The effect of polyhydroxyalkanoates in Pseudomonas chlororaphis PA23 biofilm formation, stress endurance, and interaction with the protozoan predator Acanthamoeba castellanii.

Pseudomonas chlororaphis PA23 is a biocontrol agent capable of protecting canola against the fungal pathogen Sclerotinia sclerotiorum. In addition to producing antifungal compounds, this bacterium synthesizes and accumulates polyhydroxyalkanoate (PHA) polymers as carbon and energy storage compounds. Because the role of PHA in PA23 physiology is currently unknown, we investigated the impact of this polymer on stress resistance, adherence to surfaces, and interaction with the protozoan predator Acanthamoeba castellanii. Three PHA biosynthesis mutants were created, PA23phaC1, PA23phaC1ZC2, and PA23phaC1ZC2D, which accumulated reduced PHA. Our phenotypic assays revealed that PA23phaC1ZC2D produced less phenazine (PHZ) compared with the wild type (WT) and the phaC1 and phaC1ZC2 mutants. All three mutants exhibited enhanced sensitivity to UV irradiation, starvation, heat stress, cold stress, and hydrogen peroxide. Moreover, motility, exopolysaccharide production, biofilm formation, and root attachment were increased in strains with reduced PHA levels. Interaction studies with the amoeba A. castellanii revealed that the WT and the phaC1 and phaC1ZC2 mutants were consumed less than the phaC1ZC2D mutant, likely due to decreased PHZ production by the latter. Collectively these findings indicate that PHA accumulation enhances PA23 resistance to a number of stresses in vitro, which could improve the environmental fitness of this bacterium in hostile environments.

Pseudomonas chlororaphis PA23 metabolites protect against protozoan grazing by the predator Acanthamoeba castellanii.

BACKGROUND: Pseudomonas chlororaphis strain PA23 is a biocontrol agent that is able to protect canola against the pathogenic fungus Sclerotinia sclerotiorum. This bacterium secretes a number of metabolites that contribute to fungal antagonism, including pyrrolnitrin (PRN), phenazine (PHZ), hydrogen cyanide (HCN) and degradative enzymes. In order to be successful, a biocontrol agent must be able to persist in the environment and avoid the threat of grazing predators. The focus of the current study was to investigate whether PA23 is able to resist grazing by the protozoan predator Acanthamoeba castellanii (Ac) and to define the role of bacterial metabolites in the PA23-Ac interaction. METHODS: Ac was co-cultured with PA23 WT and a panel of derivative strains for a period of 15 days, and bacteria and amoebae were enumerated on days 1, 5, 10 and 15. Ac was subsequently incubated in the presence of purified PRN, PHZ, and KCN and viability was assessed at 24, 48 and 72 h. Chemotactic assays were conducted to assess whether PA23 compounds exhibit repellent or attractant properties towards Ac. Finally, PA23 grown in the presence and absence of amoebae was subject to phenotypic characterization and gene expression analyses. RESULTS: PRN, PHZ and HCN were found to contribute to PA23 toxicity towards Ac trophozoites, either by killing or inducing cyst formation. This is the first report of PHZ-mediated toxicity towards amoebae. In chemotaxis assays, amoebae preferentially migrated towards regulatory mutants devoid of extracellular metabolite production as well as a PRN mutant, indicating this antibiotic has repellent properties. Co-culturing of bacteria with amoebae led to elevated expression of the PA23 phzI/phzR quorum-sensing (QS) genes and phzA and prnA, which are under QS control. PHZ and PRN levels were similarly increased in Ac co-cultures, suggesting that PA23 can respond to predator cues and upregulate expression of toxins accordingly. CONCLUSIONS: PA23 compounds including PRN, PHZ and HCN exhibited both toxic and repellent effects on Ac. Co-culturing of bacteria and amoebae lead to changes in bacterial gene expression and secondary metabolite production, suggesting that PA23 can sense the presence of these would-be predators and adjust its physiology in response.

Quorum sensing and the anaerobic regulator (ANR) control polyhydroxyalkanoate (PHA) production in Pseudomonas chlororaphis PA23.

Pseudomonas chlororaphis PA23 is a biocontrol agent that, in addition to producing antifungal compounds, synthesizes polyhydroxyalkanoate (PHA) polymers as a carbon and energy sink. Quorum sensing (QS) and the anaerobic regulator (ANR) are required for PA23-mediated fungal suppression; however, the role of these regulators in PHA production is unknown. Strains lacking either QS or ANR accumulated less PHA polymers when propagated on Ramsay's minimal medium (RMM) with glucose or octanoate as the carbon source. In the acyl-homoserine lactone (AHL)-deficient background, all six of the genes in the pha locus (phaC1, phaC2, phaZ, phaD, phaF, phaI) showed reduced expression in RMM glucose, and all except phaC2 were repressed in RMM octanoate. Although changes in gene activity were observed in the anr mutant, they were less pronounced. Analysis of the promoter regions for QS- and ANR-binding consensus sequences revealed putative phzboxes upstream of phaZ and phaI, but no anr boxes were identified. Our findings indicate that altered pha gene expression likely contributes to the lower PHA accumulation in the QS- and ANR-deficient strains, which may be in part indirectly mediated. This study is the first to show that mcl-PHA production is under QS and ANR control.