The Processes And Mechanisms Of Microbial Outer Membrane C-type Cytochrome Mediated Extracellular Electron Transfer

The extracellular electron transfer(EET)in microorganisms plays a critical role in the biogeochemical cycle of iron(Fe),which not only couples the biogeochemical cycles of carbon,nitrogen,and other elements but also directly drives the degradation of organic pollutants,the migration and transformation of heavy metals,and the decreases in greenhouse gas emissions.Therefore,to understand these environmental processes,the relevant mechanism of this process needs to be analyzed.The active center of the EET process is the outer membrane(OM)c-type cytochromes(c-Cyts).Whether through direct membrane contact,nanowires,or indirect electron exchange with an extracellular substrate through a shuttle,the essence of these processes is the electron exchange between the OM c-Cyts and an extracellular substrate.Therefore,it is of great significance to study the mechanism of microbial OM c-Cyts in the process of Fe reduction and oxidation.The mechanistic understanding of these electron transfer processes at the molecular level will lay the foundation for improving the biotechnological applications of microorganisms with EET capability.In this study,diffuse-transmission UV/Vis(DT-UV/Vis)spectroscopy was used to record the spectra of OM c-Cyts in living bacterial cells of the dissimilar metal-reducing bacteria Shewanella oneidensis MR-1 and the neutrophilic nitrate-reducing,Fe(II)-oxidizing bacteria Pseudogulbenkiania sp.strain 2002.Combining a series of batch experiments,electrochemical and chemical analysis,and molecular biology techniques and other methods,the cells of Shewanella oneidensis MR-1 grown without electron acceptors were investigated to test the hypothesis that these bacteria would release soluble extracellular polymeric substances(EPS)containing the OM c-Cyts,and highlight the geochemical importance of this process in environments lacking available electron acceptors.It was demonstrated that the c-Cyts produced and released by MR-1 likely played a role in EET under electron acceptor-limited conditions.In addition,the biological oxidation of Fe(II)was demonstrated using the EPS of Pseudogulbenkiania sp.strain 2002 containing an OM c-Cyts.The main conclusions are summarized as follows:(1)By comparison to normal UV/Vis spectroscopy,it was determined that DT-UV/Vis spectroscopy detected the OM c-Cyts spectra in a suspension of live bacterial cells in situ.Because interference from cell surface scattering was removed,the DT-UV/Vis showed a higher absorbance of c-Cyts and lower background compared to normal UV/Vis.The two extinction coefficients of oxidized c-Cyt(410 nm and 419 nm)and the three extinction coefficients of reduced c-Cyt(410 nm,419 nm,and 552 nm)were observed.Using this method and the obtained c-Cyts extinction coefficients,the redox transformation kinetics of c-Cyts under anoxic conditions were examined in the presence of various electron acceptors.These results suggested that the in situ spectral analysis of the OM proteins of intact cells using DT-UV/Vis spectroscopy appeared to be a promising method to investigate microbial metal reduction processes in living cells.(2)In the absence of dissolved electron acceptors,c-Cyts and other dissolved extracellular organic compounds(EOCs)were released from Shewanella oneidensis MR-1 cells.In this process,most of the cells ruptured and underwent apoptosis,but some cells survived.Before these cells released dissolved EOCs,their OM formed nanowires and vesicles using extensions of the OM;the regulation of gene expression of this process was similar to the formation of nanowires during conditions where electron acceptors were limited.Because most of the gene expression in this process was consistent with that of OM vesicle and nanowire formation under electron acceptorlimited conditions,these data suggest that the OM vesicles and nanowires formed during the soluble release of c-Cyts,and the dissolved EOCs may have been released from debris of broken cells,as well as from OM extensions.These findings provide a new insight in the understanding of the survival of MR-1 cells in conditions where electron receptors are absent.(3)Based on the above kinetic modeling analysis,the elementary reaction mechanism of chromium(Cr)(VI)reduction by MR-1 cells could be quantitatively described as involving bound-Cyts,flavins,and released-Cyts.These results suggested that the relative contribution of EOCs(MW > 3000)containing released-Cyts mediated higher EET than low MW(< 3000 Da)soluble EOCs containing flavins.Therefore,EOCs(MW > 3000)containing released-Cyts produced by MR-1 played an important role in the EET process.These findings provided a new understanding of extracellular electron shuttling and the metabolic strategy of metal-reducing bacteria under electron acceptorlimited conditions.(4)DT-UV/Vis spectroscopy directly demonstrated the existence of the OM c-Cyts in Pseudogulbenkiania sp.strain 2002 cells.Using the EPS containing OM c-Cyts of sp.strain 2002,Fe(II)was oxidized,but the OM c-Cyts could not directly react with nitrate or nitrite.After adding the EPS containing OM c-Cyts,soluble c-Cyts mediated Fe(II)oxidation,which became nucleation sites in the process.These data also indicated that the OM c-Cyts of nitrate-reducing,Fe(II)-oxidizing bacteria could directly mediate the biological Fe(II)oxidation process,and also play a key role in the encrustation effect on the cell surface.These results provided an in-depth understanding of the diverse enzymatic pathways involved in the nitrate reduction and Fe(II)oxidation processes in the natural environment.