Extracellular reduction of uranium via Geobacter conductive pili as a protective cellular mechanism

Abstract

The in situ stimulation of Fe(III) oxide reduction by Geobacter bacteria leads to the concomitant precipitation of hexavalent uranium [U(VI)] from groundwater. Despite its promise for the bioremediation of uranium contaminants, the biological mechanism behind this reaction remains elusive. Because Fe(III) oxide reduction requires the expression of Geobacter's conductive pili, we evaluated their contribution to uranium reduction in Geobacter sulfurreducens grown under pili-inducing or noninducing conditions. A pilin-deficient mutant and a genetically complemented strain with reduced outer membrane c-cytochrome content were used as controls. Pili expression significantly enhanced the rate and extent of uranium immobilization per cell and prevented periplasmic mineralization. As a result, pili expression also preserved the vital respiratory activities of the cell envelope and the cell's viability. Uranium preferentially precipitated along the pili and, to a lesser extent, on outer membrane redox-active foci. In contrast, the pilus-defective strains had different degrees of periplasmic mineralization matching well with their outer membrane c-cytochrome content. X-ray absorption spectroscopy analyses demonstrated the extracellular reduction of U(VI) by the pili to mononuclear tetravalent uranium U(IV) complexed by carbon-containing ligands, consistent with a biological reduction. In contrast, the U(IV) in the pilin-deficient mutant cells also required an additional phosphorous ligand, in agreement with the predominantly periplasmic mineralization of uranium observed in this strain. These findings demonstrate a previously unrecognized role for Geobacter conductive pili in the extracellular reduction of uranium, and highlight its essential function as a catalytic and protective cellular mechanism that is of interest for the bioremediation of uranium-contaminated groundwater.

Fig. 1.

Reduction of U(VI) to U(IV) (A) and TEM images of unstained whole cells showing the subcellular localization of the U deposits in the WT_P+ (B), WT_P− (C), PilA⁻ (D), and pRG5::pilA (E) strains. (Scale bar, 0.5 μm.)

Fig. 2.

U L_III-edge EXAFS spectra (symbols) and models (line). (A) Magnitude of Fourier transform spectra are offset for clarity. (B and C) Real part of Fourier transform of WT_P+ (B) and PilA⁻ (C). The components of the model are shown offset beneath the total model. (B and C Insets) U(IV) moiety consistent with the measured EXAFS spectra. U(IV), light gray; O, red; C, black; P, dark gray.

Fig. 3.

Effect of U(VI) exposure on cell vitality (A) and viability (B). (A) Vitality was measured as bacterial reductase (respiratory) activity with the RedoxSensor dye in resting cells of the pili-expressing (WT_P+ and pRG5::pilA) and nonexpressing (WT_P− and PilA⁻) strains and expressed as the ratio of relative fluorescence units emitted by cells incubated with (U⁺) or without (U⁻) U. (B) Growth recovery of resting cells of the pRG5::pilA (black), WT_P+ (dark gray), and WT_P− (light gray) after 6 h of U exposure (circles) in comparison with controls without U (lines).