Requirement for ubiquinone downstream of cytochrome(s) b in the oxygen-terminated respiratory chains of Escherichia coli K-12 revealed using a null mutant allele of ubiCA.
An Escherichia coli knockout ubiCA mutant has been constructed using a gene replacement method and verified using both Southern hybridization and PCR. The mutant, which was unable to synthesize ubiquinone (Q), showed severely diminished growth yields aerobically but not anaerobically with either nitrate or fumarate as terminal electron acceptors. Low oxygen uptake rates were demonstrated in membrane preparations using either NADH or lactate as substrates. However, these rates were greatly stimulated by the addition of ubiquinone-1 (Q-1). The rate of electron transfer to those oxidase components observable by photodissociation of their CO complexes was studied at sub-zero temperatures. In the ubiCA mutant, the reduced form of haemoproteins--predominantly cytochrome b595--was reoxidized significantly faster in the presence of oxygen than in a Ubi+ strain, indicating the absence of Q as electron donor. Continuous multiple-wavelength recordings of the oxidoreduction state of cytochrome(s) b during steady-state respiration showed greater reduction in membranes from the ubiCA mutant than in wildtype membranes. A scheme for the respiratory electron-transfer chain in E. coli is proposed, in which Q functions downstream of cytochrome(s) b.
Balle
Britta Sø
BS
Department of Molecular Biology and Biotechnology, Krebs Institute for Biomolecular Research, The University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK.|Division of Life Sciences, King's College London, Campden Hill Road, London W8 7AH, UK.
Poole
Robert K
RK
Department of Molecular Biology and Biotechnology, Krebs Institute for Biomolecular Research, The University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK.
D000332:Aerobiosis; D019883:Alkyl and Aryl Transferases / Q000235:genetics* / Q000378:metabolism; D000483:Alleles; D000693:Anaerobiosis; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D003573:Cytochrome b Group / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics; D004579:Electron Transport; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D005650:Fumarates / Q000378:metabolism; D019344:Lactic Acid / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D009566:Nitrates / Q000378:metabolism; D010084:Oxidation-Reduction; D007652:Oxo-Acid-Lyases / Q000235:genetics* / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D010957:Plasmids; D016133:Polymerase Chain Reaction; D014451:Ubiquinone / Q000235:genetics / Q000378:metabolism*
D003573:Cytochrome b Group; D004269:DNA, Bacterial; D005650:Fumarates; D009566:Nitrates; D009243:NAD; D014451:Ubiquinone; D019344:Lactic Acid; C032005:fumaric acid; D019883:Alkyl and Aryl Transferases; C021386:4-hydroxybenzoate polyprenyltransferase; D007652:Oxo-Acid-Lyases; C075960:chorismate pyruvate lyase; D010100:Oxygen