Stringent Control of Fatty Acid Synthesis in Escherichia coli: POSSIBLE REGULATION OF ACETYL COENZYME A CARBOXYLASE BY ppGpp

Abstract

Abstract The effect of amino acid starvation on the rate of fatty acid synthesis was examined in stringent (CP 78, rel+) and relaxed (CP 79, rel-) isogenic strains of Escherichia coli (leu-, his-, arg-, thr-, thi-). Rates of incorporation of [U-14C]glucose, [1-14C]acetate, and 3H2O into chloroform-methanol-soluble lipids (g95% phospholipid) were instantly reduced 50 to 60% by leucine starvation of stringent, but not relaxed, cells. That this depressed rate of lipid labeling was not due to leucine starvation-induced diversion of labeled fatty acyls into chloroform-methanol-insoluble form (e.g. lipopolysaccharide, etc.) is indicated by the fact that the rate of labeling from [1-14C]acetate of the lipid extract of saponified cells was decreased to the same extent by leucine deprivation. With leucine-deprived rel+ cells, the rate of [1-14C]acetate incorporation into phosphatidylethanolamine and cardiolipin was more drastically curtailed than into phosphatidylglycerol, while the incorporation pattern in rel- cells (± leucine) was similar to that of nonstarved rel+ cells. An elevated turnover rate of fatty acyl groups due to amino acid starvation cannot account for the decreased rate of lipid labeling by various precursors, since no detectable loss of 14C-activity from fatty acyls labeled during growth or leucine deprivation occurs during subsequent growth or leucine starvation in the presence of unlabeled precursor. Only minor amounts of labeled lipid are secreted by stringent or relaxed cells grown in the presence of [1-14C]acetate; leucine deficiency has no significant effect on the rate of labeling of these extracellular lipids. These results lead to the conclusion that fatty acid synthesis per se is subject to stringent control being partially suppressed, i.e. 50 to 60%, by amino acid starvation in rel+ strains of E. coli. Amino acid starvation of stringent, but not relaxed, strains of E. coli is known to activate ribosomal synthesis of ppGpp and pppGpp. The resultant accumulation of (p)ppGpp appears to mediate stringent control of fatty acid synthesis by blocking the initial committed step of the pathway, i.e. the carboxylation of acetyl-CoA. Of the two catalytic components of the acetyl-CoA carboxylase system, namely biotin carboxylase and carboxyltransferase, only the latter component is inhibited by physiological concentrations (up to 4 mm) of ppGpp. Two carboxyltransferase-catalyzed reactions—(a) carboxyl transfer from malonyl-CoA to the model acceptor, d-biotin methyl ester, and (b) carboxyl carrier protein-dependent malonyl-CoA-[1-14C]acetyl-CoA exchange—are inhibited 50 to 60% at saturating concentrations (1.0 to 1.2 mm) of (p)ppGpp. Inhibition is specific in that GTP, GDP, GMP, ATP, ADP, AMP, and cyclic adenosine 3':5'-monophosphate have no effect at comparable concentrations. The binding of Mn2+ by ppGpp was investigated by electron paramagnetic resonance to evaluate the possible metal scavenging effect of the guanosine nucleotide on the metaldependent reactions (i.e. biotin and acetyl-CoA carboxylation) catalyzed by the biotin carboxylase component of acetylCoA carboxylase. Kd values determined at pH 6.8 for the dissociation of Mn2+ from the binary (Mn·ppGpp) and ternary (Mn2·ppGpp) complexes are 1.1 µm and 65 µm, respectively, the former being tighter and the latter an order of magnitude weaker than Mn2+ dissociation from Mn·ATP. Whereas the biotin carboxylase-catalyzed reaction per se (carboxylation of free d-biotin) is not inhibited, 2.5 mm Mn·ppGpp inhibits both acetyl-CoA carboxylation and malonyl-CoA-[1-14C]acetyl-CoA exchange 50 to 60%. These results suggest that stringent control of fatty acid synthesis in E. coli is mediated through the inhibitory action of (p)ppGpp on the carboxyltransferase component of the acetyl-CoA carboxylase system.