Cyclic-di-GMP Regulates the Quorum-Sensing System and Biocontrol Activity of Pseudomonas fluorescens 2P24 through the RsmA and RsmE Proteins.

Pseudomonas fluorescens 2P24 is a rhizosphere bacterium that protects many crop plants against soilborne diseases caused by phytopathogens. The PcoI/PcoR quorum-sensing (QS) system and polyketide antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) are particularly relevant to the strain's biocontrol potential. In this study, we investigated the effects of c-di-GMP on the biocontrol activity of strain 2P24. The expression of the Escherichia coli diguanylate cyclase (YedQ) and phosphodiesterase (YhjH) in P. fluorescens 2P24 significantly increased and decreased the cellular concentration of c-di-GMP, respectively. The production of the QS signals N-acyl homoserine lactones (AHLs) and 2,4-DAPG was negatively regulated by c-di-GMP in 2P24. The regulatory proteins RsmA and RsmE were positively regulated by c-di-GMP. Genomic analysis revealed that 2P24 has 23 predicted proteins that contain c-di-GMP-synthesizing or -degrading domains. Among these proteins, C0J56_12915, C0J56_13325, and C0J56_27925 contributed to the production of c-di-GMP and were also involved in the regulation of the QS signal and antibiotic 2,4-DAPG production in P. fluorescens Overexpression of C0J56_12915, C0J56_13325, and C0J56_27925 in 2P24 impaired its root colonization and biocontrol activities. Taken together, these results demonstrated that c-di-GMP played an important role in fine-tuning the biocontrol traits of P. fluorescensIMPORTANCE In various bacteria, the bacterial second messenger c-di-GMP influences a wide range of cellular processes. However, the function of c-di-GMP on biocontrol traits in the plant-beneficial rhizobacteria remains largely unclear. The present work shows that the QS system and polyketide antibiotic 2,4-DAPG production are regulated by c-di-GMP through RsmA and RsmE proteins in P. fluorescens 2P24. The diguanylate cyclases (DGCs) C0J56_12915, C0J56_13325, and C0J56_27925 are especially involved in regulating the biocontrol traits of 2P24. Our work also demonstrated a connection between the Gac/Rsm cascade and the c-di-GMP signaling pathway in P. fluorescens.

Pleiotropic effects of RsmA and RsmE proteins in Pseudomonas fluorescens 2P24.

BACKGROUND: Pseudomonas fluorescens 2P24 is a rhizosphere bacterium that produces 2,4-diacetyphloroglucinol (2,4-DAPG) as the decisive secondary metabolite to suppress soilborne plant diseases. The biosynthesis of 2,4-DAPG is strictly regulated by the RsmA family proteins RsmA and RsmE. However, mutation of both of rsmA and rsmE genes results in reduced bacterial growth. RESULTS: In this study, we showed that overproduction of 2,4-DAPG in the rsmA rsmE double mutant influenced the growth of strain 2P24. This delay of growth could be partially reversal when the phlD gene was deleted or overexpression of the phlG gene encoding the 2,4-DAPG hydrolase in the rsmA rsmE double mutant. RNA-seq analysis of the rsmA rsmE double mutant revealed that a substantial portion of the P. fluorescens genome was regulated by RsmA family proteins. These genes are involved in the regulation of 2,4-DAPG production, cell motility, carbon metabolism, and type six secretion system. CONCLUSIONS: These results suggest that RsmA and RsmE are the important regulators of genes involved in the plant-associated strain 2P24 ecologic fitness and operate a sophisticated mechanism for fine-tuning the concentration of 2,4-DAPG in the cells.

Citrate Synthase GltA Modulates the 2,4-Diacetylphloroglucinol Biosynthesis of Pseudomonas fluorescens 2P24 and is Essential for the Biocontrol Capacity.

Carbon metabolism is critical for microbial physiology and remarkably affects the outcome of secondary metabolite production. The production of 2,4-diacetylphloroglucinol (2,4-DAPG), a bacterial secondary metabolite with a broad spectrum of antibiotic activity, is a major mechanism used by the soil bacterium Pseudomonas fluorescens 2P24 to inhibit the growth of plant pathogens and control disease occurrence. Strain 2P24 has evolved a complex signaling cascade to regulate the production of 2,4-DAPG. However, the role of the central carbon metabolism in modulating 2,4-DAPG production has not been fully determined. In this study, we report that the gltA gene, which encodes citrate synthase, affects the expression of the 2,4-DAPG biosynthesis gene and is essential for the biocontrol capacity of strain 2P24. Our data showed that the mutation of gltA remarkably decreased the biosynthesis of 2,4-DAPG. Consistent with this result, the addition of citrate in strain 2P24 resulted in increased 2,4-DAPG production and decreased levels of RsmA and RsmE. In comparison with the wild-type strain, the gltA mutant was severely impaired in terms of biocontrol activity against the bacterial wilt disease of tomato plants caused by Ralstonia solanacearum. Moreover, the gltA mutant exhibited increased antioxidant activity, and the expression of oxidative, stress-associated genes, including ahpB, katB, and oxyR, was significantly upregulated in the gltA mutant compared to the wild-type strain. Overall, our data indicate that the citrate synthase GltA plays an important role in the production of 2,4-DAPG and oxidative stress and is required for biocontrol capacity.

Effect of the Monothiol Glutaredoxin GrxD on 2,4-Diacetylphloroglucinol Biosynthesis and Biocontrol Activity of Pseudomonas fluorescens 2P24.

Pseudomonas fluorescens 2P24 is a plant root-associated bacterium that suppresses several soilborne plant diseases due to its production of the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG). The biosynthesis of 2,4-DAPG is controlled by many regulatory elements, including the global regulator of the Gac/Rsm regulon and the pathway-specific repressor PhlF. In this work, a novel genetic element grxD, which encodes the monothiol glutaredoxin GrxD, was identified and characterized in the production of 2,4-DAPG in P. fluorescens 2P24. Our data showed that the mutation of grxD remarkably decreased 2,4-DAPG production. GrxD lost its ability to alter the production of 2,4-DAPG when the active-site CGFS motif of GrxD was mutated by site-directed mutagenesis. Further studies showed that the RsmA and RsmE proteins were essential for the GrxD-mediated regulation of 2,4-DAPG and exoprotease production. In addition, our data revealed that the deletion of grxD increased the expression of phlF, which negatively regulated the production of 2,4-DAPG. In addition, the grxD mutant was severely impaired in the biocontrol effect against the bacterial wilt of tomato. Overall, our results indicated that the monothiol glutaredoxin GrxD is involved in the production of 2,4-DAPG of P. fluorescens by influencing the Gac/Rsm global signaling pathway and transcriptional regulator PhlF and is essential for the biocontrol properties.