Anaerobic respiration using Fe(3+), S(0), and H(2) in the chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans

Abstract

The chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans has been known as an aerobe that respires on iron and sulfur. Here we show that the bacterium could chemolithoautotrophically grow not only on H(2)/O(2) under aerobic conditions but also on H(2)/Fe(3+), H(2)/S(0), or S(0)/Fe(3+) under anaerobic conditions. Anaerobic respiration using Fe(3+) or S(0) as an electron acceptor and H(2) or S(0) as an electron donor serves as a primary energy source of the bacterium. Anaerobic respiration based on reduction of Fe(3+) induced the bacterium to synthesize significant amounts of a c-type cytochrome that was purified as an acid-stable and soluble 28-kDa monomer. The purified cytochrome in the oxidized form was reduced in the presence of the crude extract, and the reduced cytochrome was reoxidized by Fe(3+). Respiration based on reduction of Fe(3+) coupled to oxidation of a c-type cytochrome may be involved in the primary mechanism of energy production in the bacterium on anaerobic iron respiration.

FIG. 1.

Chemolithoautotrophic growth of A. ferrooxidans on aerobic and anaerobic respiration with H₂ as the electron donor. (A) Time-dependent changes in cell density of strain IFO 14262 aerobically respired in the presence (•) or absence (○) of H₂ and those of JCM 7811 anaerobically respired using Fe³⁺ as an electron acceptor in the presence (▴) or absence (▵) of H₂. (B) Time-dependent changes in the concentrations of total iron (open symbols) and Fe²⁺ (closed symbols) in the presence (○ and •) or absence (▵ and ▴) of bacteria; in this case, strain JCM 7811 anaerobically respired by using Fe³⁺ as an electron acceptor. The datum points are averages of three independent determinations with the standard deviations.

FIG. 2.

Iron-reducing activity of A. ferrooxidans strain JCM 7811 grown on anaerobic iron respiration. (A) Lineweaver-Burk plot of iron-reducing activity of the cells on hydrogen. (B) Effects of respiratory inhibitors on iron-reducing activity of the cells. The rate of iron reduction at 2.0 mM of Fe³⁺ in the presence of HOQNO, hydrazine sulfate, 2,3-dimercapto-1-propanol, or PCMS at 100 μM was expressed as relative activity of control without the inhibitors. The datum points are averages of three independent determinations with the standard deviations.

FIG. 3.

Chemolithoautotrophic growth of A. ferrooxidans strain JCM 7811 on anaerobic S⁰ respiration with H₂ as the electron donor. Shown are time-dependent changes in cell density in the presence of H₂ (•) or N₂ (▴) with S⁰ and in the presence of H₂ without S⁰ (▪). The datum points are averages of three independent determinations with the standard deviations.

FIG. 4.

Expression of a soluble, acid-stable cytochrome in strain JCM 7811 anaerobically cultured with H₂ as the electron donor and Fe³⁺ as the electron acceptor. (A) CBB-stained SDS-polyacrylamide gel. Lanes 1 and 2 were loaded with the respective supernatants from crude extracts of cells grown with Fe²⁺/O₂ and H₂/Fe³⁺; lane 3 shows the cytochrome purified from the anaerobic extract loaded into lane 2. (B) Absorbance spectra of the oxidized (Ox; dashed line) and reduced (Red; solid line) forms of the cytochrome. The inset shows the difference spectrum representing the absolute spectrum of the cytochrome obtained by subtracting the spectrum of the oxidized form from that of the reduced form.

FIG. 5.

Redox response of the cytochrome in cell extract and Fe³⁺. (A) Absorbance spectra of the crude extract of the cells grown on H₂/Fe³⁺(solid line) and the oxidized form of the purified cytochrome (dashed line). (B) Absorbance spectra of the mixture containing the crude extract and the oxidized form of the purified cytochrome with (dashed line) or without (solid line) Fe³⁺.

FIG. 6.

CBB-stained SDS-polyacrylamide gel showing the levels of the cytochrome in crude extracts of cells grown with H₂/O₂ (lane 1), H₂/S⁰ (lane 2), or S⁰/Fe³⁺ (lane 3). The arrow indicates the 28-kDa protein of the cytochrome.