The two-component regulators GacS and GacA influence accumulation of the stationary-phase sigma factor sigma(S) and the stress response in Pseudomonas fluorescens Pf-5

Three global regulators are known to control antibiotic production by Pseud omonas fluorescens. A two-component regulatory system comprised of the sens or kinase GacS (previously called ApdA or LemA) and GacA, a member of the F ixJ family of response regulators, is required for antibiotic production. A mutation in rpoS, which encodes the stationary-phase sigma factor sigma(S) , differentially affects antibiotic production and reduces the capacity of stationary-phase cells of P. fluorescens to survive exposure to oxidative s tress. The gacA gene of P. fluorescens Pf-5 was isolated, and the influence of gacS and gacA on rpoS transcription, sigma(S) levels, and oxidative str ess response of Pf-5 was determined. We selected a gacA mutant of Pf-5 that contained a single nucleotide substitution within a predicted alpha-helica l region, which is highly conserved among the FixJ family of response regul ators. At the entrance to stationary phase, sigma(S) content in gacS and ga cA mutants of Pf-5 was less than 20% of the wild-type level. Transcription of rpoS, assessed with an rpoS-lacZ transcriptional fusion, was positively influenced by GacS and GacA, an effect that was most evident at the transit ion between exponential growth and stationary phase. Mutations in gacS and gacA compromised the capacity of stationary-phase cells of Pf-5 to survive exposure to oxidative stress. The results of this study provide evidence fo r the predominant roles of GacS and GacA in the regulatory cascade controll ing stress response and antifungal metabolite production in P. fluorescens.