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.