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

• Published in: mBio 2024-11, Vol.15 (11), p.e0243024
• Main Authors: Zhao, Li, Zhou, Shi-Yu, Fu, Yu, Shen, Jin-Long, Yin, Bin-Cheng, You, Di, Ye, Bang-Ce
• Format: Article
• Language: English
• Subjects: (p)ppGpp; Acetylation; Bacteriology; cAMP-CRP; Cyclic AMP Receptor Protein - genetics; Cyclic AMP Receptor Protein - metabolism; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Gene Expression Regulation, Bacterial; GTP Pyrophosphokinase; Guanosine Pentaphosphate - metabolism; Guanosine Tetraphosphate - metabolism; Ligases - genetics; Ligases - metabolism; protein acetylation; Pyrophosphatases - genetics; Pyrophosphatases - metabolism; Research Article; Ribosomal Proteins; transcriptional regulation; translational regulation
• ISSN: 2150-7511; 2150-7511
• DOI: 10.1128/mbio.02430-24

Gene expression and proper downstream cellular functions upon facing environmental shifts depend on the combined and cooperative regulation of genetic networks. Here, we identified cAMP receptor protein (CRP) as a master regulator of (p)ppGpp (guanosine tetra- and penta-phosphate) homeostasis. Via CRP-mediated direct transcriptional regulation of the (p)ppGpp synthetase/hydrolase RelA and SpoT, cAMP-CRP stimulates pervasive accumulation of (p)ppGpp under glucose-limiting conditions. Notably, CRP exerts a nonclassical property as a translational regulator through YfiQ-dependent acetylation of ribosome protein S1 at K247, which further enhances the translation of RelA, SpoT, and CRP itself. From a synthetic biology perspective, this self-activating feedback loop for (p)ppGpp synthesis highlights the function of RP- ediated ual nhancement (CMDE) in controlling bacterial gene expression, which enables stable activation of genetic circuits. CMDE applied in synthetic circuits leads to a stable increase in -coumaric acid, cinnamic acid, and pinosylvin production. Our findings showed that CRP-mediated dual circuits for (p)ppGpp regulation enable robust activation that could address bioproduction and other biotechnological needs.IMPORTANCETranscriptional-translational coordination is fundamental for rapid and efficient gene expression in most bacteria. Here, we uncovered the roles of cAMP-CRP in this process. We found that CRP distinctly increases RelA and SpoT transcription and translation, and that acetylation of S1 at K247 accelerates the self-activation of the leading CRP under glucose-limiting conditions. We further found that elevated (p)ppGpp significantly impedes the formation of the cAMP-CRP complex, an active form responsible for transcriptional activation. A model was created in which cAMP-CRP and (p)ppGpp cooperate to dynamically modulate the efficiency of transcriptional-translational coordination responses to stress. More broadly, productive activation in synthetic circuits was achieved through the application of RP- ediated ual nhancement (CMDE), promising to inspire new approaches for the development of cell-based biotechnologies.