A switch-rheostat circuit governs quorum sensing homeostasis in a phytopathogen

Pseudomonas fuscovaginae, a wide-host-range plant pathogen of several cereal and grass species, possesses two canonical N-acyl homoserine (AHL)-based quorum sensing (QS) systems called PfsI/R and PfvI/R, both of which are inactive under laboratory conditions but active in planta. The pfsI-pfsR intergenic region encodes for RsaM, a putative protein that has since been hypothesized to act as a repressor switch of this QS circuit. In the present study, we demonstrate that the stringent repression exerted on the PfsI/R system depends entirely on the divergent promoter/intergenic pfsR-rsaM region rather than RsaM itself. Remarkably, this regulatory switch element stringently represses the expression of both the pfsR and rsaM genes. We further show for the first time that RsaM is endogenously expressed and functions as a negative regulator modulating the PfsI/R circuit instead of preventing its activation. Taken together, our results evidence a unique two-tiered/hierarchical repression of a QS system, provided by a master repressor switch and a repressor modulator. ImportancePseudomonas fuscovaginae is a globally occurring plant pathogen that employs AHL QS to regulate virulence. In this bacterium, QS signaling circuits display a rather unusual feature: the lack of activation at high cell densities under standard laboratory conditions. A hypothetical regulator named RsaM was previously linked to this phenomenon as a repressor switch acting on the PfsI/R AHL QS system in the absence of an unknown signal or stimulus. In this study, we show that a regulatory element within the pfsR-rsaM intergenic region acts as the primary switch of the PfsI/R QS system, independently of RsaM. Conversely, RsaM functions as a post-activation modulator that fine-tunes the QS response. This study advances our understanding of the regulatory configurations of unconventional QS systems by revealing a two-tier control mechanism in which a master regulatory switch governs circuit activation, while the previously uncharacterized protein RsaM controls signaling output once the system is engaged.