A GacS-mediated regulatory framework coordinates bacterial competition and rhizosphere microbiome remodeling in Pseudomonas fluorescens

Plant growth-promoting Pseudomonas bacteria are promising biocontrol agents whose regulatory and effector genes shape bacterial traits involved in interactions with host plants and soil microorganisms. However, previous research has predominantly examined individual genes or traits in isolation, often making it difficult to compare their relative contributions within a single strain. This reductionist approach limits our understanding of how beneficial bacteria simultaneously engage in interactions with plants, suppress pathogens, and shape microbial communities. To address this gap, we selected 16 representative genes and gene clusters in P. fluorescens 2P24 based on genome annotation and previous knowledge of three PGPR-relevant functional layers: protein secretion systems, regulatory pathways, and secondary metabolite biosynthesis. This panel included the major annotated protein secretion systems in strain 2P24, selected regulatory systems with known or predicted roles in bacterial physiology and ecological interactions, and secondary-metabolite-related genes/gene clusters such as phl (2,4-DAPG biosynthetic gene cluster). We then examined how deletion of these genes or gene clusters affected bacterial traits, plant immune elicitation, and soil or rhizosphere bacterial community composition. Through comprehensive genetic and phenotypic analyses, we identified six genes and gene clusters— lapBCE , gacS , phoP , rsmX , fliC , and phl —that are essential for fundamental bacterial physiological processes and ecological fitness. More importantly, we discovered two regulatory modules, GacS-Phl and GacS-FliC, that coordinately govern multi-interface ecological interactions in the rhizosphere. Specifically, GacS regulates the production of 2,4-diacetylphloroglucinol, an antimicrobial compound that directly suppresses competing soil-dwelling Streptomyces populations. Simultaneously, GacS modulates flagellin expression, and flagellin acts as a microbe-associated molecular pattern triggering plant immune responses that indirectly reshape the composition and structure of rhizosphere microbiome communities. This study establishes a coordinated regulatory model that integrates global regulatory networks, microbe-associated molecular patterns, and antimicrobial secondary metabolites in mediating complex ecological interactions. Our findings reveal that beneficial Pseudomonas strains employ a GacS-mediated regulatory framework to simultaneously manage multiple ecological interfaces, providing mechanistic insights into how these bacteria balance plant-beneficial activities with competitive strategies. This regulatory framework offers a theoretical foundation for rational design and application of microbial biocontrol agents for sustainable agricultural practices. Video Abstract