GacA uses two distinct regulatory mechanisms to control the biosynthesis of 2,4-diacetylphloroglucinol in Pseudomonas protegens Pf-5

ABSTRACT Antibiotic 2,4-diacetylphloroglucinol (DAPG) is produced by many plant-associated beneficial bacteria of the Pseudomonas genus and plays an important role in plant disease control due to its broad antimicrobial activity against different pathogens. DAPG biosynthesis is activated by the conserved GacA/GacS two-component regulatory system. Here, we report that a Δ gacA mutant of Pseudomonas protegens Pf-5, lacking both DAPG production and pathogen inhibition in culture, controlled pea Aphanomyces root rot in a DAPG-dependent manner in the greenhouse. DAPG production of the Δ gacA mutant could be restored by exogenous phloroglucinol (PG), the first intermediate in DAPG biosynthesis, by culturing the Δ gacA mutant with either PG or PG-producing bacteria. We identified a new Gac-dependent promoter of phlD . Gene expression assays demonstrated that GacA is required to activate the phlD promoter. In vitro binding assays showed that the RNA-binding protein RsmE bound directly to the leader mRNA of phlA , another DAPG biosynthetic gene converting PG into DAPG, indicating that GacA regulates phlA expression post-transcriptionally. No detectable binding activity was observed between RsmE and the phlD leader mRNA. These results show that GacA regulates DAPG biosynthesis at multiple steps via different mechanisms and elucidate a novel layer of Gac-Rsm regulation in secondary metabolism. Targeted PG supplementation and/or partner microbe interactions may help to enhance the disease control efficacy and stability of the DAPG-producing bacteria. IMPORTANCE Antibiotic production is important for many beneficial bacteria to inhibit plant pathogens and control plant diseases. Understanding the molecular mechanism of how bacteria regulate antibiotic production can help improve the disease control effect. Previous studies have shown that the production of 2,4-diacetylphloroglucinol (DAPG) is activated by a global regulator GacA in strains of Pseudomonas spp. In this work, we found that two different regulatory mechanisms are used by GacA to regulate the DAPG production in Pseudomonas protegens Pf-5. Specifically, GacA regulates the expression of phlD and phlA , two DAPG core biosynthetic genes, at transcriptional and post-transcriptional levels, respectively. DAPG production of the Pf-5 mutant lacking GacA could be restored by amendment of phloroglucinol (PG), an intermediate of DAPG biosynthesis. The Δ gacA mutant protected pea plants in soil from root rot disease caused by an oomycete pathogen, Aphanomyces euteiches, that can be inhibited by DAPG. Results of this study advanced our understanding of the molecular mechanisms that regulate antibiotic production of plant beneficial bacteria and suggest that PG amendment may be used to improve the disease control stability and efficacy of DAPG-producing bacteria in the field.