ROLE OF LIPID IN THE FORMATION AND FUNCTION OF THE RESPIRATORY SYSTEM OF STAPHYLOCOCCUS AUREUS

Anaerobically growing Staphylococcus aureus, when aerated vigorously, forms a membrane-bound electron transport system that consists of primary dehydrogenases, cytochromes, and cytochrome oxidases. Concomitant with this, there are changes in the composition of the membrane lipids, with increases in glucolipids, vitamin K2 isoprenologues, and phospholipids. The inhibitors benzo(a) pyrene or piperonyl butoxide blocked both the lipid changes and the formation of the electron transport system, suggesting that the lipid changes are an obligatory part of the process. Further evidence of the involvement of lipids in the formation of the respiratory system comes from the study of mutants. A glycerol auxotroph, when deprived of glycerol, stops net phospholipid biosynthesis and forms a defective cytochrome system. Depriving a menadione mutant also forms a defective respiratory system. Present activity is directed toward isolating fractions of the membranes in which the lipid and respiratory pigment changes are occurring. Staphylococcus aureus is an ideal organism in which to study the formation of the membrane-bound electron transport system, since it can grow glycolitically in the absence of oxygen if glucose is present, or can utilize a respiratory system if it is aerated.1-3 If exponentially growing cells are incubated in the absence of air but the presence of glucose, no detectable respiratory system is f ~ r m e d . ~ If such a culture is aerated, a functional respiratory system is formed (FIGURE 1). This involves the synthesis of the primary dehydrogenases, the cytochromes, and cytochrome oxidases, which are readily detectable by difference spectroscopy.3 Since the organism is also capable of efficient oxidative phosphorylation (P. Keyser and D. C. White; unpublished data) and active transport of amino acids,4* the necessary coupling factors are also present in the membrane. Under the conditions of growth utilized in these experiments a cytochrome system with ratios of pigments consistent with the environment of growth is fully active within about 1.5 hours after the onset of aeration. The electron transport system consists of the pigments that form a multienzyme complex, which must have structural integrity to function. Disruption of their spatial relationships by organic solvents interrupts electron transport. The fact that absorbance changes on reduction of the pigments are a measure of function, and are readily measured in living cells, makes the function of the electron transport system a convenient multienzyme-membrane complex to study. In addition to the respiratory system, the staphylococcal membrane also contains lipids. Methods for the quantitative assay of the phospholipids,B* fatty acids,s carotenoids: and vitamin K, isoprenologues lo of the S . aureus membrane were developed. With these methods in hand it was possible to