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

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• Cyclic adenosine monophosphate (cAMP) is a key second messenger regulating gene expression via the transcriptional regulator GlxR in *Corynebacterium glutamicum*.
• Adenylate cyclase CyaB synthesizes cAMP, and its absence impacts cellular processes.
• Understanding cAMP's role is crucial for optimizing metabolic functions in *C. glutamicum*.

Purpose of the Study:

• To investigate the physiological consequences of reduced intracellular cAMP levels in *C. glutamicum*.
• To elucidate the role of the cAMP-GlxR regulatory system in energy metabolism.
• To identify mechanisms underlying growth inhibition in *cyaB* deletion mutants.

Main Methods:

• Construction and characterization of a *cyaB* deletion mutant (Δ*cyaB*) in *C. glutamicum*.
• Growth analysis on various carbon sources, including acetate, with and without cAMP supplementation.
• Measurement of membrane potential and sensitivity to protonophores (e.g., CCCP).
• Isolation and characterization of suppressor mutants and genetic analysis of GlxR.

Main Results:

• The Δ*cyaB* mutant exhibited severe growth inhibition on acetate, which was reversed by cAMP or *cyaB* expression.
• Growth inhibition was linked to acetate's uncoupling activity and a reduced membrane potential in the mutant.
• A suppressor mutation in GlxR (Ala131Thr) restored growth, underscoring the importance of the cAMP-GlxR pathway.
• Impaired membrane potential correlated with decreased expression of the cytochrome *bc*₁-*aa*₃ supercomplex.

Conclusions:

• The cAMP-GlxR system is essential for regulating energy metabolism and maintaining membrane potential in *C. glutamicum*, particularly under acetate stress.
• Reduced cAMP levels compromise the proton-motive force, affecting ATP synthesis and transport.
• Targeting the cAMP-GlxR pathway offers potential strategies for enhancing *C. glutamicum* performance in specific metabolic conditions.