Characterization of an electron conduit between bacteria and the extracellular environment

Abstract

A number of species of Gram-negative bacteria can use insoluble minerals of Fe(III) and Mn(IV) as extracellular respiratory electron acceptors. In some species of Shewanella, deca-heme electron transfer proteins lie at the extracellular face of the outer membrane (OM), where they can interact with insoluble substrates. To reduce extracellular substrates, these redox proteins must be charged by the inner membrane/periplasmic electron transfer system. Here, we present a spectro-potentiometric characterization of a trans-OM icosa-heme complex, MtrCAB, and demonstrate its capacity to move electrons across a lipid bilayer after incorporation into proteoliposomes. We also show that a stable MtrAB subcomplex can assemble in the absence of MtrC; an MtrBC subcomplex is not assembled in the absence of MtrA; and MtrA is only associated to the membrane in cells when MtrB is present. We propose a model for the modular organization of the MtrCAB complex in which MtrC is an extracellular element that mediates electron transfer to extracellular substrates and MtrB is a trans-OM spanning beta-barrel protein that serves as a sheath, within which MtrA and MtrC exchange electrons. We have identified the MtrAB module in a range of bacterial phyla, suggesting that it is widely used in electron exchange with the extracellular environment.

Fig. 1.

SDS/PAGE analysis of Mtr proteins. (A and B) Two replica samples were resolved by 12% (wt/vol) SDS/PAGE and stained for heme-dependent peroxidase activity (A) or with Coomassie blue (B). Lane 1, molecular mass markers (from top of gel: 100, 75, 50, 37.5, 25, and 15 kDa); lane 2, MtrCAB; lane 3, MtrAB. (C) Soluble and insoluble fractions of S. oneidensis WT and ΔmtrB strains probed using MtrA- and MtrB-specific antibodies.

Fig. 2.

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.

Fig. 3.

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.

Fig. 4.

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.

Fig. 5.

MtrAB homologues in a range of phyla and OM electron transport systems.