Intracytoplasmic membranes develop inGeobacter sulfurreducensunder thermodynamically limiting conditions

Abstract

Geobacter sulfurreducens is an electroactive bacterium capable of reducing metal oxides in the environment and electrodes in engineered systems. 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. 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 at low potentials, whereas cells grown at higher potentials 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.

Widespread extracellular electron transfer pathways for charging microbial cytochrome OmcS nanowires via periplasmic cytochromes PpcABCDE

“…The most affected residues were determined using amino acid-specific chemical shift mapping 42 (see Methods for details). Analysis of the PpcA backbone residues affected by the presence of OmcS (Δδ combined > Δδ cut-off ) shows two different extents of variation: residues Ile 4 , Ala 8 , Val 13 , Lys 14 , Phe 15 , Lys 18 , Ala 46 , His 47 , Gly 48 are the most affected ones by the presence of OmcS (Δδ combined > 1.5Δδ cut-off ), followed by Asp 3 , His 20 , Cys 30 , His 31 , Lys 37 , Lys 52 , Cys 54 , Thr 63 and Cys 68 (Δδ combined > Δδ cut-off ). The most affected residues comprise a region near heme IV (Ala 8 , Val 13 , Ala 46 , His 47 , Gly 48 ) and in between hemes I and III (Ile 4 , Lys 14 , Phe 15 , Lys 18 ) (Fig.…”

“…For interaction experiments in the oxidized state, isotopically labeled 15 N PpcA (overexpressed and purified according to the protocol discussed previously 68 ) was resuspended in 8 mM potassium phosphate buffer with KCl (20 mM final ionic strength), pH 7, prepared in H 2 O/ 2 H 2 O (92%/ 8%) and NMR spectra were acquired in the absence and presence of OmcS (PpcA:OmcS heme ratio 1:4). PpcA's backbone assignment in the absence and presence of OmcS is reported in Supplementary Table 2.…”

“…All 1D 1 H-NMR spectra were acquired with 3.3 k data points and a spectral width of 24 kHz, with 256 scans. For backbone interaction experiments, 2D 1 H, 15 N-HSQC spectra were acquired with 2048 (t 2 ) × 256 (t 1 ) data points, with a sweep width of 11.4 k ( 1 H) and 2.4 k ( 15 N) and 32 scans. 1 H chemical shifts were referenced to 2,2-dimethyl-2-silapentane-5-sulfonate at 0 ppm 69 .…”

“…where Δδ H and Δδ N correspond, respectively, to the chemical shift change in the 1 H and 15 N dimensions in the absence and presence of OmcS and w i is the quotient between the gyromagnetic ratio of 15 N and 1 H to compensate for scaling differences 42 . The most affected residues were selected using a threshold based on the procedure described previously 42 : the cut-off value was determined iteratively by determining the standard deviation, σ corr , from the dataset and excluding the residues which presented Δδ combined three times higher than the calculated…”

“…Although nanowires transport electrons received from within the cell to external acceptors, there are large spatial, kinetic and thermodynamic barriers to move electrons from the inner-membrane to the outer-surface nanowires 14 . The periplasmic compartment in G. sulfurreducens is significantly larger (>40 nm) than that of most microbes (<30 nm) 15 , which is too large for direct electron transfer limited to ~1.5 nm 16 . In addition to this large spatial barrier, cells pass electrons rapidly 17 (>10 6 s −1 ) to outer-surface nanowires despite the kinetic barriers of a crowded periplasmic environment 18 and slow (<10 5 s -1 ) electron diffusion within monomeric periplasmic cytochromes 19,20 , which will require at least 10-times higher abundance of periplasmic cytochromes than nanowires to match the rate.…”