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

Previously, it was shown that an aconitase (citB ) null mutation results in a vast overaccumulation of citrate in the culture fluid of growingBacillus subtilis cells, a phenotype that causes secondary effects, including the hyperexpression of thecitB 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 ]). ThecitB2 mutant was a glutamate auxotroph and accumulated citrate, while thecitB7 mutant was a glutamate prototroph. Unexpectedly, thecitB7 strain also accumulated citrate. Both mutant strains exhibited overexpression of thecitB promoter and accumulated high levels of aconitase protein. These strains and thecitB 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 thecitB null mutant, due at least in part to a >3-fold increase in the stability of thecitZ transcript compared to the wild type. PurifiedB. subtilis aconitase bound to thecitZ 5′ leader RNAin vitro , but the mutant proteins did not. Together, these data suggest that wild-type aconitase binds to and destabilizes thecitZ transcript in order to maintain proper cell homeostasis by preventing the overaccumulation of citrate.