Cytochrome gene expression shifts in Geobacter sulfurreducens to maximize energy conservation in response to changes in redox conditions.

Previous studies have identified that Geobacter sulfurreducens has three different electron transfer pathways for respiration, and it switches between these pathways to adapt to the redox potential of its electron acceptor. However, only a small fraction of the electron carriers from each pathway have been identified. In this study, we combined electrochemical and gene expression data to identify electron carriers in the inner membrane, periplasm, outer membrane, and exterior of the cell that may be induced by the use of the three different electron transfer pathways. Cyclic voltammetry was performed on thin biofilms grown on anodes poised at different redox potentials, providing a quantitative assessment of the relative use of three electron-transfer pathways in each condition (catalytic midpoint potentials (EKAs) of -0.227 V [Low], -0.15 V [Medium], -0.1 V [High] vs. SHE). Transcriptomic analyses as a function of electrochemical signals or fumarate utilization showed differential induction in inner membrane (Medium: cbcL), periplasmic (Low: ppcB/ppcE, Medium: ppcA), outer membrane (Low: extA/extC, Medium: extJ/extK, Fumarate: extF/extG), and extracellular (Medium: omcZ, High/Fumarate: omcS/omcT) cytochromes, suggesting the pathway signals are associated with complex transcriptomic responses in genes across the electron transfer pathway. Our method combining electrochemical modeling and transcriptomics could be adapted to better understand electron transport in other electroactive organisms with complex metabolisms.

Intracytoplasmic membranes develop in Geobacter sulfurreducens under thermodynamically limiting conditions.

Geobacter sulfurreducens is an electroactive bacterium capable of reducing metal oxides in the environment and electrodes in engineered systems1,2. Geobacter sp. are the keystone organisms in electrogenic biofilms, as their respiration consumes fermentation products produced by other organisms and reduces a terminal electron acceptor e.g. iron oxide or an electrode. To respire extracellular electron acceptors with a wide range of redox potentials, G. sulfurreducens has a complex network of respiratory proteins, many of which are membrane-bound3-5. We have identified intracytoplasmic membrane (ICM) structures in G. sulfurreducens. This ICM is an invagination of the inner membrane that has folded and organized by an unknown mechanism, often but not always located near the tip of a cell. Using confocal microscopy, we can identify that at least half of the cells contain an ICM when grown on low potential anode surfaces, whereas cells grown at higher potential anode surfaces or using fumarate as electron acceptor had significantly lower ICM frequency. 3D models developed from cryo-electron tomograms show the ICM to be a continuous extension of the inner membrane in contact with the cytoplasmic and periplasmic space. The differential abundance of ICM in cells grown under different thermodynamic conditions supports the hypothesis that it is an adaptation to limited energy availability, as an increase in membrane-bound respiratory proteins could increase electron flux. Thus, the ICM provides extra inner-membrane surface to increase the abundance of these proteins. G. sulfurreducens is the first Thermodesulfobacterium or metal-oxide reducer found to produce ICMs.

Geobacter sulfurreducens' Unique Metabolism Results in Cells with a High Iron and Lipid Content.

Geobacter sulfurreducens is a ubiquitous iron-reducing bacterium in soils, and in engineered systems, it can respire an electrode to produce measurable electric current. Its unique metabolism, heavily dependent on an extensive network of cytochromes, requires a unique cell composition. In this work, we used metallomics, cell fraction and elemental analyses, and transcriptomics to study and analyze the cell composition of G. sulfurreducens. Elemental composition studies (C, H, O, N, and ash content) showed high C:O and H:O ratios of approximately 1.7:1 and 0.25:1, indicative of more reduced cell composition that is consistent with high lipid content. Our study shows that G. sulfurreducens cells have a large amount of iron (2 ± 0.2 µg/g dry weight) and lipids (32 ± 0.5% dry weight/dry weight) and that this composition does not change whether the cells are grown with a soluble or an insoluble electron acceptor. The high iron concentration, higher than similar microorganisms, is attributed to the production of cytochromes that are abundant in transcriptomic analyses in both solid and soluble electron acceptor growth. The unique cell composition of G. sulfurreducens must be considered when growing this microorganism for lab studies and commercial applications. IMPORTANCE Geobacter sulfurreducens is an electroactive microorganism. In nature, it grows on metallic minerals by transferring electrons to them, effectively "breathing" metals. In a manmade system, it respires an electrode to produce an electric current. It has become a model organism for the study of electroactive organisms. There are potential biotechnological applications of an organism that can bridge the gap between biology and electrical signal and, as a ubiquitous iron reducer in soils around the world, G. sulfurreducens has an impact on the global iron cycle. We measured the concentrations of metals, macromolecules, and basic elements in G. sulfurreducens to define this organism's composition. We also used gene expression data to discuss which proteins those metals could be associated with. We found that G. sulfurreducens has a large amount of lipid and iron compared to other bacteria-these observations are important for future microbiologists and biotechnologists working with the organism.