Altering the ratio of phenazines in Pseudomonas chlororaphis (aureofaciens) strain 30-84: effects on biofilm formation and pathogen inhibition

Abstract

Pseudomonas chlororaphis strain 30-84 is a plant-beneficial bacterium that is able to control take-all disease of wheat caused by the fungal pathogen Gaeumannomyces graminis var. tritici. The production of phenazines (PZs) by strain 30-84 is the primary mechanism of pathogen inhibition and contributes to the persistence of strain 30-84 in the rhizosphere. PZ production is regulated in part by the PhzR/PhzI quorum-sensing (QS) system. Previous flow cell analyses demonstrated that QS and PZs are involved in biofilm formation in P. chlororaphis (V. S. R. K. Maddula, Z. Zhang, E. A. Pierson, and L. S. Pierson III, Microb. Ecol. 52:289-301, 2006). P. chlororaphis produces mainly two PZs, phenazine-1-carboxylic acid (PCA) and 2-hydroxy-PCA (2-OH-PCA). In the present study, we examined the effect of altering the ratio of PZs produced by P. chlororaphis on biofilm formation and pathogen inhibition. As part of this study, we generated derivatives of strain 30-84 that produced only PCA or overproduced 2-OH-PCA. Using flow cell assays, we found that these PZ-altered derivatives of strain 30-84 differed from the wild type in initial attachment, mature biofilm architecture, and dispersal from biofilms. For example, increased 2-OH-PCA production promoted initial attachment and altered the three-dimensional structure of the mature biofilm relative to the wild type. Additionally, both alterations promoted thicker biofilm development and lowered dispersal rates compared to the wild type. The PZ-altered derivatives of strain 30-84 also differed in their ability to inhibit the fungal pathogen G. graminis var. tritici. Loss of 2-OH-PCA resulted in a significant reduction in the inhibition of G. graminis var. tritici. Our findings suggest that alterations in the ratios of antibiotic secondary metabolites synthesized by an organism may have complex and wide-ranging effects on its biology.

FIG. 1.

(A) Simplified PZ biosynthetic pathway. PZs are derived from chorismate precursors, of which PCA is the primary PZ compound synthesized. Subsequently PCA is hydroxylated by the phzO product, a monooxygenase. (B) Liquid cultures of the PZ derivative mutants compared to wild-type 30-84. Cultures were grown in AB-CAA with selection in 96-well plates. Images of strains 30-84, 30-84O*, and 30-84PCA after 24 h are shown.

FIG. 2.

Effect of altered PZ ratio on the initial attachment stage of biofilm formation. The early attachment phase of biofilm formation by strains 30-84, 30-84PCA, and 30-84O* was observed and analyzed in a flow cell assay by taking light microscope images at early time points. Five random images for each treatment and time point combination were taken. Each experiment was repeated twice, and representative data are presented. Representative images were taken 45 min and 6 h after the start of medium flow. All the images were obtained at a magnification of ×1,000 using an Olympus BX60 light microscope.

FIG. 3.

Effect of altered PZ ratio on biofilm architecture. Mature biofilm architecture was observed and analyzed in a flow cell assay using fluorescently labeled derivatives of strains 30-84, 30-84PCA, and 30-84O*. (A and B) Bird's-eye (A) and cross-sectional (B) three-dimensional images obtained from CLSM of 5-day-old mature biofilms of wild-type 30-84, 30-84PCA, and 30-84O*. Random Z-series image stacks were obtained using a Nikon E800 CLSM at a magnification of ×400 and rendered to obtain three-dimensional images using Volocity software. (C) Basal view of the 5-day-old mature biofilms of wild-type 30-84, 30-84PCA, and 30-84O*. Images were taken using an Olympus BX60 light microscope at a magnification of ×100.