| 摘要: | 異化金屬還原菌(dissimilatory metal reducing bacteria, DMRB)常驅動自然沉積物系統(即底泥與地下含水層)中的污染物移動與轉化，是極重要的環境微生物族群之一。事實上，某些DMRB如Shewanella oneidensis MR-1已知可在好氧及厭氧環境的低汞濃度下(ppb)，進行與汞抗性操作子(mer operon)無關的生物性汞還原作用，但其運作機制截至目前為止仍有待確認。有鑒於深入了解MR-1的汞還原機制對於未來相關環境系統的管理與整治可能有所助益，本研究即以MR-1的Mtr胞外呼吸途徑(即the metal-reducing respiratory patehway)為模型，進行厭氧培養試驗，以探討MR-1在生長遲滯狀態下與溶解性二價汞的互動。試驗結果發現，當菌/汞比log (cell/mol)為17.42時，有最多的汞還原發生(約40%)，但當菌/汞比高於18.2時，還原情況則有所抑制，取而代之的是多數Hg(II)與菌表面硫醇(-SH)的結合；而當同時利用MR-1野生菌株，及調控外膜表面多血紅素細胞色素蛋白(MtrC, OmcA)和調控黃素類化合物排出蛋白(bacterial FAD exporter, Bfe)的基因剔除後的突變菌ΔmtrC/omcA與Δbfe為模式生物時，發現MR-1在生長遲滯的狀態下更傾向利用外膜細胞色素蛋白、以直接接觸的方式將Hg(II)還原，因電子穿梭(electron-shuttle)所成的還原效應反而較不顯著，且當調控培養液的主要汞物種，使其由HgEDTA2-轉為更易與表面淨電荷為負電的MR-1細胞接觸的Hg(NH3)32+時，Hg(II)還原情形也有所提升，進一步支持此狀態下直接接觸Hg(II)還原的重要性；當系統額外加入內源性與外源性電子穿梭物riboflavin (1 μM)、AQDS (30 μM)、tetracycline (0.45 μM)時，就統計結果而言均無顯著增加MR-1對Hg(II)的還原情形，再次暗示著MR-1在生長遲滯狀態下可能是以直接接觸主導著Hg(II)的還原作用。不過，若以平均值而言，riboflavin與AQDS仍有略微增加Hg(II)還原的情形，但tetracyclin在不影響MR-1的生長狀態下，並無觀察到任何Hg(II)還原情形變化。有趣的是，當系統內存有不同濃度梯度的溶氣時，氧氣似乎會作為系統內另一電子接受者而影響汞的還原，推斷可能與兩者的還原電位及在系統內的濃度佔比有關。這些結果說明MR-1在不同環境下對胞外的金屬轉化會隨環境變化有所調整，這些策略皆可相當程度的影響汞在環境中的型態轉化與分佈。;Dissimilatory metal reducing bacteria (DMRB) often drive the movement and transformation of pollutants in natural sedimentary systems and are considered one of the most important environmental microorganisms. In fact, some DMRB model strains such as Shewanella oneidensis MR-1 are known to be able to perform biological Hg(II) reduction independent of mer activities in aerobic and anaerobic environments at low Hg(II) concentrations (ppb). Yet, the underlying mechanism is still unclear. Therefore, an in-depth understanding of the mechanism of Hg(II) reduction by MR-1 may improve environmental management in the future. This study uses the metal-reducing respiratory pathway of MR-1 as a model to explore the interaction between cells and soluble Hg(II) during growth-arrested states. It was found that when the cell number to Hg concentration at a log scale was 17.42, Hg(II) reduction was the most significant (~ 40%). However, when the log (cell/mol) was higher than 18.2, Hg(II) reduction was inhibited, and most of the Hg(II) were bound to the bacterial surface thiols (-SH). Using wild-type MR-1 and its mutants as model strains, we observed that extracellular Hg(II) reduction was largely attributed to the direct contact pathway for growth-arrested cells. This direct contact pathway was also supported by the results from the external spike of both endogenous (i.e., 1 μM riboflavin) and exogenous (i.e., 30 μM AQDS and 0.45 μM tetracycline) electron mediating compounds, as well as from the charge manipulation of Hg(II) species, because we did not observe a significant change in the reduction of Hg(II) after adding endogenous or exogenous electron shuttles, and the reduction of Hg(NH3)32+ was significantly higher than that of HgEDTA2- for MR-1. Lastly, the presence of strong oxidizing agents like dissolved oxygen at elevated levels exhibited an inhibiting effect on this microbially-mediated extracellular Hg(II) reduction, which might be due to the inherent higher reduction potential of oxygen and the concentration ratio of oxygen and electron shuttles in the system. Together, this study shows that the reduction of Hg(II) by MR-1 in different environments will be case specific, which can affect the transformation and distribution of mercury in the environment to a considerable extent. |
