电化学检测氘动力学同位素对希瓦氏菌 oneidensis MR-1 细胞外电子传输的影响
Summary
在这里, 我们提出了一个全细胞电化学实验的协议, 以研究质子传输的贡献, 通过外膜细胞色素复合体在希瓦氏菌 oneidensis MR-1 的细胞外电子转运率。
Abstract
在细菌外膜中嵌入的c型细胞色素复合物的直接电化学检测 (外膜c型细胞色素复合物;OM c-青旅) 最近出现作为一种新的全细胞分析方法来表征细菌电子运输从呼吸道链到细胞外部, 称为细胞外电子转运 (EET)。研究了 EET 反应过程中电子流的途径和动力学, 研究了与 EET 相关的阳离子输送的影响的全细胞电化学方法尚未建立。在本研究中, 本文介绍了通过 OM c-中青旅使用模型微生物 (希瓦氏菌 oneidensis MR-1) 检测 EET 上氘动力学同位素效应 (绢) 的生物化学技术的例子。如果 EET 通过 OM c-青旅作为微生物电流生产中的速率限制步骤, 则可以获得 EET 过程的绢。为此, 在添加 D2O 之前, 将上清液替换为含有足够数量的电子捐献者的新鲜介质, 以支持上游代谢反应的速率, 并从统一的浮游细胞中移除工作电极上的单层生物膜。此外, 还介绍了通过 OM c-青旅法确定微生物电流生产中 EET 速率限制步骤的替代方法。研究质子转运动力学的全细胞电化学检测技术可应用于其它活性微生物菌株。
Introduction
在一个完整的细菌细胞中, 直接表征氧化还原蛋白的电化学技术最近出现, 自从发现了金属还原微生物菌株, 如S. oneidensis MR-1 或地杆菌能 sulfurreducens PCA,其中有外膜 c 型细胞色素复合体 (OM c-青旅) 暴露于细胞外部1,2,3,4,5。OM c-青旅介导从呼吸道链到位于 extracellularly 的固体基底的电子传输。此传输称为胞外电子传输 (EET)1,6 , 是新兴生物技术 (如微生物燃料电池6) 的关键过程。因此, 为了了解潜在的 EET 动力学和机制, 以及它与微生物生理学的联系, OM c-中青旅已使用全细胞电化学47, 结合显微术进行了研究。8,9, 光谱学10,11, 分子生物学2,4。相比之下, 研究 EET 相关的阳离子传输 (例如,质子) 对活细胞中 EET 动力学的影响的方法几乎没有建立, 尽管质子在细菌膜中的转运具有关键作用, 在信号、稳态和能源生产12,13,14。在本研究中, 我们描述了一个技术, 以检查质子传输对 EET 动力学的影响, 在S. oneidensis MR-1 细胞使用全细胞电化学测量, 这需要确定的速率限制步骤在微生物电流生产15。
一个直接的方法来评估质子传输对相关 EET 的贡献是氘动能同位素效应 (绢)。绢是可观察的作为电子转移动力学的变化在替换质子与氘离子, 代表质子传输对电子转移动力学的影响16。绢本身的理论已得到很好的建立使用电化学测量与纯化酶17。但是, 由于oneidensis中的当前生产 MR-1 来自多个、不同和波动的进程18, 因此不能简单地将 EET 标识为速率限制过程。为了观察质子传输过程与 EET 耦合的绢, 我们需要确认微生物电流的产生是通过 OM c-中青旅对电极的电子传输来限制的。为此, 在绢测量前, 我们在最佳 pH 值和温度条件下, 用含有高浓度乳酸的新鲜培养基取代上清液;这个替换服务两个角色: (1) 它提高了上游代谢过程的速率与 EET 相比, (2) 省略了在工作电极上从S. oneidensis MR-1 的单层生物膜上释放的上清液中的游泳细胞 (铟锡掺杂氧化物 (ITO) 电极)。所提供的详细协议旨在帮助新的从业者保持并确认 EET 过程是速率确定步骤。
Protocol
1. 在 ITO 电极上形成oneidensis MR-1 的单层生物膜 (图 1) 注: 为了防止电化学反应器与其他微生物的污染, 电化学反应器的所有介质、装置和元件应预先消毒。当使用oneidensis MR-1 单元格并构造电化学反应器时, 所有程序都应在干净的工作台上进行。 oneidensis MR-1 细胞的培养注: 在以前报告的4的条件下, 在 ITO 电极…
Representative Results
25 h 的潜在应用在 +0.4 V (对她), 在 ITO 玻璃的工作电极上形成单层生物膜, 以前是通过扫描电子显微镜或共聚焦显微镜4确认的。在单层生物膜形成过程中, 从oneidensis MR-1 的当前生产的代表性时间过程显示在图 2中。尽管电流在每次测量中都有改变, 但如果单层生物膜均匀形成, 则产生的电流不会从图 2中的?…
Discussion
与蛋白质电化学相比, 我们的全细胞电化学检测具有多种技术优势。蛋白质纯化需要多步骤的耗时程序, 但我们的全细胞方法在细胞培养后需要一天的自组织生物膜形成。为了实现 OM c-青旅和电极之间的稳定交互, 我们只需要对电极表面进行杀菌和清洗;它不需要电极修改来组织蛋白质的方向4,例如, S. oneidensis MR-1 本质上附着在电极上, 同时将 OM c-青旅定?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作在财政上得到了资助, 为特别促进研究从日本促进科学学会 (jsp) KAKENHI 授予数字 24000010, 17H04969 和 JP17J02602, 美国海军研究全球办公室 (N62909-17-1-2038)。Y.T. 是一个 jsp 研究研究员, 并通过该项目的领导研究生学校 (功绩) 支持的 jsp。
Materials
| Glass cylinder | N/A | N/A | Custom-made, used as the electrochemical reactor |
| PTFE cover and base | N/A | N/A | Custom-made, used as a cover and a foundation of the electrochemical reactor |
| Buthyl rubber | N/A | N/A | Custom-made, inserted between each component of electrochemical reactor |
| Septa | GL Science | 3007-16101 | Used as an injection port of electrochemical reactor |
| Indium tin-doped oxide (ITO) electrode | GEOMATEC | No.0001 | Used as a working electrode, 5Ω/sq |
| Ag/AgCl KCl saturated electrode | HOKUTO DENKO | HX-R5 | Used as a reference electrode, Φ0.30mm |
| Platinum wire | The Nilaco Cooporation | PT-351325 | Used as a counter electrode |
| Luria-Bertani (LB) Broth, Miller | Becton, Dichkinson and Company | 244620 | Medium for precultivation of S. oneidensis MR-1 |
| Bacto agar | Becton, Dichkinson and Company | 214010 | |
| Anthraquinone-1-sulfonate (α-AQS) | TCI | A1428 | |
| Flavin mononucleotide (FMN) | Wako | 184-00831 | |
| NaHCO3 | Wako | 191-01305 | Used for defined medium (DM) |
| CaCl2 · 2H2O | Wako | 031-00435 | Used for DM |
| NH4Cl | Wako | 011-03015 | Used for DM |
| MgCl2 · 6H2O | Wako | 135-00165 | Used for DM |
| NaCl | Wako | 191-01665 | Used for DM |
| 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (HEPES) | DOJINDO | 346-08235 | Used for DM |
| Sodium Lactate Solution | Wako | 195-02305 | |
| Bacto Yeast Extract | Becton, Dichkinson and Company | 212750 | |
| Deuterium oxide (D, 99.9%) | Cambridge Isotope Laboratories, Inc. | DLM-4-PK | Additive for kinetic isotope effect experiments |
| Incubator | TOKYO RIKAKIKAI CO. LTD. | LTI-601SD | Used for precultivation |
| Shaker | TAITEC | NR-3 | Used for precultivation |
| Autoclave machine | TOMY SEIKO CO. LTD. | LSX-500 | Used for sterilization of the electrochemical reactor and the medium |
| Clean bench | SANYO | MCV-91BNF | Used to prevent the contamination of the electrochemical reactor and the medium with other microbes |
| Centrifuge separator | Eppendorf | 5430R | Rotational speed upto 6000×g is required |
| Nitrogen gas generator | Puequ CO. LTD. | PNTN-2 | Nitrogen gas cylinder can also be used instead of gas generator |
| UV-vis spectrometer | SHIMADZU | UV-1800 | Used for optimization of cell density |
| Potentiostat | BioLogic | VMP3 | Used for biofilm formation and kinetic isotope effect experiments |
| Thermal water circulator | AS ONE | TR-1A | Used for maintanance of temperature of electrochemcial reactor |
| Faraday cage | HOKUTO DENKO | HS-201S | Used for electrochemical experiments |
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Cite This Article
Tokunou, Y., Hashimoto, K., Okamoto, A. Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1. J. Vis. Exp. (134), e57584, doi:10.3791/57584 (2018).