Study of cytochrome bo function in Vitreoscilla using a cyo(-) knockout mutant.
The bacterium, Vitreoscilla, produces a delta mu(Na+) across its membrane during respiration. A key enzyme for this function is the cytochrome bo terminal oxidase which, when incorporated into synthetic proteoliposomes, pumps Na(+) across the membrane upon the addition of a substrate. A Vitreoscilla cytochrome bo knock out (cyo(-)) mutant was isolated by transposon mutagenesis using pUT-mini-Tn5Cm. The membranes of this mutant lacked the characteristic 416 nm peak and 432 nm trough in CO difference spectra, which are clearly visible in spectra of the Vitreoscilla wild-type, but peaks at 627, 560, and 530 nm in reduced minus oxidized difference spectra indicate that cytochrome bd is still present. The specific NADH oxidase and ubiquinol-1 oxidase activities of the cyo(-) mutant membranes were less than those of Vitreoscilla wild-type and Escherichia coli membranes, and the stimulation of these activities of the mutant and E. coli membranes by 75 mM NaCl was approximately 50% less than that of Vitreoscilla wild-type membranes. The ubiquinol-1 oxidase activity of the cyo(-) mutant membranes was inhibited by 10 mM KCN to a lesser degree than that of the Vitreoscilla wild-type and E. coli membranes (50, 80, and 85%, respectively). This result is also consistent with the cyo(-) mutant membrane fragments containing only the cytochrome bd terminal oxidase, which is known to be less sensitive to KCN. Although the maximum respiration and growth of the cyo(-) mutant were less than those of the wild-type, this mutant is still capable of growing with cytochrome bd alone.
Kim
K J
KJ
Division of Biology, Department of Biological, Chemical, and Physical Sciences, Illinois Institute of Technology, Chicago, IL 60616, USA.
Chi
P Y
PY
Hwang
K W
KW
Stark
B C
BC
Webster
D A
DA
D002455:Cell Division / Q000235:genetics; D002462:Cell Membrane / Q000378:metabolism; D003573:Cytochrome b Group; D003580:Cytochromes / Q000235:genetics* / Q000378:metabolism; D004251:DNA Transposable Elements; D029968:Escherichia coli Proteins; D020868:Gene Silencing; D005818:Genetic Engineering / Q000379:methods; D009097:Multienzyme Complexes / Q000378:metabolism; D009154:Mutation; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D011808:Quinone Reductases / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D019802:Succinic Acid / Q000378:metabolism; D020589:Vitreoscilla / Q000235:genetics* / Q000378:metabolism*
D003573:Cytochrome b Group; D003580:Cytochromes; D004251:DNA Transposable Elements; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; C017168:cytochrome bo, E coli; D012964:Sodium; D019802:Succinic Acid; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; D011808:Quinone Reductases; C030914:ubiquinol-1 oxidase