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Applied and Environmental Microbiology, August 2000, p. 3142-3150, Vol. 66, No. 8
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Controlling Instability in gacS-gacA Regulatory Genes during Inoculant Production of Pseudomonas fluorescens Biocontrol Strains

Brion K. Duffydagger ,* and Geneviève Défago

Phytopathology Group, Institute for Plant Sciences, Swiss Federal Institute of Technology, CH-8092 Zürich, Switzerland

Received 22 February 2000/Accepted 16 May 2000

Secondary metabolism in fluorescent pseudomonads is globally regulated by gacS, which encodes a membrane-bound sensor kinase, and gacA, which encodes a transcriptional response regulator. Spontaneous mutation in either gene blocked biosynthesis of the antimicrobial compounds hydrogen cyanide, 2,4-diacetylphloroglucinol, pyoluteorin, and pyrrolnitrin by the model biocontrol strain Pseudomonas fluorescens CHA0. Spontaneous mutants also had altered abilities to utilize several carbon sources and to increase medium pH compared with the wild type, suggesting that gacS and gacA influence primary as well as secondary bacterial metabolism. Inoculant efficacy for biocontrol was significantly reduced by contamination with regulatory mutants which accumulated during inoculum production. Spontaneous mutants accumulated in all 192 separate liquid cultures examined, typically at a frequency of 1% or higher after 12 days. During scale-up in a simulated industrial fermentation process, mutants increased exponentially and accounted for 7, 23, and 61% of the total viable cells after transfer to 20-, 100-, and 500-ml preparations, respectively. GacS- and GacA- mutants had identical phenotypes and occurred at the same frequency, indicating that the selective pressures for the two mutants were similar. We developed a simple screening method for monitoring inoculant quality based on the distinctive appearance of mutant colonies (i.e., orange color, enlarged diameter, hyperfluorescence). Mutant competitiveness was favored in a nutrient-rich medium with a high electrolyte concentration (nutrient broth containing yeast extract). We were able to control mutant accumulation and to clean up contaminated cultures by using certain mineral amendments (i.e., zinc, copper, cobalt, manganese, and ammonium molybdate) or by diluting media 1/10. Spontaneous mutants and genetic constructs had the same response to culture conditions. Zinc and medium dilution were also effective for improving the genetic stability of other P. fluorescens biocontrol strains obtained from Ghana and Italy.

* Corresponding author. Mailing address: Phytopathology Group, Institute for Plant Sciences, Swiss Federal Institute of Technology, Universitätstrasse 2, CH-8092 Zürich, Switzerland. Phone: 411-632-4836. Fax: 411-632-1108 or 411-632-1092. E-mail: brion.duffy{at}ipw.agrl.ethz.ch.

dagger Present address: Food Safety and Health Unit, Agricultural Research Service, U.S. Department of Agriculture, Albany, CA 94710.

Applied and Environmental Microbiology, August 2000, p. 3142-3150, Vol. 66, No. 8
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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