ABSTRACTPseudomonas aeruginosais 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 inP. aeruginosa. To gain further insights into the direct targets of RpoNin 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, andpqsR) and the T6SS (hcpAandhcpB). The direct targets of RpoN have been verified by electrophoretic mobility shifts assays (EMSA),luxreporter assay, reverse transcription-quantitative PCR, and phenotypic detection. TheΔrpoN::Tc mutant resulted in the reduced production of pyocyanin, motility, and proteolytic activity. However, the production of rhamnolipids and biofilm formation were higher in theΔrpoN::Tc mutant than in the wild type. In summary, the results indicated that RpoN had direct and profound effects on QS and the T6SS.IMPORTANCEAs a global regulator, RpoN controls a wide range of biological pathways, including virulence inP. aeruginosaPAO1. 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 inP. aeruginosaand other bacteria.
Expression of the Type VI Secretion System 1 Component Hcp1 Is Indirectly Repressed by OpaR inVibrio parahaemolyticus
