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 hybridizatio n and PCR. The mutant, which was unable to synthesize ubiquinone (Q), showed severely diminished growth yields aerobically but not anaerobic ally with either nitrate or fumarate as terminal electron accepters. L ow oxygen uptake rates were demonstrated in membrane preparations usin g either NADH or lactate as substrates. However, these rates were grea tly stimulated by the addition of ubiquinone-1 (Q-1). The rate of elec tron transfer to those oxidase components observable by photodissociat ion of their CO complexes was studied at sub-zero temperatures. In the ubiCA mutant, the reduced form of haemoproteins predominantly cytochr ome b(595)-was reoxidized significantly faster in the presence of oxyg en than in a Ubi(+) strain, indicating the absence of Q as electron do nor. Continuous multiple-wavelength recordings of the oxidoreduction s tate of cytochrome(s) b during steady-state respiration showed greater reduction in membranes from the ubiCA mutant than in wildtype membran es. A scheme for the respiratory electron-transfer chain in E. coli is proposed, in which Q functions downstream of cytochrome(s) b.