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Regulation of 2,4-diacetylphloroglucinol biosynthesis and biocontrol capacity by the BolA family protein IbaG in Pseudomonas fluorescens 2P24.

ABSTRACT: The monothiol glutaredoxin GrxD plays an essential role in the biosynthesis of the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) and the biocontrol capacity of the soil bacterium Pseudomonas fluorescens 2P24. However, the detailed mechanism underlying GrxD-mediated activation of the production of 2,4-DAPG remains unclear. Here, we found that GrxD directly interacted with IbaG, a BolA protein family member. The mutation of ibaG significantly decreased 2,4-DAPG production. Furthermore, expressing ibaG restored the production of 2,4-DAPG in the grxD ibaG double mutant to wild-type levels in the presence of dithiothreitol, suggesting that IbaG was required for GrxD-mediated regulation of 2,4-DAPG production. Transcriptome sequencing analyses revealed that IbaG plays a global role in gene regulation by affecting the expression of numerous genes throughout the genome. We also demonstrated that IbaG is an important regulator of several cellular processes, including swarming motility, biofilm formation, siderophore production, and acid resistance. Altogether, our data suggest that IbaG has an essential role in 2,4-DAPG production, motility, and biofilm formation. We also propose a regulatory mechanism linking GrxD to 2,4-DAPG production via IbaG. IMPORTANCE The production of 2,4-diacetylphloroglucinol (2,4-DAPG) is positively influenced by the monothiol glutaredoxin GrxD in Pseudomonas fluorescens 2P24. However, the regulatory mechanism underlying GrxD-mediated regulation of 2,4-DAPG biosynthesis is mostly uncharacterized. Here, we show the function of the BolA-like protein IbaG in 2,4-DAPG biosynthesis. We also demonstrate that GrxD directly interacts with IbaG and influences the redox state of IbaG. Altogether, this work provides new insights into the role of the highly conserved IbaG protein in regulating 2,4-DAPG synthesis, biofilm formation, and other biocontrol traits of P. fluorescens.

SUBMITTER: Dong Q

PROVIDER: S-EPMC10580914 | biostudies-literature | 2023 Sep

REPOSITORIES: biostudies-literature

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Regulation of 2,4-diacetylphloroglucinol biosynthesis and biocontrol capacity by the BolA family protein IbaG in Pseudomonas fluorescens 2P24.

Dong Qiuling Q Yan Qing Q Zhang Bo B Zhang Li-Qun LQ Wu Xiaogang X

Microbiology spectrum 20230919

The monothiol glutaredoxin GrxD plays an essential role in the biosynthesis of the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) and the biocontrol capacity of the soil bacterium Pseudomonas fluorescens 2P24. However, the detailed mechanism underlying GrxD-mediated activation of the production of 2,4-DAPG remains unclear. Here, we found that GrxD directly interacted with IbaG, a BolA protein family member. The mutation of ibaG significantly decreased 2,4-DAPG production. Further ...[more]

PMID: 37724877

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Project description:The antimicrobial metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) contributes to the capacity of Pseudomonas fluorescens strain CHA0 to control plant diseases caused by soilborne pathogens. A 2, 4-DAPG-negative Tn5 insertion mutant of strain CHA0 was isolated, and the nucleotide sequence of the 4-kb genomic DNA region adjacent to the Tn5 insertion site was determined. Four open reading frames were identified, two of which were homologous to phlA, the first gene of the 2,4-DAPG biosynthetic operon, and to the phlF gene encoding a pathway-specific transcriptional repressor. The Tn5 insertion was located in an open reading frame, tentatively named phlH, which is not related to known phl genes. In wild-type CHA0, 2, 4-DAPG production paralleled expression of a phlA'-'lacZ translational fusion, reaching a maximum in the late exponential growth phase. Thereafter, the compound appeared to be degraded to monoacetylphloroglucinol by the bacterium. 2,4-DAPG was identified as the active compound in extracts from culture supernatants of strain CHA0 specifically inducing phlA'-'lacZ expression about sixfold during exponential growth. Induction by exogenous 2,4-DAPG was most conspicuous in a phlA mutant, which was unable to produce 2, 4-DAPG. In a phlF mutant, 2,4-DAPG production was enhanced severalfold and phlA'-'lacZ was expressed at a level corresponding to that in the wild type with 2,4-DAPG added. The phlF mutant was insensitive to 2,4-DAPG addition. A transcriptional phlA-lacZ fusion was used to demonstrate that the repressor PhlF acts at the level of transcription. Expression of phlA'-'lacZ and 2,4-DAPG synthesis in strain CHA0 was strongly repressed by the bacterial extracellular metabolites salicylate and pyoluteorin as well as by fusaric acid, a toxin produced by the pythopathogenic fungus Fusarium. In the phlF mutant, these compounds did not affect phlA'-'lacZ expression and 2, 4-DAPG production. PhlF-mediated induction by 2,4-DAPG and repression by salicylate of phlA'-'lacZ expression was confirmed by using Escherichia coli as a heterologous host. In conclusion, our results show that autoinduction of 2,4-DAPG biosynthesis can be countered by certain bacterial (and fungal) metabolites. This mechanism, which depends on phlF function, may help P. fluorescens to produce homeostatically balanced amounts of extracellular metabolites.

Project description:IntroductionPseudomonas fluorescens 2P24 was isolated from soil of natural decay associated with wheat take-all and it can effectively control soil-borne diseases caused by a variety of plant pathogens. 2,4-diacetylphloroglucinol (2,4-DAPG), is produced by P. fluorescens 2P24 and plays an important role in the prevention and control of plant diseases. To understand the resistant mechanism, in this study, we conducted experiments to explore the regulation role of rpoZ in the synthesis of the antibiotic 2,4-DAPG and regulation of QS system.MethodsA random mini-Tn5 mutagenesis procedure was used to screen regulators for phlA transcription in stain PM901, which containing a phlA∷lacZ transcriptional fusion reporter plasmid. We identified 12 insertion mutants could significantly change phlA gene expression. By analyzing the amino acid sequences of the interrupted gene, we obtained a mutant strain Aa4-29 destroyed the rpoZ gene, which encodes the omiga subunit. We constructed the plasmid of rpoZ mutant (pBBR-△rpoZ) transformed into competent cells of P. fluorescens 2P24 by electro-transformation assay. The strains of P. fluorescens 2P24/pBBR, 2P24-△rpoZ/pBBR, 2P24-△rpoZ/pBBR-rpoZ were used to evaluate the regulation role of rpoZ in 2,4-DAPG production and quorum sensing system.ResultsAccording to β-galactosidase activity, we found that rpoZ positively regulated the expression of phlA (a synthesis gene of 2,4-DAPG) and PcoI (a synthesis gene of PcoI/PcoR QS signal system) at the transcriptional level. The production of 2,4-DAPG antibiotic and signal molecule AHL was influenced by rpoZ. Further, rpoZ was involved in regulating rsmA expression. RpoZ also has a certain regulatory effect on rpoS transcription, but no effect on the transcription of phlF, emhABC and emhR. According to the biocontrol assay, P. fluorescens 2P24 strains with rpoZ showed obvious antagonism ability against the Rhizoctonia solani in cotton, while the mutant strain of rpoZ lost the biocontrol effect. RpoZ had a significant effect on the swimming and biofilm formation in P. fluorescens 2P24.ConclusionOur data showed that rpoZ was an important regulator of QS system, 2,4-DAPG in P. fluorescens 2P24. This may imply that P. fluorescens 2P24 has evolved different regulatory features to adapt to different environmental threats.

Dataset Information

Regulation of 2,4-diacetylphloroglucinol biosynthesis and biocontrol capacity by the BolA family protein IbaG in Pseudomonas fluorescens 2P24.

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Regulation of 2,4-diacetylphloroglucinol biosynthesis and biocontrol capacity by the BolA family protein IbaG in <i>Pseudomonas fluorescens</i> 2P24.

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