Regulation of production of the antifungal metabolite 2,4-diacetylphloroglucinol in Pseudomonas fluorescens F113: genetic analysis of phlF as a transcriptional repressor The GenBank accession number for the sequence reported in this paper is AF129856.

Abstract

The antifungal metabolite 2,4-diacetylphloroglucinol plays a major role in the biocontrol capabilities of Pseudomonas fluorescens. The phloroglucinol biosynthetic locus of P. fluorescens F113 has been isolated previously. From nucleotide sequence data, a putative regulator gene (phlF) was identified upstream and divergently transcribed from the phlACBD phloroglucinol biosynthetic genes. PhlF shows similarity to various transcriptional repressors in the EMBL database and exhibits a helix-turn-helix motif in its amino acid sequence. phlF was cloned into an expression vector and the PhlF protein product was purified. Gel retardation experiments demonstrated PhlF to be a DNA-binding protein and showed that it binds to the phlA-phlF intergenic region. Introduction of phlF into P. fluorescens F113 in multiple copies resulted in repression of phloroglucinol production in this strain. This effect was mediated at the transcription level since the expression of a phloroglucinol biosynthetic gene fusion in this background was equally repressed. Furthermore, the inactivation of phlF results in derepression of phloroglucinol production in this strain.

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

“…We have previously reported the purification of a histidine-tagged PhlF repressor (23.6 kDa) (12). A protein of smaller size (18 kDa) invariably copurified with the histidine-tagged PhlF protein.…”

“…For these reasons we optimized the purification conditions to purify only the full-length PhlF. A number of parameters were modified relative to the previous protocol (12). The procedure is detailed below, the major modifications being the use of a different strain of E. coli and different inducing conditions.…”

“…To characterize the transcriptional activity of the phlACBD and phlF genes, we used pCU107 and pCU109 constructs bearing lacZ transcriptionally fused with phlACBD and phlF, respectively (11,12). The kinetics of expression of biosynthetic fusions in different backgrounds were monitored by performing ␤-galactosidase assays (40).…”

Detection of and Response to Signals Involved in Host-Microbe Interactions by Plant-Associated Bacteria

“…Upstream of the phlACBD operon is a divergently transcribed phlF gene, which encodes a TetR-type repressor of DAPG synthesis (105). The dimeric PhlF protein binds to the phlA promoter region, and this binding is prevented by the addition of DAPG (105,445). Thus, DAPG positively controls its own biosynthesis, and the fact that the signal is made by bacteria and is diffusible is reminiscent of the autoinduction systems of various bacteria (154).…”

“…In P. fluorescens CHA0, the four DAPG biosynthesis genes, phlACBD, are organized as an operon, which is followed by a gene coding for a putative efflux protein (phlE). Upstream of the phlACBD operon is a divergently transcribed phlF gene, which encodes a TetR-type repressor of DAPG synthesis (105). The dimeric PhlF protein binds to the phlA promoter region, and this binding is prevented by the addition of DAPG (105,445).…”