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Expression and Content of Terminal Oxidases in Azotobacter Vinelandii Grown with Excess NH₄+ are Modulated by O₂ Supply

Rita D'mello^1,

Diane Purchase^1,

Robert K. Poole^1,,3

¹Division of Life Sciences, King's College London, Campden Hill Road, London W8 7AH, UK
²Nitrogen Fixation Laboratory, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK

ABSTRACT

The influence of the rate of O₂ supply to batch cultures on the contents of cytochromes bd and ‘o’ in NH₄⁺-grown Azotobacter vinelandii has been investigated. Difference spectra at room temperature (reduced + CO minus reduced) were recorded for whole cells of a wild-type strain and mutants which either lacked or over-produced the cytochrome bd-type terminal oxidase encoded by cydAB. A Tn5-B20 insertion in cydB in the former mutant also provided a means of monitoring cydAB gene expression from measurements of β-galactosidase activity. The content of cytochrome d in the wild-type, and the expression of cydAB-lacZ, in the mutant, increased as the O₂ supply was raised, suggesting that O₂ regulates cydAB expression even in the absence of diazotrophy. In a strain carrying a mutation in cydR, a regulatory gene upstream of cydAB, and which over-produces cytochrome bd, the responses to O₂ supply during growth at different O₂ supply rates were reversed. Changes in the content of a haemoprotein detectable in low temperature photodissociation spectra, and attributed to cytochrome b₅₉₅ -the high-spin cytochrome b component of the cytochrome bd complex - followed the changes in cytochrome d levels. CO difference spectra of both the wild-type strain and the cytochrome bd-deficient mutant revealed a haemoprotein with spectral characteristics similar to cytochrome o, the levels of which increased as the O₂ supply was raised. These results are discussed with reference to previous reports of cytochrome changes in cells grown under N₂-fixing conditions.

³Author for correspondence: Robert K. Poole. Tel: + 44 114 282 4447. Fax: +44 114 2^-2 8697.

Present address: The Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, The University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK.

Present address: School of Environmental Science and Engineering, Middlesex University, Bounds Green Road, London N11 2NQ, UK.

Present address: Department of Paediatrics, Imperial College School of Medicine at St Mary's, London W2 1PG, UK.