本文選題：Shewanella + oneidensis�。� 參考：《華南理工大學(xué)》2016年博士論文

【摘要】：微生物胞外呼吸是近年來(lái)新發(fā)現(xiàn)的一種特殊無(wú)氧呼吸方式,細(xì)胞色素c在微生物胞外呼吸過(guò)程中起到至關(guān)重要的作用,其作用主要是作為電子傳遞載體或末端還原酶。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胞外呼吸電子傳遞鏈中關(guān)鍵的電子傳遞蛋白已經(jīng)被分離和純化,但是每個(gè)蛋白在礦物生物還原和生物礦化過(guò)程中的特定功能仍然沒(méi)有被完全理解,而且MR-1以不同類(lèi)型基質(zhì)作為胞外受體進(jìn)行胞外還原時(shí),其活體細(xì)胞中細(xì)胞色素c尤其是外膜細(xì)胞色素c在胞外呼吸中的氧化還原狀態(tài)變化仍鮮有報(bào)道。本論文研究了敲除不同細(xì)胞色素c的MR-1突變體對(duì)水鐵礦生物還原和生物礦化的影響,并利用漫透射光譜法研究了不同培養(yǎng)條件下MR-1以金屬鉻、醌和可溶性螯合鐵為胞外電子受體時(shí)外膜細(xì)胞色素c的氧化還原狀態(tài)變化。本研究揭示了MR-1電子傳遞鏈中細(xì)胞色素c在鐵氧化物生物還原和生物礦化中的作用,有助于理解微生物與礦物相互作用過(guò)程。利用光譜法研究原位活體細(xì)胞色素c與胞外受體的反應(yīng)能夠加深我們對(duì)微生物胞外呼吸過(guò)程的理解。主要結(jié)論包括:(1)研究了MR-1野生型(wild type)和敲除不同細(xì)胞色素c的突變體(ΔmtrA、Δmtr C、ΔmtrD、ΔmtrF、ΔomcA和ΔcymA)對(duì)水鐵礦生物還原和生物礦化的影響。結(jié)果表明:ΔmtrD、ΔmtrF和ΔomcA處理組還原水鐵礦速率與野生型MR-1還原速率保持相似水平,而ΔcymA和ΔmtrA處理組還原水鐵礦的能力顯著下降。水鐵礦次生礦物用X射線衍射、傅里葉變換紅外光譜和掃描電鏡進(jìn)行表征。結(jié)果表明,所有處理在剛開(kāi)始2天中以針鐵礦和赤鐵礦為次生礦物的主要形式,接著6天后在wild type、ΔmtrD、ΔmtrF和ΔomcA四個(gè)處理組中赤鐵礦完全轉(zhuǎn)化為磁鐵礦;而ΔmtrC處理中磁鐵礦從第6天開(kāi)始出現(xiàn);然而,ΔmtrA和ΔcymA處理中在6天培養(yǎng)期間沒(méi)有磁鐵礦出現(xiàn)。根據(jù)基元反應(yīng)的熱力計(jì)算,確定了生物還原較為合理的電子轉(zhuǎn)移途徑和相位變化。(2)利用漫透射-UV/Vis光譜法研究了不同培養(yǎng)條件下Shewanella oneidensis MR-1活體細(xì)胞色素c還原Cr(VI)的原位光譜動(dòng)力學(xué)。結(jié)果表明:還原態(tài)細(xì)胞色素c在開(kāi)始階段迅速下降,然后逐漸恢復(fù)到初始水平。Cr(VI)還原率隨著初始Cr(VI)濃度的增加而下降,Cr(III)被確認(rèn)為是MR-1還原Cr(VI)的還原產(chǎn)物。Cr(III)的存在大幅抑制了Cr(VI)還原和還原態(tài)細(xì)胞色素c的恢復(fù)速率,表明Cr(III)可能抑制了細(xì)胞生長(zhǎng)。Cr(VI)還原速率隨著細(xì)胞濃度的增加而增加。最高Cr(VI)還原速率和最快還原態(tài)細(xì)胞色素c恢復(fù)速率出現(xiàn)在pH 7.0、30°C和以乳酸作為電子供體時(shí)。氧氣的存在強(qiáng)烈地抑制了Cr(VI)還原,表明氧氣可能作為電子受體與Cr(VI)競(jìng)爭(zhēng)。(3)利用漫透射-UV/Vis光譜法研究了不同培養(yǎng)條件下MR-1外膜細(xì)胞色素c還原醌的動(dòng)力學(xué)過(guò)程。結(jié)果表明:還原態(tài)細(xì)胞色素c在反應(yīng)開(kāi)始時(shí)迅速下降,然后逐漸恢復(fù)到初始水平。醌類(lèi)物質(zhì)AQDS(anthraquinone-2,6-disulfonate)還原速率隨著初始AQDS濃度的增加而下降。AQDS還原速率隨著細(xì)胞濃度的增加而增加。最高的AQDS還原速率和最快的細(xì)胞色素c恢復(fù)速率在30°C和pH 7.0條件下得到。并對(duì)不同培養(yǎng)條件下MR-1外膜細(xì)胞色素c還原醌的過(guò)程進(jìn)行了熱力學(xué)分析,結(jié)果表明最適溫度和pH可能提高M(jìn)R-1細(xì)胞的代謝,從而提高M(jìn)R-1對(duì)乳酸的消耗率進(jìn)而提高了胞內(nèi)電子傳遞和胞外電子傳遞時(shí)的電子輸出效率。(4)采用漫透射UV/Vis光譜法研究了厭氧條件下Shewanella oneidensis MR-1活體細(xì)胞中細(xì)胞色素c還原不同類(lèi)型螯合鐵的原位光譜動(dòng)力學(xué)。結(jié)果表明,厭氧條件下,無(wú)MR-1存在時(shí),Fe(III)-檸檬酸鐵不發(fā)生還原反應(yīng)。當(dāng)無(wú)檸檬酸鐵存在時(shí),MR-1外膜細(xì)胞色素c不發(fā)生氧化還原反應(yīng)。隨著Fe(III)-檸檬酸鐵的還原,還原性的細(xì)胞色素c在反應(yīng)起始階段迅速下降,然后逐漸恢復(fù)到初始狀態(tài)。與野生型MR-1相比,敲除了MR-1電子傳遞鏈中的關(guān)鍵蛋白(例如CymA、MtrA和MtrC等)的處理中,檸檬酸鐵還原率和Heme_(red)的消耗速率和恢復(fù)速率均不同程度的受到影響。MR-1活體細(xì)胞中細(xì)胞色素c還原不同類(lèi)型螯合鐵時(shí)顯示出不同的鐵還原速率、Heme_(red)消耗速率和恢復(fù)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.
【學(xué)位授予單位】：華南理工大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：Q93

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本文編號(hào)：1977998