Autoinduction of 2,4-Diacetylphloroglucinol Biosynthesis in the Biocontrol Agent Pseudomonas fluorescens CHA0 and Repression by the Bacterial Metabolites Salicylate and Pyoluteorin

Abstract

The antimicrobial metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) contributes to the capacity of Pseudomonas fluorescens strain CHA0 to control plant diseases caused by soilborne pathogens. A 2,4-DAPG-negative Tn5 insertion mutant of strain CHA0 was isolated, and the nucleotide sequence of the 4-kb genomic DNA region adjacent to the Tn5 insertion site was determined. Four open reading frames were identified, two of which were homologous to phlA, the first gene of the 2,4-DAPG biosynthetic operon, and to the phlF gene encoding a pathway-specific transcriptional repressor. The Tn5 insertion was located in an open reading frame, tentatively named phlH, which is not related to known phl genes. In wild-type CHA0, 2,4-DAPG production paralleled expression of a phlA-lacZ translational fusion, reaching a maximum in the late exponential growth phase. Thereafter, the compound appeared to be degraded to monoacetylphloroglucinol by the bacterium. 2,4-DAPG was identified as the active compound in extracts from culture supernatants of strain CHA0 specifically inducing phlA-lacZ expression about sixfold during exponential growth. Induction by exogenous 2,4-DAPG was most conspicuous in a phlA mutant, which was unable to produce 2,4-DAPG. In a phlF mutant, 2,4-DAPG production was enhanced severalfold and phlA-lacZ was expressed at a level corresponding to that in the wild type with 2,4-DAPG added. The phlF mutant was insensitive to 2,4-DAPG addition. A transcriptional phlA-lacZ fusion was used to demonstrate that the repressor PhlF acts at the level of transcription. Expression of phlA-lacZ and 2,4-DAPG synthesis in strain CHA0 was strongly repressed by the bacterial extracellular metabolites salicylate and pyoluteorin as well as by fusaric acid, a toxin produced by the pythopathogenic fungus Fusarium. In the phlF mutant, these compounds did not affect phlA-lacZ expression and 2,4-DAPG production. PhlF-mediated induction by 2,4-DAPG and repression by salicylate of phlA-lacZ expression was confirmed by using Escherichia coli as a heterologous host. In conclusion, our results show that autoinduction of 2,4-DAPG biosynthesis can be countered by certain bacterial (and fungal) metabolites. This mechanism, which depends on phlF function, may help P. fluorescens to produce homeostatically balanced amounts of extracellular metabolites.Certain root-associated strains of fluorescent Pseudomonas spp. produce and excrete metabolites that are inhibitory to soilborne plant pathogens (13, 24, 52). Among these metabolites, 2,4-diacetylphloroglucinol (2,4-DAPG) has received particular attention because of its production by a wide range of pseudomonads used for the biological control of root diseases (13,26,50,52). 2,4-DAPG is a phenolic compound with broadspectrum antifungal, antibacterial, antihelminthic, and phytotoxic activity (13,25,52). A 2,4-DAPG biosynthetic gene cluster is conserved among numerous 2,4-DAPG-producing pseudomonads isolated from soils that are naturally suppressive to take-all of wheat, black root rot of tobacco, and tomat...

The putative permease PhlE of Pseudomonas fluorescens F113 has a role in 2,4-diacetylphloroglucinol resistance and in general stress tolerance

“…5). As well as acting as the precursor of PHL synthesis, MAPG is also known to be a degradation product of PHL (Schnider- Keel et al, 2000;A. Abbas, unpublished results).…”

“…The available data strongly suggest that PhlA, PhlC, PhlB and PhlD function in a multienzyme complex (Bangera & Thomashow, 1999;Delany, 1999). Expression of the phlACBD operon is subject to complex regulation (Aarons et al, 2000;Abbas et al, 2002;Bangera & Thomashow, 1999;Chou et al, 1993;Corbell & Loper, 1995;Delany, 1999; Delany et al, 2000;Duffy & Defago, 1999;Schnider-Keel et al, 2000). In addition, PHL is an autoregulator, positively influencing its own biosynthesis (Abbas et al, 2002;Schnider-Keel et al, 2000).…”

Regulation of Bacterial Virulence by Csr (Rsm) Systems

“…P. fluorescens induces systemic resistance phenotypes in plants and secretes lytic enzymes (phospholipase C, tryptophan side chain oxidase, and exoprotease) and antimicrobial compounds (pyoluteorin and 2,4-diacetylphloroglucinol) (118,(159)(160)(161)(162)(163)(164). The GacS/GacA (BarA/ UvrY) TCS controls the expression of genes involved in the production/secretion of these factors, including the genes for 2,4-diacetylphloroglucinol biosynthesis, phlACBDE, the major exoprotease, aprA, and hydrogen cyanide synthase, hcnABC (119,160,(163)(164)(165). In the closely related pathogen P. aeruginosa, GacA activates the expression of RsmY and RsmZ sRNAs, and GacA from P. fluorescens follows a similar trend via RsmX, RsmY, and RsmZ (74,76).…”

The Chloride Ion Is an Environmental Factor Affecting the Biosynthesis of Pyoluteorin and 2,4-Diacetylphloroglucinol inPseudomonassp. YGJ3

“…Binding of PLT to a transcriptional repressor PhlF has been identified by Schnider-Keel et al with a DAPG-negative mutant of P. fluorescens CHA0. 14) In this study we showed for the first time that Cl À stimulated PLT production and thereby strongly repressed DAPG production. The results indicate that the biosynthesis of PLT and that of DAPG are mutually closely linked by Cl À .…”