Intracytoplasmic membranes develop in Geobacter sulfurreducens under thermodynamically limiting conditions

Abstract

Geobacter sulfurreducens is an electroactive bacterium capable of reducing metal oxides in the environment and electrodes in engineered systems^1,2. 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-bound^3-5. 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.

Fig. 1. Transmission electron microscopy of anode-grown G. sulfurreducens.

Plunge frozen, freeze substituted plastic embedded TEM micrographs of G. sulfurreducens collected from an anode biofilm poised at −0.07 V vs. SHE. ICM structures present as parallel bands in one area of a cell. a, c, and e show ICM in cells sliced perpendicular to the major axis, while b, d, and f show ICM in cells sliced parallel to the major axis where the ICM is located near the tip of the cell. Micrographs were collected on an FEI TF20. Scale bars: 100 nm.

Fig. 2. 3D segmentation of ICM in anode-grown G. sulfurreducens.

a Tomogram slices illustrating how 3D models were created via tomogram segmentation of ICM located in the tip of a G. sulfurreducens cell with the inner membrane, outer membrane, ICM, and several nanowires modeled from the tomogram. The scale bar is 100 nm. b–d 3D models of three separate cells displaying ICM near the tip of each cell. These cells were grown at −0.07 V vs. SHE directly on a grid.

Fig. 3. G. sulfurreducens invagination of the inner membrane.

Cryotomograph slices and 3D model of a G. sulfurreducens cell grown at –0.17 V vs SHE on a holey-carbon/gold cryo-EM grid as anode for 24 h. Cryotomogram shows how ICM seems to form by invagination of the inner membrane. Top – selected tomogram slices in the z axis displaying sections of the invagination. Bottom – 3D model of the inner membrane and outer membrane through (bottom left) the entire thickness of the modeled volume and (bottom right) the model sliced approximately in half in the z axis to show the ICM profile at a different depth. Scale bar: 100 nm.

Fig. 4. ICM frequency in G. sulfurreducens measured by confocal microscopy.

a Fraction of cells with an ICM in G. sulfurreducens under different growth conditions. Each dot represents a unique image with an average of 113 cells, see Supplemental Table 1 for raw counts. The three potentials – −0.17, −0.07, and +0.07 – represent cells collected from anodes poised at the respective potentials vs. SHE. The ‘fumarate’ condition cells were grown planktonically with fumarate as the electron acceptor. Statistical testing consisted of a two-tailed t-test with Hochberg multiple comparison correction. *(ρ < 0.05), ***(ρ ≤ 0.001), ****(ρ ≤ 0.0001). b Sum projection of confocal z-stacks of G. sulfurreducens cells grown on an electrode at -0.07 V vs. SHE with examples of ICM annotated. c Sum projection of cells grown with fumarate as an electron acceptor exemplifying typical cell morphology when ICM is not present. Both cell images were cropped from larger images taken at 100X magnification using Nile red as a phospholipid-selective fluorophore. See zoomed insets in Supplemental Fig. 5.