Extracellular electron transfer to Fe(III) oxides by the hyperthermophilic archaeon Geoglobus ahangari via a direct contact mechanism

Abstract

The microbial reduction of Fe(III) plays an important role in the geochemistry of hydrothermal systems, yet it is poorly understood at the mechanistic level. Here we show that the obligate Fe(III)-reducing archaeon Geoglobus ahangari uses a direct-contact mechanism for the reduction of Fe(III) oxides to magnetite at 85°C. Alleviating the need to directly contact the mineral with the addition of a chelator or the electron shuttle anthraquinone-2,6-disulfonate (AQDS) stimulated Fe(III) reduction. In contrast, entrapment of the oxides within alginate beads to prevent cell contact with the electron acceptor prevented Fe(III) reduction and cell growth unless AQDS was provided. Furthermore, filtered culture supernatant fluids had no effect on Fe(III) reduction, ruling out the secretion of an endogenous mediator too large to permeate the alginate beads. Consistent with a direct contact mechanism, electron micrographs showed cells in intimate association with the Fe(III) mineral particles, which once dissolved revealed abundant curled appendages. The cells also produced several heme-containing proteins. Some of them were detected among proteins sheared from the cell's outer surface and were required for the reduction of insoluble Fe(III) oxides but not for the reduction of the soluble electron acceptor Fe(III) citrate. The results thus support a mechanism in which the cells directly attach and transfer electrons to the Fe(III) oxides using redox-active proteins exposed on the cell surface. This strategy confers on G. ahangari a competitive advantage for accessing and reducing Fe(III) oxides under the extreme physical and chemical conditions of hot ecosystems.

Fig 1

Effect of electron shuttles (AQDS) or metal chelators (NTA) on the reduction of Fe(III) oxides by G. ahangari grown at 85°C. (A) Stimulation of Fe(III) oxide reduction [calculated as HCl-extractable Fe(II)] with 50 μM AQDS (squares) and 4 mM NTA (triangles) in reference to cultures with no additions (circles). Controls lacking electron donor (pyruvate, 10 mM) are also shown (open symbols). (Inset) Electron diffraction pattern of the reduced mineral, which is consistent with magnetite. (B) Doubling times [calculated from the rates of HCl-extractable Fe(II) production] as a function of the AQDS concentration present in the medium. The horizontal dashed line marks the doubling time in cultures without AQDS. (C) Soluble Fe(II) and Fe(III) in filtered supernatant fluids from stationary-phase [ca. 17 mM HCl-extractable Fe(II)] cultures with no additions or supplemented with NTA. All data are averages and standard deviations for five replicates.

Fig 2

(A) Growth (dashed lines) and Fe(III) reduction (solid lines) of G. ahangari with entrapped Fe(III) oxides in the presence (squares) or absence (circles) of 50 μM AQDS. Controls lacking electron donor (pyruvate) are also shown (open symbols). The data points represent averages and standard deviations of triplicate cultures. (B) CLSM micrographs of biofilms formed on the Fe(III) oxides exposed on the bead's surface in cultures with or without (inset) AQDS supplementation. Live cells are stained in green and dead cells in red. Bar, 50 μm. (C) Reduction of free Fe(III) oxides (10 mM) by washed cells resuspended in fresh MM medium (open symbols) or in filter-sterilized supernatant fluids (solid symbols) with 10 μM (squares) or without (circles) AQDS.

Fig 3

TEM micrographs of negatively stained cells of G. ahangari from cultures grown with free Fe(III) oxides (A) or Fe(III) citrate (B and C). Partial solubilization of cell-associated iron particles with oxalate reveals abundant filaments, some still associated with the mineral particles (B). At higher magnification, the cell's flagellum (Fl) and curled filaments (Cu) are revealed (C).

Fig 4

(A and B) Coomassie blue-stained (A) and heme-stained (B) SDS-PAGE of whole-cell (lanes 1 to 4, decreasing concentrations of protein) and sheared outer surface (lane 6) proteins extracted from G. ahangari. Lane 5 shows protein markers and numbers at right (panel A) show their relative molecular masses in kilodaltons. Arrows in panel B indicate 4 discrete heme-containing proteins sheared from the outer surface. (C to E) Effect of shearing (sheared, solid symbols; untreated, open symbols) on the reduction of 10 mM Fe(III) oxides (C), production of extracellular appendages by oxalate-treated cells (D), and growth with Fe(III) citrate (E). Axes in panel E are the same as those in panel C.