Systematic Identification of CpxRA-Regulated Genes and Their Roles in Escherichia coli Stress Response

The two-component system CpxRA can sense environmental stresses and regulate transcription of a wide range of genes for the purpose of adaptation. Despite extensive research on this system, the identification of the CpxR regulon is not systematic or comprehensive. Herein, genome-wide screening was performed using a position-specific scoring matrix, resulting in the discovery of more than 10,000 putative CpxR binding sites, which provides an extensive and selective set of targets based on sequence. More than half of the candidate genes ultimately selected (73/97) were experimentally confirmed to be CpxR-regulated genes through experimental analysis. These genes are involved in various physiological functions, indicating that the CpxRA system regulates complex cellular processes. The study also found for the first time that the CpxR-regulated genes ydeE, xylE, alx, and galP contribute to Escherichia coli resistance to acid stress, whereas prlF, alx, casA, yacH, ydeE, sbmA, and ampH contribute to E. coli resistance to cationic antimicrobial peptide stress. Among these CpxR-regulated genes, ydeE and alx responded to both stressors. In a similar way, a cationic antimicrobial peptide is capable of directly activating the periplasmic domain of CpxA kinase in vitro, which is consistent with the CpxA response to acid stress. These results greatly expand our understanding of the CpxRA-dependent stress response network in E. coli. IMPORTANCE CpxRA system is found in many pathogens and plays an essential role in sensing environmental signals and transducing information inside cells for adaptation. It usually regulates expression of specific genes in response to different environmental stresses and is important for bacterial pathogenesis. However, systematically identifying CpxRA-regulated genes and elucidating the regulative role of CpxRA in bacteria responding to environmental stress remains challenging. This study discovered more than 10,000 putative CpxR binding sites based on sequence. This bioinformatics approach, combined with experimental assays, allowed the identification of many previously unknown CpxR-regulated genes. Among the novel 73 CpxRA-regulated genes identified in this study, the role of nine of them in contributing to E. coli resistance to acid or cationic antimicrobial peptide stress was studied. The potential correlation between these two environmental stress responses provides insight into the CpxRA-dependent stress response network. This also improves our understanding of environment-bacterium interaction and Gram-negative pathogenesis.