Nitric oxide signaling and transcriptional control of denitrification genes in Pseudomonas stutzeri

The expression of denitrification by a facultatively anaerobic bacterium re quires as exogenous signals a low oxygen tension concomitant with an N oxid e. We have studied the role of nitric oxide (NO), nitrous oxide (N2O), and nitrite as signal molecules for the expression of the denitrification appar atus of Pseudomonas stutzeri, Transcriptional kinetics of structural genes were monitored by Northern blot analysis in a 60-min time frame after cells mere exposed to an N oxide signal. To differentiate the inducer role of NO from that of nitrite, mRNA kinetics were monitored under anoxic conditions in a nirF strain, where NO generation from nitrite is prevented because of a defect in heme D-1 biosynthesis, NO-triggered responses were monitored f rom the nirSTB operon (encoding cytochrome cd(1) nitrite reductase), the no rCB operon (encoding NO reductase), nosZ (encoding nitrous oxide reductase) , and nosR (encoding a putative regulator). Transcription of nirSTB and nor CB was activated by 5 to 50 nM NO, whereas the nosZ promoter required about 250 nM, Nitrite at 5 to 50 nN elicited no response. At a threshold concent ration of 650 nM N2O, we observed in the anoxic cell the transient appearan ce of nosZ and nosR transcripts. Constant levels of transcripts of both gen es were observed in an anoxic cell sparged with N2O, NO at 250 nM stimulate d in this cell type the expression of nos genes severalfold, The transcript ion factor DnrD, a member of the FNR-CRP family, was found to be part of th e NO-triggered signal transduction pathway. However, overexpression of dnrD in an engineered strain did not result in NirS synthesis, indicating a nee d for activation of DnrD, NO modified the transcriptional pattern of the dn rD aperon by inducing the transcription of dnrN and dnrO, located upstream of dnrD, Insertional mutagenesis of dnrN altered the kinetic response of th e nirSTB operon towards nitrite, Our data establish NO and DnrD as key elem ents in the regulatory network of denitrification in P, stutzeri. The NO re sponse adds to the previously identified nitrate-nitrite response mediated by the NarXL two component system for the expression of respiratory nitrate reductase encoded by the narGHJI operon.