Role of Pyruvate Carboxylase, Phosphoenolpyruvate Carboxykinase, and Malic Enzyme during Growth and Sporulation of Bacillus subtilis

Abstract

Abstract In extracts of Bacillus subtilis, CO2 fixation occurs primarily through the apparently constitutive enzyme carboxylase, which is strongly activated by acetyl-CoA. This enzyme is necessary for growth on glucose but is not required for sporulation, as was established with a carboxylase mutant. The malic enzyme can use either NAD or, less effectively, NADP as cofactor. The ratio of these activities remains constant through enzyme purification and during enzyme induction by l-malate. Enzyme synthesis is not repressed by glucose. Malic enzyme and carboxylase form a pyruvate shunt to the citric acid cycle, which apparently is necessary during growth on malate for the production of oxalacetate in substrate amounts; malic dehydrogenase functions mainly to provide energy via the citric acid cycle. A specific and sensitive [γ-32P]ATP assay for P-enolpyruvate carboxykinase has been developed. Using this assay, a purified enzyme preparation gave a Km for oxalacetate of about 25 µm. Enzyme synthesis is repressed by glucose. P-enolpyruvate carboxykinase mutants have established that the enzyme is needed for gluconeogenesis and, under normal growth conditions, for sporulation. Sporulation can be restored by the continuous feeding of gluconate.