A dual program for CRP-mediated regulation in bacterial alarmone (p)ppGpp

Area of Science:

• Molecular Biology
• Bacterial Physiology
• Synthetic Biology

Background:

• Gene expression and cellular functions rely on coordinated genetic network regulation, especially during environmental shifts.
• The stringent response, mediated by (p)ppGpp (guanosine tetra- and penta-phosphate), is crucial for bacterial adaptation.
• cAMP receptor protein (CRP) is a known transcriptional regulator, but its role in coordinating gene expression under stress is less understood.

Purpose of the Study:

• To identify master regulators of (p)ppGpp homeostasis and understand their role in bacterial stress response.
• To elucidate the mechanisms by which CRP controls both transcription and translation of key stress-related genes.
• To explore the application of CRP-mediated regulatory circuits in synthetic biology for enhanced bioproduction.

Main Methods:

• Investigated CRP's direct transcriptional regulation of (p)ppGpp synthetase/hydrolase genes (RelA and SpoT).
• Examined CRP's non-classical translational regulatory role via YfiQ-dependent acetylation of ribosome protein S1.
• Applied CRP-mediated dual enhancement (CMDE) in synthetic circuits to assess its impact on metabolite production.

Main Results:

• Identified CRP as a master regulator of (p)ppGpp homeostasis, stimulating its accumulation under glucose limitation.
• Demonstrated CRP's dual role: direct transcriptional activation of RelA, SpoT, and itself, and enhanced translation via S1 acetylation.
• Showcased CMDE's ability to create a self-activating feedback loop, leading to stable increases in p-coumaric acid, cinnamic acid, and pinosylvin production in synthetic circuits.

Conclusions:

• CRP orchestrates bacterial stress responses through a novel dual transcriptional-translational regulatory mechanism impacting (p)ppGpp levels.
• The CRP-mediated dual enhancement (CMDE) provides a robust self-activating feedback loop for stable genetic circuit activation.
• CMDE offers a promising strategy for advancing cell-based biotechnologies and bioproduction applications.