Shewanella oneidensis MR-1外膜细胞色素c介导的胞外电子传递过程研究

发布时间：2018-06-04 16:08

本文选题：Shewanella + oneidensis　；参考：《华南理工大学》2016年博士论文

【摘要】：微生物胞外呼吸是近年来新发现的一种特殊无氧呼吸方式,细胞色素c在微生物胞外呼吸过程中起到至关重要的作用,其作用主要是作为电子传递载体或末端还原酶。Shewanella oneidensis MR-1是目前研究最多的胞外呼吸模式菌之一。Shewanella oneidensis MR-1胞外呼吸电子传递链中关键的电子传递蛋白已经被分离和纯化,但是每个蛋白在矿物生物还原和生物矿化过程中的特定功能仍然没有被完全理解,而且MR-1以不同类型基质作为胞外受体进行胞外还原时,其活体细胞中细胞色素c尤其是外膜细胞色素c在胞外呼吸中的氧化还原状态变化仍鲜有报道。本论文研究了敲除不同细胞色素c的MR-1突变体对水铁矿生物还原和生物矿化的影响,并利用漫透射光谱法研究了不同培养条件下MR-1以金属铬、醌和可溶性螯合铁为胞外电子受体时外膜细胞色素c的氧化还原状态变化。本研究揭示了MR-1电子传递链中细胞色素c在铁氧化物生物还原和生物矿化中的作用,有助于理解微生物与矿物相互作用过程。利用光谱法研究原位活体细胞色素c与胞外受体的反应能够加深我们对微生物胞外呼吸过程的理解。主要结论包括:(1)研究了MR-1野生型(wild type)和敲除不同细胞色素c的突变体(ΔmtrA、Δmtr C、ΔmtrD、ΔmtrF、ΔomcA和ΔcymA)对水铁矿生物还原和生物矿化的影响。结果表明:ΔmtrD、ΔmtrF和ΔomcA处理组还原水铁矿速率与野生型MR-1还原速率保持相似水平,而ΔcymA和ΔmtrA处理组还原水铁矿的能力显著下降。水铁矿次生矿物用X射线衍射、傅里叶变换红外光谱和扫描电镜进行表征。结果表明,所有处理在刚开始2天中以针铁矿和赤铁矿为次生矿物的主要形式,接着6天后在wild type、ΔmtrD、ΔmtrF和ΔomcA四个处理组中赤铁矿完全转化为磁铁矿;而ΔmtrC处理中磁铁矿从第6天开始出现;然而,ΔmtrA和ΔcymA处理中在6天培养期间没有磁铁矿出现。根据基元反应的热力计算,确定了生物还原较为合理的电子转移途径和相位变化。(2)利用漫透射-UV/Vis光谱法研究了不同培养条件下Shewanella oneidensis MR-1活体细胞色素c还原Cr(VI)的原位光谱动力学。结果表明:还原态细胞色素c在开始阶段迅速下降,然后逐渐恢复到初始水平。Cr(VI)还原率随着初始Cr(VI)浓度的增加而下降,Cr(III)被确认为是MR-1还原Cr(VI)的还原产物。Cr(III)的存在大幅抑制了Cr(VI)还原和还原态细胞色素c的恢复速率,表明Cr(III)可能抑制了细胞生长。Cr(VI)还原速率随着细胞浓度的增加而增加。最高Cr(VI)还原速率和最快还原态细胞色素c恢复速率出现在pH 7.0、30°C和以乳酸作为电子供体时。氧气的存在强烈地抑制了Cr(VI)还原,表明氧气可能作为电子受体与Cr(VI)竞争。(3)利用漫透射-UV/Vis光谱法研究了不同培养条件下MR-1外膜细胞色素c还原醌的动力学过程。结果表明:还原态细胞色素c在反应开始时迅速下降,然后逐渐恢复到初始水平。醌类物质AQDS(anthraquinone-2,6-disulfonate)还原速率随着初始AQDS浓度的增加而下降。AQDS还原速率随着细胞浓度的增加而增加。最高的AQDS还原速率和最快的细胞色素c恢复速率在30°C和pH 7.0条件下得到。并对不同培养条件下MR-1外膜细胞色素c还原醌的过程进行了热力学分析,结果表明最适温度和pH可能提高MR-1细胞的代谢,从而提高MR-1对乳酸的消耗率进而提高了胞内电子传递和胞外电子传递时的电子输出效率。(4)采用漫透射UV/Vis光谱法研究了厌氧条件下Shewanella oneidensis MR-1活体细胞中细胞色素c还原不同类型螯合铁的原位光谱动力学。结果表明,厌氧条件下,无MR-1存在时,Fe(III)-柠檬酸铁不发生还原反应。当无柠檬酸铁存在时,MR-1外膜细胞色素c不发生氧化还原反应。随着Fe(III)-柠檬酸铁的还原,还原性的细胞色素c在反应起始阶段迅速下降,然后逐渐恢复到初始状态。与野生型MR-1相比,敲除了MR-1电子传递链中的关键蛋白(例如CymA、MtrA和MtrC等)的处理中,柠檬酸铁还原率和Heme_(red)的消耗速率和恢复速率均不同程度的受到影响。MR-1活体细胞中细胞色素c还原不同类型螯合铁时显示出不同的铁还原速率、Heme_(red)消耗速率和恢复速率。
[Abstract]:Extracellular respiration of microorganism is a special anaerobic respiration in recent years. Cytochrome C plays a vital role in the process of extracellular respiration of microorganisms. Its role is mainly as an electron transfer carrier or the terminal reductase,.Shewanella oneidensis MR-1, one of the most widely studied exo respiratory mode bacteria,.Shewanella The key electron transfer proteins in the oneidensis MR-1 exo exo electron transport chain have been separated and purified, but the specific functions of each protein in the process of mineral biological reduction and biomineralization are still not fully understood, and the cells in the living cells of MR-1 with different types of matrix as extracellular matrix. The changes in the redox state of the pigment C, especially the outer membrane cytochrome C in the exo respiratory respiration, are still rarely reported. In this paper, the effects of the MR-1 mutant with different cytochrome c on the biologic reduction and biomineralization of the iron ore were studied, and the metal chromium, quinone and soluble chelation of MR-1 under different culture conditions were studied under different culture conditions. The changes in the redox state of the outer membrane cytochrome c when iron is an extracellular electron acceptor. This study reveals the role of cytochrome C in the biological reduction and mineralization of iron oxide in the MR-1 electron transfer chain, which helps to understand the process of interaction between microbes and minerals. The study of the in situ cytochrome C and the extracellular receptor by spectroscopic method The main conclusions are as follows: (1) the effects of the MR-1 wild type (wild type) and the mutant (delta mtrA, delta MTR C, Delta mtrD, Delta mtrF, Delta omcA and delta cymA) on the biologic reduction and biomineralization of the iron ore are studied. The rate of reduction of iron ore was similar to that of the wild type MR-1 reduction rate, while the ability to reduce the reduction of iron ore was significantly decreased in the delta cymA and delta mtrA treatment groups. The secondary minerals of the iron ore were characterized by X ray diffraction, Fourier transform infrared spectroscopy and scanning electron microscopy. The results showed that all the sites were treated with goethite and hematite at the beginning of the first 2 days. The main form of raw mineral, after 6 days, hematite was completely converted into magnetite in wild type, Delta mtrD, Delta mtrF and delta omcA, and magnetite in the delta mtrC treatment began to appear from sixth days; however, there was no magnetite coming out during the 6 day incubation period of delta mtrA and delta cymA. According to the thermodynamic calculation of the element reaction, the biological return was determined. The original more reasonable electron transfer pathway and phase change. (2) the in situ spectroscopy of Shewanella oneidensis MR-1 in vivo cytochrome c reduction Cr (VI) under different culture conditions was studied by the diffuse transmission -UV/Vis spectroscopy. The results showed that the reduced cytochrome C was rapidly decreased at the beginning stage, and then gradually recovered to the initial level.Cr (V). I) reduction rate decreased with the increase of initial Cr (VI) concentration, and Cr (III) was confirmed as the reduced product.Cr (III) of MR-1 reduction Cr (VI), which significantly inhibited the recovery rate of Cr (VI) reduction and reductive cytochrome reductive rates, indicating that the reduction rate of cell growth may be inhibited by the increase of cell concentration. The rate of reduction and the fastest reduced state of cytochrome c appeared at pH 7.0,30 [C] and lactic acid as an electron donor. The presence of oxygen strongly inhibited the reduction of Cr (VI), indicating that oxygen might compete with Cr (VI) as an electron acceptor. (3) the cytochrome c of the MR-1 outer membrane under different culture conditions was studied by the diffuse -UV/Vis spectrum method. The kinetic process of the original quinone showed that the reduction of cytochrome c rapidly decreased at the beginning of the reaction and then gradually recovered to the initial level. The reduction rate of the AQDS (anthraquinone-2,6-disulfonate) of quinone substance decreased with the increase of the initial AQDS concentration and increased with the increase of cell concentration. The highest AQDS reduction. The rate and the fastest cytochrome c recovery rate were obtained under the conditions of 30 C and pH 7. The thermodynamic analysis of the reduction of quinones by MR-1 outer membrane cytochrome C was carried out. The results showed that the optimum temperature and pH may improve the metabolism of MR-1 cells, thus increasing the consumption rate of MR-1 against lactic acid and thus increasing the intracellular electron transmission. Electron output efficiency of delivery and extracellular electron transfer. (4) in situ spectroscopy of the reduction of different types of chelating iron by cytochrome C in Shewanella oneidensis MR-1 living cells under anaerobic conditions was studied by diffuse transmission UV/Vis spectroscopy. The results showed that Fe (III) - iron citrate did not occur when there was no MR-1 presence in the anaerobic condition. When no citrate iron exists, the MR-1 outer membrane cytochrome C does not redox reaction. With the reduction of Fe (III) - iron citrate, the reductive cytochrome c rapidly decreases at the beginning of the reaction and then gradually returns to the initial state. Compared with the wild type MR-1, the key proteins (such as CymA, MtrA and Mt) in the MR-1 electron transfer chain are knocked out. In the treatment of rC, the reduction rate of iron citrate and the consumption rate of Heme_ (red) and the recovery rate were affected to varying degrees by the reduction of different types of chelating iron by cytochrome C in.MR-1 living cells. The rate of iron reduction and the rate of Heme_ (red) consumption and recovery were different.
【学位授予单位】：华南理工大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q93

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