Global control in Pseudomonas fluorescens mediating antibiotic synthesis and suppression of black root rot of tobacco ( secondary metabolites / hydrogen cyanide / 2 , 4 - diacetylphloroglucinol / two - component regulatory systems / uvrC ) JACQUES

Pseudomonas fluorescens CHAO colonizes plant roots, produces several secondary metabolites in stationary growth phase, and suppresses a number of plant diseases, including Thielaviopsis basicola-induced black root rot of tobacco. We discovered that mutations in a P. fluorescens gene named gacA (for global antibiotic and cyanide control) pleiotropically block the production of the secondary metabolites 2,4-diacetylphloroglucinol (Phl), HCN, and pyoluteorin. The gacA mutants of strain CHAO have a drastically reduced ability to suppress black root rot under gnotobiotic conditions, supporting the previous observations that the antibiotic Phl and HCN individually contribute to the suppression of black root rot. The gacA gene is directly followed by a uvrC gene. Double gacA-uvrC mutations render P. fluorescens sensitive to UV irradiation. The gacA-uvrC cluster is homologous to the orf-2 (= uvrY)-uvrC operon of Escherichia coli. The gacA gene specifies a trans-active 24-kDa protein. Sequence data indicate that the GacA protein is a response regulator in the FixJ/DegU family of two-component regulatory systems. Expression of the gacA gene itself was increased in stationary phase. We propose that GacA, perhaps activated by conditions of restricted growth, functions as a global regulator of secondary metabolism in P. fluorescens. Soil bacteria of the genus Pseudomonas synthesize a wide variety of antibiotic compounds (1-3). There is growing evidence that some of these compounds play an important role in the biological control of plant diseases (2-5). Strains of Pseudomonas fluorescens that effectively suppress root diseases caused by soil-borne fungi have been isolated from plant roots in various parts of the world. Most, if not all, of these strains synthesize one or several antibiotic compounds (3). Phenazine-1-carboxylic acid, an antibiotic having activity against phytopathogenic fungi, is produced by the fluorescent Pseudomonas strains 2-79 and 30-84 in the rhizosphere of wheat. Phenazine-nonproducing mutants have a significantly reduced ability to suppress take-all, a wheat disease caused by Gaeumannomyces graminis var. tritici (6-8). 2,4Diacetylphloroglucinol (Phl) is another antibiotic whose production by P. fluorescens can be detected in the rhizosphere ofwheat (9). A mutant ofP.fluorescens strain CHAO blocked in Phl synthesis lacks part ofthe suppressive effect on take-all of wheat and on black root rot of tobacco. In vitro, the Phlmutant shows diminished inhibition of the corresponding fungal pathogens, G. graminis var. tritici and Thielaviopsis basicola (2, 9). HCN (10), oomycin (5, 11), pyoluteorin (12), and pyrrolnitrin (13) are further antibiotic compounds that are produced by rhizosphere isolates of Pseudomonas species and that may contribute to the suppression of root diseases. Conditions of restricted growth or stationary phase generally favor the production of antibiotics and other secondary metabolites. In Bacillus and Streptomyces species global control elements involved in antibiotic synthesis have been identified (14-17). In Pseudomonas species, by contrast, little is known about the regulatory mechanisms governing antibiotic production. We have therefore decided to characterize a mutant ofP. fluorescens CHAO that is pleiotropically defective in the synthesis of three secondary metabolitesPhl, pyoluteorin, and HCN. We have previously shown that Phl as well as cyanide are important for the suppression of black root rot of tobacco by strain CHAO (9, 10), whereas pyoluteorin probably helps suppress Pythium ultimuminduced root disease ofcucumber (18). Here we show that the pleiotropic loss of the three secondary metabolites in P. fluorescens CHAO is due to a mutation in a putative activator$ of a two-component regulatory system. This defect essentially disables P. fluorescens to suppress black root rot of tobacco. MATERIALS AND METHODS Microorganisms and Plasmids. Derivatives of P. fluorescens CHAO (19) isolated in this study are shown in Fig. 1. The sources of Escherichia coli strains used for cloning and of T. basicola have been given previously (10, 19). Recombinant plasmids (Fig. 1) were constructed in cosmid pVK100 (21) and mobilized with the helper plasmid pME497 (10, 22) from E. coli to P. fluorescens. TnS-259 mutagenesis was carried out with pME12 (22). Expression vectors pMS119EH and pMS119HE were kindly provided by E. Lanka (MaxPlanck-Institut fur Molekulare Genetik, Berlin). Growth Conditions and DNA Manipulation. Growth media have been described (10, 19, 22). Standard methods were used for plasmid extraction by alkaline lysis and for cloning (23). Nucleotide sequences were determined for both strands by the chain-termination method with Sequenase Version 2.0 and 7-deaza-dGTP (United States Biochemical) according to the manufacturer's instructions; single-stranded DNAs generated from derivatives ofM13mpl8 and M13mpl9 (24) were purified on Sephaglas (PhagePrep Kit, Pharmacia). Gene replacements in P. fluorescens were performed with a ColE1based, mobilizable suicide vector (details to be given elsewhere) essentially as described (10). The CLUSTAL and FASTSCAN programs (PC-Gene, Release 6.5; IntelliGenetics) were used to align amino acid sequences and to find homologies with proteins in the Swiss-Prot data bank. Abbreviations: IPTG, isopropyl /-D-thiogalactoside; ORF, open reading frame; Phl, 2,4-diacetylphloroglucinol. tPresent address: Department of Plant Pathology, University of Wisconsin, 1630 Linden Drive, Madison, WI 53706. §To whom reprint requests should be addressed. $The sequence reported in this paper has been deposited in the GenBank data base (accession no. M80913). 1562 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 89 (1992) 1563