Arch. Microbiol. 107, 49 55 (1976) Archives of Microbiology © by Springer-Verlag 1976 Altered Phospholipid Metabolism in a Temperature-Sensitive Mutant of a Thermophilic Bacillus LUBA L. KOSTIW and KENNETH A. SOUZA Planetary Biology Division, Ames Research Center, NASA, Moffett Field, California 94035, U.S.A. Abstract. The phospholipid metabolism of a temper- ature-sensitive mutant of a thermophilic bacillus was studied after the shift from a permissive (58 ° C) to a restrictive (65°C) growth temperature. During the short period of growth of the mutant at 65°C, the proportions of cardiolipin and its 3-acyl derivative (lyso-cardiolipin) increased, and the proportions of phosphatidylglycerol and phosphatidylethanolamine decreased on cell dry weight basis. In 32p incorporation and turnover experiments, phosphatidylglycerol showed the most rapid uptake and loss of the label. Turnover of cardiolipin, limited to a short period, ceased 18 rain after the shift, as did the turnover of phosphatidylethanolamine. In the absence of net phospholipid synthesis, there was a quantitative con- version of phosphatidylglycerol to cardiolipin and an increase in the proportion of lyso-cardiolipin. Chlor- amphenicol, added to the medium at the time of the shift, reduced the rate of phospholipid synthesis, prevented the increase in the proportions of cardiolipin and lyso-cardiolipin, and slowed the decrease in the proportions of the other two phospholipids. The results indicated a defect in the regulatory mecha- nism(s) of phospholipid metabolism in the mutant at the restrictive temperature. Key words ." Phospholipids - Membrane - Mutant - Temperature - Thermophiie. The previous communication (Souza et al., 1974) described a temperature-sensitive mutant (TS-13) of a thermophilic bacillus (WT) which was unable tO Nonstandard Abbreviations. WT = parental strain, thermophilic bacillus; TS-13 = temperature-sensitive mutant of a thermophilic bacillus; CL = cardiolipin; PG = phosphatidylglycerol; PE = phos- phatidylethanolamine; 1-CL = lyso-cardiolipin. maintain cellular integrity when shifted to a restrictive temperature, 65 ° C. It was shown that the mutant had an altered fatty acid composition of lower thermal stability at the permissive temperature, 58°C, and that it was unable to adjust its phospholipid com- position in a manner analogous to the parent at all the growth temperatures tested. These results, there- fore, implicated lipid synthesis, and consequently membrane synthesis, as the possible site of the lesion in the mutant. Phospholipids are major components of cell mem- branes. In several temperature-sensitive lysis mutants of mesophiles, defective membrane synthesis has been attributed to an altered phospholipid metabolism (Hechemy and Goldfine, 1971; Henning et al., 1969; Steenbakkers et al., 1973). Therefore, the phospholipid metabolism in TS-13 was investigated after the shift from a permissive (58 ° C) to a restrictive (65 ° C) growth temperature. This paper describes an altered phospho- lipid composition and metabolism in TS-13, as com- pared to that in WT. MATERIALS AND METHODS Materials. KHz32po4, specific activity 500 mCi/mM, was supplied by New England Nuclear Corp., Boston, MA. Phospholipid standards were obtained from Supelco, Inc., Bellefonte, PA. Phos- pholipase A was purchased from Sigma Chem. Co., St. Louis, MO. Bacteria. The source of the thermophilic bacillus (YTG-2), identified as a strain of Bacillus stearothermophilus and designated as WT, and the selection of its temperature-sensitive mutant, designated as TS-13, which were used in this study, have been de- scribed (Souza et al., 1974). Growth Conditions. Both strains were grown on Tryptic Soy Broth (Difco, 10 g/l) with supplements as described (Souza et al., 1974). Temperature shift experiments were carried out as follows. The medium (1.2 or 1.6 I), preheated to 58°C, was inoculated with 50 ml of an overnight culture grown at 58°C and incubated aero- bically in a water bath at the same temperature until an absorbance (A) of 0.40 at 450 nm was reached. At that time, appropriate aliquots (200- 400 ml) were poured into 1-1 flasks preheated to 65 ° C, which assured that the new temperature was reached in not more than
