Bacterial Exudates: Characteristics and Role in Selenium Reduction

The goals of this research are to further our understanding of interactions between soluble bacterial exudates and selenium, and to determine how these interactions may affect the environmental behavior of Se. In Chapter 2, I discuss a study that tests whether soluble bacterial exudates can reduce aqueous Se(IV). Exudate solutions were isolated from three different model species of bacteria and tested for reductive activity to added Se(IV). The results show that some bacterial exudates reduce Se(IV), but that those from Shewanella oneidensis can not unless the cells are grown in the presence of aqueous Se(IV). The results also demonstrate that Se(IV) reduction by each of the exudate solutions involves exudate-hosted sulfhydryl binding. Chapter 3 describes potentiometric titration experiments that were used to measure the concentrations of proton-active sites on the exudate molecules produced by several bacterial species as a function of soak time, and the chapter also reports the first measurements of sulfhydryl site concentrations on exudate molecules. The total binding site concentrations of the exudates are primarily controlled by DOC concentration, unlike sulfhydryl site concentrations, suggesting that production of sulfhydryl-site bearing exudates is primarily an active process. The research reported in Chapter 4 uses comparative proteomics to identify candidate proteins that are responsible for the inducible Se(IV) reductase activity of soluble S. oneidensis exudates. Combining the control of S. oneidensis exudates’ inducible activity with knowledge of required characteristics for Se(IV) reduction allows us to narrow the list to six candidate proteins for further investigation. The research in this dissertation demonstrates for the first time the ability of soluble bacterial exudates to reduce Se(IV) and suggests that Se(IV) binding onto sulfhydryl sites controls the reduction reaction. The research characterizes the total concentration of binding sites and the concentration of sulfhydryl sites specifically on exudate molecules, and it identifies six candidate proteins that are responsible for the observed Se(IV) reduction. Overall, the research presented here improves our understanding of Se(IV)-exudate reactions, and could be useful in refining environmental models of selenium behavior and developing bioremediation methods for selenium contamination.本学位论文的研究目标在于深化对可溶性细菌渗出液（soluble bacterial exudates）与硒之间相互作用的理解，并明确此类相互作用如何影响硒的环境行为。第二章将介绍一项探究可溶性细菌渗出液是否能够还原水相四价硒（Se(IV)）的研究：研究从三种不同模式细菌菌株中分离得到渗出液溶液，并针对其对添加的四价硒的还原活性开展测试。结果显示，部分细菌渗出液可还原四价硒，但源自希瓦氏菌（Shewanella oneidensis）的渗出液则无此活性，除非菌体在水相四价硒存在的条件下进行培养。此外，研究结果还表明，各类渗出液对四价硒的还原过程均涉及渗出液所承载的巯基（sulfhydryl）结合作用。第三章介绍了电位滴定法（potentiometric titration）实验，该实验用于测量不同细菌菌株产生的渗出液分子上质子活性位点的浓度随浸泡时间的变化关系，同时本章还首次报道了渗出液分子上巯基位点浓度的测定结果。渗出液的总结合位点浓度主要受溶解性有机碳（dissolved organic carbon, DOC）浓度调控，而巯基位点浓度则无此规律，这表明携带巯基位点的渗出液的产生主要是一个主动过程。第四章的研究采用比较蛋白质组学（comparative proteomics）方法，旨在鉴定出负责可溶性希瓦氏菌（S. oneidensis）渗出液可诱导四价硒还原酶活性的候选蛋白。结合希瓦氏菌渗出液可诱导活性的调控机制，以及四价硒还原所需的特征条件，我们可将候选蛋白范围缩小至六种，以供后续深入研究。本学位论文的研究首次证实了可溶性细菌渗出液还原四价硒的能力，并表明四价硒与巯基位点的结合是调控该还原反应的关键。本研究对渗出液分子上的总结合位点浓度以及特异性巯基位点浓度进行了表征，同时鉴定出六种与观测到的四价硒还原活性相关的候选蛋白。总体而言，本研究深化了我们对四价硒与渗出液之间反应过程的认知，可为优化硒行为的环境模型以及开发硒污染生物修复（bioremediation）方法提供参考依据。