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类型 基础研究 预答辩日期 2017-11-10
开始(开题)日期 2016-06-02 论文结束日期 2017-11-01
地点 东南大学电子所楼北103生医学院会议室 论文选题来源 国家自然科学基金项目     论文字数 7.5 (万字)
题目 基于细胞色素c相互作用的产电微生物胞外电子传递机制研究
主题词 胞外电子传递,细胞色素c,蛋白质-蛋白质相互作用,转录调控作用,生物网络
摘要 希瓦氏菌(Shewanella)和地杆菌(Geobacter)等产电微生物可以将细胞内产生的电子传递到细胞的外部,还原胞外的不可溶性固体电子受体,这一过程称为胞外电子传递(Extracellular Electron Transfer,EET)。通过EET过程,产电微生物可以在电极表面生长,并能够完成微生物燃料电池(Microbial Fuel Cells,MFC)的电流输出。然而,产电效率太低限制了MFC技术的推广应用,对产电微生物特有的EET机制的研究将是发展MFC技术的一个关键环节。细胞色素c (Cytochrome C,C-type Cytochrome)在EET过程中发挥了重要作用,是其中不可缺少的一类关键蛋白质。一般认为,从内(细胞质)膜通过周质、外膜到细胞外部空间,细胞色素c可以形成EET途径。 产电微生物的细胞色素c具有丰富的多样性,可以形成不同的组合,构成不同的EET途径以实现有效的电子传递。另一方面,细胞内的生物学过程大多是通过蛋白质之间的相互作用完成的,通过构建蛋白质-蛋白质相互作用(Protein-Protein Interaction,PPI)网络来研究特定的生物学过程是非常有效的。因此,本论文以希瓦氏菌属的Shewanella oneidensis MR-1作为主要的研究对象,基于细胞色素c的相互作用和相关的生物网络对产电微生物的EET机制进行研究。这不仅有助于我们认识和阐明EET过程的分子机制,还有助于我们形成关于EET分子机制的科学假设,例如:识别在EET过程中发挥重要作用的细胞色素c和相关的蛋白质,挖掘与特定EET过程相关的功能模块,等等。研究结果将为通过基因工程技术改造产电微生物、提高产电微生物与电极之间的电子传递效率提供科学依据和理论指导。论文研究主要进展体现在以下方面。 1. 构建了Shewanella oneidensis MR-1的基因组尺度细胞色素c网络,识别了网络中的关键蛋白质和功能模块,并推断出潜在的EET途径。细胞色素c可以作为传递电子的载体,在EET过程中发挥了重要的作用。我们获取了Shewanella oneidensis MR-1中所有41个细胞色素c的蛋白质相互作用信息,构建了一个蛋白质作用网络(即细胞色素c网络),并研究了该网络的结构特征和功能意义。首先,我们使用多种网络中心化方法分析了细胞色素c网络中的关键蛋白质,并综合各种不同方法得到的结果确定了网络中的十大关键蛋白质。其中七个与希瓦氏菌的电流产生有关,表明Shewanella oneidensis MR-1产生电力的能力可能是源自其细胞色素c网络独特的结构。其次,通过网络模块化分析,我们从网络中获得了5个模块。亚细胞定位分析表明这些模块中的蛋白质大多分布在多样化的细胞区室中,反映了它们形成EET途径的潜力。特别地,我们发现组成经典MtrCAB途径的主要细胞色素c (CymA,MtrA,MtrC和OmcA)都在同一个模块中(Mtr-like的模块)。最后,结合蛋白质的亚细胞定位和操纵子分析,我们发现可以从这个Mtr-like的模块中推断已知的和新候选的EET途径,表明可以从这样的细胞色素c网络中获得潜在的EET途径。 2. 构建了Shewanella oneidensis MR-1的基因组尺度电子传递网络,识别了网络中的不同功能部分,发现了辅助EET途径的细胞色素c。EET途径主要是由细胞色素c和其它一些参与电子传递过程的蛋白质组成的。我们构建了一个基因组尺度的电子传递网络,它包含了Shewanella oneidensis MR-1中454个可能与电子传递有关的蛋白质以及这些蛋白质之间的2276对相互作用。使用网络的k-shell分解方法,我们识别并分析了Shewanella oneidensis MR-1电子传递网络中的不同部分。我们发现该网络前三层的蛋白质主要分布在细胞质和细胞内膜,这些蛋白质可以用于负责在各种不同的环境条件下把电子传递到醌池。网络其它各层的蛋白质广泛地分布于所有的五个细胞区室(细胞质、内膜、周质、外膜和细胞外),这确保了Shewanella oneidensis MR-1的EET能力。特别地,我们论证了网络第四层的蛋白质负责处理EET过程,剩余各层中的细胞色素c可以用于辅助EET过程。总的来说,这些结果表明Shewanella oneidensis MR-1的电子传递网络中存在不同的功能部分,EET过程是由这些不同功能部分相互合作而高效完成的。 3. 构建了13种希瓦氏菌的整合转录调控和蛋白交互网络,识别了高保守的网络模体,发现了网络模体“共调控的PPI”在EET过程中起到重要作用。转录调控作用(Transcriptional Regulatory Interaction,TRI)控制了基因的表达水平,进而决定了蛋白质的生成;而蛋白质之间的相互作用介导了细胞的大部分功能。通过整合转录调控和蛋白交互这两种类型的相互作用,我们构建了13种不同希瓦氏菌的整合型生物网络,并从网络模体的角度对它们进行了分析。结果表明,只有7到11种不同的(三节点)网络子图是这些整合型网络中的网络模体。我们发现,与只包含单一类型相互作用的分子生物网络类似,包含两种类型相互作用的整合型生物网络中的网络模体也是进化保守的。随后,我们识别了高保守的网络模体,并讨论了它们的生物学功能。特别地,我们发现网络模体“共调控的PPI”对EET过程特别重要。从结构上来看,这种模体除了调控成对的蛋白质编码基因之外,还需要确保它们能够形成相互作用。进一步的功能分析表明,这种网络模体与细胞中蛋白质利用的“提前准备”模式之间有一定的关系,这将有助于细胞迅速响应环境的变化。此外,通过GO富集分析和蛋白质域富集分析,我们论证了II型辅因子(网络模体“TRI Interacting With A Third Protein”中的辅因子)主要实现了Shewanella oneidensis MR-1信号传递的功能。 4. 构建了Shewanella oneidensis MR-1中EET途径相关的转录调控模块,识别了其中辅因子(蛋白质)的信号传递功能,发现了关键的信号蛋白质。为了利用各种不同的胞外电子受体,Shewanella oneidensis MR-1需要使用复杂的调控机制来激活相应的EET途径。通过整合EET基因和相关的转录因子,我们构建了与EET途径相关的转录调控模块。随后,我们分析了这些模块中的辅因子,结果发现这些模块中均富含有大量的信号蛋白质。此外,我们论证了多样化的信号蛋白质(或信号域)可用于协调不同的EET途径,并讨论了与经典MtrCAB途径有关的转录调控模块中信号蛋白质的功能。特别地,我们论证了信号蛋白质SO_2145和SO_1417在厌氧条件下激活EET途径的过程中扮演着重要的角色。总体而言,这些结果表明信号蛋白质在EET基因的转录调控过程中有着重要的作用,在研究希瓦氏菌的EET途径时应当充分考虑涉及其中的信号蛋白质。
英文题目 RESEARCHES ON THE EXTRACELLULAR ELECTRON TRANSFER MECHANISM OF ELECTRICIGENS BASED ON THE INTERACTIONS OF CYTOCHROME C
英文主题词 extracellular electron transfer,cytochrome c,protein-protein interaction,transcriptional regulatory interaction,biological network
英文摘要 The electricigens such as Shewanella and Geobacter can transfer the electrons from the intracellular environment to the extracellular space to reduce the extracellular insoluble solid electron acceptors, this process is known as the extracellular electron transfer (EET). Via EET process, these bacteria can grow on electrode surfaces and make current output of microbial fuel cells (MFC). However, the low efficiency of electricity production limits the popularization and application of MFC technologies, and the research on the EET mechanism of electricigens is a key step in the development of MFC technologies. C-type cytochromes play an important role in the EET process, and they are essential proteins in such process. Typically, from the inner (cytoplasmic) membrane through periplasm and outer membrane to extracellular space, they could form EET pathways. C-type cytochromes of electricigens have a rich diversity and could form different combinations, and thus constitute different EET pathways to achieve efficient electron transfer. On the other hand, the biological processes in a cell are mostly performed by protein-protein interactions, the construction of protein interaction network to study the specific biological process will be very effective. Therefore, using Shewanella oneidensis MR-1 as the main research object, we will focus on the EET mechanism of electricigens based on the interactions of c-type cytochromes and related biological networks in this thesis. The works will not only help us to understand and elucidate the molecular mechanism of the EET process, but will also contribute to the formation of the hypothesis about the molecular mechanism of the EET process, such as discovering the key c-type cytochromes and related proteins that play an important role in the EET process, or mining the functional modules that associate with the specific EET process, and so on. The research results will provide scientific basis and theoretical guidance for the improvement of electricigens with genetic engineering technology, and for improving the electron transfer efficiency between electricigens and electrode. The main research progress of this dissertation is embodied in the following aspects. 1. We constructed a genome-scale c-type cytochrome network of Shewanella oneidensis MR-1, identified the key proteins and functional modules, and inferred potential EET pathways from the network. C-type cytochromes can be used as carriers to transfer electrons, which play an important role in the EET process. We obtained the protein interaction information for all 41 c-type cytochromes in Shewanella oneidensis MR-1, constructed a protein interaction network (i.e., c-type cytochrome network), and studied its structural characteristics and functional significance. Firstly, we studied the key proteins in the c-type cytochrome network with multi-centrality measures, and by integrating the results from different measures, we identified the top 10 key proteins of the network. Seven of them are associated with electricity production in the bacteria, which suggests that the ability of Shewanella oneidensis MR-1 to produce electricity might be derived from the unique structure of the c-type cytochrome network. Then, we obtained 5 modules from the network by modularity analysis. The subcellular localization study has shown that the proteins in these modules all have diversiform cellular compartments, which reflects their potential to form EET pathways. In particular, we found that the main c-type cytochromes for constituting the MtrCAB pathway (CymA, MtrA, MtrC and OmcA) were all in the same module (the Mtr-like module). At last, combination of protein subcellular localization and operon analysis, the well-known and new candidate EET pathways are obtained from the Mtr-like module, indicating that potential EET pathways could be obtained from such a c-type cytochrome network. 2. We constructed a genome-scale electron transfer network of Shewanella oneidensis MR-1, identified distinct functional parts in the network, and found the c-type cytochromes which were involved in aiding EET pathways. The EET pathways mainly consist of c-type cytochromes, along with some other proteins that are involved in the electron transfer process. We constructed a genome-scale electron transfer network, which containing 2276 interactions among 454 electron transfer related proteins in Shewanella oneidensis MR-1. Using the k-shell decomposition method, we identified and analyzed distinct parts in the electron transfer network of Shewanella oneidensis MR-1. We found that the proteins in the top three shells of the network were mainly located in the cytoplasm and inner membrane; these proteins can be responsible for transferring electrons into the quinone pool in a wide variety of environmental conditions. In most of the other shells, proteins broadly located throughout the five cellular compartments (cytoplasm, inner membrane, periplasm, outer membrane, and extracellular space), which ensured the important EET ability of Shewanella oneidensis MR-1. Specifically, we demonstrated that the fourth shell was responsible for EET and the c-type cytochromes in the remaining shells of the electron transfer network were involved in aiding EET. Taken together, our results showed that there were distinct functional parts in the electron transfer network of Shewanella oneidensis MR-1, and the EET process could achieve high efficiency through cooperation through such an electron transfer network. 3. We constructed the integrated transcriptional regulation and protein interaction networks of 13 Shewanella species, identified the highly conserved network motifs, and found that the motif “Co-regulated PPI” played central roles in the EET process. Transcriptional regulatory interactions control the expression levels of genes and therefore the generation rate of proteins; and the interactions between the proteins mediate the most of the cellular function. By integrating the transcriptional regulation interactions and the protein-protein interactions, we constructed the integrated networks of 13 Shewanella species, and analyzed these networks from the perspective of network motifs. The results shown that only 7 to 11 different (three-node) network sub-graphs were network motifs in these integrated networks. We have further shown that the network motifs were also evolutionary conserved in these integrated networks, just like that their roles in many molecule networks that only contained single type of interaction. Then, we identified the highly conserved network motifs and discussed the functional significance of these motifs. In particularly, we found that the network motif “Co-regulated PPI” was an important motif that involved in the Shewanella EET process. Structurally, in addition to regulating paired protein-coding genes, this motif also needs to ensure them to interact with each other. Further functional analysis showed that there was a certain relationship between the motif “Co-regulated PPI” and the “standby mode” of proteins in the cell, which will be helpful for cells to rapidly response to environmental changes. Furthermore, through the GO enrichment analysis and protein domain enrichment analysis, we demonstrated that the type II cofactors, those involved in the motif “TRI Interacting With A Third Protein”, mainly carried out a signalling role in Shewanella oneidensis MR-1. 4. We constructed the transcriptional regulatory modules that were involved in the EET pathways of Shewanella oneidensis MR-1, identified the signaling roles of the cofactors (proteins), and found the key signal proteins. To utilize various extracellular electron acceptors, Shewanella oneidensis MR-1 employed complex regulatory mechanisms that can be involved in eliciting the relevant EET pathways. By integrating the EET genes and related transcriptional factors, we constructed the transcriptional regulatory modules that were involved in the EET pathways. Then, we analyzed the cofactors in these modules, and showed that signal proteins were overabundant in these modules. Furthermore, we further demonstrated that diverse signal proteins (or signal domains) reconciled different EET pathways, and we also discussed the functional roles of the signal proteins that were drawn into the MtrCAB pathway. In particularly, we found that the signal proteins SO_2145 and SO_1417 played central roles in triggering the EET pathways under anaerobic environments. These results suggested that signal proteins had a profound impact on the transcriptional regulation of the EET genes, and they should be fully considered in studying Shewanella EET pathways.
学术讨论
主办单位时间地点报告人报告主题
东南大学生医学院生物信息组 2014年1月 生物电子学国家重点实验室 丁德武 A half story of cytochromes PPI network
东南大学生医学院生物信息组 2014年3月 生物电子学国家重点实验室 丁德武 Topological features reflect the potential EET pathways in cytochrome c PPI network
东南大学生医学院生物信息组 2015年4月 生物电子学国家重点实验室 丁德武 Controllability of a structural electron transfer network
东南大学生医学院生物信息组 2015年10月 生物电子学国家重点实验室 丁德武 Exploring the EET mechanisms in S. oneidensis MR-1 through an electron carrier network
东南大学生医学院生物信息组 2015年12月 生物电子学国家重点实验室 丁德武 K-shell analysis reveals distinct functional parts in an electron transfer network & its implications for EET
东南大学生医学院生物信息组 2016年3月 生物电子学国家重点实验室 丁德武 Network topology, disordered regions and expression data to probe EET mechanisms
东南大学生医学院生物信息组 2016年10月 生物电子学国家重点实验室 丁德武 Co-factors reconcile multiple signals in EET pathways
东南大学生医学院生物信息组 2017年1月 生物电子学国家重点实验室 丁德武 Inferring direct associations in biological networks
     
学术会议
会议名称时间地点本人报告本人报告题目
2014年生物电子学与生物光子学联合学术论坛 2014年11月 苏州 Identifying the potential extracellular electron transfer pathways from a c-type cytochrome network
2015年江苏省生物信息学年会 2015年7月 苏州 The structure and function of electron transfer network in Shewanella oneidensis MR-1
     
代表作
论文名称
Identifying the potential extracellular electron transfer pathways from a c-type cytochrome network
K-shell Analysis Reveals Distinct Functional Parts in an Electron Transfer Network and Its Implicati
 
答辩委员会组成信息
姓名职称导师类别工作单位是否主席备注
许晓风 正高 教授 博导 南京师范大学
宋晓峰 正高 教授 博导 南京航空航天大学
王俊 正高 教授 博导 南京邮电大学
王进科 正高 教授 博导 东南大学
顾万君 正高 教授 博导 东南大学
      
答辩秘书信息
姓名职称工作单位备注
刘宏德 副高 副教授 东南大学