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
B. Soballe et Rk. Poole, 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, Microbiology, 144, 1998, pp. 361-373
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.