Metabolic Imbalance and Sporulation in an Isocitrate Dehydrogenase Mutant of Bacillus subtilis

A Bacillus subtilis mutant with a deletion in the citC gene, encoding isocitrate dehydrogenase, the third enzyme of the tricarboxylic acid branch of the Krebs cycle, exhibited reduced growth yield in broth medium and had greatly reduced ability to sporulate compared to the wild type due to a block at stage I, i.e., a failure to form the polar division septum. In early stationary phase, mutant cells accumulated intracellular and extracellular concentrations of citrate and isocitrate that were at least 15-fold higher than in wild-type cells. The growth and sporulation defects of the mutant could be partially bypassed by deletion of the major citrate synthase gene ( citZ ), by raising the pH of the medium, or by supplementation of the medium with certain divalent cations, suggesting that abnormal accumulation of citrate affects survival of stationary-phase cells and sporulation by lowering extracellular pH and chelating metal ions. While these genetic and environmental alterations were not sufficient to allow the majority of the mutant cell population to pass the stage I block (lack of asymmetric septum formation), introduction of the sof-1 mutant form of the Spo0A transcription factor, when coupled with a reduction in citrate synthesis, restored sporulation gene expression and spore formation nearly to wild-type levels. Thus, the primary factor inhibiting sporulation in a citC mutant is abnormally high accumulation of citrate, but relief of this metabolic defect is not by itself sufficient to restore competence for sporulation.