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

Abstract

2,4-Diacetylphloroglucinol (PHL) is the primary determinant of the biological control activity of Pseudomonas fluorescens F113. The operon phlACBD encodes enzymes responsible for PHL biosynthesis from intermediate metabolites. The phlE gene, which is located downstream of the phlACBD operon, encodes a putative permease suggested to be a member of the major facilitator superfamily with 12 transmembrane segments. PhlE has been suggested to function in PHL export. Here the sequencing of the phlE gene from P. fluorescens F113 and the construction of a phlE null mutant, F113-D3, is reported. It is shown that F113-D3 produced less PHL than F113. The ratio of cell-associated to free PHL was not significantly different between the strains, suggesting the existence of alternative transporters for PHL. The phlE mutant was, however, significantly more sensitive to high concentrations of added PHL, implicating PhlE in PHL resistance. Furthermore, the phlE mutant was more susceptible to osmotic, oxidative and heat-shock stresses. Osmotic stress induced rapid degradation of free PHL by the bacteria. Based on these results, we propose that the role of phlE in general stress tolerance is to export toxic intermediates of PHL degradation from the cells. INTRODUCTIONMany Pseudomonas fluorescens strains produce 2,4-diacetylphloroglucinol (PHL) (Bangera & Thomashow, 1996;Boruah & Kumar, 2002;Nowak-Thompson et al., 1994;Reddi & Borovkov, 1970; Sharifi-Tehrani et al., 1998). This phenolic molecule has broad-spectrum antifungal (Levy et al., 1992;Schoonbeek et al., 2002;Tomas-Lorente et al., 1989;Weller & Cook, 1983), antibacterial (Levy et al., 1992), antihelminthic (Bowden et al., 1965; Harrison et al., 1993) and phytotoxic (Keel et al., 1992;Reddi et al., 1969) activities. PHL is the primary determinant of biological control by P. fluorescens F113 (Shanahan et al., 1992). Synthesis of PHL is directed by the phlACBD genes, which are believed to constitute an operon (Bangera & Thomashow, 1996Delany, 1999). PhlD shows structural similarities with plant chalcone synthase, also called polyketide synthase type III, and is necessary but not sufficient for the synthesis of monoacetylphloroglucinol (MAPG) (Bangera & Thomashow, 1999). PhlA, PhlC and PhlB are necessary and sufficient for the transacetylation of MAPG to produce PHL (Bangera & Thomashow, 1999;Shanahan et al., 1992). 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).Micro-organisms have developed various ways to resist the toxic effects of the secondary metabolites they produce. These mechanisms include expo...

Characterization of PhlG, a Hydrolase That Specifically Degrades the Antifungal Compound 2,4-Diacetylphloroglucinol in the Biocontrol Agent Pseudomonas fluorescens CHA0

“…DAPG is synthesized and accumulated until the early stationary growth phase, as is typical for secondary metabolites (1,3,8,31,39). Thereafter, the metabolite appears to be degraded by the producing bacterium, with MAPG temporarily accumulating as an intermediary product of the degradation process (39).…”

“…The products of the phlACB genes also mediate the conversion of MAPG to DAPG (4). The phlE gene located immediately downstream of the phlACBD operon encodes a putative transmembrane permease (4) which appears to be implicated in DAPG resistance (1). The divergently transcribed phlF gene located adjacent to phlA codes for a pathway-specific transcriptional repressor of the DAPG biosynthetic operon (4,10,39).…”

Evolutionary History of the phl Gene Cluster in the Plant-Associated Bacterium Pseudomonas fluorescens

“…A. Moynihan and J. P. Morrissey, unpublished data). The 8-kb cluster involved in the biosynthesis, regulation, export, and degradation of 2,4-DAPG consists of eight genes, phlHGFACBDE, and is conserved at the organizational level in 2,4-DAPG-producing strains (1,2,5,7,13,31,57). The key biosynthetic gene is phlD, which displays interesting similarity to genes for plant chalcone synthases.…”

Molecular and Catalytic Properties of 2,4-Diacetylphloroglucinol Hydrolase (PhlG) from <i>Pseudomonas</i> sp. YGJ3

“…[2][3][4] The DAPG-producing bacterium itself must be tolerant toward DAPG. This is due to both PhlE, a pump protein that discharges DAPG out of the cell, 5) and PhlG, a hydrolase that converts DAPG to less toxic monoacetylphloroglucinol (MAPG) according to the following equation: DAPG þ H 2 O ! MAPG þ CH 3 COO À þ H þ .…”