Characterization of Interactions between the Transcriptional Repressor PhlF and Its Binding Site at the phlA Promoter in Pseudomonas fluorescens F113

Abstract

The phlACBD genes responsible for the biosynthesis of the antifungal metabolite 2,4-diacetylphloroglucinol (PHL) by the biocontrol strain Pseudomonas fluorescens F113 are regulated at the transcriptional level by the pathway-specific repressor PhlF. Strong evidence suggests that this regulation occurs mainly in the early logarithmic phase of growth. First, the expression of the phlF gene is relatively high between 3 and 13 h of growth and relatively low thereafter, with the phlACBD operon following an opposite expression profile. Second, the kinetics of PHL biosynthesis are specifically altered in the logarithmic phase in a P. fluorescens F113 phlF mutant. The phlA-phlF intergenic region presents a complex organization in that phlACBD is transcribed from a 70 RNA polymerase-dependent promoter that is likely to overlap the promoter of the divergently transcribed phlF gene. The repression by PhlF is due to its interaction with an inverted repeated sequence, phO, located downstream of the phlA transcriptional start site. Cross-linking experiments indicate that PhlF can dimerize in solution, and thus PhlF may bind phO as a dimer or higher-order complex. Furthermore, it is now demonstrated that certain regulators of PHL synthesis act by modulating PhlF binding to phO. PHL, which has previously been shown to be an autoinducer of PHL biosynthesis, interacts with PhlF to destabilize the PhlF-phO complex. Conversely, the PhlF-phO complex is stabilized by the presence of salicylate, which has been shown to be an inhibitor of phlA expression.2,4-Diacetylphloroglucinol (PHL) is a phenolic molecule produced by many fluorescent pseudomonad bacteria (3,23,42,47,55,56,61,63). It has antifungal (28, 55, 58), antibacterial (28), antihelminthic (7, 21), and phytotoxic (23, 48) activities. PHL is a polyketide synthesized by condensation of three molecules of acetyl coenzyme A with one molecule of malonyl coenzyme A to produce the precursor monoacetylphloroglucinol, which is subsequently transacetylated to generate PHL (37,55).The genes involved in the biosynthesis of PHL have been cloned and sequenced from a number of Pseudomonas strains (3,4,11,17,37,55,61). Genetic and sequence data show that the phl locus is comprised of a number of transcriptional units. The structural genes, phlA, phlC, phlB, and phlD, are transcribed as a single operon (phlACBD), with the putative permease gene, phlE, probably transcribed from its own promoter further downstream. The transcriptional repressor, phlF, is located upstream of the phlACBD operon and is transcribed in the opposite direction (4,11,54).

The TetR Family of Regulators

“…Of these, six TFRs, i.e., ActR, KijA8, LanK, PhlF, SimR, and VarR, have been shown to bind the products of the biosynthetic pathways in which they are encoded (25,(75)(76)(77)(78)(79). These TFRs primarily regulate the expression of efflux pumps required for antibiotic export but may also regulate the expression of late-stage biosynthetic genes.…”

“…Biosynthesis of 2,4-diacetylphloroglucinol is of interest, as it occurs via a type III polyketide synthase (PKS) thought to be rare in bacteria (90). PhlF binds to the intergenic region between phlF and phlA, repressing expression of the phlABCD operon (75). DNA binding is enhanced in the presence of salicylate and disrupted by the biosynthetic product of the cluster 2,4-diacetylphloroglucinol.…”

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

“…The divergently transcribed phlF gene located adjacent to phlA codes for a pathway-specific transcriptional repressor of the DAPG biosynthetic operon (4,10,39). The repression by the TetR-like regulator PhlF is due to its interaction with specific binding sites in the phlA promoter region (2,16). DAPG acts as the signal that dissociates PhlF from the phlA promoter, thereby autoinducing its own biosynthesis (2,16,39).…”

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

“…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).…”