Selection and Characterization of an E. coli Mutant Defective in Membrane Lipid Biosynthesis

Abstract

Abstract. A procedure is described for the selection of temperature-sensitive phospholipid mutants based upon radiation suicide of the wild-type organisms by tritiated L-glycerol-3-phosphate incorporated in the phospholipids.One of these mutants possesses an acyltransferase activity much more thermolabile than that of its parent. This mutant ceases growth and phospholipid biosynthesis immediately upon shift to a nonpermissive temperature although DNA, RNA, and protein continue to be synthesized. The phenotype of this mutant appears due to a single mutation by reversion analysis and by enzymatic analysis of temperature-resistant revertants.Introduction. Biological membranes are composed mainly of lipid and protein. About one third of the dry mass of the E. coli cell membrane is lipid' all of which is phospholipid.2 Thus, it is obvious that phospholipids play a major role in the structure of this membrane. Phospholipids also have a functional role in membranes as they are required for activity by certain membraneassociated enzymes.3 Hence, a study of the roles of phospholipids in membrane systems should prove vital to understanding membrane structure and function.One approach to delineating the roles of membrane phospholipids is through the study of structural and functional changes in biological membranes resulting from alterations in the fatty acid composition of the phospholipids. The isolation of E. coli auxotrophs defective in the biosynthesis of unsaturated fatty acids has facilitated such studies because the unsaturated acyl moieties of the phospholipids of these organisms can be dramatically varied.4" Another approach is the isolation of mutants defective in the biosynthesis of various species of membrane phospholipids since these mutants might permit similar studies attempting to correlate changes in specific phospholipids with resulting membrane changes. Such mutants have not been available.The existence of unsaturated fatty acid auxotrophs demonstrates that unsaturated fatty acids and phospholipids fulfill an indispensable function in cellular metabolism. Since to our knowledge E. coli cannot be effectively supplemented with intact phospholipids, a method for selecting mutants of this bacterium conditionally defective (temperature-sensitive) in phospholipid biosynthesis has been developed. In this paper we report this selection procedure

Thermal Regulation of the Fatty Acid Composition of Lipopolysaccharides and Phospholipids of Proteus mirabilis

“…Resembling other microbial systems [23,24], the downshift in growth temperature of P. mirabilis markedly increased the content of the unsaturated liexadecenoic (16: 1) and octadecenoic (18: 1) fatty acids of its phospholipids at the expense of the saturated fatty acids ( Table 2). The mechanisms responsible for temperature-induced fatty acid alterations were extensively investigated in E. coli [6], where they were found to operate at the level of both phosphatidic and fatty acid biosynthesis.…”

Biogenesis of membrane lipids: mutants of Escherichia coli with temperature-sensitive phosphatidylserine decarboxylase.

“…1 and 2). Until recently, the only phospholipid mutants available were affected early in the pathway at the level of phosphatidic acid synthesis (3)(4)(5)(6)(7). Reports have recently appeared, however, describing the isolation of mutants of Escherichia coli defective in some early stage of the formation of phosphatidylethanolamine, the predominant phospholipid in E. coli (8)(9)(10).…”

Effects of Dietary Fat on Lipid Composition of Serum and Erythrocytes of the Swine and in Vitro Incorporation of Fatty Acids Into Erythrocyte Membranes

“…The author specurates that the palmitic acid incorporated into 2 position might be transferred to 2 position during long (8 hr) incubation, because the fatty acid was incorporated into both positions while linoleic acid was incorporated exclusively into 2 position. These facts also seem to indicate the existence of different types of acyltransferase, as shown by CRONAN et al (50). At present, the multiplicity of the enzyme action needs further investigation.…”

“…(50), species, and so forth. Furthermore, it is generally said that fatty acids at 2 position of glyceryl phospholipids are easily transferred to 1 position.…”