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

A soluble enzyme activity which catalyzes the synthesis of acyl-acyl carrier protein from acyl carrier proteins, a long chain fatty acid, and ATP has been demonstrated in E. coil. The reaction requires high concentrations of both Ca++ and Mg++ for activity, and cleaves ATP to AMP and PP-. The fatty acyl product has been identified as acyl-acyl carrier protein by its solubility, thioester linkage, molecular weight, charge, and biological activity. Several palmitic acid (10,000 cpm/nmol); 2 mg/ml Triton X-100 and crude enzyme (0-0.1 mg) in a total volume of 0.1 ml. After incubation at 370 for 10 min, incorporation of fatty acid into acyl-ACP was assayed by either of two methods. The first method was solvent extraction to remove free fatty acid as described by Mancha et al. (9). A second and less cumbersome method was to pipet the reaction mix onto a filter paper disc (Whatman 3MM, 2.4 cm diameter). The discs were hen washed twice in a beaker containing chloroform-methanol-acetic acid (1:2:0.3, vol/vol) in order to remove free fatty acid (fatty acyl-CoA is also removed). Under these conditions, the enzyme assay gave a linear response with both protein and time. Blank values (minus enzyme, ACP, or ATP) showed <5% of the incorporation given by complete reaction mixtures. A unit of enzyme activity is a nmol of acyl-ACP formed per minute.Separation of ACP-SH and Acyl-ACP. Standard incubation mixtures, in which about 30% of the [3H]pantothenate-ACP-SH (4100 cpm/nmol) was converted to ['4C]palmityl-[3H]pantothenate-ACP by enzyme purified through the ammonium sulfate step, were extracted to remove free fatty acid and then acid precipitated at pH 3.5. The precipitate was resuspended in 1 ml of 0.1 M Tris-HCI buffer at pH 8.0 which contained 1 M KCI (to prevent nonspecific binding), 1 mM NaEDTA, and a 10-fold excess of dithiothreitol. After reduction, the ACP-SH in the mixture was then converted to its thionitrobenzoic acid derivative by addition of a 10-fold excess (over dithiothreitol) of 5,5'-dithiobis(2-nitrobenzoic) acid (DTNB). Excess DTNB and the other reaction products were removed from the protein fraction via a Sephadex G-25 column. The ACP derivatives from the void of the G-25 column were then applied to a 5 ml column of DTNB-agarose (10). The DTNB-agarose column was slowly (0.1 ml/min) eluted with the Tris-KCI-EDTA buffer.This treatment removed >95% of the ACP-SH in a parallel sample incubated without ATP. The ratio of palmitate to ACP in the eluate was then determined by dual label scintillation counting, by using the appropriate standards.Electrophoresis. Gels of 12% acrylamide were run in 0.1% sodium dodecyl sulfate (NaDodSO4) as previously described (8,11) except that sample preparation and quantitation were done as described by .Chromatography. The Sephadex G-75 column (1 X 36 cm,

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