Nitric Oxide Signaling and Transcriptional Control of Denitrification Genes inPseudomonas stutzeri

Abstract

The expression of denitrification by a facultatively anaerobic bacterium requires as exogenous signals a low oxygen tension concomitant with an N oxide. We have studied the role of nitric oxide (NO), nitrous oxide (N₂O), and nitrite as signal molecules for the expression of the denitrification apparatus ofPseudomonas stutzeri. Transcriptional kinetics of structural genes were monitored by Northern blot analysis in a 60-min time frame after cells were 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 anirFstrain, where NO generation from nitrite is prevented because of a defect in heme D₁biosynthesis. NO-triggered responses were monitored from thenirSTBoperon (encoding cytochromecd₁nitrite reductase), thenorCBoperon (encoding NO reductase),nosZ(encoding nitrous oxide reductase), andnosR(encoding a putative regulator). Transcription ofnirSTBandnorCBwas activated by 5 to 50 nM NO, whereas thenosZpromoter required about 250 nM. Nitrite at 5 to 50 nM elicited no response. At a threshold concentration of 650 nM N₂O, we observed in the anoxic cell the transient appearance ofnosZandnosRtranscripts. Constant levels of transcripts of both genes were observed in an anoxic cell sparged with N₂O. NO at 250 nM stimulated in this cell type the expression ofnosgenes severalfold. The transcription factor DnrD, a member of the FNR-CRP family, was found to be part of the NO-triggered signal transduction pathway. However, overexpression ofdnrDin an engineered strain did not result in NirS synthesis, indicating a need for activation of DnrD. NO modified the transcriptional pattern of thednrDoperon by inducing the transcription ofdnrNanddnrO, located upstream ofdnrD. Insertional mutagenesis ofdnrNaltered the kinetic response of thenirSTBoperon towards nitrite. Our data establish NO and DnrD as key elements in the regulatory network of denitrification inP. stutzeri. The NO response adds to the previously identified nitrate-nitrite response mediated by the NarXL two-component system for the expression of respiratory nitrate reductase encoded by thenarGHJIoperon.