolism. The isolated hybrid plasmids have been mapped by restriction endonuclease analysis. The presence and location of other genetic markers have also been established. The above data, along with analysis of deletion derivatives of these plasmids and subcloning of appropriate restriction fragments, have established the position of the pgsA locus on the hybrid plasmids. From this data, the position of the pgsA locus has been determined to le between flaI and uvrC on the E. coli genetic map.