High-yield purification of cytochrome aa3 and cytochrome caa3 oxidases from Bacillus subtilis plasma membranes

Abstract

When grown in aerated shaking culture, Bacillus subtilis expresses two different haem A-containing terminal oxidases: cytochrome aa3-quinol oxidase and cytochrome caa3 oxidase. This paper describes a high-yield conventional procedure for purifying the two haem A-containing oxidases from the same aerobic culture of Bacillus subtilis. Yields of close to 40% of the total haem A are achieved and about 6 mg of each of the purified oxidases is obtained from 4 litres of liquid culture. Both of the purified enzymes have two subunits, with apparent molecular masses of 71.6 kDa and 34.3 kDa for the cytochrome caa3 oxidase, and 67.6 kDa and 37.2 kDa for aa3-quinol oxidase. These features are in agreement with the sequence data for the corresponding structural genes in the aa3 and caa3 operons of B. subtilis. Some spectral and enzymic features of the two purified oxidases are reported that are consistent with the inclusion of both of these enzymes as members of the cytochrome oxidase superfamily.

Expression, Purification, and Characterization of Recombinant Forms of Membrane-Bound Cytochromec-550nm fromBacillus subtilis

“…The protein concentration was determined (22) and purified protein samples were run on SDS-PAGE (21). Detection of the heme C moiety of cytochrome c on SDS-PAGE was via a peroxidase reaction (23).…”

“…Cultures of strains containing either plasmid were supplemented with 15 g chloramphenicol per milliliter. B. subtilis cultures were harvested and membrane proteins extracted as in (21).…”

Characterization of YpmQ, an Accessory Protein Required for the Expression of Cytochrome c Oxidase in Bacillus subtilis

“…Western Blots with Anti-His Tag and Anti-caa 3 Antibodies-Membrane proteins of B. subtilis were extracted in Triton X-100 as described (22). Extracted proteins were separated by SDS-PAGE (32) and blotted to PVDF membrane using a mini trans-blot cell (Bio-Rad).…”

“…All E. coli strains were grown on LB medium at 37°C; LB agar also contained 1.5% agar (Difco). All B. subtilis strains were grown on super rich medium (22) or tryptose blood agar base (Difco) at 37°C. Antibiotic concentrations for phenotype testing and routine growth were as follows: 100 g/ml ampicillin, 5 g/ml chloramphenicol, 10 g/ml kanamycin, 10 g/ml tetracycline, 7 g/ml Nm, 1 100 g/ml spectinomycin, and 75 g/ml streptomycin.…”

The 3 Terminal Oxidase of

“…The caa 3 -type oxidases are a class of copper-heme oxidases, which, at variance with the aa 3 -type enzymes, include a c heme containing domain. Terminal oxidases of this type have been identified in several microorganisms, such as Thermus thermophilus (1), PS3 (2), Bacillus subtilis (3), Bacillus firmus OF4 (4), and Bacillus FTU (5). A caa 3 -type cytochrome c oxidase has been also identified and purified to homogeneity from the thermophilic bacterium Bacillus stearothermophilus (6).…”