Cloning and DNA sequencing of the <em> fbc</em> operon encoding the cytochrome <em>bc</em>1 complex from <em>Rhodobacter sphaeroides</em>.

The ubiquinol: cytochrome-c oxidoreductase (cytochrome bc₁ complex) is a central component of the mitochondrial respiratory chain as well as the respiratory and/or photosynthetic systems of numerous prokaryotic organisms. In Rhodobacter sphaeroides, the bc₁ complex has a dual function. When the cells are grown photosynthetically, the bc₁ complex is present in the intracytoplasmic membrane and is a critical component of the cyclic electron transport system. When the cells are grown in the dark in the presence of oxygen, the same bc₁ complex is a necessary component of the cytochrome-c₂-dependent respiratory chain. The fact that the bc₁ complex from R. sphaeroides has been extensively studied, plus the ability to manipulate this organism genetically, makes this an ideal system for using site-directed mutagenesis to address questions relating to the structure and function of the bc₁ complex. In the current work, the cloning and complete sequence of the fbc operon from R. sphaeroides is reported. As in other bacteria, this operon contains three genes, encoding the Rieske 2Fe-2S subunit, the cytochrome b subunit, and the cytochrome c₁ subunit. Recombination techniques were used to delete the entire fbc operon from the chromosome. The resulting strain cannot grow photosynthetically, but can grow aerobically utilizing a quinol oxidase. Photosynthetic growth is restored by providing fbc operon on a plasmid, and the reappearance of the protein subunits and the spectroscopic features due to the bc₁ complex are also demonstrated. Finally, a mutation is introduced within the gene encoding the cytochrome b subunit which is predicted to confer resistance to the inhibitor myxothiazol. It is shown that the resulting strain contains a functional bc₁ complex which, as expected, is resistant to the inhibitor. Hence, this system is suitable for the detailed characterization of the bc₁ complex, combining site-directed mutagenesis with the biochemical and biophysical techniques which have been previously developed for the study of photosynthetic bacteria. [ABSTRACT FROM AUTHOR]