Tn5 mutagenesis and complementation analysis were used to clone a 6-kb genomic fragment required for biosynthesis of 2,4-diacetylphloroglucinol (Phi) from fluorescent Pseudomonas sp. strain F113. A recombinant plasmid, pCU203, containing this region partially complemented a Phl production-negative mutant (F113G22) derived from strain F113. When sugar beet seeds were sown into an unsterilized soil, in which sugar beet was subject to damping-off by Pythium ultimum, the emergence of sugar beet seeds inoculated with strain F113 was significantly greater than that of seeds inoculated with F113G22. Transfer of pCU203 into eight other Pseudomonas strains conferred the ability to synthesize Phl in only one of these strains, Pseudomonas sp. strain M114. Strain M114(pCU203) showed enhanced antagonism towards P. ultimum in vitro and significantly increased the emergence of sugar beet seeds in the same soil compared with emergence induced by the parent strain M114.

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 potato cyst nematode Globodera rostochiensis is an important pest of potato (Solanum tuberosum). Pseudomonas fluorescens F113, which produces 2,4-diacetylphloroglucinol (DAPG), was investigated as a potential biocontrol agent against G. rostochiensis. Exposure of nematode cysts to the pseudomonad, under in vitro conditions or in soil microcosms, almost doubled the ability of the eggs to hatch. The percentage of mobile juveniles was reduced threefold following their incubation in the presence of the pseudomonad, both in vitro and in soil. Results obtained with a transposon-induced DAPG-negative biosynthetic mutant of F113 and its complemented derivative with restored DAPG synthesis showed that the ability of strain F113 to produce DAPG was responsible for the increase in hatch ability and the reduction in juvenile mobility. Similar effects on egg hatch ability and juvenile mobility of G. rostochiensis were obtained in vitro by incubating nematode cysts and juveniles, respectively, in the presence of synthetic DAPG. DAPG-producing P. fluorescens F113 is proposed as a potential biocontrol inoculant for the protection of potato crops against the potato cyst nematode.

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...

Erwinia carotovora subspecies atroseptica is the agent of soft rot of potato and causes important crop damage in Europe. Synthetic 2,4‐diacetylphloroglucinol (DAPG) inhibited the growth of E. carotovora subsp. atroseptica under in vitro conditions and Pseudomonas fluorescens F113, which produces DAPG, was studied for biocontrol of E. carotovora subsp. atroseptica. Wild‐type F113 (or the spontaneous rifampicin‐resistant mutant F113Rif) inhibited growth of E. carotovora subsp. atroseptica on solid medium, displayed bactericidal activity towards the pathogen in liquid medium, and prevented Erwinia‐mediated rotting of wounded potato tuber under in vitro conditions. F113Rif reduced the population size of E. carotovora subsp. atroseptica in soil and on potato tuber dice in competition experiments carried out with unplanted soil and soil planted with diced potato tubers, respectively. Co‐inoculation of potato tuber seeds with F113Rif and E. carotovora subsp. atroseptica reduced Erwinia contamination of the seed tubers compared with single inoculation with the pathogen. F113G22 is a Tn5::lacZY‐induced DAPG‐negative biosynthetic derivative of F113 and showed no antibiosis towards E. carotovora subsp. atroseptica in vitro. In contrast to F113Rif, F113G22 did not inhibit Erwinia‐mediated rotting of wounded potato tuber in vitro, did not influence survival of E. carotovora subsp. atroseptica in unplanted soil or soil planted with potato tuber dice and did not reduce Erwinia contamination of potato seed tubers. F113G22(pCU203) is a complemented derivative with restored DAPG‐producing ability. F113G22(pCU203) had similar effects against E. carotovora subsp. atroseptica as F113 (or F113Rif) under in vitro conditions and in soil microcosms. The results indicate that P. fluorescens F113 is a promising biocontrol agent against the potato soft rot agent E. carotovora subsp. atroseptica and suggest that the pseudomonad's ability to produce DAPG is a key factor in its inhibition of the pathogen.