Ristovic, N. | Search bioRxiv

bioRxiv2026

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.

microbiology↗

bioRxiv2026

RsaM is not a switch but a built-in modulator of quorum sensing in Pseudomonas fuscovaginae

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. A Tn5 mutant insertion in the pfsI and pfsR intergenic region was previously reported to trigger the PfsI/R system. This region contains coding sequences for RsaM, a putative protein that has since been hypothesized to play a central role in imposing repression on the PfsI/R system. Putative rsaM genes/RsaM proteins negatively controlling AHL QS systems have also been reported in several other bacterial species. In the present study, we report for the first time the endogenous expression of an RsaM family protein and determine its position within the PfsI/R regulatory circuit. We found that RsaM is not produced in the wild-type P. fuscovaginae and does not play a role in keeping the PfsI/R system in a quiescent state. The expression of RsaM is instead triggered upon targeted mutations in the pfsR-rsaM intergenic region, which concomitantly activate the transcription of both the pfsI and pfsR genes. Moreover, we demonstrated that RsaM attenuates the PfsI/R circuit upon its activation. Taken together, our results evidenced that RsaM does not function as a repressor switch of the PfsI/R system, but behaves as a built-in modulator that prevents overactivation of this circuit once it is triggered. ImportancePseudomonas fuscovaginae is a globally occurring plant pathogen that employs AHL QS to regulate virulence. In this bacterium, QS signalling 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 possible repressor switch acting on the PfsI/R AHL QS system in the absence of an unknown signal or stimulus. In this study, we demonstrated that the expression of RsaM and activation of the PfsI/R system both depend on the disruption of the rsaM and pfsR divergent intergenic region, and that RsaM functions as a negative modulator of this circuit, rather than as its master repressor. This study unveils the functional position of a novel protein regulator and broadens our understanding of regulatory configurations governing QS circuits.

microbiology↗