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 ΔgacA 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 ΔgacA mutant could be restored by exogenous phloroglucinol (PG), the first intermediate in DAPG biosynthesis, by culturing the ΔgacA 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.