Strain 2P24 Research Articles

(p)ppGpp interacts with TrmE and regulates the PcoI/PcoR quorum-sensing system of Pseudomonas fluorescens 2P241

The efficient colonization of plant-beneficial Pseudomonas spp. is a prerequisite for their biocontrol capacity. Prior work revealed that the PcoI/PcoR quorum-sensing (QS) system plays a pivotal role in the root colonization of P. fluorescens 2P24. During the colonization, strain 2P24 has faced diverse impacts from plant-derived reactive oxygen species and other environmental stress. However, the molecular mechanism by which the PcoI/PcoR QS system is regulated under unfavored conditions remains unclear. Thus, in this study, the role of the (p)ppGpp synthetase RelA and the bifunctional (p)ppGpp synthase/hydrolase SpoT in the PcoI/PcoR QS system of P. fluorescens was investigated. Our data indicated that the deficiency of relA and spoT genes remarkably improved the expression of the pcoI gene, whereas the mutation of the spoT gene significantly repressed the expression of the pcoI gene. We further demonstrated that the regulation of the PcoI/PcoR QS system by (p)ppGpp was dependent on the function of the trmE gene, which encodes a tRNA modification GTPase. Furthermore, the mutation of relA, spoT, or both significantly influenced the motility, biofilm formation, oxidative stress, osmotic tolerance, and rhizosphere colonization. Collectively, our data indicated that the (p)ppGpp signaling pathway mediated by the relA gene and spoT gene was important to the function of the PcoI/PcoR QS system and had important implications for the understanding of the molecular mechanism of (p)ppGpp in epiphytic fitness via TrmE of P. fluorescens.

Pseudomonas fluorescens 2P24: A root beneficial bacterium with an excellent ability to remove multiple nitrogen sources

The nitrogen removal capacity of the root-associated beneficial bacterium Pseudomonas fluorescens 2P24 was investigated in synthetic wastewater and actual wastewater. The results revealed that strain 2P24 removed 97.63 % of ammonium and 100 % of nitrate and nitrite within 48 h when ammonium, nitrate, or nitrite served as the sole nitrogen source (initial concentrations of 100 mg/L). At an ammonium to nitrate to nitrite ratio of 1:1:1, ammonium, nitrate, and nitrite were all removed with efficiencies exceeding 98 %. In addition, strain 2P24 exhibited high nitrogen removal performance in actual wastewater, removing 79.52 % of ammonium and 100 % of nitrate from unsterilized domestic sewage in 10 h. Moreover, regardless of whether the strain was grown in the presence of a sole nitrogen source or mixed nitrogen sources, there was no hazardous nitrite accumulation after 48 h of cultivation. Collectively, the findings indicated that strain 2P24 has potential applications as a standby bacterium for the biological treatment of wastewater.

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.

Characterization and metabolic pathway of Pseudomonas fluorescens 2P24 for highly efficient ammonium and nitrate removal

The ammonium and nitrate removal performance and metabolic pathways of a biocontrol strain, Pseudomonas fluorescens 2P24, were investigated. Strain 2P24 could completely remove 100 mg/L ammonium and nitrate, with removal rates of 8.27 mg/L/h and 4.29 mg/L/h, respectively. During these processes, most of the ammonium and nitrate were converted to biological nitrogen via assimilation, and only small amounts of nitrous oxide escaped. The inhibitor allylthiourea had no impact on ammonium transformation, and diethyl dithiocarbamate and sodium tungstate did not inhibit nitrate removal. Intracellular nitrate and ammonium were detectable during the nitrate and ammonium transformation process, respectively. Moreover, the nitrogen metabolism functional genes (glnK, nasA, narG, nirBD, nxrAB, nirS, nirK, and norB) were identified in the strain. All results highlighted that P. fluorescens 2P24 is capable of assimilatory and dissimilatory nitrate reduction, ammonium assimilation and oxidation, and denitrification.

Characterization of the SPI-1 Type III Secretion System in Pseudomonas fluorescens 2P24.

Pseudomonas fluorescens 2P24 is a plant growth-promoting rhizobacterium (PGPR) isolated from wheat take-all decline soil. Genomic analysis of strain 2P24 revealed the presence of a complete SPI-1 type III secretion system (T3SS) gene cluster on the chromosome with an organization and orientation similar to the SPI-1 T3SS gene clusters of Salmonella enterica and P. kilonensis F113. Phylogenetic analysis revealed that the SPI-1 T3SS gene cluster of strain 2P24 might be obtained from Salmonella and Shigella by horizontal gene transfer. Two transcriptional regulator homologs of HilA and InvF were found from the SPI-1 T3SS gene cluster of strain 2P24. HilA regulated the expression of the structural genes positively, such as invG, sipB, sipD, prgI, and prgK. Prediction of transcriptional binding sites and RNA-seq analysis revealed 14 genes were up-regulated by InvF in strain 2P24. Exploring potential roles of SPI-1 T3SS revealed that it was not associated with motility. However, 2P24ΔinvF reduced resistance against Fusarium graminearum significantly. 2P24ΔhilA enhanced formation of biofilm significantly at 48 h. All three mutants 2P24ΔhilA, 2P24ΔinvF, and 2P24ΔinvE-C reduced the chemotactic responses to glucose significantly. Finally, the determination of SPI-1 mutants to trigger innate immunity in Nicotiana benthamiana showed that 2P24ΔinvE-C reduced the ability to induce the production of reactive oxygen species compared with the wild type strain 2P24.

Fic Proteins Inhibit the Activity of Topoisomerase IV by AMPylation in Diverse Bacteria

The Fic (filamentation induced by cyclic AMP) domain is a widely distributed motif with a conserved sequence of HPFx[D/E]GN[G/K]R, some of which regulate cellular activity by catalyzing the transfer of the AMP moiety from ATP to protein substrates. Some Fic proteins, including Fic-1 from the soil bacterium Pseudomonas fluorescens strain 2P24, have been shown to inhibit bacterial DNA replication by AMPylating the subunit B of DNA gyrase (GyrB), but the biochemical activity and cellular target of most Fic proteins remain unknown. Here, we report that Fic-2, which is another Fic protein from strain 2P24 and Fic-1 AMPylate the topoisomerase IV ParE at Tyr109. We also examined Fic proteins from several phylogenetically diverse bacteria and found that those from Yersinia pseudotuberculosis and Staphylococcus aureus AMPylate ParE and GrlB, the counterpart of ParE in Gram-positive bacteria, respectively. Modification by Fic-1 of P. fluorescens and FicY of Y. pseudotuberculosis inhibits the relaxation activity of topoisomerase IV. Consistent with the inhibition of ParE activity, ectopic expression of these Fic proteins causes cell filamentation akin to the canonical par phenotype in which nucleoids are assembled in the center of elongated cells, a process accompanied by the induction of the SOS response. Our results establish that Fic proteins from diverse bacterial species regulate chromosome division and cell separation in bacteria by targeting ParE.

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

BackgroundPseudomonas 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.ResultsIn 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.ConclusionsThese 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.

Characterization the role of GacA-dependent small RNAs and RsmA family proteins on 2,4-diacetylphloroglucinol production in Pseudomonas fluorescens 2P24

Pseudomonas fluorescens 2P24 is a plant-beneficial rhizobacteria that controls many root diseases caused by soil-borne pathogens, and the production of the antibiotic compound 2,4-diacetylphloroglucinol (2,4-DAPG) is essential for its biocontrol ability. In the present study, we investigated the regulatory mechanism acting on the production of 2,4-DAPG by the GacA-dependent small non-coding RNAs (sRNAs) and RsmA/E proteins in strain 2P24. Our results showed that the GacS-GacA system regulates the expression of the phlACBD locus, which is responsible for 2,4-DAPG production, by inducing the expression of rsmX, rsmX1, rsmY, and rsmZ. A novel GacA-regulated sRNA, RgsA, was found to negatively regulate 2,4-DAPG production. Activation of the phlACBD locus by the GacS-GacA system is mediated through RsmA and RsmE proteins (but not RsmI), which inhibit phlACBD translation by binding to the putative RsmA/E recognition element in the phlACBD leader. Taken together, our results suggested that in P. fluorescens 2P24, the GacS-GacA system controls the cellular 2,4-DAPG levels in the cell by fine-tuning the function of sRNAs in P. fluorescens.

Effect of reducing the activity of respiratory chain on biosynthesis of poly(3-hydroxybutyrate-co-lactate) in Escherichia coli

Poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)] belongs to the polyhydroxyalkanoates (PHA) family and possesses promising properties including biocompatibility and biodegradability. In this study, we directly synthesized P(3HB-co-LA) with glucose by introducing the β-ketothiolase and acetoacetyl-CoA reductase from Ralstonia eutropha, the engineered propionate CoA transferase from Clostridium propionicum and the engineered polyhydroxyalkanoate synthase from Pseudomonas fluorescens strain 2P24 into Escherichia coli. The polymer content was 83.9% (W/W), and the molar percentage of lactate reached 1.6%. On this basis, in order to accumulate lactate, we reduced the activity of respiratory chain by deleting the ubiX gene, which is involved in the synthesis of coenzyme Q8. Moreover, we removed the dld gene to avoid the conversion of lactate to pyruvate during the fermentation. With these manipulations, the molar percentage of lactate in the polymer was improved to 14.1%, with an 81.7% (W/W) of polymer content. The test results indicated that the strategy of reducing the activity of respiratory chain effectively increased the lactate units in the polymer, and it contributed a new approach to change the content of monomer components in the polymer.

Transcriptional Regulator PhlH Modulates 2,4-Diacetylphloroglucinol Biosynthesis in Response to the Biosynthetic Intermediate and End Product.

Certain strains of biocontrol bacterium Pseudomonas fluorescens produce the secondary metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) to antagonize soilborne phytopathogens in the rhizosphere. The gene cluster responsible for the biosynthesis of 2,4-DAPG is named phlACBDEFGH and it is still unclear how the pathway-specific regulator phlH within this gene cluster regulates the metabolism of 2,4-DAPG. Here, we found that PhlH in Pseudomonas fluorescens strain 2P24 represses the expression of the phlG gene encoding the 2,4-DAPG hydrolase by binding to a sequence motif overlapping with the -35 site recognized by σ70 factors. Through biochemical screening of PhlH ligands we identified the end product 2,4-DAPG and its biosynthetic intermediate monoacetylphloroglucinol (MAPG), which can act as signaling molecules to modulate the binding of PhlH to the target sequence and activate the expression of phlG Comparison of 2,4-DAPG production between the ΔphlH, ΔphlG, and ΔphlHG mutants confirmed that phlH and phlG impose negative feedback regulation over 2,4-DAPG biosynthesis. It was further demonstrated that the 2,4-DAPG degradation catalyzed by PhlG plays an insignificant role in 2,4-DAPG tolerance but contributes to bacterial growth advantages under carbon/nitrogen starvation conditions. Taken together, our data suggest that by monitoring and down-tuning in situ levels of 2,4-DAPG, the phlHG genes could dynamically modulate the metabolic loads attributed to 2,4-DAPG production and potentially contribute to rhizosphere adaptation.IMPORTANCE 2,4-DAPG, which is synthesized by biocontrol pseudomonad bacteria, is a broad-spectrum antibiotic against bacteria, fungi, oomycetes, and nematodes and plays an important role in suppressing soilborne plant pathogens. Although most of the genes in the 2,4-DAPG biosynthetic gene cluster (phl) have been characterized, it is still not clear how the pathway-specific regulator phlH is involved in 2,4-DAPG metabolism. This work revealed the role of PhlH in modulating 2,4-DAPG levels by controlling the expression of 2,4-DAPG hydrolase PhlG in response to 2,4-DAPG and MAPG. Since 2,4-DAPG biosynthesis imposes a metabolic burden on biocontrol pseudomonads, it is expected that the fine regulation of phlG by PhlH offers a way to dynamically modulate the metabolic loads attributed to 2,4-DAPG production.

PBT1, a novel antimicrobial protein from the biocontrol agent Bacillus subtilis XF-1 against Plasmodiophora brassicae

Plasmodiophora brassicae is a Plasmodiophorid and obligate biotrophic protist in the eukaryotic kingdom of Rhizaria. It causes clubroot disease of cruciferous crops by producing resting spores that can survive in the soil for more than ten years. Although up to date there is no effective chemical control but now a strain of the bacterium Bacillus subtilis XF-1, isolated from China, has been found to inhibit P. brassicae and the mycelial growth of other plant-pathogenic fungi, such as Magnaporthe oryzae, Curvularia lunata,Fusarium oxysporum and so on. The antimicrobial activity of this biocontrol bacterium lies in its water-soluble protein (PBT1) which was isolated and purified by means of molecular sieve and affinity chromatography. The single band of the purified protein has a molecular mass of 25 kDa, estimated by a sodium dodecyl sulfate- polyacrylamide gel electrophoresis and it was heat resistant. The result of the sequencing proved PBT1 to be a new protein. Recombinant plasmid pQE81L-PBT1 was constructed using the coding gene of Pbt1 and plasmid PQE81-L, and expressed in Escherichia coli BL21. The fusion protein of BL21 exhibited significant detrimental effects on P. brassicae spores. In addition, pQE81L-PBT1 was successfully inserted into Pseudomonas fluorescens strain 2P24 which developed antagonistic effect to the clubroot pathogen that was not suppressed by 2P24 alone. The results showed that PBT1 protein might play a key role in the biocontrol of clubroot disease of crucifers.

Supramolecular Structure and Functional Analysis of the Type III Secretion System in Pseudomonas fluorescens 2P24

The type III secretion system (T3SS) of plant and animal bacterial pathogens directs the secretion and injection of proteins into host cells. Some homologous genes of T3SS were found also in non-pathogenic bacteria, but the organization of its machinery and basic function are still unknown. In this study, we identified a T3SS gene cluster from the plant growth-promoting Pseudomonas fluorescens 2P24 and isolated the corresponding T3SS apparatus. The T3SS gene cluster of strain 2P24 is similar organizationally to that of pathogenic P. syringae, except that it lacks the regulator hrpR and the hrpK1 and hrpH genes, which are involved in translocation of proteins. Electron microscopy revealed that the T3SS supramolecular structure of strain 2P24 was comprised of two distinctive substructures: a long extracellular, filamentous pilus, and a membrane-embedded base. We show that strain 2P24 deploys a harpin homolog protein, RspZ1, to elicit a hypersensitive response when infiltrated into Nicotiana tabacum cv. xanthi leaves with protein that is partially purified, and by complementing the hrpZ1 mutation of pHIR11. The T3SS of strain 2P24 retained ability to secrete effectors, whereas its effector translocation activity appeared to be excessively lost. Mutation of the rscC gene from 2P24 T3SS abolished the secretion of effectors, but the general biocontrol properties were unaffected. Remarkably, strain 2P24 induced functional MAMP-triggered immunity that included a burst of reactive oxygen species, strong suppression of challenge cell death, and disease expansion, while it was not associated with the secretion functional T3SS.

Posttranscriptional regulation of 2,4-diacetylphloroglucinol production by GidA and TrmE in Pseudomonas fluorescens 2P24.

Pseudomonas fluorescens 2P24 is a soilborne bacterium that synthesizes and excretes multiple antimicrobial metabolites. The polyketide compound 2,4-diacetylphloroglucinol (2,4-DAPG), synthesized by the phlACBD locus, is its major biocontrol determinant. This study investigated two mutants defective in antagonistic activity against Rhizoctonia solani. Deletion of the gidA (PM701) or trmE (PM702) gene from strain 2P24 completely inhibited the production of 2,4-DAPG and its precursors, monoacetylphloroglucinol (MAPG) and phloroglucinol (PG). The transcription of the phlA gene was not affected, but the translation of the phlA and phlD genes was reduced significantly. Two components of the Gac/Rsm pathway, RsmA and RsmE, were found to be regulated by gidA and trmE, whereas the other components, RsmX, RsmY, and RsmZ, were not. The regulation of 2,4-DAPG production by gidA and trmE, however, was independent of the Gac/Rsm pathway. Both the gidA and trmE mutants were unable to produce PG but could convert PG to MAPG and MAPG to 2,4-DAPG. Overexpression of PhlD in the gidA and trmE mutants could restore the production of PG and 2,4-DAPG. Taken together, these findings suggest that GidA and TrmE are positive regulatory elements that influence the biosynthesis of 2,4-DAPG posttranscriptionally.

Effect of the hfq gene on 2,4-diacetylphloroglucinol production and the PcoI/PcoR quorum-sensing system in Pseudomonas fluorescens 2P24

Pseudomonas fluorescens 2P24 is an effective biological control agent of a number of soilborne plant diseases caused by pathogenic microorganisms. Among a range of secondary metabolites produced by strain 2P24, the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) is the major determinant of its disease-suppressive capacity. In this study, we performed random mutagenesis using mini-Tn5 in order to screen for the transcriptional regulators of the phlA gene, a biosynthase gene responsible for 2,4-DAPG production. The mutant PMphlA23 with significantly decreased phlA gene expression was identified from approximately 10,000 insertion colonies. The protein sequence of the interrupted gene has 84% identity to Hfq, a key regulator important for stress resistance and virulence in Pseudomonas aeruginosa. Genetic inactivation of hfq resulted in decreased expression of phlA and reduced production of 2,4-DAPG. Furthermore, the hfq gene was also required for the expression of pcoI, a synthase gene for the LuxI-type quorum-sensing signaling molecule N-acyl-homoserine lactone. Additionally, the hfq mutation drastically reduced biofilm formation and impaired the colonization ability of strain 2P24 on wheat rhizospheres. Based on these results, we propose that Hfq functions as an important regulatory element in the complex network controlling environmental adaption in P. fluorescens 2P24.

The resistance-nodulation-division efflux pump EmhABC influences the production of 2,4-diacetylphloroglucinol in Pseudomonas fluorescens 2P24

The polyketide metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) plays a major role in the biological control of soil-borne plant diseases by Pseudomonas fluorescens 2P24. Two mutants (PM810 and PM820) with increased extracellular accumulation of 2,4-DAPG were isolated using transposon mutagenesis. The disrupted genes in these two mutants shared >80 % identity with the genes of the EmhR-EmhABC resistance-nodulation-division (RND) efflux system of P. fluorescens cLP6a. The deletion of emhA (PM802), emhB (PM803) or emhC (PM804) genes in strain 2P24 increased the extracellular accumulation of 2,4-DAPG, whereas the deletion of the emhR (PM801) gene decreased the biosynthesis of 2,4-DAPG. The promoter assay confirmed the elevated transcription of emhABC in the EmhR disrupted strain (PM801) and an indirect negative regulation of 2,4-DAPG biosynthetic locus transcription by the EmhABC efflux pump. Induction by exogenous 2,4-DAPG led to remarkable differences in transcription of chromosome-borne phlA : : lacZ fusion in PM901 and PM811 (emhA(-)) strains. Additionally, the EmhABC system in strain 2P24 was involved in the resistance to a group of toxic compounds, including ampicillin, chloramphenicol, tetracycline, ethidium bromide and crystal violet. In conclusion, our results suggest that the EmhABC system is an important element that influences the production of antibiotic 2,4-DAPG and enhances resistance to toxic compounds in P. fluorescens 2P24.

Differential control of the PcoI/PcoR quorum-sensing system in Pseudomonas fluorescens 2P24 by sigma factor RpoS and the GacS/GacA two-component regulatory system

A quorum-sensing (QS) locus PcoI/PcoR had been identified previously in the biological control bacterium Pseudomonas fluorescens 2P24. In this paper, we investigated the upstream regulators that influence the transcription of the N-acyl homoserine lactone (AHL) biosynthase gene pcoI using a chromosomal pcoIColon, two colonslacZ fusion reporter strain. Stationary sigma factor RpoS was identified as a negative regulator of QS system using a random mini-Tn5 mutant procedure. Furthermore, deletion mutagenesis and complementation experiments demonstrated that the two-component system GacS/GacA positively regulated the QS system by upregulating pcoI transcription. However, compared with the gacA or gacS mutant, introduction of a second mutation of rpoS in the gacA(-) or gacS(-) background did not lead to further change in the transcriptional expression of the pcoI gene or the synthesis of AHL. Our results suggest that in strain 2P24, RpoS could only play its negative regulatory role on the pcoI gene under a functional GacS/GacA system background.

Quorum-sensing system influences root colonization and biological control ability in Pseudomonas fluorescens 2P24

Pseudomonas fluorescens 2P24 is a biocontrol agent isolated from a wheat take-all decline soil in China. This strain produces several antifungal compounds, such as 2,4-diacetylphloroglucinol (2,4-DAPG), hydrogen cyanide and siderophore(s). Our recent work revealed that strain 2P24 employs a quorum-sensing system to regulate its biocontrol activity. In this study, we identified a quorum-sensing system consisting of PcoR and PcoI of the LuxR-LuxI family from strain 2P24. Deletion of pcoI from 2P24 abolishes the production of the quorum-sensing signals, but does not detectably affect the production of antifungal metabolites. However, the mutant is significantly defective in biofilm formation, colonization on wheat rhizosphere and biocontrol ability against wheat take-all, whilst complementation of pcoI restores the biocontrol activity to the wild-type level. Our data indicate that quorum sensing is involved in regulation of biocontrol activity in P. fluorescens 2P24.