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

Abstract

Certain strains of biocontrol bacterium <i>Pseudomonas fluorescens</i> 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 <i>phlACBDEFGH</i> and it is still unclear how the pathway-specific regulator <i>phlH</i> within this gene cluster regulates the metabolism of 2,4-DAPG. Here, we found that PhlH in <i>Pseudomonas fluorescens</i> strain 2P24 represses the expression of the <i>phlG</i> gene encoding the 2,4-DAPG hydrolase by binding to a sequence motif overlapping with the -35 site recognized by σ⁷⁰ 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 <i>phlG</i> Comparison of 2,4-DAPG production between the Δ<i>phlH</i>, Δ<i>phlG</i>, and Δ<i>phlHG</i> mutants confirmed that <i>phlH</i> and <i>phlG</i> 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 <i>in situ</i> levels of 2,4-DAPG, the <i>phlHG</i> genes could dynamically modulate the metabolic loads attributed to 2,4-DAPG production and potentially contribute to rhizosphere adaptation.<b>IMPORTANCE</b> 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 (<i>phl</i>) have been characterized, it is still not clear how the pathway-specific regulator <i>phlH</i> 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 <i>phlG</i> by PhlH offers a way to dynamically modulate the metabolic loads attributed to 2,4-DAPG production.