Abstract
In Pseudomonas fluorescens strain CHA0, the response regulator gene gacA controls expression of extracellular enzymes and antifungal secondary metabolites, which are important for this strain's biocontrol activity in the plant rhizosphere. Two Tn5 insertion mutants of strain CHA0 that had the same pleiotropic phenotype as gacA mutants were complemented by the gacS sensor kinase gene of P. syringae pv. syringae as well as that of P. fluorescens strain Pf-5, indicating that both transposon insertions had occurred in the gacS gene of strain CHA0. This conclusion was supported by Southern hybridisation using a gacS probe from strain Pf-5. Overexpression of the wild-type gacA gene partially compensated for the gacS mutation, however, the overexpressed gacA gene was not stably maintained, suggesting that this is deleterious to the bacterium. Strain CHA0 grown to stationary phase in nutrient-rich liquid media for several days accumulated spontaneous pleiotropic mutants to levels representing 1.25% of the population; all mutants lacked key antifungal metabolites and extracellular protease. Half of 44 spontaneous mutants tested were complemented by gacS, the other half were restored by gacA. Independent point and deletion mutations arose at different sites in the gacA gene. In competition experiments with mixtures of the wild type and a gacA mutant incubated in nutrient-rich broth, the mutant population temporarily increased as the wild type decreased. In conclusion, loss of gacA function can confer a selective advantage on strain CHA0 under laboratory conditions.
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Aigle B, Schneider D, Morilhat C, Vandewiele D, Dary A, Holl A-C, Simonet J-M & Decaris B (1996) An amplifiable and deletable locus of Streptomyces ambofaciens RP181110 contains a vary large gene homologous to polyketide synthase genes. Microbiology 142: 2815-2824
Albright LM, Huala E & Ausubel FM (1989) Prokaryotic signal transduction mediated by sensor and regulator protein pairs. Annu. Rev. Genet. 23: 311-336
Blumer C, Heeb S, Pessi G & Haas D (1999) Global GacA-steered control of cyanide and exoprotease production in Pseudomonas fluorescens involves specific ribosome binding sites. Proc. Natl. Acad. Sci. USA 96: 14073-14078
Corbell N & Loper JE (1995) A global regulator of secondary metabolite production in Pseudomonas fluorescens Pf-5. J. Bacteriol. 177 6230-6236
Del Sal G, Manfioletti G & Schneider C (1988) A one-tube plasmid DNA mini-preparation suitable for sequencing. Nucleic Acids Res. 16: 9878
Duffy BK & Défago G (1998) A Fusarium pathogenicity factor blocks antibiotic biosynthesis by antagonistic pseudomonads. IOBC WPRS Bull. 21(9): 145-148
Duffy BK & Défago G (1995) Influence of cultural conditions on spontaneous mutations in Pseudomonas fluorescens CHA0. Phytopathology 85: 1146
Duffy BK & Défago G (1999) Environmental factors modulating antibiotic and siderophore biosynthesis by Pseudomonas fluorescens biocontrol strains. Appl. Environ. Microbiol. 65: 2429-2438
Eriksson ARB, Andersson RA, Pirhonen M & Palva ET (1998) Two-component regulators involved in the global control of virulence in Erwinia carotovora subsp. carotovora. Mol. Plant-Microbe Interact. 11: 743-752
Frederick RD, Chin J, Bennetzen JL & Handa AK (1997) Identification of a pathogenicity locus, rpfA, in Erwinia carotovora subsp. carotovora that encodes a two-component sensor-regulator protein. Mol. Plant-Microbe Interact. 10: 407-415
Gaffney TD, Lam ST, Ligon J, Gates K, Frazelle A, DiMaio J, Hill S, Goodwin S, Torkewitz N, Allshouse AM, Kempf H-J & Becker JO (1994) Global regulation of expression of antifungal factors by a Pseudomonas fluorescens biological control strain. Mol. Plant-Microbe Interact. 7: 455-463
Gerhardt P, Murray RGE, Costilow RN, Nester EW, Wood WA, Krieg NR & Phillips GB (1981) Manual of Methods for General Bacteriology. American Society for Microbiology, Washington, DC
Grewal SIS, Han B & Johnstone K (1995) Identification and characterization of a locus which regulates multiple functions in Pseudomonas tolaasii, the cause of brown blotch disease of Agaricus bisporus. J. Bacteriol. 177: 4658-4668
Gubba S, Xie Y-H & Das A (1995) Regulation of Agrobacterium tumefaciens virulence gene expression: Isolation of a mutation that restores virGD52E function. Mol. Plant-Microbe Interact. 8: 788-791
Haas D, Blumer C & Keel C (2000) Biocontrol ability of fluorescent pseudomonads genetically dissected: importance of positive feedback regulation. Curr. Opin. Biotechnol. 11: 290-297
Handelsman J & Stabb EV (1996) Biocontrol of soilborne plant pathogens. Plant Cell 8: 1855-1869
Hirano SS, Ostertag EM, Savage SA, Baker LS, Willis DK & Upper CD (1997) Contribution of the regulatory gene lemA to field fitness of Pseudomonas syringae pv. syringae. Appl. Environ. Microbiol. 63: 4304-4312
Hrabak EM & Willis DK (1992) The gacS gene required for pathogenicity of Pseudomonas syringae pv. syringae on bean is a member of a family of two-component regulators. J. Bacteriol. 174: 3011-3020
Jin S, Prusti RK, Roitsch T, Ankenbauer RG & Nester EW (1990) Phosphorylation of the VirG protein of Agrobacterium tumefaciens by the autophosphorylated VirA protein: Essential role in biological activity of VirG. J. Bacteriol. 172: 4945-4950
Johnston C, Pegues DA, Hueck CJ, Lee CA & Miller SI (1996) Transcriptional activation of Salmonella typhimurium invasion genes by a member of the phosphorylated response-regulator superfamily. Mol. Microbiol. 22: 715-727
Keel C & Défago G (1997) Interactions between beneficial soil bacteria and root pathogens: Mechanisms and ecological impact. In: Gange AC & Brown VK (Eds). Multitrophic interactions in terrestrial systems (pp 27-46). Blackwell Scientific Publishers, London
Keel C, Voisard C, Berling CH, Kahr G & Défago G (1989) Iron suf-ficiency, a prerequisite for the suppression of tobacco black root rot by Pseudomonas fluorescens strain CHA0 under gnotobiotic conditions. Phytopathology 79: 584-589
Keel C, Schnider U, Maurhofer M, Voisard C, Laville J, Burger U, Wirthner P, Haas D & Défago G (1992) Suppression of root diseases by Pseudomonas fluorescens CHA0: importance of the bacterial secondary metabolite 2,4-diacetylphloroglucinol. Mol. Plant-Microbe Interact. 5: 4-13
King EO, Ward MK & Raney DE (1954) Two simple media for the demonstration of pyocyanin and fluorescin. J. Lab. Clin. Med. 44: 301-307
Kitten T, Kinscherf TG, McEvoy JL & Willis DK (1998) A newlyidentified regulator is required for virulence and toxin production in Pseudomonas syringae. Mol. Microbiol. 28: 917-930
Laville J, Voisard C, Keel C, Maurhofer M, Défago G & Haas D (1992) Global control in Pseudomonas fluorescens mediating antibiotic synthesis and suppression of black root rot of tobacco. Proc. Natl. Acad. Sci. USA 89: 1562-1566
Liao C-H, McCallus DE, Wells JM, Tzean S-S & Kang G-Y (1996) The repB gene required for production of extracellular enzymes and fluorescent siderophores in Pseudomonas viridiflava is an analog of the gacA gene of Pseudomonas syringae. Can. J. Microbiol 42: 177-182
Ligon JM, Hill DS, Hammer PE & Torkewitz NR (1999) Genetic modifications of Pseudomonas that enhance biological disease control. Acta Horticulturae 504: 53-60
Maurhofer M, Keel C, Haas D & Défago G (1994) Pyoluteorin production by Pseudomonas fluorescens strain CHA0 is involved in the suppression of Pythium damping-off of cress but not of cucumber. Eur. J. Plant Pathol. 100: 221-232
Moore JB, Shiau S-P & Reitzer LJ (1993) Alterations of highly conserved residues in the regulatory domain of nitrogen regulator I (NtrC) of Escherichia coli. J. Bacteriol. 175: 2692-2701
Oberhänsli T, Défago G & Haas D (1991) Indole-3-acetic acid (IAA) synthesis in the biocontrol of strain CHA0 of Pseudomonas fluorescens: role of tryptophan side chain oxidase. J. Gen. Microbiol. 137: 2273-2279
Parkinson JS & Kofoid EC (1992) Communication modules in bacterial signaling proteins. Annu. Rev. Genet. 26: 71-112
Perraud A-L, Weiss V & Gross R (1999) Signalling pathways in two-component phosphorelay systems. Trends Microbiol. 7: 115-120
Pirrung MC (1999) Histidine kinases and two-component signal transduction systems. Chemistry & Biology 6: R167-R175
Reimmann C, Beyeler M, Latifi A, Winteler H, Foglino M, Lazdunski A & Haas D (1997) The global activator GacA of Pseudomonas aeruginosa PAO positively controls the production of the autoinducer N-butyryl-homoserine lactone and the formation of the virulence factors pyocyanin, cyanide, and lipase. Mol. Microbiol. 24: 309-319
Reyrat J-M, David M, Batut J & Boistard P (1994) FixL of Rhizobium meliloti enhances the transcriptional activity of a mutant FixJD54N protein by phosphorylation of an alternate residue. J. Bacteriol. 176: 1969-1976
Rich JJ, Kinscherf TG, Kitten T & Willis DK (1994) Genetic evidence that the gacA gene encodes the cognate response regulator for the gacS sensor in Pseudomonas syringae. J. Bacteriol. 176: 7468-7475
Sacherer P, Défago G & Haas D (1993) Extracellular protease and phospholipase C are controlled by the global regulatory gene gacA in the biocontrol strain Pseudomonas fluorescens CHA0. FEMS Microbiol. Lett. 116: 155-160
Sambrook J, Fritsch EF & Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
Sanger F, Nicklen S & Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74: 5463-5467
Scheeren-Groot EP, Rodenburg KW, Den Dulk-Ras A, Turk SCHJ & Hooykaas PJJ (1994) Mutational analysis of the transcriptional activator VirG of Agrobacterium tumefaciens. J. Bacteriol. 176: 6418-6426
Schnider-Keel U, Seematter A, Maurhofer M, Blumer C, Duffy B, Gigot-Bonnefoy C, Reimmann C, Notz R, Défago G, Haas D & Keel C (2000) Autoinduction of 2,4-diacetylphloroglucinol biosynthesis in the biocontrol agent Pseudomonas fluorescens CHA0 and repression by the bacterial metabolites salicylate and pyoluteorin. J. Bacteriol. 182: 1215-1225
Stanisich VA & Holloway BW (1972) A mutant sex factor of Pseudomonas aeruginosa. Genet. Res. 19: 91-108
Staskawicz B, Dahlbeck D, Keen N & Napoli C (1987) Molecular characterization of cloned avirulence genes from race 0 and race 1 of Pseudomonas syringae pv. glycinea. J. Bacteriol. 169: 5789-5794
Thomashow LS & Weller DM (1995) Current concepts in the use of introduced bacteria for biological disease control. In: Stacey G & Keen N (Eds). Plant-microbe interactions, vol. 1 (pp 187-235). Chapman & Hall, New York, NY
Voisard C, Rella M & Haas D (1988) Conjugative transfer of plasmid RP1 to soil isolates of Pseudomonas fluorescens is facilitated by certain large RP1 deletions. FEMS Microbiol. Lett. 55: 9-14
Voisard C, Keel C, Haas D & Défago G (1989) Cyanide production by Pseudomonas fluorescens helps suppress black root rot of tobacco under gnotobiotic conditions. EMBO J. 8: 351-358
Voisard C, Bull CT, Keel C, Laville J, Maurhofer M, Schnider U, Défago G & Haas D (1994) Biocontrol of root diseases by Pseudomonas fluorescens CHA0: current concepts and experimental approaches. In: O'Gara F, Dowling D & Boesten B (Eds). Molecular ecology of rhizosphere microorganisms (pp 67-89). VCH Publishers, Weinheim, Germany
Vogel HJ & Bonner D (1956) Acetylornithinase of Escherichia coli: Partial purification and some properties. J. Biol. Chem. 218: 97-106
Weller DM (1988) Biological control of soilborne plant pathogens in the rhizosphere with bacteria. Annu. Rev. Phytopathol. 26: 379-407
Whistler CA, Corbell NA, Sarniguet A, Ream W & Loper JE (1998) The two-component regulators GacS and GacA influence accumulation of the stationary-phase sigma factor σS and the stress response in Pseudomonas fluorescens Pf-5. J. Bacteriol. 180: 6635-6641
Yanisch-Perron C, Vieira J & Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33: 103-119
Zambrano MM & Kolter R (1996) GASPing for life in stationary phase. Cell 86: 181-184
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Bull, C.T., Duffy, B., Voisard, C. et al. Characterization of spontaneous gacS and gacA regulatory mutants of Pseudomonas fluorescens biocontrol strain CHA0. Antonie Van Leeuwenhoek 79, 327–336 (2001). https://doi.org/10.1023/A:1012061014717
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DOI: https://doi.org/10.1023/A:1012061014717