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 (N 2 O), and nitrite as signal molecules for the expression of the denitrification apparatus of Pseudomonas 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 a nirF strain, where NO generation from nitrite is prevented because of a defect in heme D 1 biosynthesis. NO-triggered responses were monitored from the nirSTB operon (encoding cytochrome cd 1 nitrite reductase), the norCB operon (encoding NO reductase), nosZ (encoding nitrous oxide reductase), and nosR (encoding a putative regulator). Transcription of nirSTB and norCB was activated by 5 to 50 nM NO, whereas the nosZ promoter required about 250 nM. Nitrite at 5 to 50 nM elicited no response. At a threshold concentration of 650 nM N 2 O, we observed in the anoxic cell the transient appearance of nosZ and nosR transcripts. Constant levels of transcripts of both genes were observed in an anoxic cell sparged with N 2 O. NO at 250 nM stimulated in this cell type the expression of nos genes 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 of dnrD in an engineered strain did not result in NirS synthesis, indicating a need for activation of DnrD. NO modified the transcriptional pattern of the dnrD operon by inducing the transcription of dnrN and dnrO, located upstream of dnrD. Insertional mutagenesis of dnrN altered the kinetic response of the nirSTB operon towards nitrite. Our data establish NO and DnrD as key elements in the regulatory network of denitrification in P. 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 the narGHJI operon.Nitric oxide (NO) is generated and reduced by bacterial denitrification. The NO generator in the denitrifying cell is respiratory nitrite reductase, which is either the tetraheme cytochrome cd 1 nitrite reductase, encoded by the nirS gene, or the Cu-containing nitrite reductase, encoded by the nirK gene (for a review, see reference 54). Although both nitrite reductases exhibit some oxygen reductase activity, there is no evidence that this property would attribute to them a dual function in anaerobic and aerobic respiratory metabolism. The concept of NO as a bacterial signal molecule has its roots in observations of nitrite reductase mutants, which exhibit low levels of NO reduction (18, 38, 52). During genetic studies of heme D 1 biosynthesis, we found that mutagenesis of nir genes other than nirS, irrespective of their encoded functi...

Functional Domains of NosR, a Novel Transmembrane Iron-Sulfur Flavoprotein Necessary for Nitrous Oxide Respiration

“…When the optical density at 660 nm was approximately 0.3, the culture was shifted to denitrifying conditions. Total RNA was extracted by the hot phenol method from cells that were frozen in liquid nitrogen, and Northern blotting was performed as described previously (45). Hybridization and detection were done with a digoxigenin-labeled nosZ probe (47) according to the instructions for the EasyHyb system (Roche Diagnostics).…”

“…Unfortunately, the mechanism of DnrD activation is still obscure. We can sum up the current situation only thus far: nosR transcription acts positively on its own promoter and activates downstream genes without an operon structure (1,13,21,38,45).…”

“…The nos genes of Pseudomonas stutzeri are arranged in three transcriptional units, comprising the monocistronic nosR and nosZ genes and the nosD operon (13,21,45). Tn5 insertions in the 5Ј region of nosR caused a loss of the nosZ transcript, which led to the consideration of NosR as a regulatory component.…”

“…nosR expression, in turn, depends on the transcription factor DnrD of the Crp-Fnr regulator family. DnrD provides a denitrification-specific signal in response to NO (45). A paralogue of nosR, the nirI gene, has been studied for Paracoccus denitrificans.…”

The yjeB ( nsrR ) Gene of Escherichia coli Encodes a Nitric Oxide-Sensitive Transcriptional Regulator

“…The ytfE homologue of Salmonella enterica serovar Typhimurium (designated nipC) was identified in a screen for promoters inducible by acidified nitrite (23). Homologues of ytfE are also NO inducible in the denitrifying organisms Ralstonia eutropha and Pseudomonas stutzeri, which make NO as an intermediate of denitrification (29,44). Despite its widespread nature and conserved expression pattern, the function of the ytfE gene product remains unknown.…”