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Study of cytochrome bo function in Vitreoscilla using a cyo(-) knockout mutant
1Division of Biology, Department of Biological, Chemical, and Physical Sciences, Illinois Institute of Technology, Chicago, IL 60616, USA.
Journal of Biochemistry
|July 6, 2000
Summary
Vitreoscilla bacteria utilize cytochrome bo oxidase for sodium ion transport. A mutant lacking this enzyme still grows using cytochrome bd, demonstrating its essential role in respiration.
Area of Science:
- Microbiology
- Biochemistry
- Molecular Biology
Background:
- Vitreoscilla bacteria generate a sodium ion gradient (delta mu(Na+)) across their membrane during respiration.
- Cytochrome bo terminal oxidase is crucial for this sodium ion pumping function.
Purpose of the Study:
- To investigate the role of cytochrome bo oxidase in Vitreoscilla respiration.
- To characterize a Vitreoscilla cytochrome bo knockout mutant (cyo(-)).
Main Methods:
- Isolation of a Vitreoscilla cyo(-) mutant using transposon mutagenesis (pUT-mini-Tn5Cm).
- Spectroscopic analysis (CO difference spectra, reduced minus oxidized difference spectra) of mutant and wild-type membranes.
- Enzyme activity assays (NADH oxidase, ubiquinol-1 oxidase) and KCN inhibition studies.
Main Results:
- The cyo(-) mutant membranes lacked characteristic cytochrome bo spectral peaks but retained cytochrome bd.
- NADH and ubiquinol-1 oxidase activities were reduced in the cyo(-) mutant, with less NaCl stimulation.
- Cytochrome bd oxidase in the mutant showed lower KCN sensitivity compared to wild-type and E. coli.
Conclusions:
- The cytochrome bo oxidase is essential for optimal sodium ion transport and respiration in Vitreoscilla.
- The cyo(-) mutant demonstrates that Vitreoscilla can grow using only cytochrome bd terminal oxidase.
- Cytochrome bd oxidase is less sensitive to KCN inhibition than cytochrome bo.