Affiliation:
1. Department of Fundamental Microbiology, University of Lausanne, CH-1015 Lausanne, Switzerland
Abstract
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 Δ
phlA
mutant of strain CHA0 was completely degraded, and MAPG was temporarily accumulated. In contrast, DAPG was not degraded in cultures of a Δ
phlA
Δ
phlG
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
k
cat
of 33 s
−1
and a
K
m
of 140 μM at 30°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.
Publisher
American Society for Microbiology
Subject
Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology
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