微生物胞外长距离电子传递网络研究进展

[目的] 解析一株从黄河三角洲湿地甲烷氧化富集物中分离获得的甲烷氧化菌伴生菌的生理学及电化学特性,并探究该菌株对甲烷氧化过程的影响。[方法] 使用高通量测序技术解析甲烷氧化富集物的菌群结构,采用稀释涂布法、平板划线法分离甲烷氧化菌的伴生菌,通过16S rRNA基因测序技术进行菌株初步鉴定。利用扫描电子显微镜观察菌株形态,并通过气相色谱（gas chromatography,GC）检测伴生菌利用甲烷情况及对甲烷氧化菌氧化甲烷效率的影响。采用双室微生物燃料电池（microbial fuel cells,MFCs）及差分脉冲伏安法（differential pulse voltammetry,DPV）检测菌株的电化学活性。[结果] 黄河三角洲湿地土壤甲烷氧化富集物主要的好氧甲烷氧化菌为甲基杆菌属Methylobacter,同时还发现一些伴生菌。分离得到一株甲醇利用菌P7,其16S rRNA基因序列与恶臭假单胞菌Pseudomonasputida的相似性达99.79%。扫描电镜结果显示该菌株为杆状,长约1.5-2.5μm,宽度约为0.5μm。GC检测结果显示,该菌株不能利用甲烷,但与甲烷氧化菌共培养时,可以促进甲烷氧化（P<0.05）。双室MFCs检测结果显示该菌株具有电活性,最大电流输出密度为28 mA/m²,DPV检测结果显示该菌株主要的氧化峰和还原峰分别位于-0.17 V和-0.25 V。[结论] 本研究从黄河三角洲湿地甲烷氧化富集物中获得一株具有电活性的甲烷氧化菌的伴生菌恶臭假单胞菌Pseudomonas putida P7,该菌株可以促进甲烷氧化。本研究加深了对甲烷氧化过程中伴生菌的生理学特性及功能的认识。     

矿物电子能量协同微生物胞外电子传递与生长代谢

微生物的电子传递过程在生命进化和生物地球化学循环中发挥着关键作用。近年来,随着微生物电子传递研究的深入开展,微生物纳米导线、导电生物被膜及种间电子传递等多种新型的微生物胞外电子传递机制不断被发现,微生物电子传递的距离也从纳米级拓展至厘米级。这些微生物的长距离电子传递过程环环相扣、相互协同,从而构成长距离电子传递网络,并在物质循环和能量转化中共同发挥作用。微生物长距离电子传递网络的结构功能及其调控机制已成为多个学科共同关注的焦点。本文以电子传递的距离为主线,对不同尺度的微生物长距离电子传递过程及网络研究的新进展进行综述,包括纳米尺度的电子传递网络（周质空间和外膜表层）、微米至毫米尺度的电子传递网络（纳米导线、细胞间电子和导电生物被膜）、厘米尺度的电子传递网络（电缆细菌）等,并分析了该研究现存的主要问题和下一步的发展方向,以期为进一步推进微生物长距离电子传递网络理论和应用研究提供科学参考。     

Stimulated and co-operative electron transfer in energy conversion and catalysis.

A new mechanism of electron transfer, stimulated electron transfer, is postulated, in which an electronic feedback is drastically increasing both the rate of electron transfer and the propagation of free energy along electron transferring molecular pathways. In principle, the idea of pushing a system far from equilibrium to achieve a high reaction rate and co-operative phenomena is applied to molecular electron transfer. The effect is calculated from a semiclassical kinetic model of a chain redox reaction with autocatalytic feedback on individual rate constants, where the steps have subsequently been minimized to obtain a continuous electron transfer pathway with electronic feedback. The influence of inhomogeneities and asymmetries in the electron transfer path and of vectorial components (electrical field, gradient of redox potential) are discussed as well as the acceleration of individual and multiple electron transfer as a function of feedback. Examples of autocatalytic feedback are provided including mechanisms involving electron transfer proteins and multi-centre electron transfer catalysts. Such a phenomenon can be described for molecular and interfacial electron transfer in analogy to stimulated and coherent light emission. The results suggest that autocatalytic or stimulated electron transfer may be a key to the understanding of efficient electron transfer and co-operative multi-electron transfer catalysis in biology and a challenge for fuel production mechanisms in artificial photosynthesis and fuel cycles.     

EPR study of electron transport in the cyanobacterium Synechocystis sp. PCC 6803: oxygen-dependent interrelations between photosynthetic and respiratory electron transport chains

In this work, we investigated electron transport processes in the cyanobacterium Synechocystis sp. PCC 6803, with a special emphasis focused on oxygen-dependent interrelations between photosynthetic and respiratory electron transport chains. Redox transients of the photosystem I primary donor P700 and oxygen exchange processes were measured by the EPR method under the same experimental conditions. To discriminate between the factors controlling electron flow through photosynthetic and respiratory electron transport chains, we compared the P700 redox transients and oxygen exchange processes in wild type cells and mutants with impaired photosystem II and terminal oxidases (CtaI, CydAB, CtaDEII). It was shown that the rates of electron flow through both photosynthetic and respiratory electron transport chains strongly depended on the transmembrane proton gradient and oxygen concentration in cell suspension. Electron transport through photosystem I was controlled by two main mechanisms: (i) oxygen-dependent acceleration of electron transfer from photosystem I to NADP(+), and (ii) slowing down of electron flow between photosystem II and photosystem I governed by the intrathylakoid pH. Inhibitor analysis of P700 redox transients led us to the conclusion that electron fluxes from dehydrogenases and from cyclic electron transport pathway comprise 20-30% of the total electron flux from the intersystem electron transport chain to P700(+).     

Mechanisms for regulating electron transfer in multi-centre redox proteins.

Protein-mediated electron transfer is a key process in nature. Many of the proteins involved in such electron transfers are complex and contain a number of redox-active cofactors. The very complexity of these multi-centre redox proteins has made it difficult to fully understand the various electron transfer events they catalyse. This is sometimes because the electron transfer steps themselves are gated or coupled to other processes such as proton transfer. However, with the molecular structures of many of these proteins now available it is possible to probe these electron transfer reactions at the molecular level. It is becoming apparent that many of these multi-centre redox proteins have rather subtle and elegant ways for regulating electron transfer. The purpose of this article is to illustrate how nature has used different approaches to control electron transfer in a number of different systems. Illustrative examples include: thermodynamic control of electron transfer in flavocytochromes b(2) and P450 BM3; a novel control mechanism involving calmodulin-binding-dependent electron transfer in neuronal nitric oxide synthase; the probable gating of electron transfer by ATP hydrolysis in nitrogenase; conformational gating of electron transfer in cytochrome cd(1); the regulation of electron transfer by protein dynamics in the cytochrome bc(1) complex; and finally the coupling of electron transfer to proton transfer in cytochrome c oxidase.     

微生物种间电子传递的研究进展

种间电子传递可促进微生物发生共代谢,因而在地球生物化学循环和环境污染修复中具有重要意义。根据电子传递方式的不同可将种间电子传递分为直接种间电子传递（direct interspecies electron transfer,DIET）和间接种间电子传递（mediated interspecies electron transfer,MIET）,其中,直接种间电子传递由于易发生、效率高而受到更加广泛的关注。本文总结了近年来关于种间电子传递的研究进展,阐述了种间电子传递的途径,比较了DIET和MIET的优缺点,并对开发更多具有种间电子传递功能的微生物提出了建议,以期加深人们对于种间电子传递的理解,并对未来该领域的研究提供参考。     

Oxygen requirement of photosynthetic CO2 assimilation

In the absence of electron acceptors and of oxygen a proton gradient was supported across thylakoid membranes of intact spinach chloroplasts by far-red illumination. It was decreased by red light. Inhibition by red light indicates effective control of cyclic electron flow by Photosystem II. Inhibition was released by oxygen which supported a large proton gradient. Oxygen appeared to act as electron acceptor simultaneously preventing over-reduction of electron carriers of the cyclic electron transport pathway. It thus has an important regulatory function in electron transport. Under anaerobic conditions, the inhibition of electron transport caused by red illumination could also be released and a large proton gradient could be established by oxaloacetate, nitrite and 3-phosphoglycerate, but not by bicarbonate. In the absence of oxygen, ATP levels remained low in chloroplasts illuminated with red light even when bicarbonate was present. They increased when electron acceptors were added which could release the over-reduction of the electron transport chain. Inhibition of electron transport in the presence of bicarbonate was relieved and CO2-fixation was initiated by oxygen concentrations as low as about 10 microM. Once CO2 fixation was initiated, very low oxygen levels were sufficient to sustain it. The results support the assumption that pseudocyclic electron transport is necessary to poise the electron transport chain so that a proper balance of linear and cyclic electron transport is established to supply ATP for CO2 reduction.     

Low-energy electron penetration range in liquid water

Monte Carlo simulations of electron tracks in liquid water are performed to calculate the energy dependence of the electron penetration range at initial electron energies between 0.2 eV and 150 keV, including the subexcitation electron region (<7.3 eV). Our calculated electron penetration distances are compared with available experimental data and earlier calculations as well as with the results of simulations using newly reported amorphous ice electron scattering cross sections in the range approximately 1-100 eV.     

Identification of cells by backscattered electron imaging of silver stained bulk tissues in scanning electron microscopy

Anuran tadpole tail muscle was stained en bloc by a modified light microscope silver stain for light microscopy and freeze-fractured in liquid nitrogen after partial dehydration with ethanol. The fractured specimens were observed in both secondary electron and backscattered electron modes in a scanning electron microscope. Since the cell nuclei specifically stained with silver provided high contrast against the unstained background due to atomic number contrast of backscattered electron image, various cells were easily identified by a comparison of secondary electron images and compositional images of backscattered electron signals.     

Intra-molecular electron transfer in proteins

Intra-molecular electron transfer is a key process, which is of prime importance, in photosynthesis, mitochondrial electron transfer and the action of many multi-centre enzymes. This mini-review considers the possible mechanisms of intra-molecular electron transfer in proteins and reviews the recent developments relating to possible electron tunnelling and electron hopping processes within di-heme cytochrome c peroxidase.     

Oxygen requirement of photosynthetic CO2 assimilation

In the absence of electron acceptors and of oxygen a proton gradient was supported across thylakoid membranes of intact spinach chloroplasts by far-red illumination. It was decreased by red light. Inhibition by red light indicates effective control of cyclic electron flow by Photosystem II. Inhibition was released by oxygen which supported a large proton gradient. Oxygen appeared to act as electron acceptor simultaneously preventing over-reduction of electron carriers of the cyclic electron transport pathway. It thus has an important regulatory function in electron transport. Under anaerobic conditions, the inhibition of electron transport caused by red illumination could also be released and a large proton gradient could be established by oxaloacetate, nitrite and 3-phosphoglycerate, but not by bicarbonate. In the absence of oxygen, ATP levels remained low in chloroplasts illuminated with red light even when bicarbonate was present. They increased when electron acceptors were added which could release the over-reduction of the electron transport chain. Inhibition of electron transport in the presence of bicarbonate was relieved and CO₂-fixation was initiated by oxygen concentrations as low as about 10 μM. Once CO₂ fixation was initiated, very low oxygen levels were sufficient to sustain it. The results support the assumption that pseudocyclic electron transport is necessary to poise the electron transport chain so that a proper balance of linear and cyclic electron transport is established to supply ATP for CO₂ reduction.     

Regulation of photosynthetic electron transport and photophosphorylation in intact chloroplasts and leaves of Spinacia oleracea L.

Oxygen ist reduced by the electron transport chain of chloroplasts during CO₂ reduction. The rate of electron flow to oxygen is low. Since antimycin A inhibited CO₂-dependent oxygen evolution, it is concluded that cyclic photophosphorylation contributes ATP to photosynthesis in chloroplasts which cannot satisfy the ATP requirement of CO₂ reduction by electron flow to NADP and to oxygen. Inhibition of photosynthesis by antimycin A was more significant at high than at low light intensities suggesting that cyclic photophosphorylation contributes to photosynthesis particularly at high intensities. Cyclic electron flow in intact chloroplasts is under the control of electron acceptors. At low light intensities or under far-red illumination it is decreased by substrates which accept electrons from photosystem I such as oxaloacetate, nitrite or oxygen. Obviously, the cyclic electron transport pathway is sensitive to electron drainage. In the absence of electron acceptors, cyclic electron flow is supported by far-red illumination and inhibited by red light. The inhibition by light exciting photosystem II demonstrated that the cyclic electron transport pathway is accessible to electrons from photosystem II. Inhibition can be relieved by oxygen which appears to prevent over-reduction of electron carriers of the cyclic pathway and thus has an important regulatory function. The data show that cyclic electron transport is under delicate redox control. Inhibition is caused both by excessive oxidation and by over-reduction of electron carriers of the pathway.     

Ultrastructure of cells cultured on polycarbonate membranes.

A method is described for preparing undisturbed cell cultures for both scanning and transmission electron microscopy. Cells were propagated on polycarbonate membranes with pores of 0.2 micrometer or less. Cultured cells together with their supports were prepared for both scanning electron microscopy and transmission electron microscopy using routine methods. For transmission electron microscopy a rapid schedule of infiltration and polymerization was used. The method described in this report yielded good results and it allowed the fine structure of cultured cells to be viewed in situ by both scanning electron microscopy and transmission electron microscopy.     

Electron energy balance and ionization in the channel of a stationary plasma thruster

The paper presents results of numerical simulations of the electron dynamics in the field of the azimuthal and longitudinal waves excited in the channel of a stationary plasma thruster (SPT). The simulations are based on the experimentally determined wave characteristics. The simulation results show that the azimuthal wave displayed as ionization instability enhances electron transport along the thruster channel. It is established that the electron transport rate in the azimuthal wave increases as compared to the rate of diffusion caused by electron scattering from neutral atoms in proportion to the ratio between the times of electron? neutral collisions responsible for ionization and elastic electron scattering, respectively. An expression governing the plasma conductivity is derived with allowance for electron interaction with the azimuthal wave. The Hall parameter, the electron component of the discharge current, and the electron heating power in the thruster channel are calculated for two model SPTs operating with krypton and xenon. The simulation results agree well with the results of experimental studies of these two SPTs.     

Identification of Cells by Backscattered Electron Imaging of Silver Stained Bulk Tissues in Scanning Electron Microscopy

Anuran tadpole tail muscle was stained en bloc by a modified light microscope silver stain for light microscopy and freeze-fractured in liquid nitrogen after partial dehydration with ethanol. The fractured specimens were observed in both secondary electron and backscattered electron modes in a scanning electron microscope. Since the cell nuclei specifically stained with silver provided high contrast against the unstained background due to atomic number contrast of backscattered electron image, various cells were easily identified by a comparison of secondary electron images and compositional images of backscattered electron signals.     

Ultrastructure of Cells Cultured on Polycarbonate Membranes

A method is described for preparing undisturbed cell cultures for both scanning and transmission electron microscopy. Cells were propagated on polycarbonate membranes with pores of 0.2 pm or less. Cultured cells together with their supports were prepared for both scanning electron microscopy and transmission electron microscopy using routine methods. For transmission electron microscopy a rapid schedule of infiltration and polymerization was used. The method described in this report yielded good results and it allowed the fine structure of cultured cells to be viewed in situ by both scanning electron microscopy and transmission electron microscopy.     

Measurements of mesophyll conductance,photosynthetic electron transport and alternative electron sinks of field grown wheat leaves

Photosynthetic electron transport drives the carbon reduction cycle, the carbon oxidation cycle, and any alternative electron sinks such as nitrogen reduction. A chlorophyll fluorescence— based method allows estimation of the total electron transport rate while a gas-exchange-based method can provide estimates of the electron transport needed for the carbon reduction cycle and, if the CO₂ partial pressure inside the chloroplast is accurately known, for the carbon oxidation cycle. The gas-exchange method cannot provide estimates of alternative electron sinks. Photosynthetic electron transport in flag leaves of wheat was estimated by the fluorescence method and gasexchange method to determine the possible magnitude of alternative electron sinks. Under non-photorespiratory conditions the two measures of electron transport were the same, ruling out substantial alternative electron sinks. Under photorespiratory conditions the fluorescence-based electron transport rate could be accounted for by the carbon reduction and carbon oxidation cycle only if we assumed the CO₂ partial pressure inside the chloroplasts to be lower than that in the intercellular spaces of the leaves. To further test for the presence of alternative electron sinks, carbon metabolism was inhibited by feeding glyceraldehyde. As carbon metabolism was inhibited, the electron transport was inhibited to the same degree. A small residual rate of electron transport was measured when carbon metabolism was completely inhibited which we take to be the maximum capacity of alternative electron sinks. Since the alternative sinks were small enough to ignore, the comparison of fluorescence and gas-exchange based methods for measuring the rate of electron transport could be used to estimate the mesophyll conductance to CO₂ diffusion. The mesophyll conductance estimated this way fell as wheat flag leaves senesced. The age-related decline in photosynthesis may be attributed in part to the reduction of mesophyll conductance to CO₂ diffusion and in part to the estimated decline of ribulose 1,5-bisphosphate carboxylase amount.     

EPR study of electron transport in the cyanobacterium Synechocystis sp. PCC 6803: Oxygen-dependent interrelations between photosynthetic and respiratory electron transport chains

In this work, we investigated electron transport processes in the cyanobacterium Synechocystis sp. PCC 6803, with a special emphasis focused on oxygen-dependent interrelations between photosynthetic and respiratory electron transport chains. Redox transients of the photosystem I primary donor P700 and oxygen exchange processes were measured by the EPR method under the same experimental conditions. To discriminate between the factors controlling electron flow through photosynthetic and respiratory electron transport chains, we compared the P700 redox transients and oxygen exchange processes in wild type cells and mutants with impaired photosystem II and terminal oxidases (CtaI, CydAB, CtaDEII). It was shown that the rates of electron flow through both photosynthetic and respiratory electron transport chains strongly depended on the transmembrane proton gradient and oxygen concentration in cell suspension. Electron transport through photosystem I was controlled by two main mechanisms: (i) oxygen-dependent acceleration of electron transfer from photosystem I to NADP⁺, and (ii) slowing down of electron flow between photosystem II and photosystem I governed by the intrathylakoid pH. Inhibitor analysis of P700 redox transients led us to the conclusion that electron fluxes from dehydrogenases and from cyclic electron transport pathway comprise 20-30% of the total electron flux from the intersystem electron transport chain to P700⁺.     

Concerning a dual function of coupled cyclic electron transport in leaves

Coupled cyclic electron transport is assigned a role in the protection of leaves against photoinhibition in addition to its role in ATP synthesis. In leaves, as in reconstituted thylakoid systems, cyclic electron transport requires “poising,” i.e. availability of electrons at the reducing side of photosystem I (PSI) and the presence of some oxidized plastoquinone between photosystem II (PSII) and PSI. Under self-regulatory poising conditions that are established when carbon dioxide limits photosynthesis at high light intensities, and particularly when stomata are partially or fully closed as a result of water stress, coupled cyclic electron transport controls linear electron transport by helping to establish a proton gradient large enough to decrease PSII activity and electron flow to PSI. This brings electron donation by PSII, and electron consumption by available electron acceptors, into a balance in which PSI becomes more oxidized than it is during fast carbon assimilation. Avoidance of overreduction of the electron transport chain is a prerequisite for the efficient protection of the photosynthetic apparatus against photoinactivation.     

Electron allocation to alternative substrates of Azotobacter nitrogenase is controlled by the electron flux through dinitrogenase

The electron flux through dinitrogenase (MoFe protein, protein containing Mo and Fe) from Azotobacter vinelandii controls the relative effectiveness of alternative substrates as electron acceptors in the nitrogenase system. The electron flux through dinitrogenase reductase (Fe protein) or the concentration of MgATP do not directly control electron allocation but rather control it via their influence on the electron flux through dinitrogenase. Kinetic properties of substrate reduction were studied as a function of the electron flux through dinitrogenase. N₂ was most effective at high electron fluxes, whereas H⁺ was the most effective acceptor at very low rates of electron flow through dinitrogenase. The K_m for acetylene was dependent on the electron flux through dinitrogenase, whereas the K_m for N₂ was much less sensitive to this electron flux. The lag period before the onset of acetylene reduction was proportional to the turnover time of dinitrogenase, and was approx. 12 times greater than the dinitrogenase turnover time. pH has effects on the electron allocation to substrates beyond that expected from the effect of pH on the electron flux; thus, pH may alter the relative ability of the nitrogenase enzyme system to reduce alternative substrates.