Comparative Multi‐Omics Survey Reveals Novel Specialized Metabolites and Biosynthetic Gene Clusters Under GacS Control in Pseudomonas donghuensis Strain SVBP6

Abstract

Diverse microbiota species thrive in the rhizosphere where the biosynthesis of antimicrobial metabolites drives plant-microbe and microbe-microbe interactions. Pseudomonas donghuensis SVBP6, an isolate from an agriculture field, has a broad antimicrobial profile; previous work showed that the well-conserved Gac-Rsm pathway regulates biosynthesis of the antifungal compound 7-hydroxytropolone (7-HT). However, 7-HT does not fully explain the strain’s antimicrobial activity under Gac-Rsm control. Here, we combined comparative transcriptomic, proteomic, and metabolomic approaches to identify novel GacS-dependent biosynthetic gene clusters (BGC) and/or extracellular specialized metabolites. Our data revealed a broad impact of GacS on gene expression and extracellular metabolite profile of SVBP6. At both the mRNA and polypeptide levels, specialized metabolism was the main affected functional category in the gacS mutant. The major extracellular MS/MS spectral families promoted by GacS were fatty acid amides, fatty acids, and alkaloids. We found that in addition to 7-HT and its BGC, GacS was required for the production of the antimicrobial compound pseudoiodinine and to positively control expression of the corresponding BGC. We also detected GacS-dependent production of 2,3,4-trihydro-β-carboline-1-one, which may add to the antimicrobial arsenal of SVBP6. Further, transcriptomics and proteomics pinpointed several GacS-activated gene clusters with biosynthetic functional features, that had escaped in silico genome mining tools. Altogether, our data shows that combining gacS loss-of-function mutants with a comparative multi-omics analysis in Pseudomonas isolates is a promising strategy to uncover bioactive metabolites and/or their BGCs. Discovery of novel natural products is important for harnessing the potential of microbiota to improve crop health by improving plant growth and inhibiting pathogens.