| Abstract: | Soil bacteria are heavily consumed by protozoan predators, and many bacteria have evolved defense strategies such as the production of toxic exometabolites. However, the production of toxins is energetically costly and therefore is likely to be adjusted according to the predation risk to balance the costs and benefits of predator defense. We investigated the response of the biocontrol bacterium Pseudomonas fluorescens CHA0 to a common predator, the free-living amoeba Acanthamoeba castellanii. We monitored the effect of the exposure to predator cues or direct contact with the predators on the expression of the phlA, prnA, hcnA, and pltA genes, which are involved in the synthesis of the toxins, 2,4-diacetylphloroglucinol (DAPG), pyrrolnitrin, hydrogen cyanide, and pyoluteorin, respectively. Predator chemical cues led to 2.2-, 2.0-, and 1.2-fold increases in prnA, phlA, and hcnA expression, respectively, and to a 25% increase in bacterial toxicity. The upregulation of the tested genes was related to the antiprotozoan toxicity of the corresponding toxins. Pyrrolnitrin and DAPG had the highest toxicity, suggesting that bacteria secrete a predator-specific toxin cocktail. The response of the bacteria was elicited by supernatants of amoeba cultures, indicating that water-soluble chemical compounds were responsible for induction of the bacterial defense response. In contrast, direct contact of bacteria with living amoebae reduced the expression of the four bacterial toxin genes by up to 50%, suggesting that protozoa can repress bacterial toxicity. The results indicate that predator-prey interactions are a determinant of toxin production by rhizosphere P. fluorescens and may have an impact on its biocontrol potential.Bacterial communities are heavily consumed by protozoan predators (30), and predation is a major force shaping the structure of microbial communities in both aquatic and terrestrial ecosystems (34, 35). The competitiveness of bacteria strongly depends on their ability to avoid predation (9, 22), and many species have developed defense mechanisms such as the production of toxins, which reduces mortality by repelling or killing predators (21, 24). Toxin production, however, is energetically costly, and defense theory predicts that prey species should optimize the investment in defense according to the resources available and the predation risk (40), for example, in response to predator-associated chemical cues (4, 15). In bacteria the production of defense traits is tightly regulated by various sensor cascades (11), and defense mechanisms, such as the formation of inedible morphotypes or microcolonies, can be elicited in the presence of predators (45).Toxin production is one of the most powerful defense strategies, and in the present study we tested whether bacteria can also modulate the production of toxic secondary metabolites in response to protozoan predators. We investigated the chemical communication between the soil bacterium Pseudomonas fluorescens CHA0 and the bacterivorous amoeba Acanthamoeba castellanii, a ubiquitous soil protist feeding on a wide range of bacterial species (33). P. fluorescens CHA0 effectively colonizes the rhizosphere of plants and produces various extracellular toxins including pyrrolnitrin (PRN), 2,4-diacetylphloroglucinol (DAPG), hydrogen cyanide (HCN), and pyoluteorin (PLT) (18). These toxins reduce predation pressure and enhance the competitiveness of the bacteria in the rhizosphere (22) but also act antagonistically against plant pathogens, thereby promoting plant growth (11).The production of secondary metabolites by P. fluorescens is a dynamic process that depends on environmental factors, such as nutrient availability (12), cell density (18), or the presence of phytopathogens (27). We hypothesized that P. fluorescens is also able to sense predators and responds by increasing the expression of toxin genes.Predators or competitors susceptible to toxins can adopt counterstrategies to repress their production. For example, the fungal pathogen Fusarium can inhibit the production of DAPG by pseudomonads (26), and we hypothesized that A. castellanii can counteract prey defense by inhibiting bacterial toxin production.We investigated the effects of predator-prey interactions on the regulation of the production of the extracellular toxins DAPG, PLT, PRN, and HCN using a set of autofluorescent green fluorescent protein (GFP)- and mCherry-based reporter fusions (2, 32). Autofluorescent reporter fusions allow in situ measurement of gene expression patterns and have been applied to monitor the expression of antifungal genes in the rhizosphere (10, 32). The response of the bacteria to predators or associated signal molecules was investigated in batch experiments and on barley roots. |
