Cloning and DNA sequencing of the fbc operon encoding the cytochrome bc 1 complex from Rhodobacter sphaeroides Characterization of fbc deletion mutants and complementation by a site‐specific mutational variant

Chang‐Hyon ‐H YUN, Rose BECI, Antony R. CROFTS, Samuel KAPLAN, Robert B Gennis

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)399-411
Number of pages13
JournalEuropean Journal of Biochemistry
Volume194
Issue number2
DOIs
StatePublished - Dec 1990

Fingerprint

Rhodobacter sphaeroides
Cloning
Cytochromes
Operon
DNA Sequence Analysis
Organism Cloning
Mutagenesis
Gene encoding
Cytochromes b
Electron Transport
DNA
Bacteria
Cytochromes c1
Protein Subunits
Chromosomes
Oxidoreductases
Plasmids
Genetic Recombination
Oxygen
Genes

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Cloning and DNA sequencing of the fbc operon encoding the cytochrome bc 1 complex from Rhodobacter sphaeroides Characterization of fbc deletion mutants and complementation by a site‐specific mutational variant",
abstract = "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.",
author = "YUN, {Chang‐Hyon ‐H} and Rose BECI and CROFTS, {Antony R.} and Samuel KAPLAN and Gennis, {Robert B}",
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T1 - Cloning and DNA sequencing of the fbc operon encoding the cytochrome bc 1 complex from Rhodobacter sphaeroides Characterization of fbc deletion mutants and complementation by a site‐specific mutational variant

AU - YUN, Chang‐Hyon ‐H

AU - BECI, Rose

AU - CROFTS, Antony R.

AU - KAPLAN, Samuel

AU - Gennis, Robert B

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