Functional Characterization of the Quorum Sensing Regulator RsaL in the Plant-Beneficial Strain Pseudomonas putida WCS358

Abstract

ABSTRACTIn many bacteria, quorum sensing (QS) systems rely on a signal receptor and a synthase producingN-acyl-homoserine lactone(s) as the signal molecule(s). In some species, thersaLgene, located between the signal receptor and synthase genes, encodes a repressor limiting signal synthase expression and hence signal molecule production. Here we investigate the molecular mechanism of action of the RsaL protein in the plant growth-promoting rhizobacteriumPseudomonas putidaWCS358 (RsaLWCS). InP. putidaWCS358, RsaLWCSdisplayed a strong repressive effect on the promoter of the QS signal synthase gene,ppuI, while it did not repress the same promoter inPseudomonas aeruginosa. DNase I protection assays showed that purified RsaLWCSspecifically binds toppuIon a DNA region overlapping the predicted σ70-binding site, but such protection was observed only at high protein concentrations. Accordingly, electrophoretic mobility shift assays showed that the RsaLWCSprotein was not able to form stable complexes efficiently with a probe encompassing theppuIpromoter, while it formed stable complexes with the promoter oflasI, the gene orthologous toppuIinP. aeruginosa. This difference seems to be dictated by the lower dyad symmetry of the RsaLWCS-binding sequence on theppuIpromoter relative to that on thelasIpromoter. Comparison of the results obtainedin vivoandin vitrosuggests that RsaLWCSneeds a molecular interactor/cofactor specific forP. putidaWCS358 to repressppuItranscription. We also demonstrate that RsaLWCSregulates siderophore-mediated growth limitation of plant pathogens and biofilm formation, two processes relevant for plant growth-promoting activity.