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

Abstract

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.

Keywords: 2,4-diacetylphloroglucinol; Pseudomonas; interspecies signaling; pyoluteorin; transcriptional repressor.

Fig 1

Effect of PLT on growth and 2,4-DAPG production of P. fluorescens 2P24 by regulating the transcription of 2,4-DAPG-related genes. (A) Effects of 20 and 50 µM PLT on the cell growth of P. fluorescens 2P24. The initial cell turbidity was 0.1 at 600 nm. (B) HPLC quantitative analysis of 2,4-DAPG in P. fluorescens 2P24 after the addition of 20 µM PLT. (C) Relative expression levels of phlA, phlD, and phlG were quantified by RT-qPCR using RNA extracted from P. fluorescens 2P24 in the absence or presence of 20 µM PLT, respectively, at 18 h post-inoculation. (D) Relative expression levels of phlA and phlG were quantified by RT-qPCR using RNA extracted from 2P24ΔphlD in the absence or presence of 20 µM PLT, respectively, at 18 h post-inoculation. (E) Relative expression levels of phlA and phlD were quantified by RT-qPCR using RNA extracted from 2P24ΔphlG in the absence or presence of 20 µM PLT, respectively, at 18 h post-inoculation. Error bars denote standard deviations of three independent replicates (n = 3). Statistical analyses were performed using t-test and two-way analysis of variance. *P < 0.05; **P < 0.01; ***P < 0.001. ns, non-significant; RT-qPCR, reverse transcription quantitative PCR; WT, wild type.

Fig 2

Transcription regulators PhlF and PhlH can bind PLT for regulating the phl biosynthesis gene cluster. (A and C) Electrophoretic mobility shift assays of 2P24_PhlH (A) or 2P24_PhlF (C) with the upstream region of phlG (free DNA) in the increasing concentrations (0–500 µM) of PLT or with the upstream region of phlG (free DNA) in the rising concentrations (0–1 mM) of PLT, respectively. (B and D). The binding affinities of 2P24_PhlH (B) or 2P24_PhlF (D) with PLT were evaluated using ITC analysis. The binding curves corrected for the dilution effects were fit to a one-site binding model, and the K_d values were calculated by the NanoAnalyze software. (E) Cartoon representation of 2P24_PhlH in complex with the docked PLT. 2P24_PhlH is in light gray. The ligand-binding tunnel is the hydrophobic tunnel in ligand recognition and is yellow as a transparent surface. 2,4-DAPG, phloretin, and PLT are shown as sticks in cyan, green, and yellow, respectively. (F) 2P24_PhlF model calculated using AlphaFold and cartoon representation of model 2P24_PhlF in complex with the docked PLT. Model 2P24_PhlF is in light green. The ligand-binding pocket is predicted ligand recognition and is light gray as a transparent surface. The bound docked PLT is shown as sticks in green. All structure figures were prepared with PyMOL.

Fig 3

In response to PLT, PhlH regulates the expression of phlG, while PhlF regulates the expression of phlA. (A) The fluorescent activity of the pSEVA225T-EGFP-PphlG plasmid was evaluated in WT and ΔphlH strains to test the expression of phlG. The bars illustrate the relative fluorescent units (RFU) normalized to a 1 mL culture with OD₆₀₀ = 1. (B) The fluorescent activity of the pSEVA225T-EGFP-PphlA plasmid was evaluated in WT and ΔphlF strains to test the expression of phlA. (C–E) 2,4-DAPG production of 2P24ΔphlH (C), 2P24ΔphlF (D), and 2P24ΔphlHΔphlF (E) strains grown in KB medium with or without 20 µM PLT. Error bars denote standard deviations of three independent replicates (n = 3). Statistical analyses were performed using t-test and two-way analysis of variance. *P < 0.05, **P < 0.01, ***P < 0.001. ns, non-significant.

Fig 4

The PhlF and PhlH share similarities with the ligand-binding domain (LBD). (A) Phylogenetic tree of TetR-type repressors PhlH and PhlF in representative strains form Pseudomonas spp. constructed using the neighbor-joining method. (B) Multiple sequence alignment of PhlH and PhlF proteins from representative Pseudomonas spp. Residues involved in binding 2,4-DAPG are shown in green boxes. The alignment is performed using MultAlin (33) and ESPript (34). The secondary structural elements of 2P24_PhlH are displayed at the top of the alignment. (C) The superposition of 2,4-DAPG-bound PhlH (PDB code 7E1N) (gray) and model PLT-bound PhlF (green) dimers is shown in the cartoon representation (top). The N-terminal DNA-binding domain (DBD) comprises three helices, with helices α2 and α3 forming the conventional helix-turn-helix-DNA-binding motif. The LBD is composed of the remaining helices. 2,4-DAPG and the bound docked PLT are shown as sticks in gray and green, respectively. A close-up view of the interaction mode between the AlphaFold-predicted PhlF (shown in transparent green) and the docked PLT (shown in dark green) is displayed at the bottom. The PLT-binding pocket, along with aromatic residues in the vicinity of PLT (highlighted in green), is shown as a stick, illustrating the potential interactions involved in the ligand-binding process. All structure figures were prepared with PyMOL.

Fig 5

Schematic representation of TetR repressors PhlH and PhlF from the 2,4-DAPG biosynthetic gene cluster positively regulated the hydrolysis of 2,4-DAPG and negatively regulated 2,4-DAPG synthesis in response to interspecies signal PLT.