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...

Fig. 2. Alignment of the deduced amino acid sequences of the fnr homologues from P. stutzeri and fnr from E. coli. The DnrE product is derived from the second potential start site of translation (see Fig. 5). The alignment was performed with the CLUSTAL 4 programme of the DNASIS 2.5 software. The helix±turn±helix region (HTH) is overlined; the dashed line indicates the dimerization domain; the triangle points to the critical position for dimer stabilization. B, cysteine residues for [4Fe±4S] cluster ligation; X, amino acids for DNA contact; W, RNA polymerase contact.

SummaryPseudomonas stutzeri is a facultative anaerobic bacterium with the capability of denitrification. In searching for regulators that control the expression of this trait in response to oxygen withdrawal, we have found an unprecedented multiplicity of four genes encoding transcription factors of the FNR family. The fnrA gene encodes a genuine FNR-type regulator, which is expressed constitutively and controls the cytochrome cbb 3 -type terminal oxidase (the cco operon), cytochrome c peroxidase (the ccp gene) and the oxygenindependent coproporphyrinogen III oxidase (the hemN gene), in addition to its previously demonstrated role in arginine catabolism (the arc operon). The fnr homologues dnrD, dnrE and dnrS encode regulators of a new subgroup within the FNR family. Their main distinctive feature is the lack of cysteine residues for complexing the [4Fe-4S] centre of redox-active FNRtype regulators. However, they form a phylogenetic lineage separate from the FixK branch of FNR proteins, which also lack this cysteine signature. We have studied the expression of the dnr genes under aerobic, oxygen-limited and denitrifying conditions. DnrD is a key regulator of denitrification by selective activation of the genes for cytochrome cd 1 nitrite reductase and NO reductase. The dnrD gene is part of the 30 kb region carrying denitrification genes of P. stutzeri. Transcrip

Cleavage of chromosomal DNA from Pseudomonas aeruginosa PA0 b y Spel and DpnI has been used together with PFGE and Southern hybridization to establish the map location of the following principal denitrification genes: narGH (encoding the large and small subunits of respiratory nitrate reductase), nirS (cytochrome-cd, nitrite reductase), nirE (uroporphyrinogen-Ill methyltransferase for haem d, biosynthesis), norCB (nitric-oxide reductase complex), nosZ (nitrous-oxide reductase) and nosA (an outer-membrane protein and OprC homologue). The study also included several genes related to anaerobic or microaerophilic metabolism: napA (encoding the catalytic subunit of the periplasmic nitrate reductase), ccoN (catalytic subunit of the cytochrome-cbb, oxidase), hemN (oxygen-independent coproporphyrinogen-Ill oxidase), an fnr-like regulatory gene, and azu and fdxA (electron carriers azurin and ferredoxin, respectively). Genes necessary for denitrif ication are concentrated a t 20 to 36 min on the P. aeruginosa chromosome, where they form three separate loci, the nir-nor, nar and nos gene clusters. Genomic DNA of Pseudomonas stutzeri ZoBell was also subjected to Spel restriction and Southern analysis to assign denitrif ication genes to individual fragments. A homologue of nosA encoding a putative component of the Cu-processing apparatus for nitrous-oxide reductase was identified. In both P. aeruginosa and P, stutzeri there is evidence for the linkage of anr (fnrA) with hemN and ccoN; and for the presence of a napA gene.

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