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Cloning and DNA sequencing of the fbc operon encoding the cytochrome bc1 complex from Rhodobacter sphaeroides. Characterization of fbc deletion mutants and complementation by a site-specific mutational variant
| Content Provider | Scilit |
|---|---|
| Author | Yun, Chang-Hyon Beci, Rose Crofts, Antony R. Kaplan, Samuel Gennis, Robert B. |
| Copyright Year | 1990 |
| Description | Journal: Journal of Biological Inorganic Chemistry The ubiquinol: cytochrome‐c oxidoreductase (cytochrome $bc_{1}$ 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 $bc_{1}$ complex has a dual function. When the cells are grown photosynthetically, the $bc_{1}$ 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 $bc_{1}$ complex is a necessary component of the $cytochrome‐c_{2}$‐dependent respiratory chain. The fact that the $bc_{1}$ 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 $bc_{1}$ 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 $c_{1}$ 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 $bc_{1}$ 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 $bc_{1}$ complex which, as expected, is resistant to the inhibitor. Hence, this system is suitable for the detailed characterization of the $bc_{1}$ complex, combining site‐directed mutagenesis with the biochemical and biophysical techniques which have been previously developed for the study of photosynthetic bacteria. |
| Related Links | http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1990.tb15633.x/pdf |
| Ending Page | 411 |
| Page Count | 13 |
| Starting Page | 399 |
| ISSN | 09498257 |
| e-ISSN | 14321327 |
| DOI | 10.1111/j.1432-1033.1990.tb15633.x |
| Journal | Journal of Biological Inorganic Chemistry |
| Issue Number | 2 |
| Volume Number | 194 |
| Language | English |
| Publisher | Wiley-Blackwell |
| Publisher Date | 1990-12-01 |
| Access Restriction | Open |
| Subject Keyword | Journal: Journal of Biological Inorganic Chemistry Biotechnology and Microbiology Encoding the Cytochrome Directed Mutagenesis |
| Content Type | Text |
| Resource Type | Article |
| Subject | Biochemistry Inorganic Chemistry |
