Crystal Structure and Computational Analyses Provide Insights into the Catalytic Mechanism of 2,4-Diacetylphloroglucinol Hydrolase PhlG from Pseudomonas fluorescens

2,4-Diacetylphloroglucinol hydrolase PhlG from Pseudomonas fluorescens catalyzes hydrolytic carbon-carbon (C–C) bond cleavage of the antibiotic 2,4-diacetylphloroglucinol to form monoacetylphloroglucinol, a rare class of reactions in chemistry and biochemistry. To investigate the catalytic mechanism of this enzyme, we determined the three-dimensional structure of PhlG at 2.0 Å resolution using x-ray crystallography and MAD methods. The overall structure includes a small N-terminal domain mainly involved in dimerization and a C-terminal domain of Bet v1-like fold, which distinguishes PhlG from the classical α/β-fold hydrolases. A dumbbell-shaped substrate access tunnel was identified to connect a narrow interior amphiphilic pocket to the exterior solvent. The tunnel is likely to undergo a significant conformational change upon substrate binding to the active site. Structural analysis coupled with computational docking studies, site-directed mutagenesis, and enzyme activity analysis revealed that cleavage of the 2,4-diacetylphloroglucinol C–C bond proceeds via nucleophilic attack by a water molecule, which is coordinated by a zinc ion. In addition, residues Tyr¹²¹, Tyr²²⁹, and Asn¹³², which are predicted to be hydrogen-bonded to the hydroxyl groups and unhydrolyzed acetyl group, can finely tune and position the bound substrate in a reactive orientation. Taken together, these results revealed the active sites and zinc-dependent hydrolytic mechanism of PhlG and explained its substrate specificity as well.     

Characterization of PhlG, a hydrolase that specifically degrades the antifungal compound 2,4-diacetylphloroglucinol in the biocontrol agent Pseudomonas fluorescens CHA0

The potent antimicrobial compound 2,4-diacetylphloroglucinol (DAPG) is a major determinant of biocontrol activity of plant-beneficial Pseudomonas fluorescens CHA0 against root diseases caused by fungal pathogens. The DAPG biosynthetic locus harbors the phlG gene, the function of which has not been elucidated thus far. The phlG gene is located upstream of the phlACBD biosynthetic operon, between the phlF and phlH genes which encode pathway-specific regulators. In this study, we assigned a function to PhlG as a hydrolase specifically degrades DAPG to equimolar amounts of mildly toxic monoacetylphloroglucinol (MAPG) and acetate. DAPG added to cultures of a DAPG-negative DeltaphlA mutant of strain CHA0 was completely degraded, and MAPG was temporarily accumulated. In contrast, DAPG was not degraded in cultures of a DeltaphlA DeltaphlG double mutant. To confirm the enzymatic nature of PhlG in vitro, the protein was histidine tagged, overexpressed in Escherichia coli, and purified by affinity chromatography. Purified PhlG had a molecular mass of about 40 kDa and catalyzed the degradation of DAPG to MAPG. The enzyme had a kcat of 33 s(-1) and a Km of 140 microM at 30 degrees C and pH 7. The PhlG enzyme did not degrade other compounds with structures similar to DAPG, such as MAPG and triacetylphloroglucinol, suggesting strict substrate specificity. Interestingly, PhlG activity was strongly reduced by pyoluteorin, a further antifungal compound produced by the bacterium. Expression of phlG was not influenced by the substrate DAPG or the degradation product MAPG but was subject to positive control by the GacS/GacA two-component system and to negative control by the pathway-specific regulators PhlF and PhlH.     

Characterization of PhlG, a Hydrolase That Specifically Degrades the Antifungal Compound 2,4-Diacetylphloroglucinol in the Biocontrol Agent Pseudomonas fluorescens CHA0

The potent antimicrobial compound 2,4-diacetylphloroglucinol (DAPG) is a major determinant of biocontrol activity of plant-beneficial Pseudomonas fluorescens CHA0 against root diseases caused by fungal pathogens. The DAPG biosynthetic locus harbors the phlG gene, the function of which has not been elucidated thus far. The phlG gene is located upstream of the phlACBD biosynthetic operon, between the phlF and phlH genes which encode pathway-specific regulators. In this study, we assigned a function to PhlG as a hydrolase specifically degrades DAPG to equimolar amounts of mildly toxic monoacetylphloroglucinol (MAPG) and acetate. DAPG added to cultures of a DAPG-negative ΔphlA mutant of strain CHA0 was completely degraded, and MAPG was temporarily accumulated. In contrast, DAPG was not degraded in cultures of a ΔphlA ΔphlG double mutant. To confirm the enzymatic nature of PhlG in vitro, the protein was histidine tagged, overexpressed in Escherichia coli, and purified by affinity chromatography. Purified PhlG had a molecular mass of about 40 kDa and catalyzed the degradation of DAPG to MAPG. The enzyme had a k_cat of 33 s⁻¹ and a K_m of 140 μM at 30°C and pH 7. The PhlG enzyme did not degrade other compounds with structures similar to DAPG, such as MAPG and triacetylphloroglucinol, suggesting strict substrate specificity. Interestingly, PhlG activity was strongly reduced by pyoluteorin, a further antifungal compound produced by the bacterium. Expression of phlG was not influenced by the substrate DAPG or the degradation product MAPG but was subject to positive control by the GacS/GacA two-component system and to negative control by the pathway-specific regulators PhlF and PhlH.     

Isolation and characterization of Pseudomonas brassicacearum J12 as an antagonist against Ralstonia solanacearum and identification of its antimicrobial components

A bacterial strain, J12, isolated from the rhizosphere soil of tomato plants strongly inhibited the growth of phytopathogenic bacteria Ralstonia solanacearum. Strain J12 was identified as Pseudomonas brassicacearum based on its 16S rRNA gene sequence. J12 could produce 2,4-diacetylphloroglucinol (2,4-DAPG), hydrogen cyanide (HCN), siderophore(s) and protease. The maximum growth and antagonistic activity were recorded at 30°C and pH 8. Glucose and tryptone were used as the most suitable carbon and nitrogen sources, respectively. Strain J12 significantly suppressed tomato bacteria wilt by 45.5% in the greenhouse experiment. The main antimicrobial compound of J12 was identified as 2,4-diacetylphloroglucinol (2,4-DAPG) by HPLC-ESI-MS analysis. The gene cluster phlACBD, which is responsible for 2,4-DAPG production, was identified and expressed in the bacterial strain Escherichia coli DH5α.     

抗生素2,4-二乙酰基间苯三酚作为荧光假单胞菌2P24菌株生防功能因子的实证分析

荧光假单胞杆菌2P24菌株分离自小麦全蚀病自然衰退土壤,它是酚类抗生素2,4-二乙酰基间苯三酚(2,4-DAPG)的高产菌,对多种土传病害具有较好的防治能力。利用同源重组构建2,4-DAPG合成基因的定位突变体,并对突变体进行基因互补,通过检测突变菌株和恢复突变菌株抗生素产量和生防效果确定2,4-DAPG在菌株2P24生防功能中的作用。实验中,定位突变体丧失产生抗生素和拮抗病原菌的能力,而恢复突变体的抗生素产量和拮抗能力均恢复至野生菌水平。在对番茄青枯病的防病试验中,2,4-DAPG突变体的防效低且下降快,而恢复突变体的生防能力与野生菌相当,且效果稳定。由此可确定2,4-DAPG是菌株2P24防治番茄青枯病的主要因子,在防效上起关键作用。     

Flavonoids repress the production of antifungal 2,4-DAPG but potentially facilitate root colonization of the rhizobacterium Pseudomonas fluorescens

In the well-known legume–rhizobia symbiosis, flavonoids released by legume roots induce expression of the Nod factors and trigger early plant responses involved in root nodulation. However, it remains largely unknown how the plant-derived flavonoids influence the physiology of non-symbiotic beneficial rhizobacteria. In this work, we demonstrated that the flavonoids apigenin and/or phloretin enhanced the swarming motility and production of cellulose and curli in Pseudomonas fluorescens 2P24, both traits of which are essential for root colonization. Using a label-free quantitative proteomics approach, we showed that apigenin and phloretin significantly reduced the biosynthesis of the antifungal metabolite 2,4-DAPG and further identified a novel flavonoid-sensing TetR regulator PhlH, which was shown to modulate 2,4-DAPG production by regulating the expression of 2,4-DAPG hydrolase PhlG. Although having similar structures, apigenin and phloretin could also influence different physiological characteristics of P. fluorescens 2P24, with apigenin decreasing the biofilm formation and phloretin inducing expression of proteins involved in the denitrification and arginine fermentation processes. Taken together, our results suggest that plant-derived flavonoids could be sensed by the TetR regulator PhlH in P. fluorescens 2P24 and acts as important signalling molecules that strengthen mutually beneficial interactions between plants and non-symbiotic beneficial rhizobacteria.     

Antagonistic activity among 2,4-diacetylphloroglucinol-producing fluorescent Pseudomonas spp

Strains of fluorescent Pseudomonas spp. that produce 2,4-diacetylphloroglucinol (2,4-DAPG) differ in their ability to colonize roots. In this study, we screened 47 2,4-DAPG-producing strains representing17 distinct genotypes for antagonistic activity associated with the production of bacteriocins. Upon induction, over 70% of the strains inhibited the growth of other isolates in vitro. Greenhouse assays indicated that populations of sensitive strains in wheat rhizosphere soil declined more rapidly in the presence of antagonists than when introduced alone. Antagonism can influence the ability of biocontrol agents to establish and maintain effective population densities in situ.     

Evolutionary history of synthesis pathway genes for phloroglucinol and cyanide antimicrobials in plant-associated fluorescent pseudomonads

Plant-beneficial fluorescent Pseudomonas spp. play important ecological roles. Here, their evolutionary history was investigated by a multilocus approach targeting genes involved in synthesis of secondary antimicrobial metabolites implicated in biocontrol of phytopathogens. Some of these genes were proposed to be ancestral, and this was investigated using a worldwide collection of 30 plant-colonizing fluorescent pseudomonads, based on phylogenetic analysis of 14 loci involved in production of 2,4-diacetylphloroglucinol (phlACBDE, phlF, intergenic locus phlA/phlF), hydrogen cyanide (hcnABC, anr) or global regulation of secondary metabolism (gacA, gacS, rsmZ). The 10 housekeeping loci rrs, dsbA, gyrB, rpoD, fdxA, recA, rpoB, rpsL, rpsG, and fusA served as controls. Each strain was readily distinguished from the others when considering allelic combinations for these 14 biocontrol-relevant loci. Topology comparisons based on Shimodaira-Hasegawa tests showed extensive incongruence when comparing single-locus phylogenetic trees with one another, but less when comparing (after sequence concatenation) trees inferred for genes involved in 2,4-diacetylphloroglucinol synthesis, hydrogen cyanide synthesis, or secondary metabolism global regulation with trees for housekeeping genes. The 14 loci displayed linkage disequilibrium, as housekeeping loci did, and all 12 protein-coding loci were subjected to purifying selection except for one positively-selected site in HcnA. Overall, the evolutionary history of Pseudomonas genes involved in synthesis of secondary antimicrobial metabolites important for biocontrol functions is in fact similar to that of housekeeping genes, and results suggest that they are ancestral in pseudomonads producing hydrogen cyanide and 2,4-diacetylphloroglucinol.     

Identification and Characterization of a Gene Cluster for Synthesis of the Polyketide Antibiotic 2,4-Diacetylphloroglucinol from Pseudomonas fluorescens Q2-87

The polyketide metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) is produced by many strains of fluorescent Pseudomonas spp. with biocontrol activity against soilborne fungal plant pathogens. Genes required for 2,4-DAPG synthesis by P. fluorescens Q2-87 are encoded by a 6.5-kb fragment of genomic DNA that can transfer production of 2,4-DAPG to 2,4-DAPG-nonproducing recipient Pseudomonas strains. In this study the nucleotide sequence was determined for the 6.5-kb fragment and flanking regions of genomic DNA from strain Q2-87. Six open reading frames were identified, four of which (phlACBD) comprise an operon that includes a set of three genes (phlACB) conserved between eubacteria and archaebacteria and a gene (phlD) encoding a polyketide synthase with homology to chalcone and stilbene synthases from plants. The biosynthetic operon is flanked on either side by phlE and phlF, which code respectively for putative efflux and regulatory (repressor) proteins. Expression in Escherichia coli of phlA, phlC, phlB, and phlD, individually or in combination, identified a novel polyketide biosynthetic pathway in which PhlD is responsible for the production of monoacetylphloroglucinol (MAPG). PhlA, PhlC, and PhlB are necessary to convert MAPG to 2,4-DAPG, and they also may function in the synthesis of MAPG.     

Involvement of the ABC transporter BcAtrB and the laccase BcLCC2 in defence of Botrytis cinerea against the broad-spectrum antibiotic 2,4-diacetylphloroglucinol

The genetic and biochemical basis of defence mechanisms in plant pathogenic fungi against antifungal compounds produced by antagonistic microorganisms is largely unknown. The results of this study show that both degradative and non-degradative defence mechanisms enable the plant pathogenic fungus Botrytis cinerea to resist the broad-spectrum, phenolic antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG). The efflux pump BcAtrB provides the first line of defence for B. cinerea , preventing accumulation of 2,4-DAPG in the cell to toxic concentrations, whereas the extracellular laccase BcLCC2 mediates, via conversion of tannic acid, subsequent degradation of 2,4-DAPG. Expression of BcatrB is induced by 2,4-DAPG and efflux gives B. cinerea sufficient time to more effectively initiate the process of BcLCC2-mediated antibiotic degradation. This is supported by the observations that the BcatrB mutant is significantly more sensitive to 2,4-DAPG than its parental strain, and is substantially less effective in 2,4-DAPG degradation. The results of this study further showed that BcLCC2 itself is not able to degrade 2,4-DAPG, but requires tannic acid as a mediator for 2,4-DAPG degradation. To our knowledge, this is the first time that the laccase-mediator system is shown to play a role in the detoxification of a broad-spectrum antibiotic compound from bacterial origin. We postulate that yet unknown constituents present in tannic acid act as substrate(s) of BcLCC2, thereby generating radicals that mediate 2,4-DAPG degradation.     

phlD-based genetic diversity and detection of genotypes of 2,4-diacetylphloroglucinol-producing Pseudomonas fluorescens

Diversity within a worldwide collection of 2,4-diacetylphloroglucinol-producing Pseudomonas fluorescens strains was assessed by sequencing the phlD gene. Phylogenetic analyses based on the phlD sequences of 70 isolates supported the previous classification into 18 BOX-PCR genotypes (A-Q and T). Exploiting polymorphisms within the sequence of phlD, we designed and used allele-specific PCR primers with a PCR-based dilution endpoint assay to quantify the population sizes of A-, B-, D-, K-, L- and P-genotype strains grown individually or in pairs in vitro, in the rhizosphere of wheat and in bulk soil. Except for P. fluorescens Q8r1-96, which strongly inhibited the growth of P. fluorescens Q2-87, inhibition between pairs of strains grown in vitro did not affect the accuracy of the method. The allele-specific primer-based technique is a rapid method for studies of the interactions between genotypes of 2,4-diacetylphloroglucinol producers in natural environments.     

Defense responses of Fusarium oxysporum to 2,4-diacetylphloroglucinol, a broad-spectrum antibiotic produced by Pseudomonas fluorescens

A collection of 76 plant-pathogenic and 41 saprophytic Fusarium oxysporum strains was screened for sensitivity to 2,4-diacetylphloroglucinol (2,4-DAPG), a broad-spectrum antibiotic produced by multiple strains of antagonistic Pseudomonas fluorescens. Approximately 17% of the F. oxysporum strains were relatively tolerant to high 2,4-DAPG concentrations. Tolerance to 2,4-DAPG did not correlate with the geographic origin of the strains, formae speciales, intergenic spacer (IGS) group, or fusaric acid production levels. Biochemical analysis showed that 18 of 20 tolerant F. oxysporum strains were capable of metabolizing 2,4-DAPG. For two tolerant strains, analysis by mass spectrometry indicated that deacetylation of 2,4-DAPG to the less fungitoxic derivatives monoacetylphloroglucinol and phloroglucinol is among the initial mechanisms of 2,4-DAPG degradation. Production of fusaric acid, a known inhibitor of 2,4-DAPG biosynthesis in P. fluorescens, differed considerably among both 2,4-DAPG-sensitive and -tolerant F. oxysporum strains, indicating that fusaric acid production may be as important for 2,4-DAPG-sensitive as for -tolerant F. oxysporum strains. Whether 2,4-DAPG triggers fusaric acid production was studied for six F. oxysporum strains; 2,4-DAPG had no significant effect on fusaric acid production in four strains. In two strains, however, sublethal concentrations of 2,4-DAPG either enhanced or significantly decreased fusaric acid production. The implications of 2,4-DAPG degradation, the distribution of this trait within F. oxysporum and other plant-pathogenic fungi, and the consequences for the efficacy of biological control are discussed.     

Composts containing fluorescent pseudomonads suppress fusarium root and stem rot development on greenhouse cucumber

Three composts (Ball, dairy, and greenhouse) were tested for the ability to suppress the development of Fusarium root and stem rot (caused by Fusarium oxysporum f. sp. radicis-cucumerinum) on greenhouse cucumber. Dairy and greenhouse composts significantly reduced disease severity (P = 0.05), while Ball compost had no effect. Assessment of total culturable microbes in the composts showed a positive relationship between disease suppressive ability and total population levels of pseudomonads. In vitro antagonism assays between compost-isolated bacterial strains and the pathogen showed that strains of Pseudomonas aeruginosa exhibited the greatest antagonism. In growth room trials, strains of P. aeruginosa and nonantagonistic Pseudomonas maculicola, plus 2 biocontrol strains of Pseudomonas fluorescens, were tested for their ability to reduce (i) survival of F. oxysporum, (ii) colonization of plants by the pathogen, and (iii) disease severity. Cucumber seedlings grown in compost receiving P. aeruginosa and P. fluorescens had reduced disease severity index scores after 8 weeks compared with control plants without bacteria. Internal stem colonization by F. oxysporum was significantly reduced by P. aeruginosa. The bacteria colonized plant roots at 1.9 × 10(6) ± 0.73 × 10(6) CFU·(g root tissue)-1 and survival was >107 CFU·(g compost)-1 after 6 weeks. The locus for 2,4-diacetylphloroglucinol production was detected by Southern blot analysis and confirmed by PCR. The production of the antibiotic 2,4-diacetylphloroglucinol in liquid culture by P. aeruginosa was confirmed by thin layer chromatography. These results demonstrate that composts containing antibiotic-producing P. aeruginosa have the potential to suppress diseases caused by Fusarium species.     

Characterisation,genetic diversity and antagonistic potential of 2,4-diacetylphloroglucinol producing Pseudomonas fluorescens isolates in groundnut-based cropping systems of Andhra Pradesh,India

This study is focused on isolation and characterisation of 2,4-diacetylphloroglucinol (2,4-DAPG)-producing Pseudomonas fluorescens isolates from different soils of groundnut-based cropping systems in Andhra Pradesh. In our studies, 21 isolates of P. fluorescens were isolated and confirmed through various biochemical tests, of which five were tested positive for 2,4-DAPGproduction with specific primers. Biocontrol potential of these isolates on groundnut stem rot pathogen (Sclerotium rolfsii) was determined through in vitro dual culture assays. The eight isolates were found effective against S. rolfsii (up to 75% inhibition) in dual culture method. All the five 2,4-DAPG-producing Plant Growth-Promoting Rhizobacteria isolates were highly antagonistic to S. rolfsii. Genetic diversity of these P. fluorescens isolates was determined by random amplification of polymorphic DNA analysis. Overall, our results suggest that the prevalence of 2,4-DAPG-producing fluorescent Pseudomonads in different crop rhizospheres of groundnut-based cropping systems.     

Indole-3-Acetic acid production in Pseudomonas fluorescens HP72 and its association with suppression of creeping bentgrass brown patch

Pseudomonas fluorescens HP72, which suppresses the brown patch disease on bentgrass, produces several secondary metabolites, 2,4-diacetylphloroglucinol (2,4-DAPG), HCN, siderophore, and indole-3-acetic acid (IAA). In this study, IAA biosynthesis in strain HP72 was investigated. After several repeated subcultures, the spontaneous IAA low-producing mutant HP72LI was isolated. The IAA low production of the strain HP72LI was due to the low tryptophan side chain oxidase (TSO) activity. Colonization of strain HP72 on the bentgrass root induced root growth reduction, while strain HP72LI did not induce such growth reduction. The colonization ability of strain HP72 on the bentgrass root is higher than that of strain HP72LI. However, as for biocontrol ability, a significant difference in both strains was not detected. IAA production by strain HP72 may play a role in the construction of short root systems and take advantage of root colonization, but does not contribute to the biocontrol properties of P. fluorescens HP72.     

荧光假单胞杆菌2P24中gacA、dsbA和phoP/phoQ基因对小RNA因子RsmZ的转录调控

Pseudomonas fluorescens 2P24是分离自麦田的植物病害生物防治菌株,产生抗生素2, 4-二乙酰基间苯三酚（2,4-diacetylphloroglucinol;2,4-DAPG）是其主要防病机制。菌株2P24中小RNA基因rsmZ正调控抗生素2,4-DAPG的产量。【目的】本文研究上游调控因子对RsmZ转录表达的影响,以进一步理解抗生素产生机制。【方法】构建了rsmZ: : lacZ的转录融合结构,将含有该结构的报告载体转入2P24的多个调控基因缺失突变体中,检测相应的缺失基因对rsmZ转录水平的调控作用。【结果】结果表明,反应调控因子GacA对rsmZ基因的转录具有正调控作用,二硫键合成蛋白DsbA对其负调控;双因子调控系统PhoP/PhoQ突变后,rsmZ基因的转录明显滞后。【结论】小RNA基因rsmZ在菌株2P24中受到多个基因的调控,并在信号传递网络中起到重要作用。     

Differential ability of genotypes of 2,4-diacetylphloroglucinol-producing Pseudomonas fluorescens strains to colonize the roots of pea plants

Indigenous populations of 2,4-diacetylphloroglucinol (2,4-DAPG)-producing fluorescent Pseudomonas spp. that occur naturally in suppressive soils are an enormous resource for improving biological control of plant diseases. Over 300 isolates of 2,4-DAPG-producing fluorescent Pseudomonas spp. were isolated from the rhizosphere of pea plants grown in soils that had undergone pea or wheat monoculture and were suppressive to Fusarium wilt or take-all, respectively. Representatives of seven genotypes, A, D, E, L, O, P, and Q, were isolated from both soils and identified by whole-cell repetitive sequence-based PCR (rep-PCR) with the BOXA1R primer, increasing by three (O, P, and Q) the number of genotypes identified previously among a worldwide collection of 2,4-DAPG producers. Fourteen isolates representing eight different genotypes were tested for their ability to colonize the rhizosphere of pea plants. Population densities of strains belonging to genotypes D and P were significantly greater than the densities of other genotypes and remained above log 6.0 CFU (g of root)(-1) over the entire 15-week experiment. Genetic profiles generated by rep-PCR or restriction fragment length polymorphism analysis of the 2,4-DAPG biosynthetic gene phlD were predictive of the rhizosphere competence of the introduced 2,4-DAPG-producing strains.