Journal Article

Novel dual regulatory roles of RpoA in quorum sensing regulation and social behavior switching in Pseudomonas aeruginosa

Dandekar, Ajai A, Zhou, Xiaoqing, Chen, Huali, Liang, Yonglin, Qiu, Huifang, Cai, Wenjie, Dai, Weijun

2026

Overview

Understanding the social structure and evolutionary dynamics of microbial communities requires the identification and characterization of relevant mutant subpopulations. While employs quorum sensing (QS) to coordinate population-wide behaviors, the social traits of many QS mutants remain poorly defined. In this study, we developed an iterative \"targeted gene duplication followed by mutant screening\" (TGD-MS) approach to systematically identify noncanonical QS cheater mutants. We discovered that a single-nucleotide mutation in , which encodes the α subunit of RNA polymerase (RNAP), produces a QS-deficient phenotype resembling QS-null mutants. This RpoA variant mutant exhibits characteristic features of social cheating, including a competitive growth advantage in mixed populations, impaired QS-dependent virulence factor production, and attenuated pathogenicity. Structural and biochemical analyses revealed that the RpoA variant impairs RNAP binding to the promoters of core QS genes ( and ), leading to diminished QS activity. Further examination of natural RpoA variants uncovered a spectrum of QS-related phenotypes, suggesting that RpoA has a dual regulatory role in QS control. Within the C-terminal domain (α-CTD) of RpoA, we identified two distinct functional determinants that, through adaptive mutations, can acquire opposing regulatory effects on QS. This enables an environmentally dependent phenotypic switch between cooperation and cheating. Our discovery of noncanonical RpoA-mediated QS cheaters expands the framework of bacterial social evolution, demonstrating that mutations outside the canonical QS circuitry can disrupt cooperative behaviors. These findings underscore how core transcriptional machinery can be evolutionarily co-opted to modulate complex social interactions in dynamic environments.IMPORTANCETo understand how bacterial populations function and evolve, it is essential to identify socially significant subpopulations, including previously unrecognized types of cheaters. In this study, we uncover an unexpected role of RNA polymerase (RNAP) in regulating quorum sensing (QS) and QS-associated social behaviors in . Specifically, we demonstrate that the α subunit of RNAP (RpoA) is a key regulatory component in this process. A single-nucleotide mutation within the C-terminal domain of RpoA was found to alter QS activity, driving an environment-dependent transition between cooperative and cheating phenotypes. This discovery of this novel, noncanonical QS cheater mutant offers new insights into intra-population interactions, population stability, and evolutionary dynamics. These findings carry significant implications for microbial ecology and deepen our understanding of social evolution in bacterial communities.

• Publisher: American Society for Microbiology
• Subject: Bacteriology / Environmental Microbiology / Microbial Physiology and Genetics / Model Organisms / Pathogenesis / Pseudomonas Species / Quorum Sensing / Quorum Sensing and Regulation / Quorum Sensing and Virulence / Quorum Sensing in Gram-Negative Bacteria / Quorum Sensing Mechanisms / Quorum Sensing Networks / Quorum Sensing Regulatory Networks / Quorum Sensing Signal Molecules / Regulatory Networks / Research Article