The two-component regulators GacS and GacA influence accumulation of the stationary-phase sigma factor sigmaS 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 in rpoS, which encodes the stationary-phase sigma factor sigmaS, differentially affects antibiotic production and reduces the capacity of stationary-phase cells of P. fluorescens to survive exposure to oxidative stress. The gacA gene of P. fluorescens Pf-5 was isolated, and the influence of gacS and gacA on rpoS transcription, sigmaS levels, and oxidative stress response of Pf-5 was determined. We selected a gacA mutant of Pf-5 that contained a single nucleotide substitution within a predicted alpha-helical region, which is highly conserved among the FixJ family of response regulators. At the entrance to stationary phase, sigmaS content in gacS and gacA 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 transition 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 for the predominant roles of GacS and GacA in the regulatory cascade controlling stress response and antifungal metabolite production in P. fluorescens.

FIG. 1

Relative ς^S accumulation. ς^S was visualized with antibodies to E. coli ς^S (46) from Western blots of protein extracted from cultures grown in LB medium (A and B) or M9 containing 0.4% glucose (C and D). ς^S content in each lane was estimated by scanning Western blots with a densitometer. For each sample, ς^S content was normalized to total protein content, determined from Bradford assays. The normalized ς^S content (ς^S content divided by total protein content) for each sample is reported relative to the normalized ς^S content of stationary-phase cells of Pf-5 grown in the corresponding medium (B and D, lanes 2). Sample numbers correspond to extracts from cells growing exponentially (first of each pair; lanes 1, 3, 5, 7, and 9) or in early stationary phase (second of each pair; lanes 2, 4, 6, 8, and 10): 1 and 2, Pf-5; 3 and 4, JL3985 (rpoS::Tn5); 5 and 6, JL4135 (gacS::Tn5); 7 and 8, JL4477 [gacA(V203)]; 9 and 10, Pf-5 harboring pJEL5649, a multiple-copy plasmid containing rpoS cloned from Pf-5. Scanned images were reproduced for publication by using Adobe Photoshop version 4.0 (Adobe Systems Incorporated, San Jose, Calif.).

FIG. 2

Relative GacS accumulation. GacS was visualized with antibodies to GacS from P. syringae (41) on the Western blots used for quantification of ς^S (Fig. 1). Protein was extracted from cultures grown in LB medium (A and B) or M9 containing 0.4% glucose (C and D). GacS content in each lane was estimated and reported as normalized values (B and D, lanes 2). Sample numbers correspond to extracts from cells growing exponentially (first of each pair; lanes 1, 3, 5, 7, and 9) or in early stationary phase (second of each pair; lanes 2, 4, 6, 8, and 10): 1 and 2, Pf-5; 3 and 4, JL3985 (rpoS::Tn5); 5 and 6, JL4135 (gacS::Tn5); 7 and 8, JL4477 [gacA(V203)]; 9 and 10, Pf-5 harboring pJEL5649, a multiple-copy plasmid containing rpoS cloned from Pf-5. The truncated form of GacS was not quantified in JL4135 (gacS::Tn5). Scanned images were reproduced for publication by using Adobe Photoshop version 4.0 (Adobe Systems Incorporated).

FIG. 3

Growth and β-galactosidase activity of Pf-5 derivatives containing chromosomal rpoS-lacZ transcriptional fusions. OD₆₀₀ (•) and β-galactosidase activity expressed in Miller units (■) were determined at 1-h intervals from duplicate cultures grown in LB medium. Bacterial strains were JL4489 (rpoS::lacZ) (A), JL4491 (gacS::Tn5 rpoS::lacZ) (B), and JL4492 [gacA(V203) rpoS::lacZ] (C). Error bars representing standard deviations may be obscured by symbols.

FIG. 4

Survival of oxidative stress. Stationary-phase cells of P. fluorescens Pf-5 (■), JL4135 (gacS::Tn5) (•), and JL4477 [gacA(V203)] (▴) were exposed to 15 mM H₂O₂, and the numbers of culturable cells were estimated over time. Presented values are means of three replicate cultures; error bars representing standard deviations may be obscured by symbols.