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

Abstract

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 To gain further insights into the direct targets of RpoN, 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 (, , and) and the T6SS ( and ). The direct targets of RpoN have been verified by electrophoretic mobility shifts assays (EMSA), reporter assay, reverse transcription-quantitative PCR, and phenotypic detection. The ::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::Tc mutant than in the wild type. In summary, the results indicated that RpoN had direct and profound effects on QS and the T6SS. As a global regulator, RpoN controls a wide range of biological pathways, including virulence in 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 and other bacteria.

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“…Very little, but always function-specific, cross-talk was present between the sigma factors, with RpoN being the common factor that interacted with the most other tested sigma factors (notably RpoH, RpoS, FliA, AlgU, SigX, and RpoD) while also attaining the second largest regulon (680 genes), only outnumbered by the house-keeping sigma factor, RpoD. Since then, RpoN has been shown to directly interact with genes associated with quorum sensing and genes encoding the type VI secretion system with the identification of several new direct targets such as lasI, rhlR, rhlI, pqsR, pqsA, hcpA, and hcpB (38). Not only has a link been established between quorum sensing and the type VI secretion system, but also with antibiotic resistance when RpoN was shown to promote survival in the presence of tobramycin (39).…”

RpoN1 and RpoN2 play different regulatory roles in virulence traits, flagellar biosynthesis, and basal metabolism in Xanthomonas campestris

“…To investigate the function of RpoN in X. campestris , protein sequence alignments of X. campestris RpoN with RpoN proteins from Escherichia coli (Tucker et al , 2010; Wiesler et al , 2012), Pseudomonas aeruginosa (Shao et al , 2018), and Sinorhizobium meliloti (Iannino et al . , 2008) were examined (Figure 1c).…”

“…RpoN also affects bacterial QS (Cai et al , 2015). Although RpoN regulates bacterial virulence and shares similar functions in gram‐negative bacteria, the regulons of RpoN vary among bacteria (Schaefer et al , 2015; Shao et al , 2018; Xu et al , 2019). In this study, we demonstrate that RpoN1 and RpoN2 play different roles in regulation of basal metabolism, flagellar biosynthesis and virulence traits in X. campestris .…”

“…EMSAs were performed as described (Hirakawa et al , 2015; Shao et al , 2018). For RpoN gel shift assays, we used DNA fragments that included the pXCC fliC (408 bp) and pXCC fliQ (497 bp) promoter regions as probes.…”

Two Functional Fatty Acyl Coenzyme A Ligases Affect Free Fatty Acid Metabolism To Block Biosynthesis of an Antifungal Antibiotic in Lysobacter enzymogenes

“…EMSA was performed as previously described (44,45). For LeClp gel shift assays, we used DNA fragments that included the Le rpfB1 (451 bp), or Le rpfB2 (486 bp) promoter region as a probe.…”