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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.

The GacS-GacA two-component signal transduction system, which is highly conserved in gram-negative bacteria, is required for the production of exoenzymes and secondary metabolites in Pseudomonas spp. Screening of a Pseudomonas fluorescens F113 gene bank led to the isolation of a previously undefined locus which could restore secondary metabolite production to both gacS and gacA mutants of F113. Sequence analysis of this locus demonstrated that it did not contain any obvious Pseudomonas protein-coding open reading frames or homologues within available databases. Northern analysis indicated that the locus encodes an RNA (PrrB RNA) which is able to phenotypically complement gacS and gacA mutants and is itself regulated by the GacS-GacA two-component signal transduction system. Primer extension analysis of the 132-base transcript identified the transcription start site located downstream of a 70 promoter sequence from positions ؊10 to ؊35. Inactivation of the prrB gene in F113 resulted in a significant reduction of 2,4-diacetylphloroglucinol (Phl) and hydrogen cyanide (HCN) production, while increased metabolite production was observed when prrB was overexpressed. The prrB gene sequence contains a number of imperfect repeats of the consensus sequence 5-AGGA-3, and sequence analysis predicted a complex secondary structure featuring multiple putative stem-loops with the consensus sequences predominantly positioned at the single-stranded regions at the ends of the stem-loops. This structure is similar to the CsrB and RsmB regulatory RNAs in Escherichia coli and Erwinia carotovora, respectively. Results suggest that a regulatory RNA molecule is involved in GacA-GacSmediated regulation of Phl and HCN production in P. fluorescens F113.Pseudomonas fluorescens F113 was isolated as a biocontrol agent for the control of Pythium ultimum-mediated dampingoff of sugar beet (35). Inhibition of Pythium ultimum has been attributed to the production of the antimicrobial agent 2,4-diacetylphloroglucinol (Phl) (11). However, the strain also synthesizes hydrogen cyanide (HCN) and an exoprotease. These secondary metabolites and exoprotease have previously been shown to be positively regulated by the GacS (previously LemA) and GacA two-component signal transduction system (8) common to numerous Pseudomonas spp., including P. syringae (31), P. viridiflava (18), P. aeruginosa (30), and P. fluorescens (6, 13, 17, 32). Sensor proteins such as GacS are typically transmembrane proteins that respond to environmental stimuli by autophosphorylation, followed by transfer of the phosphate to the cognate response regulator, in this case GacA. The GacA response regulator contains a DNA binding motif and is thought to activate or repress genes directly by binding to the target gene promoter. However, direct binding of GacA to putative target promoters has yet to be demonstrated.Recent research in P. aeruginosa PAO (30) has revealed that the GacS-GacA signal transduction system contributes to a larger regulatory cascade involving acyl-homoserine lactone...

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