A KDPG sensor RccR governs Pseudomonas aeruginosa carbon metabolism and aminoglycoside antibiotic tolerance

Pseudomonas aeruginosa harbors sophisticated transcription factor (TF) networks to coordinately regulate cellular metabolic states for rapidly adapting to changing environments. The superior capacity in fine-tuning the metabolic states enables its success in tolerance to antibiotics and evading host immune defenses. However, the linkage among transcriptional regulation, metabolic states, and antibiotic tolerance in P. aeruginosa remains largely unclear. By screening the P. aeruginosa TF mutant library constructed by CRISPR/Cas12k-guided transposase, we identify that rccR (PA5438) is a major genetic determinant in aminoglycoside antibiotic tolerance, the deletion of which substantially enhances bacterial tolerance. We further reveal the inhibitory roles of RccR in pyruvate metabolism (aceE/F) and glyoxylate shunt pathway (aceA and glcB), and overexpression of aceA or glcB enhances bacterial tolerance. Moreover, we identify that 2-keto-3-deoxy-6-phosphogluconate (KDPG) is a signal molecule that directly binds to RccR. Structural analysis of the RccR/KDPG complex reveals the detailed interactions. Substitution of the key residues R152, K270, or R277 with alanine abolishes KDPG sensing by RccR and impairs bacterial growth with glycerol or glucose as the sole carbon source. Collectively, our study unveils the connection between aminoglycoside antibiotic tolerance and RccR-mediated central carbon metabolism regulation in P. aeruginosa, and elucidates the KDPG sensing mechanism by RccR. P. aeruginosa PAO1 wild-type and rccR-deletion mutant strains were cultured in LB medium at 37℃ and harvested at the exponential phase and stationary phase, respectively. The total RNAs were extracted using the MiniBEST Universal RNA Extraction Kit (Takara) and used as templates for reverse transcription to cDNA using the TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech Co) according to the manufacturer’s instructions. The cDNA products were subjected to transcriptome sequencing by Majorbio (Shanghai).