The expression of denitrification by a facultatively anaerobic bacterium requires as exogenous signals a low oxygen tension concomitant with an N oxide. of the FNR-CRP family, was found to be part of the NO-triggered signal transduction pathway. However, overexpression of in an engineered strain did not result in NirS synthesis, indicating a need for activation of DnrD. NO modified the transcriptional pattern of the operon by inducing the transcription 138890-62-7 IC50 of and altered the kinetic response of the operon towards nitrite. Our data establish NO and DnrD as key elements in the regulatory network of denitrification in operon. Nitric oxide (NO) is generated 138890-62-7 IC50 and reduced by bacterial denitrification. The NO generator in the denitrifying cell is respiratory nitrite 138890-62-7 IC50 reductase, which is either the tetraheme cytochrome gene, or the Cu-containing nitrite reductase, encoded by the 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 D1 biosynthesis, we found that mutagenesis of genes other than operon, which codes for the NO reductase complex. The key observation to explain this effect came from interspecies exchange of and gene products, it is possible to express in active form in a NirS? background (24). Expression of active was used in a rescue strategy to relieve the low expression of in a mutant. Since NirK and NirS proteins both generate NO, we proposed NO as an inducer of its own reductase and the existence of an NO-signaling mechanism (38, 55). Studies of the gene of (35, 45) and the gene of (46) have subsequently shown that NO-releasing compounds activate gene expression. Here we have investigated the roles of NO, N2O, and nitrite as signal molecules in the expression of denitrification genes and the interlacing of their regulons with the operon. The denitrification regulator DnrD, a member of the DNR branch of the FNR-CRP family, is necessary for the expression of the and operons in (47). A mutant nicein-150kDa possesses neither nitrite reductase nor NO reductase. We had found a complex transcriptional pattern of the region in response to denitrifying conditions. However, both the cause of the transcriptional pattern and the organization of the underlying operon remained unclear. We show here by direct transcriptional analysis that NO and DnrD fulfill key roles in expressing the nitrite-denitrifying system of and strains used in this work were derivatives of MK21 (56), a spontaneously streptomycin-resistant mutant of strain ATCC 14405. The generation of strains MK220 (strains used for propagation of plasmids were DH10B (Gibco-BRL) and JM110 (51). Vectors used for cloning and sequencing were pBluescript II SK (Stratagene), pUCP22 (49), 138890-62-7 IC50 and pBSL15 (2), with the neomycinphosphotransferase II (were grown on a synthetic, asparagine-citrate-containing (AC) medium at 30C (12). Unless stated otherwise, aerobic and 138890-62-7 IC50 denitrifying cultures were established as previously described (17). For studying mRNA kinetics in response to the addition of an N oxide, the following protocol was used. Aerobically grown cells (gyratory shaker speed set at 240 rpm) were shifted first to a low-oxygen supply (shaker speed reduced to 120 rpm) and incubated for 3 h. Anoxic conditions were then established by transferring the cells into a sealed serum flask under an argon atmosphere for about 30 min before mRNA kinetics were monitored. For anoxic N2O cells, a culture was grown first aerobically to an optical density at 660 nm (OD660) of 0.6. Cells were harvested by centrifugation, suspended with fresh AC medium in a 100-ml flask, and sparged for 3 h with a slow stream of N2O before being challenged with the NO signal. Solute concentrations of NO and N2O were calculated from published values (48). NO was synthesized from acidified nitrite in the presence of Fe(II). In a 100-ml argon-filled and then evacuated gas storage vessel, 5 ml of 1 1 M KNO2 was added slowly from a syringe to 4.5 ml of 1 1 M FeSO4 in 1 M H2SO4. The vessel was equipped with a rubber septum as the gas sampling port. Sodium nitroprusside (SNP) was purchased from Merck (Darmstadt, Germany); was cultured in Luria-Bertani medium at 37C. The following antibiotics were used at the indicated concentrations (in micrograms per milliliter): ampicillin, 100; kanamycin, 50;.