Characterization of an electron conduit between bacteria and the extracellular environment

SE analysis of the Mtr proteins. (A) Absorbance profiles of 0.7 μM MtrABC (triangles) and 2.5 μM MtrAB (circles) measured at 435 nm and 9 krpm. (B) Absorbance profiles of MtrAB and MtrC at 0.4 μM (triangles) and 0.1 μM (circles) measured at 435 and 410 nm, respectively, at 8 krpm. (C) Absorbance profiles of 10 μM MtrC (triangles) and MtrA (circles) measured at 530 nm and 8 krpm. The lines represent fits to a single species. At top are shown residual differences between the experimental data and fitted curves.

Spectropotentiometric properties of the Mtr proteins. (A) Oxidized (red spectrum) and reduced (blue spectrum) UV-vis spectra of 2 μM MtrCAB. (Inset) Successive UV-vis spectra of MtrCAB recorded during reductive titration (arrow indicates increasingly negative solution). (B) UV-vis spectra of MtrCAB MV-proteoliposomes. Black, oxidized; red, reduced for 1 min with 5 μM dithionite; blue, 1 min after addition of 1% TX100 to the dithionite-reduced sample; green, a new sample is reduced by dithionite and a spectrum collected 1 min after addition of Fe(III) citrate (100 μM). (Inset) Dithionite reduced minus oxidized difference spectrum of the MtrCAB MV-proteoliposomes (red) and purified MtrCAB in solution (black) normalized to the maximum signal at 422 nm. (C) Baseline-subtracted cyclic voltammograms for adsorbed MtrC, MtrCAB, and MtrA recorded at 30 mV s⁻¹, 0 °C with 3 krpm electrode rotation. Oxidative and reductive peaks (normalized to their respective peak currents) and the normalized integral of the oxidative peak (indicating the extent of protein reduction as a function of potential) are shown as black lines with gray fill and heavy solid lines, respectively. Circles indicate the extent of reduction for each protein as determined by spectrophotometric solution titration. (D) Comparison of the redox properties of MtrCAB, MtrC, and MtrA. (Upper) Oxidative voltammetric current per mole of protein for MtrC (dashed line) and MtrA (dotted line) and their sum (gray solid line) assuming 10 redox active single-electron hemes per protein. (Lower) Oxidative peak currents per mole of MtrCAB (black solid line) compared with the sum of those from the constituents MtrC and MtrA (gray solid line) assuming 20 redox active single-electron hemes in each case. Experimental data as in C.

Characterization of mtrA and mtrB mutants. (A) Immunoblot of cell lysates reacted with antibody specific for MtrB. Lane 1, WT MR-1; lane 2, ΔmtrB; lane 3, ΔmtrA; lane 4 ΔmtrA(mtrA_c); lane 5, ΔmtrA(mtrB_c). (B) Agarose gel electrophoresis of product generated from RT-PCR of WT (lanes 1 and 3) and ΔmtrA RNA extracts (lanes 2 and 4). RNA extracts were also treated in the absence of reverse transcriptase enzyme to verify complete digestion of contaminating DNA (lanes 3 and 4). (C–E) Ferrihydrite (FH), Fe(III)-citrate or Mn(IV)O₂ reduction kinetics of WT, ΔmtrA, ΔmtrB. Error bars represent SDs from three replicates.