In Ralstonia eutropha H16, two genes, norA and norB, form a dicistronic operon that is controlled by the NO-responsive transcriptional regulator NorR. NorB has been identified as a membrane-bound NO reductase, but the physiological function of NorA is unknown. We found that, in a NorA deletion mutant, the promoter activity of the norAB operon was increased 3-fold, indicating that NorA attenuates activation of NorR. NorA shows limited sequence similarity to the oxygen carrier hemerythrin, which contains a di-iron center. Indeed, optical and EPR spectroscopy of purified NorA revealed the presence of a di-iron center, which binds oxygen in a similar way as hemerythrin. Nitric oxide (NO)2 is an obligate intermediate of denitrification (1, 2), formed by the reduction of nitrite by nitrite reductases and subsequently converted to nitrous oxide by NO reductases. In the denitrifier Ralstonia eutropha H16, formation of the NO reductase NorB is controlled by NorR, an NObinding transcriptional activator (3, 4). The norB gene is cotranscribed with norA that encodes a protein of unknown function. Orthologs of NorA are present in many genomes of proteobacteria and firmicutes (5) and have been annotated as DnrN (Pseudomonas stutzeri), ScdA (Staphylococcus aureus), or YtfE (Escherichia coli and Salmonella enterica). The expression of DnrN, ScdA, and YtfE has been shown to be up-regulated by NO or nitrosating agents (6 -9). The non-denitrifiers S. aureus, E. coli, and S. enterica, however, do not possess the norB gene, which demonstrates that norA-like genes do not necessarily co-occur with norB. The NO-dependent expression of norA and its coexpression with norB in R. eutropha suggest a function for NorA in NO metabolism. However, it was shown previously that a nonpolar in-frame deletion of the norA gene did not affect denitrification of R. eutropha cells in terms of growth, accumulation of nitrite or nitrous oxide, or formation of dinitrogen (4). In this study we report the purification and characterization of NorA from R. eutropha. We show that NorA is an NO-binding di-iron protein that modulates the NOresponsive expression of the norAB operon in denitrifying cells of R. eutropha. EXPERIMENTAL PROCEDURESStrains and Plasmids-Strains and plasmids used in this study are listed in Table 1. E. coli JM109 was used for propagation of plasmids. E. coli S17-1 served as the donor in conjugative transfers. E. coli BL21(DE3) was used for overproduction of NorAhexahistidine fusion protein. Mutant strains of R. eutropha were constructed by a gene replacement strategy (15) using the suicide vector pLO1, and were verified by Southern blot analysis. The NorR, NorA, and NorB proteins referred to in this study are encoded by the norR1, norA1, and norB1 genes located on megaplasmid pHG1 of R. eutropha. A chromosomally encoded paralog nor gene cluster termed norR2A2B2 has not been extensively studied, but mutational analyses indicate that either of the two nor clusters is sufficient for denitrification (4).All R. eutropha strains used in this stu...

In Ralstonia eutropha H16, the nitric oxide (NO)-responsive transcriptional activator NorR controls the expression of a dicistronic operon that encodes a membrane-bound NO reductase, NorB, and a protein of unknown function, NorA. The N-terminal domain (NTD) of NorR is responsible for perception of the signal molecule, nitric oxide. Thirteen out of 29 conserved residues of the NTD were exchanged by site-directed mutagenesis. Replacement of R63, R72, D93, D96, C112, D130, or F137 strongly decreased NorR-dependent promoter activation, while the exchange of Y95 or H110 led to an increase in promoter activity compared to that of the wild type. A purified truncated NorR comprising only the NTD (NorR-NTD) contained one iron atom per molecule and was able to bind NO in the as-isolated state. Based on the iron content of NorR-NTD proteins with single amino acid replacements, residues R72, D93, D96, C112, and D130 are likely candidates for iron ligands. Residues R63, Y95, and H110 appear not to be involved in NO binding but may take part in subsequent steps of the signal transduction mechanism of NorR.The diatomic radical nitric oxide (NO) is an obligate intermediate of bacterial denitrification. NO is produced from nitrite by respiratory nitrite reductase and is converted to nitrous oxide by nitric oxide reductase (30). Also, nondenitrifiers like Escherichia coli K-12 can produce considerable amounts of NO during growth under hypoxic conditions with nitrate (6). Since NO and derived reactive nitrogen oxides act as toxic and mutagenic agents, defense mechanisms must be induced under conditions of nitrosative stress. NO-responsive transcription factors are found in the Fnr/Crp family (e.g., DnrD [31]), the Rrf2 family (e.g., NsrR [2]), and the NtrC/NifA family (e.g., NorR). The last protein has been identified in the betaproteobacterium Ralstonia eutropha H16 (21) and E. coli (15). However, genomic analysis revealed that NorR orthologs are widely distributed among beta-and gammaproteobacteria (22). Like all proteins of the 54 -dependent NtrC/NifA superfamily (25), NorR shows a typical modular structure, including an N-terminal domain (NTD), a central AAAϩ domain, and a helix-turn-helix domain for DNA binding (3,21). In a recent study, it was shown that the NTD of E. coli NorR harbors a mono-iron center that is able to bind NO as a ferrous nitrosyl (10). Those authors presented a model for the activation of NorR which postulates the existence of an inactive form of NorR, where the ATPase activity of the AAAϩ domain is blocked by the NTD. Upon binding of NO, this block is relieved and NorR switches to an active state which is competent to induce promoter activation. This hypothesis is corroborated by the fact that truncated forms of NorR from E. coli and R. eutropha that lack the NTD show signal-independent activation of gene expression (10, 21). To date, neither the iron ligands nor the residues that are involved in the switching mechanism have been identified.Given the high positional identity (45%) between the amino acid ...

Nitric oxide reduction in Ralstonia eutropha H16 is catalysed by the quinol-dependent NO reductase NorB. norB and the adjacent norA form an operon that is controlled by the sigma(54)-dependent transcriptional activator NorR in response to NO. A NorR derivative containing MalE in place of the N-terminal domain binds to a 73 bp region upstream of norA that includes three copies of the putative upstream activator sequence GGT-(N(7))-ACC. Mutations altering individual bases of this sequence resulted in an 80-90% decrease in transcriptional activation by wild-type NorR. Similar motifs are present in several proteobacteria upstream of genes encoding proteins of NO metabolism. The N-terminal domain of NorR contains a GAF module and is hypothesized to interact with a signal molecule. A NorR derivative lacking this domain activates the norAB promoter constitutively. Amino acid exchanges within the GAF module identified a cysteine residue that is essential for promoter activation by NorR. Signal sensing by NorR is negatively modulated by the iron-containing protein NorA.

No abstract

The sigma54-dependent transcriptional regulator NorR of Ralstonia eutropha H16 activates gene expression in response to nitric oxide (NO). The N-terminal domain of NorR is thought to be involved in signal perception. A C112S exchange within this domain abolished promoter activation by the mutated protein, indicating that Cys(112) is essential for the signalling mechanism of NorR. The DNA region recognized by NorR contains three copies of a conserved element termed the NorR-box. Alteration of bases within any of the NorR-boxes resulted in a significant decrease in promoter activation. Therefore all three boxes have to be recognized by NorR to activate its target promoter. NorR controls expression of an operon that encodes a redox-active non-haem-iron protein NorA and an NO reductase NorB. NorA exerts a negative effect on signal-dependent promoter activation by NorR. Optical spectroscopy of purified NorA indicates that the reduced protein can react with NO to form a ferrous nitrosyl adduct. Hence, NO binding by NorA opens up the possibility that NorA and NorR compete for NO in the cytoplasm.