Redox Potential Controls Electron Transfer Through The Inner Membrane Of Geobacter Sulfurreducens

Abstract

Harnessing energy for growth and survival is universal to all living forms. Bacteria are constantly faced with changing environment forcing them to quickly adapt to the conditions to gain maximum energy available. Acquisition of energy involves transfer of electrons from substrate that gets oxidized to the reduction of electron acceptors. Microorganisms performing extracellular electron transfer have evolved to couple oxidation of electron donors to the reduction of electron acceptors present outside the cell using a chain of redox active proteins. Geobacter sulfurreducens is one such model organism for studying extracellular electron transfer, providing unique opportunities for the development of bioelectronic devices and sensors. Despite the usefulness of G. sulfurreducens extracellular electron transfer ability in biotechnological applications, the complete electron transfer pathway still remains unknown. The factors regulating the electron transfer between different cytochromes, as well as the specific utilization of different cytochromes in energy conservation is one of the lesser studied aspects of G. sulfurreducens physiology. The work presented in this thesis includes discovery and characterization of an inner membrane cytochrome complex, CbcBA essential for respiration of electron acceptors near the thermodynamic limit of acetate respiration (< -0.21 V vs. Standard Hydrogen Electrode (SHE)). A \sigma^{54}–dependent transcription factor, BccR controlling the expression of CbcBA was also characterized. Other inner membrane cytochromes involved in redox dependent electron transfer, ImcH, and CbcL are constitutively expressed. Using genetic and electrochemical approaches, CbcL was found to function as a redox dependent switch showing oxidative inactivation above redox potentials of -0.1 V vs. SHE. Using specific mutants lacking one or more inner membrane cytochromes, cellular yields were measured corroborating earlier reported data that the ImcH-dependent electron transfer pathway supported the highest cellular yield, while the CbcL-dependent pathway supported much lower cell yields. The CbcBA-dependent pathway could not support growth under conditions tested, but was found to be needed for survival under low electron acceptor conditions. Expressing fluorescent proteins in specific inner membrane cytochrome mutants allowed studying metabolic heterogeneity of G. sulfurreducens biofilms visualized using confocal microscopy. At high redox potentials (+0.24 V vs. SHE), G. sulfurreducens utilizes ImcH-dependent pathway in cells closest to the electrode, and CbcL-dependent pathway in cells beyond 10 µm from the electrode surface. At low redox potentials (-0.13 V vs. SHE), only the CbcL-dependent pathway is utilized. The findings reported in this thesis, suggests a route for building biosensors for redox sensing.