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Research Paper |
Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín, CSIC, E-18080 Granada, Spain1
Centro de Investigación y Formación Hortícola.E-04700 El Ejido, Almería, Spain2
Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QT, UK3
Author for correspondence: Eulogio J. Bedmar. Tel: +34 958 121011. Fax: +34 958 129600. e-mail: ejbedmar{at}eez.csic.es
The genes norCBQD that encode the bc-type nitric oxide reductase from Bradyrhizobium japonicum USDA110 have been isolated and characterized. norC and norB encode the cytochrome c-containing subunit II and cytochrome b-containing subunit I of nitric oxide reductase, respectively. norQ encodes a protein with an ATP/GTP-binding motif, and the predicted norD gene product shows similarity with NorD from other denitrifiers. Mutational analysis indicates that the two structural norC and norB genes are required for microaerobic growth under nitrate-respiring conditions. A mutant strain lacking a functional norC gene also lacked the 16 kDa c-type cytochrome that is normally detectable by haem-staining of proteins from membranes of microaerobically grown wild-type cells. Expression of a transcriptional fusion of the nor promoter region to the reporter gene lacZ (PnorC–lacZ) was not detected in aerobically grown cells of USDA110, but the fusion was induced threefold when the cells were cultured under microaerobic conditions (1% O2) with either nitrite or nitric oxide, and about 18-fold when nitrate was the N oxide present in the medium. The PnorC–lacZ fusion was not expressed in the B. japonicum fixK2 mutant strain 9043, but complementation of the mutant with the fixK2 gene restored ß-galactosidase activity to levels similar to those found in the parental strain. The promoter region of the norCBQD genes has been characterized by primer extension. A major transcript initiates 45·5 bp downstream of the centre of a putative binding site for the transcription factor FixK2.
Keywords: Rhizobium, nitrate, denitrification, nitric oxide reduction, transcription, regulation
Abbreviations: FNR, fumarate and nitrate reductase regulator; SNP, sodium nitroprusside
b The GenBank accession number for the B. japonicum norCBQD genes reported in this paper is AJ132911.
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