Transcriptional analysis of the nirS gene, encoding cytochrome cd(1) nitrite reductase, of Paracoccus pantotrophus LMD 92.63

The gene for cytochrome ed, nitrite reductase of Paracoccus pantotrophus, a protein of known crystal structure, is nirS, This gene is shown to be flan ked by genes previously recognized in other organisms to encode proteins in volved in the control of its transcription (nirI) and the biosynthesis of t he d(1) cofactor (nirE). Northern blot analysis has established under anaer obic conditions that a monocistronic transcript is produced from nirS, in c ontrast to observations with other denitrifying bacteria in which arrangeme nt of flanking genes is different and the messages produced are polycistron ic. The lack of a transcript under aerobic conditions argues against a role for cytochrome ed, in the previously proposed aerobic denitrification path way in Pa, pantotrophus, A putative rho-independent transcription terminati on sequence immediately following nirS, and preceding nirE, can be identifi ed. 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 dete rmined by two complementary techniques, 5'-RACE (Rapid amplification of cDN A 5' ends) and primer extension. It is 29 bp upstream of the AUG of nirS. A n anaerobox, which presumably binds Nnr, is centred a further 41.5 bp upstr eam of the transcript start. No standard sigma(70) DNA sequence motifs can be identified, but a conserved sequence (T-T-G/C-C-C/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 nitri te to nitric oxide.