Secondary Metabolites Help Biocontrol Strain Pseudomonas fluorescens CHA0 To Escape Protozoan Grazing

Abstract

In soil ecosystems, bacteria must cope with predation activity, which is attributed mainly to protists. The development of antipredation strategies may help bacteria maintain higher populations and persist longer in the soil. We analyzed the interaction between the root-colonizing and biocontrol strain Pseudomonas fluorescens CHA0 and three different protist isolates (an amoeba, a flagellate, and a ciliate). CHA0 produces a set of antibiotics, HCN, and an exoprotease. We observed that protists cannot grow on CHA0 but can multiply on isogenic regulatory mutants that do not produce the extracellular metabolites. The in vitro responses to CHA0 cells and its exoproducts included growth inhibition, encystation, paralysis, and cell lysis. By analyzing the responses of protists to bacterial supernatants obtained from different isogenic mutants whose production of one or more exometabolites was affected and also to culture extracts with antibiotic enrichment, we observed different contributions of the phenolic antifungal compound 2,4-diacetylphloroglucinol (DAPG) and the extracellular protease AprA to CHA0 toxicity for protists and to the encystation-reactivation cycle. The grazing pressure artificially produced by a mixture of the three protists in a microcosm system resulted in reduced colonization of cucumber roots by a regulatory isogenic CHA0 mutant unable to produce toxins. These results suggest that exometabolite production in biocontrol strain CHA0 may contribute to avoidance of protist grazing and help sustain higher populations in the rhizosphere, which may be a desirable and advantageous trait for competition with other bacteria for available resources.

Soil amoebae rapidly change bacterial community composition in the rhizosphere of Arabidopsis thaliana

“…The strong and highly reproducible changes in the gacA-banding pattern further revealed a major shift in this master gene controlling antibiotics production of pseudomonads (De Souza et al, 2003). We suggest that pseudomonads quickly upregulated secondary metabolite production in response to protozoan predators, which is in accordance with Jousset et al (2006) who demonstrated that antibiotics of P. fluorescens are of particular toxicity to protozoa; and that antibiotic-producing P. fluorescens disproportionally benefit from protozoan predation when their bacterial competitors are consumed and nutrients excreted by the protozoan predators (Jousset et al, 2008).…”

“…The numbers of bacteria have been shown to decline in presence of protozoa until a dynamic equilibrium with bacterial densities of 10 5 -10 7 g À1 is reached (Danso and Alexander 1975;Habte and Alexander, 1975;Acea et al, 1988;Clarholm 1981Clarholm , 1989. However, the different bacterial taxa that constitute the rhizosphere bacterial community strongly differ in their food quality for protozoa (Bjørnlund et al, 2006;Jousset et al, 2006), suggesting taxon-specific differences in bacterial survival. In fact, studies in freshwater ecosystems uncovered a number of adaptations of bacteria against protozoan grazing, such as changes in motility, size, filament formation, surface masking or toxin production to prevent ingestion, or resistance to digestion by protozoa (Jü rgens and Matz, 2002;Pernthaler, 2005).…”

“…Soil amoebae have been shown to graze preferentially on Gram-negative bacteria (Foster and Dormaar, 1991;Andersen and Winding, 2004). Not surprising, the gacA regulated antibiotic production of pseudomonads has been found to play also a significant role in bacterial defence against protozoan predators (Jousset et al, 2006(Jousset et al, , 2008.…”

Predators promote defence of rhizosphere bacterial populations by selective feeding on non-toxic cheaters

“…Pseudomonas fluorescens CHA0 is an effective colonizer of the rhizosphere of mono-and dicotyledonous plants possessing remarkable biocontrol efficiency (Voisard et al, 1994) and high resistance to predation (Jousset et al, 2006) due to the production of an array of secondary metabolites including toxins, such as hydrogen cyanide (HCN), 2,4-diacetylphloroglucinol (DAPG) and pyoluteorin (Haas et al, 2002).…”

Evolutionary history predicts the stability of cooperation in microbial communities

“…We used the isogenic signal-blind mutant CHA0DgacS, chromosomally tagged with green fluorescent protein 43 . This mutant lacks QS and lacks extracellular proteases required for growth on proteins 30 .…”