Quantitative transcriptomic and metabolic analyses reveal the roles of RpoS and Crp in the acid resistance system 1 in Escherichia coli.

Foodborne pathogens exploit acid resistance (AR) mechanisms to survive harsh environments, such as the stomach, increasing contamination risk. While the sigma factor RpoS and cyclic AMP receptor protein (Crp) are known mediators of AR, their systemic regulatory networks remain unclear. Here, we integrated transcriptomics and metabolomics to dissect RpoS- and Crp-dependent responses in E. coli under simulated gastrointestinal neutral (pH 7.5) and acidic (pH 5.5) conditions. We identified 78 coregulated genes, with RpoS dominating energy/amino acid metabolism and Crp primarily modulating amino acid pathways. Both regulators upregulate membrane components (fatty acids, chaperones, transporters) essential for AR, with transporters NarU (RpoS-dependent) and AmtB (Crp-dependent) conferring exceptional survival at pH 2.5. Surprisingly, Crp activates flagellar genes, whereas RpoS suppresses them during acid stress. Our work provides a systems-level understanding of AR adaptation, offering translational potential for antimicrobial development and bioremediation strain engineering.IMPORTANCEEscherichia coli and other gut bacteria colonize or infect the extremely acidic environment of the gastrointestinal tract through the acid resistance mechanism. Therefore, it is necessary to investigate the acid resistance mechanism of intestinal bacteria. Although RpoS and Crp have long been found to regulate the acid resistance of E. coli, there is a lack of systematic research to analyze its regulatory network under the acidic environment. Through an integrated approach combining genomic analysis, we obtained robust research findings. These results establish a novel theoretical framework for understanding microbial adaptation to acidic environments while offering potential applications for developing new antibiotic targets against intestinal pathogens and engineering industrial bacterial strains with enhanced acid tolerance capabilities.