Positive autoregulation and signaling properties of pyoluteorin, an antibiotic produced by the biological control organism Pseudomonas fluorescens Pf-5

Abstract

Pseudomonas fluorescens Pf-5, a rhizosphere bacterium, produces a suite of secondary metabolites that are toxic to seed- and root-rotting plant pathogens. Among these are the polyketide compounds pyoluteorin and 2,4-diacetylphloroglucinol. We provide evidence that pyoluteorin production is influenced by positive autoregulation. Addition of pyoluteorin to liquid cultures of Pf-5 enhanced pyoluteorin production. In addition, pyoluteorin and 2,4-diacetylphloroglucinol mutually inhibit one another's production in Pf-5. For pyoluteorin, both positive autoregulation and negative influences on production by 2,4-diacetylphloroglucinol were demonstrated at the transcriptional level by measuring activity from transcriptional fusions of an ice nucleation reporter gene (inaZ) to three separate pyoluteorin biosynthetic genes. The occurrence of pyoluteorin autoregulation in the rhizosphere was assessed on cucumber seedlings in pasteurized soil with cross-feeding experiments. In the rhizosphere, expression of a pyoluteorin biosynthesis gene by a pyoluteorin-deficient mutant of Pf-5 was enhanced by pyoluteorin produced by coinoculated cells of Pf-5. These data establish that the polyketide pyoluteorin is an autoregulatory compound and functions as a signal molecule influencing the spectrum of secondary metabolites produced by the bacterial cell.

FIG. 1.

Structures of pyoluteorin (a) and 2,4-diacetylphloroglucinol (b).

FIG. 2.

Influence of exogenous pyoluteorin on pyoluteorin and 2,4-diacetylphloroglucinol production by derivatives of Pf-5. Cultures of Pf-5 iceC⁺ (•,○) and Pf-5 pltB::inaZ (▿,▾) were grown in nonamended NBGly (filled symbols) or in NBGly amended with 4 μg of pyoluteorin/ml (open symbols). At the indicated sampling times following inoculation, pyoluteorin concentrations (a), 2,4-diacetylphloroglucinol concentrations (b), and population densities of cultures (c) were assessed. Population sizes did not differ significantly between pyoluteorin-amended and nonamended treatments at any time point. All three panels represent data from the same experiment. Error bars denote one standard error.

FIG. 3.

Influence of exogenous 2,4-diacetylphloroglucinol on pyoluteorin production by derivatives of Pf-5. Cultures of Pf-5 iceC⁺ (•,○) and Pf-5 pltB::inaZ (▿,▾) were grown in nonamended NBGly (filled symbols) or in NBGly amended with 12.5 μg of 2,4-diacetylphloroglucinol/ml (open symbols). At the indicated sampling times following inoculation, pyoluteorin concentrations (a) and population densities of cultures (b) were assessed. Population sizes did not differ significantly between 2,4-diacetylphloroglucinol-amended and nonamended treatments at any time point. Error bars denote one standard error. Symbols denoting pyoluteorin concentrations for unamended Pf-5 pltB::inaZ cultures are occluded by those denoting 2,4-diacetylphloroglucinol-amended Pf-5 pltB::inaZ cultures.

FIG. 4.

Minimum effective concentration of pyoluteorin for autoinduction in Pf-5. Pyoluteorin concentrations (a) were measured in Pf-5 (•) cultured for 18 h in NBGly that was amended with the concentrations of pyoluteorin indicated on the abscissa. To determine how much of the recovered pyoluteorin could be attributed to the original medium amendments, pyoluteorin was also measured in cultures of the pyoluteorin-deficient strain Pf-5 pltB::inaZ (○) that had been amended with identical amounts of pyoluteorin. Filled symbols connected by a solid line (—•—) represent actual concentrations of pyoluteorin recovered from Pf-5, and filled symbols connected by a dashed line (- - -•- - -) represent pyoluteorin that resulted from biosynthesis by Pf-5, approximated as (pyoluteorin recovered from Pf-5 cultures) − (pyoluteorin recovered from Pf-5 pltB::inaZ cultures at the same amendment rate). CFU (b) were also enumerated in each sample. Pyoluteorin amendments did not significantly alter population sizes. Differing letters above symbols denote treatments significantly different from one another (P ≤ 0.05; two-sample t test). Error bars denote one standard error.

FIG. 5.

Minimum effective concentration of 2,4-diacetylphloroglucinol for repression of pyoluteorin production in Pf-5. Pyoluteorin concentrations (a) and CFU (b) were measured in Pf-5 after culturing for 18 h in NBGly that was amended with the concentrations of 2,4-diacetylphloroglucinol indicated on the abscissa. 2,4-Diacetylphloroglucinol amendments did not significantly alter population sizes. Differing letters above symbols denote treatments significantly different from one another (P ≤ 0.05; two-sample t test). Error bars denote one standard error.

FIG. 6.

Positive and negative influences by pyoluteorin and 2,4-diacetylphloroglucinol, respectively, on transcriptional activity within the pyoluteorin biosynthetic gene cluster, assessed from transcriptional fusions of the ice nucleation reporter gene (inaZ) to three pyoluteorin biosynthetic genes (pltB, pltE, and pltF). Panels a to c depict the response of individual reporter strains to exogenous pyoluteorin and 2,4-diacetylphloroglucinol. Pf-5 pltB::inaZ (▪) (a); Pf-5 pltE::inaZ (▴) (b); and Pf-5 pltF::inaZ (•) (c); were grown in unamended NBGly (solid lines) or in NBGly amended with 4 μg of pyoluteorin/ml (dotted lines) or 12.5 μg of 2,4-diacetylphloroglucinol/ml (dashed lines). Population densities (d) are shown for all strains and did not differ significantly among treatments at any time point. Asterisks above symbols denote treatment means which differ significantly from the mean of the unamended treatment at the identical time point (P ≥ 0.05; Student's t test). Error bars denote one standard error.

FIG. 7.

Autoinduction of pyoluteorin as a result of cross-feeding in the rhizosphere. A pyoluteorin-deficient reporter strain (Pf-5 pltB::inaZ) was coinoculated onto cucumber seedlings at a cell density ratio of 1:10 with each of the following inducing strains: Pf-5 (parental strain [▾]), Pf-5 pltR::aacC1 (pyoluteorin deficient [•]) and Pf-5 rpoS::lacZ (pyoluteorin overproducer [▪]). After seedlings were grown in pasteurized soil for the durations indicated, bacteria were harvested and evaluated for INA conferred by Pf-5 pltB::inaZ in the presence of each coinoculant. Values represent the mean of 10 replicate seedlings, and error bars indicate one standard error, which are obscured by the symbols for some time points.