Importance of c-Type cytochromes for U(VI) reduction by Geobacter sulfurreducens

Abstract

Background: In order to study the mechanism of U(VI) reduction, the effect of deleting c-type cytochrome genes on the capacity of Geobacter sulfurreducens to reduce U(VI) with acetate serving as the electron donor was investigated.

Results: The ability of several c-type cytochrome deficient mutants to reduce U(VI) was lower than that of the wild type strain. Elimination of two confirmed outer membrane cytochromes and two putative outer membrane cytochromes significantly decreased (ca. 50-60%) the ability of G. sulfurreducens to reduce U(VI). Involvement in U(VI) reduction did not appear to be a general property of outer membrane cytochromes, as elimination of two other confirmed outer membrane cytochromes, OmcB and OmcC, had very little impact on U(VI) reduction. Among the periplasmic cytochromes, only MacA, proposed to transfer electrons from the inner membrane to the periplasm, appeared to play a significant role in U(VI) reduction. A subpopulation of both wild type and U(VI) reduction-impaired cells, 24-30%, accumulated amorphous uranium in the periplasm. Comparison of uranium-accumulating cells demonstrated a similar amount of periplasmic uranium accumulation in U(VI) reduction-impaired and wild type G. sulfurreducens. Assessment of the ability of the various suspensions to reduce Fe(III) revealed no correlation between the impact of cytochrome deletion on U(VI) reduction and reduction of Fe(III) hydroxide and chelated Fe(III).

Conclusion: This study indicates that c-type cytochromes are involved in U(VI) reduction by Geobacter sulfurreducens. The data provide new evidence for extracellular uranium reduction by G. sulfurreducens but do not rule out the possibility of periplasmic uranium reduction. Occurrence of U(VI) reduction at the cell surface is supported by the significant impact of elimination of outer membrane cytochromes on U(VI) reduction and the lack of correlation between periplasmic uranium accumulation and the capacity for uranium reduction. Periplasmic uranium accumulation may reflect the ability of uranium to penetrate the outer membrane rather than the occurrence of enzymatic U(VI) reduction. Elimination of cytochromes rarely had a similar impact on both Fe(III) and U(VI) reduction, suggesting that there are differences in the routes of electron transfer to U(VI) and Fe(III). Further studies are required to clarify the pathways leading to U(VI) reduction in G. sulfurreducens.

Figure 1

Cell viability in previously published (A) and modified (B) U(VI) reduction assays: 1 – culture; 2 – washed cells suspended in washing buffer; 3 – cells suspended in reaction buffer (t = 0 hours); 4 – t = 1 hour; 5 – t = 2 hours; 6 – t = 3 hours; 7 – t = 4 hours. Data are means of duplicates ± SD

Figure 2

Measurement of U(VI)-reducing activity by resting (A) and killed (B) cells of the strain DL1. Final protein concentration is 0.014 mg/ml; data are means of triplicates ± SD.

Figure 3

Transmission Electron Microscopy of thin sections of the U(VI) reducing strains DL1 (A and C) and MacA (B and D) before (A, B) and after (C, D) treatment with bicarbonate and air. Bar represents 1 μm

Figure 4

U(VI)-reducing activity of wild type and mutant strains of G. sulfurreducens. Data are means of triplicates ± SD.

Figure 5

Transmission Electron Microscopy of thin sections of U(VI) reducing G. sulfurreducens. A, B and C. Periplasmic uranium accumulation by various strains of G. sulfurreducens: A – wild type (DL1), B – DLMC5 (U(VI) reduction is inhibited 60%), C – DL1-MacA (U(VI)-reduction is inhibited completely). OM – outer membrane, P – periplasm, IM – inner membrane. Bar represents 0.1 μm.

Figure 6

High-resolution TEM (HRTEM) and selected-area electron diffraction (SAED) characterization of uranium-loaded periplasm of the DL1 and DL1-MacA strains. A. representative SAED pattern from the cell wall displaying diffuse diffraction rings. Average scattering distance measured from the first diffraction ring is 2.2 Å; B and C – High magnification bright-field (BF) and dark-field (DF) TEM images of the cell wall of the strain DL1-MacA; D and E – BF and DF TEM images of the cell wall of the strain DL1; F and G – High-resolution TEM image of the cell wall of the strains DL1-Mac-1(F) and DL1 (G), showing amorphous features of the U-bearing cell wall layers. OM – outer membrane, P – periplasm, IM – inner membrane.

Figure 7

X-ray EDS spectra from the cytoplasm (A) and periplasm (B) of a U(VI)-reducing cell of the strain DL1.

Figure 8

Impact of cytochrome deletion on U(VI) and Fe(III) reduction. Data are means of triplicates ± SD.