Two Roles for Aconitase in the Regulation of Tricarboxylic Acid Branch Gene Expression in Bacillus subtilis

Abstract

ABSTRACT Previously, it was shown that an aconitase ( citB ) null mutation results in a vast overaccumulation of citrate in the culture fluid of growing Bacillus subtilis cells, a phenotype that causes secondary effects, including the hyperexpression of the citB promoter. B. subtilis aconitase is a bifunctional protein; to determine if either or both activities of aconitase were responsible for this phenotype, two strains producing different mutant forms of aconitase were constructed, one designed to be enzymatically inactive (C450S [ citB2 ]) and the other designed to be defective in RNA binding (R741E [ citB7 ]). The citB2 mutant was a glutamate auxotroph and accumulated citrate, while the citB7 mutant was a glutamate prototroph. Unexpectedly, the citB7 strain also accumulated citrate. Both mutant strains exhibited overexpression of the citB promoter and accumulated high levels of aconitase protein. These strains and the citB null mutant also exhibited increased levels of citrate synthase protein and enzyme activity in cell extracts, and the major citrate synthase ( citZ ) transcript was present at higher-than-normal levels in the citB null mutant, due at least in part to a >3-fold increase in the stability of the citZ transcript compared to the wild type. Purified B. subtilis aconitase bound to the citZ 5′ leader RNA in vitro , but the mutant proteins did not. Together, these data suggest that wild-type aconitase binds to and destabilizes the citZ transcript in order to maintain proper cell homeostasis by preventing the overaccumulation of citrate.