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Transcriptional analysis of the nirS gene, encoding cytochrome cd₁ nitrite reductase, of Paracoccus pantotrophus LMD 92.63

Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK¹
Oxford Centre for Molecular Sciences, University of Oxford, South Parks Road, Oxford OX1 3QT, UK²

Author for correspondence: Stuart J. Ferguson (Dept of Biochemistry). Tel: +44 1865 275240. Fax: +44 1865 275259. e-mail: ferguson{at}bioch.ox.ac.uk

The gene for cytochrome cd₁ nitrite reductase of Paracoccus pantotrophus, a protein of known crystal structure, is nirS. This gene is shown to be flanked by genes previously recognized in other organisms to encode proteins involved in the control of its transcription (nirI) and the biosynthesis of the d₁ cofactor (nirE). Northern blot analysis has established under anaerobic conditions that a monocistronic transcript is produced from nirS, in contrast to observations with other denitrifying bacteria in which arrangement of flanking genes is different and the messages produced are polycistronic. The lack of a transcript under aerobic conditions argues against a role for cytochrome cd₁ in the previously proposed aerobic denitrification pathway in Pa. pantotrophus. A putative rho-independent transcription termination sequence immediately following nirS, and preceding nirE, can be identified. The independent transcription of nirS and nirE indicates that it should be possible to produce site-directed mutants of nirS borne on a plasmid in a nirS deletion mutant. The transcript start point for nirS has been determined by two complementary techniques, 5'-RACE (Rapid amplification of cDNA 5' ends) and primer extension. It is 29 bp upstream of the AUG of nirS. An anaerobox, which presumably binds Nnr, is centred a further 41·5 bp upstream of the transcript start. No standard ⁷⁰ DNA sequence motifs can be identified, but a conserved sequence (T-T-G/C-C-G/C-G/C) can be found in approximately the same position (-16) upstream of the transcript starts of nirS and nirI, whose products are both involved in the conversion of nitrite to nitric oxide.

Keywords: Paracoccus pantotrophus, nitrite reductase, transcription, denitrification, promoter

Abbreviations: Fnr, fumarate nitrate reduction transcriptional activator; Nnr, nitrate nitrite reduction transcriptional activator; 5'-RACE, rapid amplification of cDNA 5' ends; TSS, transcript start site

The GenBank accession number for the sequence in this paper is U75413.

^a Present address: Department of Microbial Physiology, Faculty of Biology, BioCentrum Amsterdam, Vrije Universiteit, De Boelelaan 1087, NL-1081 HV Amsterdam, the Netherlands.

^b Present address: Oxford Centre for Environmental Biotechnology, NERC Institute of Virology and Environmental Microbiology, Mansfield Road, Oxford OX1 3SR, and Department of Engineering Science, Parks Road, Oxford OX1 3PJ, UK.

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