Characterization of PhlG, a hydrolase that specifically degrades the antifungal compound 2,4-diacetylphloroglucinol in the biocontrol agent Pseudomonas fluorescens CHA0

Abstract

The potent antimicrobial compound 2,4-diacetylphloroglucinol (DAPG) is a major determinant of biocontrol activity of plant-beneficial Pseudomonas fluorescens CHA0 against root diseases caused by fungal pathogens. The DAPG biosynthetic locus harbors the phlG gene, the function of which has not been elucidated thus far. The phlG gene is located upstream of the phlACBD biosynthetic operon, between the phlF and phlH genes which encode pathway-specific regulators. In this study, we assigned a function to PhlG as a hydrolase specifically degrades DAPG to equimolar amounts of mildly toxic monoacetylphloroglucinol (MAPG) and acetate. DAPG added to cultures of a DAPG-negative DeltaphlA mutant of strain CHA0 was completely degraded, and MAPG was temporarily accumulated. In contrast, DAPG was not degraded in cultures of a DeltaphlA DeltaphlG double mutant. To confirm the enzymatic nature of PhlG in vitro, the protein was histidine tagged, overexpressed in Escherichia coli, and purified by affinity chromatography. Purified PhlG had a molecular mass of about 40 kDa and catalyzed the degradation of DAPG to MAPG. The enzyme had a kcat of 33 s(-1) and a Km of 140 microM at 30 degrees C and pH 7. The PhlG enzyme did not degrade other compounds with structures similar to DAPG, such as MAPG and triacetylphloroglucinol, suggesting strict substrate specificity. Interestingly, PhlG activity was strongly reduced by pyoluteorin, a further antifungal compound produced by the bacterium. Expression of phlG was not influenced by the substrate DAPG or the degradation product MAPG but was subject to positive control by the GacS/GacA two-component system and to negative control by the pathway-specific regulators PhlF and PhlH.

FIG. 1.

Structures of three acylphloroglucinols produced by fluorescent pseudomonads. (A) Monoacetylphloroglucinol (MAPG); (B) 2,4-diacetylphloroglucinol (DAPG); (C) triacetylphloroglucinol (TAPG).

FIG. 2.

Physical location of phlG in the DAPG biosynthetic locus of P. fluorescens strain CHA0. The phlG gene is located upstream of phlA, i.e., the first DAPG biosynthetic gene, and is flanked by phlH and phlF which encode pathway-specific transcriptional regulators (16, 39). ▵, Region deleted in strains CHA1091 and CHA1092 and in plasmid pME8020. The genes are indicated by shaded arrows. For phlA, only the 5′ end is shown. The horizontal bars designate the fragments cloned into vector pME3087 to give pME8020, into pME6015 to give pME8030 and pME8031, and into pME6182 to give pME8039.

FIG. 3.

Requirement of PhlG function for degradation of DAPG to MAPG by P. fluorescens. The DAPG- and MAPG-negative mutants CHA631 (ΔphlA) (A) and CHA1092 (ΔphlA ΔphlG) (B) were grown at 27°C in KMBmalt broth supplemented with 100 μM DAPG. After different incubation periods, bacterial growth (ODs at 600 nm) (▵) and concentrations of DAPG (○) and MAPG (▪) were determined. Means ± the standard deviations from three independent cultures are shown. The experiment was repeated twice with similar results.

FIG. 4.

SDS-PAGE analysis of PhlG-His6 expression and purification. Lanes: 1, molecular mass standards; 2, crude cell extract (10 μg of protein) before induction; 3, crude cell extract 2 h after induction with 1 mM IPTG (12 μg of protein); 4, soluble fraction from the IPTG-induced cell extract (8 μg of protein); 5, purified PhlG-His6 (4 μg of protein). Cell extracts were prepared from E. coli BL21(DE3)/pME8032. The arrow indicates PhlG-His6. The SDS-15% PAGE gel was stained with Coomassie brilliant blue.

FIG. 5.

Time course of DAPG degradation by the purified PhlG-His6 protein. Portions of 50 μM DAPG were incubated with 16 nM purified PhlG-His6 enzyme in a reaction buffer at pH 7.0 and 30°C as detailed in Materials and Methods. DAPG (○) and MAPG (▪) concentrations were determined by HPLC at different time points after the start of the reaction. Means ± the standard deviations from three independent experiments are shown.

FIG. 6.

Effect of DAPG and MAPG on phlG expression in P. fluorescens CHA0. (A) β-Galactosidase expression of a phlG—lacZ translational fusion carried by pME8030 was determined in wild-type CHA0 with (▵) or without (•) addition of 100 μM DAPG and in the phlA mutant CHA631 (○). (B) Expression of phlG—lacZ in CHA0 with (▪) or without (•) addition of 100 μM MAPG. Bacteria were grown in OSGly medium at 30°C. DAPG and MAPG were dissolved in acetonitrile. Acetonitrile did not affect phlG expression (data not shown). Means ± the standard deviations from three replicate cultures are shown. The experiment was repeated twice with similar results.

FIG. 7.

PhlF, PhlH, and GacS control of phlG expression in P. fluorescens CHA0. β-Galactosidase expression of a phlG′-′lacZ translational fusion carried by pME8030 was determined in the wild-type CHA0 (•), the phlF mutant CHA638 (□), the phlH mutant CHA630 (▴), and the gacS mutant CHA19 (⋄). Strains were grown in OSGly medium at 30°C. Means ± the standard deviations from three replicate cultures are shown. The experiment was repeated twice with similar results.