荧光假单胞菌2P24中retS对抗生素2，4-二乙酰基间苯三酚合成的影响

假单胞菌中RetS是一个位于膜上的感应激酶,对多种基因的表达都有调控作用.在铜绿假单胞菌中,RetS可以与另一个感应激酶GacS直接互作,并抑制GacS的磷酸化.[目的]本文利用遗传学方法研究了荧光假单胞菌2P24中RetS对抗生素2,4-二乙酰基间苯三酚(2,4-DAPG)合成的影响,并对其可能的调控机制进行了初步探索.[方法]利用高压液相色谱法(HPLC)检测2P24及其衍生菌株中2,4-DAPG的产量.将Gac/Rsm 信号途径中小RNA及调控蛋白的转录报告质粒转入到菌株2P24及其retS突变菌株中,检查RetS对以上基因转录表达的影响.[结果]菌株2P24中缺失retS后未知红色素和抗生素2,4-DAPG的产量较野生型均明显升高.进一步试验表明,RetS转录水平负调控小RNA RsmX和RsmZ的表达,这说明RetS可在转录后水平影响2,4-DAPG的合成.然而,同时缺失retS和gacS或同时缺失retS和gacA之后,由retS单基因缺失所造成的未知红色素和2,4-DAPG合成量升高、小RNA转录表达增强等性状消失,而与gacS或gacA单基因缺失突变体的表型一致.[结论]以上结果说明菌株2P24中RetS是2,4-DAPG及未知红色素合成的负调控因子,并且RetS对2,4-DAPG及未知红色素合成的调控依赖于Gac/Rsm信号传递路径.     

荧光假单胞菌2P24中PcoI-PcoR群体感应系统的自体反馈调控

荧光假单胞菌2P24的PcoI-PcoR 群体感应(QS)系统信号合成基因pcoI的表达受多种因子的调控, 其中GacS-GacA双因子调控系统在转录水平正调控信号合成基因pcoI的表达。为进一步研究QS系统调控因子, 将2P24基因组文库转入gacA缺失的pcoI基因转录报告菌株PM203 (pcoI-lacZ, gacA-), 筛选可提高pcoI表达的基因。结果表明粘粒pP32-24可显著提高pcoI转录水平, 亚克隆实验证明其中的功能基因为pcoI; 外源添加标准信号分子3-氧-己酰高丝氨酸内酯(3-oxo-C8-HSL)同样可显著提高pcoI基因的表达, 表明pcoI基因的表达对自身有正调控作用。同时构建了QS系统的另外一个组分pcoR基因的缺失突变体, pcoR基因缺失后pcoI的表达和N-乙酰高丝氨酸内酯信号分子(AHL)的产量明显低于野生菌株及其互补菌株, 并显著降低该菌株的生物膜(Biofilm)形成能力。这些结果表明菌株2P24的PcoI-PcoR QS系统中, 信号合成基因pcoI的表达受自体反馈调控, pcoR基因参与pcoI基因表达的调控以及生物膜的形成。     

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。     

Effect of 2,4-Diacetylphloroglucinol Producing,Overproducing, and Nonproducing Pseudomonas fluorescens F113 in the Rhizosphere of Pea

Pseudomonas fluorescens F113lacZY and modified strains carrying different function modifications were assessed for their impact in the rhizosphere of pea. Strain F113lacZY naturally produces the anti-fungal metabolite 2,4-diacetylphloroglucinol (Phl) useful in plant disease control. The first modified strain of F113 was repressed in production of Phl, creating the Phl negative strain F113G22. The second was a plasmid based overproducer of Phl (F113Rif (pCUGP)). Both the F113lacZY and the F113Rif (pCUGP) strains increased the rhizoplane fungal populations, whereas the same strains reduced the rhizosphere soil fungal populations with respect to the control. Similar results were found with the rhizoplane and rhizosphere soil bacterial populations. The F113G22 treatment resulted in a significantly greater indigenous fluorescent Pseudomonas population than the F113lacZY and F113Rif (pCUGP) treatments and a greater total Pseudomonas population than the control, F113lacZY, and F113Rif (pCUGP) treatments. Overproduction of Phl did not affect the establishment of the introduced Pseudomonas population. None of the inocula displaced the indigenous populations, but the F113G22 inocula had an additive effect on the total Pseudomonas population. P (phosphatase), S (sulphatase), and N (urease) cycle enzyme activities were increased while C (glucosidase, NAGase) cycle activities were decreased by the F113lacZY and F113Rif (pCUGP) treatments, suggesting C leakage from the roots. Overall, most effects of inoculation compared to the wild type were found with the non-Phl-producing strain. Overproduction of Phl had little environmental effect in relation to wild-type inocula.     

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.     

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.     

Uptake of 2,4-Dichlorophenoxyacetic Acid by Pseudomonas fluorescens

Factors influencing the uptake of the sodium salt of 2,4-dichlorophenoxyacetic acid (2,4-D), under conditions in which no net metabolism occurred, were investigated in an effort to determine both the significance of "non-metabolic" uptake as a potential agent in reducing pesticide levels and the mechanisms involved. Uptake of 2,4-D was affected by pH, temperature, and the presence of other organic and inorganic compounds. Uptake was more pronounced at pH values less than 6, which implies that there may be some interaction between charged groups on the cell and the ionized carboxyl group of 2,4-D. Active transport, carrier-mediated diffusion, passive diffusion, and adsorption were considered as possible mechanisms. Though uptake was inhibited by glucose, sodium azide, and fluorodinitrobenzene (but not by uranyl ion), 2,4-D was not accumulated against a concentration gradient, a necessary consequence of an active transport system, nor was isotope counterflow found to occur. Thus, carrier-mediated diffusion was finally precluded, implying that uptake probably occurs by a two-step process: sorption onto the cell wall followed by passive diffusion into the cytoplasm.     

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.     

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.     

荧光假单胞菌2P24中gidA基因对PcoI/PcoR群体感应系统的影响

【目的】荧光假单胞菌(Pseudomonas fluorescens)2P24中PcoI/PcoR群体感应系统是调控生物膜形成与植物根部定殖能力的重要元件,同时该系统也受到多种上游因子的调控。利用遗传学方法研究P.fluorescens 2P24中gidA基因对群体感应系统的调控作用。【方法】将群体感应信号合成基因pcoI的转录报告质粒p970km-pcoIp转入菌株2P24和gidA基因突变体中以检测gidA对pcoI基因表达的影响,并利用报告菌Agrobacterium tumefaciens NTL4(pZLR4)测定菌株2P24及其衍生菌的信号N-乙酰高丝氨酸内酯(AHL)产量。【结果】gidA基因突变后pcoI基因的转录表达和AHL产量与野生型2P24相比显著降低。gidA基因突变体的游动能力没有受到明显的影响,但生物膜的形成显著低于野生型和互补菌株。小麦根部定殖实验表明,温室条件下gidA突变菌株在灭菌土和自然土中对小麦根尖和根围的定殖量较野生型和互补菌显著减少。此外,突变gidA基因并不影响菌株在LB培养基中的生长,但以葡萄糖、蔗糖、果糖、甘油、半乳糖、阿拉伯糖、甘露糖、木糖或山梨醇为唯一碳源时,gidA突变体的生长受到明显的抑制。【结论】GidA对假单胞菌2P24中的PcoI/PcoR群体感应系统、生物膜形成、定殖和碳源利用具有显著的正调控作用。     

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。     

生防假单胞菌2P24中mvaT和mvaV基因对PcoI/PcoR群体感应系统的调控作用

【目的】自小麦全蚀病自然衰退土壤分离得到的荧光假单胞菌(Pseudomonas fluorescens)2P24,可防治多种由植物病原菌引起的土传病害。菌株2P24具有群体感应(quorum-sensing,QS)系统PcoI/PcoR,该系统影响生防菌2P24生物膜的形成以及其在小麦根围的定殖能力,从而影响2P24的生防能力。本文利用遗传学方法进一步研究了2P24中QS系统的调控途径。【方法】将QS系统信号合成基因pcoI的转录报告质粒p970Gm-pcoIp转入gacA基因突变菌株PM201中,再利用Tn5转座子对该菌株进行随机突变,筛选影响pcoI基因表达的调控因子。【结果】根据菌落颜色的变化筛选到2株突变菌株。Tn5插入位点和基因序列分析表明这2个突变体中Tn5破坏了同一个基因mvaT;设计引物利用PCR方法从2P24基因组中获得mvaT基因及其同源基因mvaV。转录融合报告实验表明:与野生菌株2P24相比,mvaT及mvaV突变体中pcoI基因的表达和N-乙酰高丝氨酸内酯的产量显著提高;HPLC试验表明mvaT和mvaV基因影响抗生素2,4-二乙酰基间苯三酚的合成。细菌双杂交试验证实,MvaT蛋白和MvaV蛋白在体内发生自身互作,这两个蛋白也可相互作用。【结论】以上结果表明mvaT和mvaV参与调控生防假单胞菌2P24的PcoI/PcoR群体感应系统,并可能影响其生防功能基因的表达。     

Fusaric Acid-Producing Strains of Fusarium oxysporum Alter 2,4-Diacetylphloroglucinol Biosynthetic Gene Expression in Pseudomonas fluorescens CHA0 In Vitro and in the Rhizosphere of Wheat

The phytotoxic pathogenicity factor fusaric acid (FA) represses the production of 2,4-diacetylphloroglucinol (DAPG), a key factor in the antimicrobial activity of the biocontrol strain Pseudomonas fluorescens CHA0. FA production by 12 Fusarium oxysporum strains varied substantially. We measured the effect of FA production on expression of the phlACBDE biosynthetic operon of strain CHA0 in culture media and in the wheat rhizosphere by using a translational phlA′-′lacZ fusion. Only FA-producing F. oxysporum strains could suppress DAPG production in strain CHA0, and the FA concentration was strongly correlated with the degree of phlA repression. The repressing effect of FA on phlA′-′lacZ expression was abolished in a mutant that lacked the DAPG pathway-specific repressor PhlF. One FA-producing strain (798) and one nonproducing strain (242) of F. oxysporum were tested for their influence on phlA expression in CHA0 in the rhizosphere of wheat in a gnotobiotic system containing a sand and clay mineral-based artificial soil. F. oxysporum strain 798 (FA⁺) repressed phlA expression in CHA0 significantly, whereas strain 242 (FA⁻) did not. In the phlF mutant CHA638, phlA expression was not altered by the presence of either F. oxysporum strain 242 or 798. phlA expression levels were seven to eight times higher in strain CHA638 than in the wild-type CHA0, indicating that PhlF limits phlA expression in the wheat rhizosphere.     

Quantification of 2,4-Diacetylphloroglucinol Produced by Fluorescent Pseudomonas spp. In Vitro and in the Rhizosphere of Wheat

The broad-spectrum antibiotic 2,4-diacetylphloroglucinol (Phl) is a major determinant in the biological control of a wide range of plant diseases by fluorescent Pseudomonas spp. A protocol was developed to readily isolate and quantify Phl from broth and agar cultures and from the rhizosphere environment of plants. Extraction with ethyl acetate at an acidic pH was suitable for both in vitro and in situ sources of Phl. For soil samples, the addition of an initial extraction step with 80% acetone at an acidic pH was highly effective in eliminating polar organic soil components, such as humic and fulvic acids, which can interfere with Phl detection by high-performance liquid chromotography. The efficiency of Phl recovery from soil by a single extraction averaged 54.6%, and a second extraction added another 6.1%. These yields were substantially greater than those achieved by several standard protocols commonly used to extract polar phenolic compounds from soil. For the first time Phl was isolated from the rhizosphere environment in raw soil. Following application of Pseudomonas fluorescens Q2-87 and the Phl-overproducing strain Q2-87(pPHL5122) to the seeds of wheat, 2.1 and 2.4 (mu)g of Phl/g of root plus rhizosphere soil, respectively, were isolated from wheat grown in a Ritzville silt loam; 0.47 and 1.3 (mu)g of Phl/g of root plus rhizosphere soil, respectively, were isolated from wheat grown in a Shano silt loam. However, when the amount of Phl was calculated on the basis of cell density, Q2-87(pPHL5122) produced seven and six times more antibiotic than Q2-87 in Ritzville silt loam, and Shano silt loam, respectively.     

Quantification of 2,4-diacetylphloroglucinol-producing Pseudomonas fluorescens strains in the plant rhizosphere by real-time PCR

A real-time PCR SYBR green assay was developed to quantify populations of 2,4-diacetylphloroglucinol (2,4-DAPG)-producing (phlD+) strains of Pseudomonas fluorescens in soil and the rhizosphere. Primers were designed and PCR conditions were optimized to specifically amplify the phlD gene from four different genotypes of phlD+ P. fluorescens. Using purified genomic DNA and genomic DNA extracted from washes of wheat roots spiked with bacteria, standard curves relating the threshold cycles (C(T)s) and copies of the phlD gene were generated for P. fluorescens strains belonging to genotypes A (Pf-5), B (Q2-87), D (Q8r1-96 and FTAD1R34), and I (FTAD1R36). The detection limits of the optimized real-time PCR assay were 60 to 600 fg (8 to 80 CFU) for genomic DNA isolated from pure cultures of P. fluorescens and 600 fg to 6.0 pg (80 to 800 CFU, corresponding to log 4 to 5 phlD+ strain CFU/rhizosphere) for bacterial DNA extracted from plant root washes. The real-time PCR assay was utilized to quantify phlD+ pseudomonads in the wheat rhizosphere. Regression analysis of population densities detected by real-time PCR and by a previously described phlD-specific PCR-based dilution endpoint assay indicated a significant linear relationship (P = 0.0016, r2 = 0.2). Validation of real-time PCR assays with environmental samples was performed with two different soils and demonstrated the detection of more than one genotype in Quincy take-all decline soil. The greatest advantage of the developed real-time PCR is culture independence, which allows determination of population densities and the genotype composition of 2,4-DAPG producers directly from the plant rhizospheres and soil.