Development of a single-cell GlxR-based cAMP biosensor for Corynebacterium glutamicum

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• Cyclic adenosine monophosphate (cAMP) is a vital second messenger in numerous organisms.
• In Corynebacterium glutamicum, cAMP regulates gene expression via the GlxR transcriptional regulator, impacting approximately 200 genes.
• While CyaB is a known adenylate cyclase, the presence of additional cAMP-modulating enzymes is suspected.

Purpose of the Study:

• To develop a novel cAMP biosensor for identifying unknown cAMP-modulating enzymes in C. glutamicum.
• To establish a high-throughput screening method for detecting altered cAMP levels in bacterial populations.

Main Methods:

• Construction of a plasmid-based biosensor using the cg3195 promoter fused to the eyfp reporter gene.
• Validation of biosensor response to varying cAMP and GlxR levels.
• Utilizing fluorescence-activated cell sorting (FACS) for high-throughput screening of mutant strains.

Main Results:

• The biosensor accurately reflected changes in cAMP and GlxR levels and differentiated between wild-type and mutant strains.
• The biosensor successfully distinguished C. glutamicum strains with defects in cAMP biosynthesis or degradation.
• FACS enabled efficient sorting of mixed wild-type and mutant cell populations based on cAMP levels.

Conclusions:

• The developed cAMP biosensor is a valuable tool for discovering novel enzymes involved in cAMP metabolism in C. glutamicum.
• This biosensor facilitates high-throughput screening, accelerating the identification of mutants with altered cAMP signaling.
• The findings contribute to a deeper understanding of cAMP-mediated gene regulation in industrial microorganisms.