Transcriptional analysis of the nirS gene, encoding cytochrome cd1 nitrite reductase, of Paracoccus pantotrophus LMD 92.63.
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 sigma70 DNA sequence motifs can be identified, but a conserved sequence (T-T-GIC-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.
Saunders
Neil F W
NFW
Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK1.
Ferguson
Stuart J
SJ
Oxford Centre for Molecular Sciences, University of Oxford, South Parks Road, Oxford OX1 3QT, UK2.|Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK1.
Baker
Simon C
SC
Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK1.
D001483:Base Sequence; D015152:Blotting, Northern; D003580:Cytochromes; D017931:DNA Primers; D003576:Electron Transport Complex IV / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009572:Nitrite Reductases / Q000235:genetics* / Q000378:metabolism*; D021141:Nucleic Acid Amplification Techniques; D010230:Paracoccus / Q000201:enzymology* / Q000235:genetics*; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic
D003580:Cytochromes; D017931:DNA Primers; D009572:Nitrite Reductases; C028123:nitrite reductase (NO-forming); D003576:Electron Transport Complex IV