The Two-Component Regulators GacS and GacA Influence Accumulation of the Stationary-Phase Sigma Factor ς ^S and the Stress Response in Pseudomonas fluorescens Pf-5

Abstract

Three global regulators are known to control antibiotic production by Pseudomonas fluorescens. A two-component regulatory system comprised of the sensor kinase GacS (previously called ApdA or LemA) and GacA, a member of the FixJ family of response regulators, is required for antibiotic production. A mutation inrpoS, which encodes the stationary-phase sigma factor ςS, differentially affects antibiotic production and reduces the capacity of stationary-phase cells of P. fluorescens to survive exposure to oxidative stress. ThegacA gene of P. fluorescens Pf-5 was isolated, and the influence of gacS and gacA onrpoS transcription, ςS levels, and oxidative stress response of Pf-5 was determined. We selected a gacAmutant of Pf-5 that contained a single nucleotide substitution within a predicted α-helical region, which is highly conserved among the FixJ family of response regulators. At the entrance to stationary phase, ςS content in gacS and gacAmutants of Pf-5 was less than 20% of the wild-type level. Transcription of rpoS, assessed with anrpoS-lacZ transcriptional fusion, was positively influenced by GacS and GacA, an effect that was most evident at the transition between exponential growth and stationary phase. Mutations ingacS 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 for the predominant roles of GacS and GacA in the regulatory cascade controlling stress response and antifungal metabolite production in P. fluorescens.

Genes expressed by the biological control bacterium Pseudomonas protegens Pf‐5 on seed surfaces under the control of the global regulators GacA and RpoS

“…In our study, transcript levels of rpoS (PFL_1207) did not differ significantly between the gacA mutant and wild-type Pf-5 on seed surfaces. The large degree of overlap in the genes downregulated in both mutants may be due to posttranscriptional regulation of rpoS by GacA (Whistler et al, 1998;Heeb et al, 2005). Alternatively, GacA may have had larger effects on rpoS transcript levels at an earlier time, causing residual effects on transcript levels of downstream genes that were still apparent at the time point evaluated.…”

“…The gacA/gacS and rpoS mutants of Pf-5 were selected originally due to their altered production of the antifungal compounds pyoluteorin, DAPG and pyrrolnitrin (Corbell and Loper, 1995;Sarniguet et al, 1995;Whistler et al, 1998). gacA mutants also lack production of hydrogen cyanide (Whistler et al, 1998), rhizoxin and orfamide A (Gross et al, 2007;Hassan et al, 2010). In agreement with the known effects of these global regulators on antibiotic production by Pf-5, we observed a striking influence of GacA and RpoS on the transcript levels of secondary metabolite biosynthetic genes (Figs 3 and S2).…”

“…Pyrrolnitrin Hydrogen cyanide PFL_2577-2579 Orfamide A PFL_2142-2150 Rhizoxin analogs Whistler et al, 1998). The plt genes accounted for 16 of the 21 genes upregulated in an rpoS mutant under both conditions (Table S2 and Fig.…”

The effect of zinc limitation on the transcriptome ofPseudomonas protegens Pf‐5

“…The extract was dried under reduced pressure using a SpeedVac DNA 110 (Savant) and dissolved in 250 ml of methanol. High-performance liquid chromatography (HPLC) was used to detect the secreted secondary metabolites of interest using conditions described previously (Whistler et al, 1998;Hassan et al, 2010). HPLC analysis was performed on a Shimadzu HPLC instrument equipped with an SPD-M10A diode array detector using a Phenomenex Luna C18(2) column (4.6 ¥ 250 mm, 5 mm).…”

“…Pseudomonas protegens Pf-5 (previously called Pseudomonas fluorescens Pf-5) (Ramette et al, 2011) is a soil-inhabiting biocontrol bacterium that can suppress a wide variety of plant pathogenic bacteria, fungi and oomycetes (Howell and Stipanovic, 1979;1980;Loper et al, 2007). Pf-5 suppresses the growth of these pathogens primarily via the secretion of a range of secondary metabolites such as hydrogen cyanide, pyoluteorin, pyrrolnitrin, rhizoxin analogues and 2,4-diacetylphloroglucinol (DAPG) (Howell and Stipanovic, 1979;1980;Nowak-Thompson et al, 1994;Whistler et al, 1998;Loper et al, 2008). Genome sequencing further highlighted the biocontrol properties of Pf-5, revealing that a large proportion of its genetic information is dedicated to biocontrol functions, such as the production of secondary metabolites, a number of which were previously unknown (Paulsen et al, 2005).…”