全细胞Bioreporter方法评估交通有机污染物的生物利用度和水饱和系统
Summary
与鼻疽sartisoli RP037-MCHE全细胞bioreporter法的开发是为了检测有机污染物（ 即芴）供细菌降解菌丝桥空气填充孔隙中水不饱和模型系统主动转运后的分数。
Abstract
Bioavailability of contaminants is a prerequisite for their effective biodegradation in soil. The average bulk concentration of a contaminant, however, is not an appropriate measure for its availability; bioavailability rather depends on the dynamic interplay of potential mass transfer (flux) of a compound to a microbial cell and the capacity of the latter to degrade the compound. In water-unsaturated parts of the soil, mycelia have been shown to overcome bioavailability limitations by actively transporting and mobilizing organic compounds over the range of centimeters. Whereas the extent of mycelia-based transport can be quantified easily by chemical means, verification of the contaminant-bioavailability to bacterial cells requires a biological method. Addressing this constraint, we chose the PAH fluorene (FLU) as a model compound and developed a water unsaturated model microcosm linking a spatially separated FLU point source and the FLU degrading bioreporter bacterium Burkholderia sartisoli RP037-mChe by a mycelial network of Pythium ultimum. Since the bioreporter expresses eGFP in response of the PAH flux to the cell, bacterial FLU exposure and degradation could be monitored directly in the microcosms via confocal laser scanning microscopy (CLSM). CLSM and image analyses revealed a significant increase of the eGFP expression in the presence of P. ultimum compared to controls without mycelia or FLU thus indicating FLU bioavailability to bacteria after mycelia-mediated transport. CLSM results were supported by chemical analyses in identical microcosms. The developed microcosm proved suitable to investigate contaminant bioavailability and to concomitantly visualize the involved bacteria-mycelial interactions.
Introduction
人口稠密由多种微生物1,2的土壤如细菌。然而，在此栖息条件具有挑战性，特别是在水的供应3项。菌永久需要寻找在异构环境4的最佳条件，但没有连续水膜是导致受限可移动性5妨碍他们自由传播。此外，溶质的扩散率（ 例如，营养素）的不饱和的条件6下降低。因此，细菌和营养物往往物理上分离和营养辅助限于3。其结果是，传输载体的化学化合物，它不需要连续水相可有助于克服这些限制。事实上，很多微生物如真菌和卵菌已经开发出一种丝状增长方式，使他们通过空气填充孔隙成长从而达到和MOBIlizing也物理长距离分离营养素7和8含碳物质。他们甚至可以作为其提供糖和其他能源，以细菌9生物的运输载体。摄取和运输中菌丝生物体也已显示对疏水性有机污染物如多环芳烃（PAH）在终极腐霉 10或在丛枝菌根真菌11。由于PAH是无处不在，难溶于水的污染物在土壤中12，菌丝体介导的运输可能有助于提高生物利用度污染的潜在细菌降解。装置10而污染物输送的总量，可以通过化学直接定量，污染物通过菌丝体降解细菌和其它生物体输送的生物利用度不能容易地进行评估。
以下协议呈现给评估myce的影响的方法LIA对污染物的生物利用度要以直接的方式细菌降解;它允许收集有关污染物的微生物生态系统的时空影响的信息。我们描述了如何建立一个复杂的不饱和缩影系统通过将一个物理上分开PAH点源通过菌丝体的运输载体的PAH降解菌bioreporter模仿气水界面土壤。因为空中交通被排除，菌丝基于交通工具上的PAH的生物利用度对于细菌的效果，可以研究在一个孤立的方式。更详细地说，三环多环芳烃芴，菌丝生物体终极腐霉和细菌bioreporter 伯克霍尔德sartisoli RP037-MCHE 13中所描述的缩影设置施加。细菌B.最初构建sartisoli RP037-MCHE研究菲通量到电池14，并表示增强型绿色荧光蛋白（EGFP）的PAH通量的结果细胞中，而红色荧光mCherry表示组成。详细信息，记者建造由TECON 等 13在初步测试中给出，细菌没有透露游泳，只有非常缓慢蜂拥能力。它能够在作为致密悬浮在菌丝顶端施加缓慢迁移对终极腐霉的菌丝。因为细菌嵌入琼脂糖在以下方案，上菌丝迁移并没有出现。
使用共聚焦激光扫描显微镜（CLSM）中，bioreporter细菌，可以直接在微观可视和EGFP的表达可以相对于被量化到细胞的量（正比于mCherry信号）与该软件ImageJ的的帮助。这允许定性在不同的情景进行比较生物利用度（ 即，更高或更低）。 FLU被发现是后由P.菌丝运输生物利用霉 （ 即，它较阴性对照更高）。此外，该协议描述了如何量化通过化学手段菌丝体介导的转运的总量，并使用硅被覆的玻璃纤维（固相微萃取纤维）中相同的缩影验证污染物的生物利用度。使用此设置一个缩影成果已发表 和体育相结合的探讨霉 ，芴和B. sartisoli RP037-MCHE 15。在这里，重点在于一个详细的方法说明和协议的潜在缺陷的鉴定潜在的进一步应用提供了这方面的知识。进一步的应用可涉及各种真菌，细菌种类（ 例如，从污染的场所），以及其它污染物（ 例如，农药）或杂质的供应（ 例如，年龄土壤）。
Protocol
Representative Results
Discussion
所提出的缩影设置证明适合学习的空间分离的化学品的生物利用度，以降低摄取和运输的菌丝体后的生物。防止部分挥发性化合物的潜在的气相输送与细菌bioreporter细胞可以可视化，而不精心样品制备并因此与敏感的系统的干扰最小。同时，样品的化学分析可以容易地进行，允许有一个良好的控制获得的结果的和的总运输定量。然而，一些点必须仔细之前和在执行实验审议。很重要的一点就是保?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Funding by the German Environmental Foundation (DBU) is acknowledged. The authors thank Ute Kuhlicke for technical help with CLSM analysis and Birgit Würz, Rita Remer, and Jana Reichenbach for skilled experimental help. The authors would particularly like to thank Prof. Jan Roelof van der Meer and Dr. Robin Tecon for fruitful discussion and providing the bioreporter strain. It contributes to the ‘Chemicals in the Environment’ (CITE) research program of the Helmholtz Association.
Materials
| Name of Reagent/ Equipment | Company | Catalog Number | Comments/Description |
| Confocal Microscope | Leica | TCS SP5X, LAS AF – Version 2.6.1; or equivalent CLSM | |
| GC HP 7890 Series GC and Agilent 5975C MSD | Agilent | an equivalent GC/MS may be used | |
| GC capillary column J&W 121-5522 | Agilent | ||
| Cork borer | Fisher Scientific | 12863952 | or any other |
| Cover slips | Marienfeld | 107222 | High performance, No.1.5H |
| GC/MS insterts | WICOM | WIC 47080 | |
| GC/MS vials 2 ml | WICOM | WIC 41150 | |
| Lids / septa for screw cap vials | DIONEX | 49463 / 049464 | |
| Lids for GC/MS vials | WICOM | WIC 43948/B | |
| Objective Slides | Menzel | ordinary | |
| PDMS coated glass fibers | Polymicro Technologies, Inc. | V (PDMS) = 13.55 ± 0.02 µL m-1 | |
| Petri Dishes small / big | Greiner | 633-102 / 628-102 | |
| Screw cap vials 40 ml | DIONEX | 48783 | other glass vials may be used |
| Screw cap vials 60 ml | DIONEX | 48784 | other glass vials may be used |
| Acenaphthylene d08 | Dr. Ehrenstorfer | C 20510100 | |
| Acetone | Carl Roth | 9372.2 | |
| Activated carbon | Sigma-Aldrich | 242276-1kg | |
| Agarose | Carl Roth | 2267.4 | |
| Fluorene | Fluka | 46880 | |
| Kanamycin sulfate | Carl Roth | T832.2 | 50 mg L-1 |
| Methanol | Carl Roth | P7171 | |
| Minimal Medium: | 100 mL solution 1 + 25 mL solution 2 + 5 mL solution 3 ad. 1000 mL aqua dest | ||
| Solution 1 | |||
| Ammonium sulfate | Carl Roth | 3746.1 | 5 g L-1 |
| Magnesium chloride x 6 H2O | Carl Roth | 2189.1 | 1 g L-1 |
| Calcium nitrate x 4 H2O | Carl Roth | P740.1 | 0.5 g L-1 |
| Solution 2 | |||
| Disodium phosphate | Carl Roth | P030.1 | 55.83 g L-1 |
| Monopotassium phosphate | Carl Roth | 3904.1 | 20 g L-1 |
| Solution 3 | pH 6.0 | ||
| Disodium EDTA | MERCK | 1084180250 | 0.8 g L-1 |
| Iron(II) chloride x 4 H2O | MERCK | 1038610250 | 0.3 g L-1 |
| Cobalt(II) chloride x 6 H2O | Carl Roth | T889.3 | 4 mg L-1 |
| Manganese(II) chloride x 1 H2O | Carl Roth | 4320.2 | 10 mg L-1 |
| Copper(II) sulfate | Carl Roth | P023.1 | 1 mg L-1 |
| Sodium molybdate x 2 H2O | Carl Roth | 0274.1 | 3 mg L-1 |
| Zinc chloride | MERCK | 1088160250 | 2 mg L-1 |
| Lithium chloride | Carl Roth | P007.1 | 0.5 mg L-1 |
| Tin(II) chloride x 2 H2O | Carl Roth | 4473.1 | 0.5 mg L-1 |
| Boric acid | Riedel-de-Haen | 11606 | 1 mg L-1 |
| Potassium bromide | Carl Roth | A137.1 | 2 mg L-1 |
| Potassium iodide | Carl Roth | 6750.1 | 2 mg L-1 |
| Barium chloride | Carl Roth | 4453.1 | 0.5 mg L-1 |
| MMA | Minimal medium + agarose 0.2 % | ||
| Phenanthrene d10 | Dr. Ehrenstorfer | C 20920100 | |
| Potato Dextrose Agar: | 24 g L-1 broth + bacto-agar 1.5 %; pH 6.8 | ||
| Potato Dextrose broth | Difco/ Beckton Dickinson | 254920 | |
| Bacto-agar | Difco/ Beckton Dickinson | 214040 | |
| Sodium acetate x 3 hydr. | Carl Roth | 6779.1 | |
| Sodium sulfate | MERCK | 1066495000 | |
| Toluene | MERCK | 1083252500 | |
| mTY medium: | 3 g L-1 yeast extract, 5 g L-1 bacto tryptone and 50 mM NaCl | ||
| Yeast extract | Merck | 1037530500 | |
| Tryptone | Serva | 4864702 | |
| Sodium chloride | Carl Roth | 3957.1 | |
| imageJ with logi tool plugin | http://rsb.info.nih.gov/ij/download.html and http://downloads.openmicroscopy.org/bio-formats/4.4.10 | ||
| Pythium ultimum strain 67-1 | Obtained from the lab of Dr. Christoph Keel; Department of Fundamental Microbiology, University of Lausanne, Switzerland | ||
| Burkholderia sartisoli RP037-mChe | Obtained from the lab of Prof. Jan Roelof van der Meer; Department of Fundamental Microbiology, University of Lausanne, Switzerland |
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