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.