The high-molecular-weight cytochrome c Cyc2 of Acidithiobacillus ferrooxidans is an outer membrane protein

Abstract

A high-molecular-weight c-type cytochrome, Cyc2, and a putative 22-kDa c-type cytochrome were detected in the membrane fraction released during spheroplast formation from Acidithiobacillus ferrooxidans. This fraction was enriched in outer membrane components and devoid of cytoplasmic membrane markers. The genetics, as well as the subcellular localization of Cyc2 at the outer membrane level, therefore make it a prime candidate for the initial electron acceptor in the respiratory pathway between ferrous iron and oxygen.

FIG. 1.

Characterization of membrane fractions from A. ferrooxidans ATCC33020 by SDS-15% PAGE (27) (A and C) and by Western immunoblotting (B). Total membranes (lanes 1) and OMs (lanes 2) were prepared from A. ferrooxidans cells grown in iron medium (3) according to the method of Mizuno and Kageyama (31). In brief, cells were pelleted, washed three times with basal salts, and resuspended in 3 ml of 20% sucrose at 4°C. Spheroplasts were obtained by adding sequentially 1.5 ml of 2 M sucrose, 1.7 ml of 0.1 M Tris-HCl (pH 8.0), 0.13 ml of 1% EDTA, 0.15 ml of 10 mg of lysozyme ml⁻¹, and 10 μg of DNase ml⁻¹. The mixture was incubated at 30°C for 1 h with occasional shaking. The spheroplasts were removed by centrifugation at 15,000 × g for 15 min; OMs were recovered from the supernatant by centrifugation at 100,000 × g for 90 min at 4°C and then resuspended with distilled water. The remaining spheroplasts were resuspended in 5 mM MgCl₂ and passed three times through a French press. Total membranes were then recovered by centrifugation at 100,000 × g for 90 min at 4°C after removal of the unbroken cells and debris by low-speed centrifugation. After SDS-PAGE, gels were stained for total proteins with Coomassie blue (A) or for hemoproteins with o-dianisidine (C) (13). The positions of Cyc2 and Omp40 are indicated by arrows in panel A. (B) Western immunoblots were performed with antisera raised against Omp40 (21), purified CoxB, and purified Cyc2 (Bonnefoy et al., unpublished) and visualized with a chemiluminescence kit (ECL Western blotting reagents from Amersham Pharmacia Biotech) according to the manufacturer’s instructions. The retarded migration of the bands in the OM fraction in panels A and B is possibly due to a higher content in LPS. The LMW Electrophoresis Calibration kit from Bio-Rad was used as molecular weight markers (MW).

FIG. 2.

Spectrophotometric characterization of membrane fractions of A. ferrooxidans. (A) Room temperature optical spectra in the α, β, and Soret regions. Spectra were obtained as the difference between dithionite-reduced minus H₂O₂-oxidized samples. TM and released OM fractions were suspended at 2.4 and 2.5 mg ml⁻¹, respectively, in 20 mM β-alanine buffer (pH 3.5). (B) Optical spectra recorded at liquid nitrogen temperature (77 K) in the α-band region by using a dual-wavelength DW2000 SLM Aminco spectrophotometer with a slit of 1 nm. Wavelengths were calibrated with a Holmium filter. Spectra were obtained as the difference between dithionite-reduced minus Na₂IrCl₆-oxidized samples. TM and OM fractions were suspended at 1.4 mg ml⁻¹ in 20 mM β-alanine buffer (pH 3.5).

FIG. 3.

Localization of Cyc2 in A. ferrooxidans ATCC 33020 cells in situ by proteinase K digestion of surface proteins (41). Iron-grown bacteria (3) were pelleted and resuspended in phosphate-buffered saline containing 10 mM MgCl₂ at a final concentration of 4 × 10⁹ bacteria ml⁻¹. Proteinase K was added to a final concentration of 100 μg ml⁻¹, and the mixture was incubated for 30, 60, 90, and 120 min at 37°C. Digestion was terminated by the addition of 1 mM phenylmethylsulfonyl fluoride plus Laemmli sample buffer (27). The samples were immediately boiled at 100°C for 5 min and then subjected to SDS-15% PAGE (27). (A) Protein digestion was assessed by immunodetection on Western blots with anti-Cyc2 polyclonal antiserum (Bonnefoy et al., unpublished) and visualized with a chemiluminescence kit (ECL Western blotting reagents from Amersham Pharmacia Biotech) according to the manufacturer’s instructions. Omp40, an OM protein (B), and CoxB, an IM protein (C), were used as positive and negative controls, respectively, and analyzed with the corresponding antisera. Control experiments were performed without any protease added.