Molecular Basis of Growth Inhibition by Acetate of an Adenylate Cyclase-Deficient Mutant of Corynebacterium glutamicum.

In Corynebacterium glutamicum , cyclic adenosine monophosphate (cAMP) serves as an effector of the global transcriptional regulator GlxR. Synthesis of cAMP is catalyzed by the membrane-bound adenylate cyclase CyaB. In this study, we investigated the consequences of decreased intracellular cAMP levels in a Δ cyaB mutant. While no growth defect of the Δ cyaB strain was observed on glucose, fructose, sucrose, or gluconate alone, the addition of acetate to these growth media resulted in a severe growth inhibition, which could be reversed by plasmid-based cyaB expression or by supplementation of the medium with cAMP. The effect was concentration- and pH-dependent, suggesting a link to the uncoupling activity of acetate. In agreement, the Δ cyaB mutant had an increased sensitivity to the protonophore carbonyl cyanide m -chlorophenyl hydrazone (CCCP). The increased uncoupler sensitivity correlated with a lowered membrane potential of acetate-grown Δ cyaB cells compared to wild-type cells. A reduced membrane potential affects major cellular processes, such as ATP synthesis by F₁F _O -ATP synthase and numerous transport processes. The impaired membrane potential of the Δ cyaB mutant could be due to a decreased expression of the cytochrome bc ₁- aa ₃ supercomplex, which is the major contributor of proton-motive force in C. glutamicum. Expression of the supercomplex genes was previously reported to be activated by GlxR-cAMP. A suppressor mutant of the Δ cyaB strain with improved growth on acetate was isolated, which carried a single mutation in the genome leading to an Ala131Thr exchange in GlxR. Introduction of this point mutation into the original Δ cyaB mutant restored the growth defect on acetate. This supported the importance of GlxR for the phenotype of the Δ cyaB mutant and, more generally, of the cAMP-GlxR system for the control of energy metabolism in C. glutamicum. [ABSTRACT FROM AUTHOR]