A Study On The Extracellular Electron Transfer Processes Mediated By Outer Membrane C-type Cytochromes Of Shewanella Oneidensis MR-1

Microbial extracellular respiration is a special kind of anaerobic respiration found recently.The outer membrane c-type cytochromes?c-Cyts?of microorganism play an important role during extracellular respiration,which mainly act as electron transport carrier or terminal reductase.Shewanella oneidensis MR-1 is one of the most studied extracellular respiratory model bacteria.The key electron-transfer proteins in the extracellular respiratory electron transport chain of Shewanella oneidensis MR-1 have been isolated and purified,but the specific functions of each protein during bioreduction and biomineralization are still not fully understood.Moreover,it is still rarely reported that the change on redox state of c-type cytochrome in vivo,especially the outer membrane c-Cyts in the extracellular respiration,when different types of substrates are used as extracellular receptors for extracellular reduction of MR-1.In this study,we investigated the effects of different mutants of MR-1 on bioreduction and mineralization of ferrihydrite,and investigated the changes on redox state of outer membrane c-Cyts in MR-1 in different culture conditions with metal chromium,quinone,and soluble Fe?III?-ligand complex as extracellular electron acceptor using diffuse transmission spectroscopy.This study revealed the role of c-Cyts during the bioreduction and mineralization of iron oxide in the electron transport chain of MR-1,and provided a further understand on the interaction between microorganisms and mineral.Spectroscopy method was used to investigate the reaction between c-Cyts and extracellular receptors in vivo in situ,which provides a further understanding of microbial extracellular respiration process.Main conclusions of this study include:?1?The effects of different mutants??mtrA,?mtrC,?mtrD,?mtrF,?omcA,and ?cymA?of MR-1 on bioreduction and mineralization of ferrihydrite were investigated.The results indicate that while the reduction rates of ferrihydrite by ?mtrD,?mtrF and ?omc A are retained at a similar level to that of the wild type?wt?,the capacity to reduce ferrihydrite decreased dramatically in the mutants ?cymA and ?mtrA.Secondary minerals of ferrihydrite were characterized using X-ray diffraction,Fourier transform infrared spectra and scanning electron microscopy.The results show that goethite and hematite were the main secondary minerals in the first 2 days in all treatments,and then hematite was fully converted into magnetite after 6 days in the wt,?mtrD,?mtrF and ?omcA treatments,while magnetite began to appear from the sixth day on in the ?mtrC treatment.No magnetite,however,was observed during the 6-day ?mtrA and ?cymA incubation treatments.The plausible electron transfer pathway of bioreduction and phase transformation were also verified according to thermodynamic calculations of elementary reactions.?2?Diffuse-transmission UV/Vis spectroscopy was used to investigate the in situ spectral reaction of Cr?VI?reduction by c-Cyts in intact Shewanella oneidensis MR-1 cells under different incubation conditions.The reduced c-Cyts decreased transiently at the beginning and then recovered gradually over time.The Cr?VI?reduction rates decreased with increasing initial Cr?VI?concentrations,and Cr?III?was identified as a reduced product.The presence of Cr?III?substantially inhibited Cr?VI?reduction and the recovery of reduced c-Cyts,indicating that Cr?III?might inhibit cell growth.Cr?VI?reduction rates increased with increasing cell density.The highest Cr?VI?reduction rate and fastest recovery of c-Cyts were obtained at pH 7.0 and 30 °C,with sodium lactate serving as an electron donor.The presence of O2 strongly inhibited Cr?VI?reduction,suggesting that O2 might compete with Cr?VI?as an electron acceptor in cells.?3?Diffuse-transmission UV/Vis spectroscopy was used to investigate the in situ spectral reaction of quinone analog anthraquinone-2,6-disulfonate?AQDS?reduction by c-Cyts in intact Shewanella oneidensis MR-1 cells under different incubation conditions.The reduced c-Cyts decreased transiently at the beginning and then recovered gradually over time.The AQDS reduction rates decreased with increasing initial AQDS concentrations.AQDS reduction rates increased with increasing cell density.The highest AQDS reduction rate and fastest recovery of c-Cyts were obtained at 30 °C and pH 7.0.Thermodynamic analysis on the effects of various incubation conditions on AQDS reduction by c-Cyts in intact cells showed the most suitable temperature and pH might improve the metabolism of cells,thus leaded a high consumption rate of lactate and a high electron output via intracellular electron transport and extracellular electron transfer.?4?The situ spectral dynamics of different types of Fe?III?-ligand complex reduction by the c-type cytochrome in vivo Shewanella oneidensis MR-1 cells under anaerobic condition was investigated using diffuse transmission UV/Vis spectroscopy.The results show that Fe?III?-citrate was not reduced in the treatment without MR-1 under anaerobic conditions.The c-type cytochrome in the outer membrane of MR-1 cell did not occur redox reaction in the absence of Fe?III?-citrate.With the reduction of Fe?III?-citrate,the reduced c-type cytochrome decreased rapidly in the initial stage of the reaction,and then gradually restored to the initial state.Compared with the MR-1 wild type,the deletion of key proteins,such as CymA,MtrA and MtrC,affected differently the reduction rate of Fe?III?-citrate,and the consumption rate and recovery rate of Heme_red.Different types of Fe?III?-ligand complex reduction by the c-type cytochrome in vivo MR-1 cell presented different reduction rate of iron,consumption rate and recovery rate of Heme_red.