The cls gene of Escherichia coli is responsible for the synthesis of a major membrane phospholipid, cardiolipin, and has been proposed to encode cardiolipin synthase. This gene cloned on a pBR322 derivative was disrupted by either insertion of or replacement with a kanamycin-resistant gene followed by exchange with the homologous chromosomal region. The proper genomic disruptions were confirmed by Southern blot hybridization and a transductional linkage analysis. Both types of disruptants had essentially the same properties; cardiolipin synthase activity was not detectable, but the strains grew well, although their growth rates and final culture densities were lower than those of the corresponding wild-type strains and strains with the classical cls-l mutation. A disruptant harboring a plasmid that carried the intact cls gene grew normally.The results indicate that the cls gene and probably the cardiolipin synthase are dispensable for E. coli but may confer growth or survival advantages. Low but definite levels of cardiolipin were synthesized by all the disruptants. Cardiolipin content of the cis mutants depended on the dosage of the pss gene,-and attempts to transfer a null allele of the cls gene into a pss-l mutant were unsuccessful. We point out the possibilities of minor cardiolipin formation by phosphatidylserine synthase and of the essential nature of cardiolipin for the survival of E. coli cells.Cardiolipin, one of the major phospholipids of Escherichia coli, is unique in its structure among membrane lipids (i.e., tetraacyl structure with two phosphate groups) and has been postulated to play specific roles in membrane functions. It is synthesized from two molecules of phosphatidylglycerol by cardiolipin synthase (4, 20), which differs from its eucaryotic counterpart that utilizes CDP-diacylglycerol and phosphatidylglycerol as substrates (21). The gene cls is responsible for cardiolipin formation, and several lines of evidence indicate, though not definitely, that it is the structural gene for cardiolipin synthase (10).An E. coli mutation (cls-J) that results in a defective cardiolipin synthase has been isolated (14), and the cells with this mutation have been shown to display only minor growth phenotypes, despite decreased levels of cardiolipin (14, 18). Therefore, it has been uncertain whether the cls gene and cardiolipin are essential in E. coli cells or whether the residual low levels of cardiolipin observed maintain membrane functions that are dependent on this particular phospholipid.To determine whether the cls gene is essential and to analyze the physiological roles, if any, of this gene, an attempt to obtain a null mutation of this gene seemed most useful. This paper describes the construction, by genetic manipulations, of such mutants and their characteristic properties. The results show that the cls gene is not essential. Cardiolipin was formed at low levels even in the null cls mutants. We also present results that favor a hypothesis of secondary cardiolipin formation by phosphatidylserine synt...

A simple, specific, and sensitive assay procedure for cardiolipin synthase of Escherichia coli has been developed. This measures the radioactivity of glycerol formed from phosphatidyl [2-3H]glycerol and is mainly based on the findings that 400 mM phosphate and 0.015% Triton X-100 markedly activate the enzyme. Cardiolipin synthase was amplified 760-fold upon induction with isopropyl beta-D-thiogalactoside in cells harboring a pBR322 derivative in which the cls gene encoding this enzyme was preceded by the tac promoter. Under these conditions, cardiolipin content increased, membrane potential decreased, spheroplasts became fragile, cells lost viability, and inducer-resistant mutants appeared at a high frequency. The amplification enabled the isolation of an enzyme preparation with a specific activity approximately 10,000-times higher than that of wild-type whole cell lysate. This purification was simply achieved by extraction of the crude membrane fraction with Triton X-100 and a single phosphocellulose column chromatography. This preparation, together with the crude envelope fraction, was used to characterize the basic properties of E. coli cardiolipin synthase, some of which were utilized in setting up the assay conditions.