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

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 Delta rpoN::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 Delta rpoN::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.