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In a glycerol auxotroph of Staphylococcus aureus, the deprivation of glycerol affected the formation of certain membrane components. (i) There was synthesis of fatty acids at the predeprivation rate even though the fatty acids synthesized accumulated as free fatty acids rather than as esterified fatty acids; (ii) there was a complete cessation of phospholipid and vitamin K isoprenologue biosynthesis; (iii) there was conservation of the glycerol esters of the complex phospholipids and glucolipids; (iv) there was an immediate decrease in the rate of synthesis of monoglucoslydiglyceride (30%) and diglucosyldiglyceride (60%); (v) there was a 50% decrease in the rate of synthesis of the polar and nonpolar carotenoids; (vi) there was synthesis of protoheme, heme a, and nonspecific membrane protein at the predeprivation rate; and (vii) there was an abrupt cessation in the formation of new, functional glycine transport activity.

Thermus aquaticuscontains four major fatty acids, iso-C15(28%), iso-C16(9%), normal-C16(13%), and iso-C17(48%), when grown at 70 C, as determined by gas chromatography and mass spectrometry. Small amounts of iso-C12, normal-C12:1, iso-C13, normal-C14, iso-C14, and normal-C15:1were also detected. A change in growth temperature (50 to 75 C at 5-C intervals) affects a shift in the proportions of some of the fatty acids. The proportions of the monoenoic and branched-C17fatty acids decreased and the proportions of the higher-melting iso-C16and normal-C16fatty acids increased. Cells grown at 75 C contained 70% more total fatty acids than cells grown at 50 C. The largest increases, in absolute amounts, were in the content of iso-C16and normal-C16fatty acids, with only a 1.6-fold increase in the major iso-C15and iso-C17fatty acids. There was a 2.5-fold decrease in normal-C15:1and at least a 24-fold decrease in anteiso-C17, which is present at 50 and 55 C but not at higher temperatures. There was no difference in proportion or amount of fatty acids between exponential and stationary-phase cells grown at 70 C. When cells were grown on glutamate instead of yeast-extract and tryptone at 70 C, the total fatty acid content remained constant, but there was an increase in the proportions of iso-C16and normal-C16fatty acids concomitant with a decrease in the proportions of the iso-C15and iso-C17fatty acids.

Rabbit liver mitochondria incubated in a basic medium containing pyruvate, bicarbonate, MgSO4, and Pisynthesize and liberate to the medium malate, citrate, and also aspartate (provided NH4+is included in the medium). Only a minor amount of P-enolpyruvate is formed and liberated under these same conditions. Substrate utilization and especially metabolite production are dependent on exogenous Piand also quite dependent on exogenous bicarbonate. Exogenous ATP inhibits substrate utilization and especially inhibits total metabolite formation.

The complex lipids of Thermus aquaticusinclude phospholipids, glucolipids, carotenoids, and vitamin K2isoprenologues. The phospholipids account for 30% of the total lipids and have been identified as phosphatidylethanolamine (4%), phosphatidylglycerol (3%), phosphatidylinositol (10%), cardiolipin (3%), and phosphatidic acid (1%). The major phospholipid contained three fatty acids, a long-chain unsaturated amine, and one glycerol per phosphate and accounted for 80% of the lipid phosphate. The carotenoids accounted for 60% of the membrane lipid. The majority of the carotenoids were very polar. Mono- and diglucosyldiglyceride and the 35-, 40-, and 45-carbon vitamin K2isoprenologues were also identified. All these lipids were localized in the membrane of T. aquaticus. When the growth temperature was increased from 50 to 75 C and measured at 5 C intervals, there was a progressive increase in the total lipid content. The phospholipids increased 2-fold, the carotenoids increased 1.8-fold, and the glucolipids increased 4-fold between cells grown at 50 C and 75 C. The vitamin K2level did not change. The proportions of the individual lipids within each lipid class remained constant as the temperature of growth was raised. Metabolic studies indicated turnover of the diacyl phospholipids during pulse-chase experiments at rates comparable with mesophilic bacteria. The major phospholipid and the carotenoids did not turn over.

A study of the effects of glycerol deprivation on the content and metabolism of the phospholipids of a glycerol auxotroph of Staphylococcus aureusshowed that (i) there was an increase in the proportions of lysylphosphatidylglycerol (LPB) and a concomitant decrease in the proportion of phosphatidylglycerol. The total phospholipid content per sample and the proportion of cardiolipin did not change, but the phosphatidic acid increased transiently and then fell to pretreatment levels. (ii) The loss of 32P from the lipids during the chase in a pulse-chase experiment was essentially the same in phosphatidylglycerol, cardiolipin, and phosphatidic acid during glycerol deprivation or growth in the presence of glycerol. LPG lost half the radioactivity in slightly more than two doubling times when grown with glycerol. In the absence of glycerol, 32P accumulated in LPG for about 20 min and then stopped, after which time there was no apparent turnover. (iii) During glycerol deprivation, the initial 32P incorporation decreased sixfold compared to that of the control with glycerol. The initial incorporation into LPG decreased only 2.5-fold, whereas that of PG decreased 45-fold. (iv) During glycerol deprivation, the free fatty acid content increased from 1.2 to 12.5% of the total extractable fatty acids and then slowly decreased. The increase was largely iso- and anti-iso-branched 21-carbon-atom fatty acids. In glycerol-supplemented cultures, the major fatty acids were branched 14- to 18-carbon fatty acids. The decrease in longer chain free fatty acids after 60 min represented their esterification into lipids. (v) During glycerol deprivation ribonucleic acid synthesis and cell growth continued for 40 min and protein synthesis continued for 90 min. Then synthesis and growth stopped. (vi) After the addition of glycerol to glycerol-deprived cells, 32P and 14C-glycerol were incorporated into the phospholipids without lag; ribonucleic acid, protein synthesis, and cell growth began after a 5- to 10-min lag at the pretreatment rate. The initial rate of lipid synthesis after the addition of glycerol was three times greater than the growth rate. This rapid rate continued for about 25 min until the lipid content and proportions of LPG and phosphatidylglycerol were restored.