Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1
Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1
复制标题
DOI：
10.3791/57584
复制
发表时间：
2018-04-01
影响因子：
1.2
通讯作者：
Okamoto, Akihiro
中科院分区：
文献类型：
--
作者：
Tokunou, Yoshihide;Hashimoto, Kazuhito;Okamoto, Akihiro
直接电化学检测嵌入在细菌外膜中的c-型细胞色素复合物（外膜c-型细胞色素复合物; OM c-Cyts）最近已成为一种新的全细胞分析方法，用于表征细菌从呼吸链到细胞外部的电子传递，称为细胞外电子传递（EET）。虽然EET反应过程中的电子流的途径和动力学进行了研究，一个全细胞电化学方法来检查与EET相关的阳离子转运的影响尚未建立。在本研究中，一个例子的生化技术，以检查EET的氘动力学同位素效应（KIE）通过OM c-Cyts使用的模式微生物，希瓦氏菌oneidensis MR-1，描述。如果通过OM c-Cyts的EET作为微生物电流生产中的限速步骤，则可以获得EET过程的KIE。为此，在添加D2O之前，将上清液溶液用含有足量电子供体的新鲜培养基替换，以支持上游代谢反应的速率，并从工作电极上的均匀单层生物膜中除去嗜酸性细胞。还描述了确认微生物电流生产中的限速步骤为EET通过OM c-Cyts的替代方法。我们的全细胞电化学分析研究质子传输动力学的技术可以应用于其他电活性微生物菌株。
Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM c-Cyts) has recently emerged as a novel whole-cell analytical method to characterize the bacterial electron transport from the respiratory chain to the cell exterior, referred to as the extracellular electron transport (EET). While the pathway and kinetics of the electron flow during the EET reaction have been investigated, a whole-cell electrochemical method to examine the impact of cation transport associated with EET has not yet been established. In the present study, an example of a biochemical technique to examine the deuterium kinetic isotope effect (KIE) on EET through OM c-Cyts using a model microbe, Shewanella oneidensis MR-1, is described. The KIE on the EET process can be obtained if the EET through OM c-Cyts acts as the rate-limiting step in the microbial current production. To that end, before the addition of D2O, the supernatant solution was replaced with fresh media containing a sufficient amount of the electron donor to support the rate of upstream metabolic reactions, and to remove the planktonic cells from a uniform monolayer biofilm on the working electrode. Alternative methods to confirm the rate-limiting step in microbial current production as EET through OM c-Cyts are also described. Our technique of a whole-cell electrochemical assay for investigating proton transport kinetics can be applied to other electroactive microbial strains.