Transcriptional Regulator PhlH Modulates 2,4-Diacetylphloroglucinol Biosynthesis in Response to the Biosynthetic Intermediate and End Product

Abstract

Certain strains of biocontrol bacterium produce the secondary metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) to antagonize soilborne phytopathogens in the rhizosphere. The gene cluster responsible for the biosynthesis of 2,4-DAPG is named and it is still unclear how the pathway-specific regulator within this gene cluster regulates the metabolism of 2,4-DAPG. Here, we found that PhlH in strain 2P24 represses the expression of the gene encoding the 2,4-DAPG hydrolase by binding to a sequence motif overlapping with the -35 site recognized by σ factors. Through biochemical screening of PhlH ligands we identified the end product 2,4-DAPG and its biosynthetic intermediate monoacetylphloroglucinol (MAPG), which can act as signaling molecules to modulate the binding of PhlH to the target sequence and activate the expression of Comparison of 2,4-DAPG production between the Δ, Δ, and Δ mutants confirmed that and impose negative feedback regulation over 2,4-DAPG biosynthesis. It was further demonstrated that the 2,4-DAPG degradation catalyzed by PhlG plays an insignificant role in 2,4-DAPG tolerance but contributes to bacterial growth advantages under carbon/nitrogen starvation conditions. Taken together, our data suggest that by monitoring and down-tuning levels of 2,4-DAPG, the genes could dynamically modulate the metabolic loads attributed to 2,4-DAPG production and potentially contribute to rhizosphere adaptation. 2,4-DAPG, which is synthesized by biocontrol pseudomonad bacteria, is a broad-spectrum antibiotic against bacteria, fungi, oomycetes, and nematodes and plays an important role in suppressing soilborne plant pathogens. Although most of the genes in the 2,4-DAPG biosynthetic gene cluster () have been characterized, it is still not clear how the pathway-specific regulator is involved in 2,4-DAPG metabolism. This work revealed the role of PhlH in modulating 2,4-DAPG levels by controlling the expression of 2,4-DAPG hydrolase PhlG in response to 2,4-DAPG and MAPG. Since 2,4-DAPG biosynthesis imposes a metabolic burden on biocontrol pseudomonads, it is expected that the fine regulation of by PhlH offers a way to dynamically modulate the metabolic loads attributed to 2,4-DAPG production.

Regulation of Antibiotic Production by Signaling Molecules in Streptomyces

“…One example comes from the actinomycete Amycolatopsis mediterranei, in which rifamycin B (the endproduct of rifamycin biosynthesis) serves as an extracellular signaling molecule to regulate rifamycin export in a feedback mechanism (Lei et al, 2018). Another study showed that 2,4-diacetylphloroglucinol and its biosynthetic intermediate could serve as autoregulatory ligands to regulate its own biosynthesis in Pseudomonas fluorescens (Yan et al, 2017). These studies suggested that antibiotics and their biosynthetic intermediates could function as autoregulators to regulate their own biosynthesis, mainly through the modulation of the binding activity of CSRs to their target genes within their cognate gene clusters.…”

The Oxidoreductase DsbA1 negatively influences 2,4-diacetylphloroglucinol biosynthesis by interfering the function of Gcd in Pseudomonas fluorescens 2P24

“…Finally, phlG encodes a hydrolase that specifically degrades 2,4-DAPG to less toxic MAPG and acetate [10]. Recent studies showed that another pathway-specific transcriptional repressor, PhlH, modulates 2,4-DAPG levels by controlling the expression of the phlG gene by sensing the concentration of 2,4-DAPG and MAPG in cells [11].…”

The antitoxin MqsA homologue in Pseudomonas fluorescens 2P24 has a rewired regulatory circuit through evolution

“…Plasmids for lacZ reporters in E. coli and P. fluorescens 2P24 were constructed using pSB1C3 and pRG970Km as previously described (Yan et al, ) and the primers used to amplify the DNA fragments are listed in Supporting Information Table S2. For lacZ assays in E.coli, the pSB1C3 derived plasmids were transformed into the E. coli DH5α strain and the bacteria were grown to an OD 600 of 0.6–0.8 in LB broth at 37°C.…”

“…Plasmids for lacZ reporters in E. coli and P. fluorescens 2P24 were constructed using pSB1C3 and pRG970Km as previously described (Yan et al, 2017) and the primers used to amplify the DNA fragments are listed in Supporting Information Table S2. For lacZ assays in E. coli, the pSB1C3 derived plasmids were transformed into the E. coli DH5α strain and the bacteria were grown to an OD 600 of 0.6-0.8 in LB broth at 37 C. For lacZ assays in P. fluorescens 2P24, the pRG970Km derived plasmids were transformed into P. fluorescens 2P24 and conjugated to the wild-type strain and mutant strains by triparental mating using a helper strain (Finan et al, 1986).…”