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The flavonoid-sensing regulator AefR is involved in modulating quorum sensing through repressing the MexEF-OprN efflux pump in Pseudomonas fluorescens

ABSTRACT: Pseudomonas fluorescens is an important plant growth-promoting rhizobacteria (PGPR) for its biocontrol properties, which are mostly influenced by the quorum sensing (QS) system. Plants produce multiple metabolites like flavonoids to regulate physiology of PGPR. However, it remains under-explored how flavonoids produced by plant roots influence the QS system of PGPR. In this study, we found the TetR-family regulator AefR was capable of binding to flavonoids and could inhibit production of AHLs by suppressing the expression of mexEF-oprN in P. fluorescens 2P24. We further showed that the efflux pump MexEF-OprN could attenuate the quorum sensing system PcoR/PcoI by extruding homoserine lactone (AHLs) molecules, leading to reduced biosynthesis of secondary metabolite mupirocin. Taken together, this study identified a TetR-like regulator responding to plant flavonoids and regulating the quorum sensing, providing insights into the interaction between PGPR and plant roots.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Pseudomonas Fluorescens

TISSUE(S): Cell Culture

SUBMITTER: yu xiaoquan

LAB HEAD: He Yongxing

PROVIDER: PXD052703 | Pride | 2025-02-15

REPOSITORIES: pride

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Project description:Indole is ubiquitously synthesized by plants and bacteria and functions as an inter species signaling molecule to modulate a wide variety of cellular activities. However, it is not clear how the indole signal is perceived and responded by plant growth promoting rhizobacteria (PGPR) at the rhizosphere. Here, we demonstrated that indole enhanced antibiotic tolerance of Pseudomonas fluorescens 2P24, a PGPR well known for its biocontrol capacity. By conducting quantitative proteomic analysis, we showed that indole influences the expression of multiple genes including the emhABC operon encoding the major multidrug efflux pump in P. fluorescens 2P24. The indole-induced antibiotic tolerance was not related to bacterial dormancy or slow growth, but depended on the emhABC operon and the divergently transcribed TetR-like regulator emhR. By binding to the semi-palindromic operator sequence, EmhR repressed the expression of emhABC. It was further revealed that indole bound to EmhR and weakened the interaction between EmhR and the operator. This is consistent with our finding that indole-induced expression of the EmhABC efflux pump is dependent on EmhR. Using homology modeling and molecular dynamics simulation, we found that indole binding resulted in significantly decreased distance between the two DNA-recognizing α3 helices within the EmhR dimer, which would possibly account for its compromised DNA binding capacity. EmhR was further shown to globally influence protein expressions, especially transporters and proteins involved in the denitrification pathway. This EmhR-dependent, indole-induced antibiotic tolerance is likely to be prevalent in the Pseudomonas species, as the EmhR homologue in Pseudomonas syringae was also shown to be responsible for the indole-induced antibiotic tolerance. Taken together, our results revealed an indole-sensing transcription factor EmhR responsible for indole-induced antibiotic tolerance in Pseudomonas species and have important implications on the general mechanism for the indole sensing and responses in rhizobacteria.

Project description:Introduction: Pantoea stewartii subsp. stewartii is a proteobacterium that causes Stewart’s wilt disease in corn plants. The bacteria form a biofilm in the xylem of infected plants and produce capsule that blocks water transport, eventually causing wilt. At low cell densities, the quorum-sensing (QS) regulatory protein EsaR is known to directly repress expression of esaR itself as well as the capsular synthesis operon transcription regulator, rcsA, and a 2,5-diketogluconate reductase, dkgA. It simultaneously directly activates expression of genes for a putative small RNA, esaS, the glycerol utilization operon, glpFKX, and another transcriptional regulator, lrhA. At high bacterial cell densities, all of this regulation is relieved when EsaR binds an acylated homoserine lactone signal, which is synthesized constitutively over growth. QS-dependent gene expression is critical for the establishment of disease in the plant. Objective: However, the identity of the full set of genes controlled by EsaR/QS is unknown. A proteomic approach previously identified around 30 proteins in the QS regulon. In this study, a whole-transcriptome, next-generation sequencing analysis of rRNA-depleted RNA from QS-proficient and -deficient P. stewartii strains was performed to identify additional targets of EsaR. Result: EsaR-dependent transcriptional regulation of a subset of differentially expressed genes was confirmed by quantitative reverse transcription-PCR (qRT-PCR). Electrophoretic mobility shift assays demonstrated that EsaR directly bound 10 newly identified target promoters. Overall, the QS regulon of P. stewartii orchestrates three major physiological responses: capsule and cell envelope biosynthesis, surface motility and adhesion, and stress response. Comparison of gene expression in two strains of Pantoea stewartii, QS-proficient DC283 ES∆IR (pSVB60) and QS-deficient DC283 ES∆IR (pBBR1MCS-3) to identify those genes that are directly regulated by the master Quorum-sensing regulator, EsaR.

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The flavonoid-sensing regulator AefR is involved in modulating quorum sensing through repressing the MexEF-OprN efflux pump in Pseudomonas fluorescens

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