Characterization of spontaneous gacS and gacA regulatory mutants of Pseudomonas fluorescens biocontrol strain CHA0

In Pseudomonas fluorescens strain CHA0, the response regulator gene gacA co ntrols expression of extracellular enzymes and antifungal secondary metabol ites, which are important for this strain's biocontrol activity in the plan t rhizosphere. Two Tn5 insertion mutants of strain CHA0 that had the same p leiotropic phenotype as gacA mutants were complemented by the gacS sensor k inase gene of P. syringae pv. syringae as well as that of P. fluorescens st rain Pf-5, indicating that both transposon insertions had occurred in the g acS gene of strain CHA0. This conclusion was supported by Southern hybridis ation using a gacS probe from strain Pf-5. Overexpression of the wild-type gacA gene partially compensated for the gacS mutation, however, the overexp ressed gacA gene was not stably maintained, suggesting that this is deleter ious to the bacterium. Strain CHA0 grown to stationary phase in nutrient-ri ch liquid media for several days accumulated spontaneous pleiotropic mutant s to levels representing 1.25% of the population; all mutants lacked key an tifungal metabolites and extracellular protease. Half of 44 spontaneous mut ants 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 tempora rily increased as the wild type decreased. In conclusion, loss of gacA func tion can confer a selective advantage on strain CHA0 under laboratory condi tions.