Secondary Metabolite Production by Pseudomonas Fluorescens Strain Pf-5 Confers Protection Against Naegleria Americana In the Wheat Rhizosphere.

Bacteria employ a variety of morphological and metabolic mechanisms to avoid protozoan predation. In Pseudomonas fluorescens strains SS101 and SBW25, cyclic lipopeptide (CLP) production served as a defense mechanism that limited predation by the amoeba-flagellate Naegleria americana, and secondary metabolites produced by certain strains of P. fluorescens limited predation in vitro. We found that a combination of secondary metabolites protect P. fluorescens strain Pf-5 against predation by N. americana within the rhizosphere of wheat. Four mutants of P. fluorescens Pf-5 possessing deletions in specific secondary metabolite biosynthetic gene clusters exhibited significantly decreased fitness compared to the wild type strain when cultured in the presence of N. americana. Unlike P. fluorescens strains SS101 and SBW25, the CLP orfamide A did not appear to provide P. fluorescens Pf-5 predation protection against N. americana, as in the presence of N. americana a mutant deficient in CLP production survived at levels equivalent to the wild type strain. The Pf-5 mutants tended to have greater fitness in the wheat rhizosphere than the wild type strain when assays were conducted in pasteurized soil in the absence of predators, indicating the metabolic expense of producing these secondary metabolites. When the same assays were conducted in the presence of N. americana, mutants that overproduced the antibiotic compound pyoluteorin had similar fitness as the wild type phenotype. Survival of mutants possessing deletions in a gene producing a known insect toxin or an exoprotease was significantly diminished in the presence of N. americana relative to the wild type strain. It appears that a suite of secondary metabolites rather than one specific compound provides the greatest protection against protozoan predation in the rhizosphere.