Interspecies signaling modulates the biosynthesis of antimicrobial secondary metabolites related to biological control activities of Pseudomonas fluorescens 2P24

Signaling between rhizosphere microorganisms is crucial in bacteria interaction and communication, shaping the rhizomicrobiome. Plant growth-promoting bacterium Pseudomonas produces a spectrum of important antibiotics to inhibit plant pathogens, albeit with an associated metabolic burden. Antibiotics could function as intra- and interspecies signals at subinhibitory concentrations to coordinate gene expression and microbial behaviors. In this work, we characterized pyoluteorin as an interspecies signal that modulates the biosynthesis of 2,4-diacetylphloroglucinol (2,4-DAPG), a broad-spectrum biocontrol agent, in non-pyoluteorin-producing Pseudomonas fluorescens 2P24. We demonstrated that the key transcriptional repressor PhlF from the 2,4-DAPG biosynthetic gene cluster spontaneously senses pyoluteorin, enhancing repression of the phlA promoter activity and inhibiting 2,4-DAPG synthesis in P. fluorescens 2P24. Pyoluteorin also binds to another transcriptional repressor, PhlH, from the 2,4-DAPG biosynthetic gene cluster, subsequently releasing the transcription of phlG , which facilitates the hydrolysis of 2,4-DAPG. Both PhlF and PhlH are simultaneously involved in sensing exogenous pyoluteorin to regulate the 2,4-DAPG biosynthetic operon, playing a crucial role in controlling antibiotic metabolites in response to environmental changes. Further phylogenetic and structural analyses demonstrated that PhlH and PhlF are widely distributed across Pseudomonas spp. with conserved ligand-binding domains. The findings shed new light on the regulatory mechanism of 2,4-DAPG biosynthesis underlying interspecies signaling by pyoluteorin and provide invaluable clues for the rational design of co-inhabiting Pseudomonas spp. as biocontrol agents. IMPORTANCE Rhizosphere microorganisms release vital signals that shape microbial communities, with antibiotics at low concentrations acting as intra- and interspecies signals. However, the mechanisms of these signals in coordinating gene expression are unclear. In non-pyoluteorin-producing Pseudomonas fluorescens 2P24, pyoluteorin was identified as an interspecies signal that regulates the phl biosynthesis gene cluster for 2,4-DAPG production. TetR family repressors PhlH and PhlF were found to positively regulate 2,4-DAPG hydrolysis and negatively regulate its synthesis in response to pyoluteorin. Structural modeling and docking analyses revealed the interactions between pyoluteorin and both PhlH and PhlF, modulating gene expression. Phylogenetic analyses showed a wide distribution of PhlH and PhlF across Pseudomonas spp. with conserved ligand-binding domains. These findings deepen our understanding of interspecies signaling mechanisms and highlight the potential for designing co-inhabiting Pseudomonas spp. as effective biocontrol agents.