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Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen of humans, particularly those with cystic fibrosis. As a global regulator, RpoN controls a group of virulence-related factors and quorum-sensing (QS) genes in P. aeruginosa To gain further insights into the direct targets of RpoN in vivo , the present study focused on identifying the direct targets of RpoN regulation in QS and the type VI secretion system (T6SS). We performed chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) that identified 1,068 binding sites of RpoN, mostly including metabolic genes, a group of genes in QS ( lasI , rhlI , and pqsR ) and the T6SS ( hcpA and hcpB ). The direct targets of RpoN have been verified by electrophoretic mobility shifts assays (EMSA), lux reporter assay, reverse transcription-quantitative PCR, and phenotypic detection. The ΔrpoN ::Tc mutant resulted in the reduced production of pyocyanin, motility, and proteolytic activity. However, the production of rhamnolipids and biofilm formation were higher in the ΔrpoN ::Tc mutant than in the wild type. In summary, the results indicated that RpoN had direct and profound effects on QS and the T6SS. IMPORTANCE As a global regulator, RpoN controls a wide range of biological pathways, including virulence in P. aeruginosa PAO1. This work shows that RpoN plays critical and global roles in the regulation of bacterial pathogenicity and fitness. ChIP-seq provided a useful database to characterize additional functions and targets of RpoN in the future. The functional characterization of RpoN-mediated regulation will improve the current understanding of the regulatory network of quorum sensing and virulence in P. aeruginosa and other bacteria.

RpoN-Dependent Direct Regulation of Quorum Sensing and the Type VI Secretion System in Pseudomonas aeruginosa PAO1