A DNA Region Recognized by the Nitric Oxide-Responsive Transcriptional Activator NorR Is Conserved in β- and γ-Proteobacteria

Abstract

The 54 -dependent regulator NorR activates transcription of target genes in response to nitric oxide (NO) or NO-generating agents. In Ralstonia eutropha H16, NorR activates transcription of the dicistronic norAB operon that encodes NorA, a protein of unknown function, and NorB, a nitric oxide reductase. A constitutively activating NorR derivative (NorR), in which the N-terminal signaling domain was replaced by MalE, specifically bound to the norAB upstream region as revealed by gel retardation analysis. Within a 73-bp DNA segment protected by MalE-NorR in a DNase I footprint assay, three conserved inverted repeats, GGT-(N 7 )-ACC (where N is any base), that we consider to be NorR-binding boxes were identified. Mutations altering the spacing or the base sequence of these repeats resulted in an 80 to 90% decrease of transcriptional activation by wildtype NorR. Genome database analyses demonstrate that the GT-(N 7 )-AC core of the inverted repeat is found in several proteobacteria upstream of gene loci encoding proteins of nitric oxide metabolism, including nitric oxide reductase (NorB), flavorubredoxin (NorV), NO dioxygenase (Hmp), and hybrid cluster protein (Hcp).Bacteria have to cope with the harmful effects of free radicals that are produced as metabolic by-products or encountered in their environment. The key species superoxide, hydroxyl radical, and nitric oxide can damage DNA, lipids, and proteins. Consequently, bacteria have evolved sophisticated molecular mechanisms to sense free radicals and to activate protective systems. In Escherichia coli, OxyR and the SoxRS system regulate a number of oxidative stress proteins in response to hydrogen peroxide and superoxide, respectively (13). Both regulatory systems are also activated by nitric oxide (NO) or derived reactive nitrogen species (RNS) (16,22). However, specialized NO detoxification systems that appear to be controlled independently of OxyR and SoxRS exist. Several bacteria and fungi contain flavohemoglobins termed Hmp or Fhp that convert NO to nitrate by acting as NO dioxygenases (19,38). The flavorubredoxin NorV from E. coli catalyzes NO reduction under anaerobic and microaerobic conditions (17,20). Denitrifying bacteria possess nitric oxide reductases (Nor) that convert nitric oxide produced by nitrite reductase (Nir) to nitrous oxide. The ammonia oxidizer Nitrosomonas europaea also contains a Nor (5) and induces biofilm formation upon exposure to NO (44). A subfamily of Nor enzymes that oxidize quinols (qNor) is also found in pathogenic bacteria like Neisseria species (1, 24), the phototrophic nondenitrifier Synechocystis sp. strain PCC6803 (7), and the archaeon Pyrobaculum aerophilum (15).Apparently, RNS-sensing regulatory systems that control expression of NO-detoxifying enzymes in bacteria are diverse. Transcription of the NO dioxygenase gene hmp is derepressed upon inactivation of the oxygen sensor Fnr by RNS in E. coli (9), whereas transcription of hmp is activated by the RNS-

Desulfovibrio gigas Flavodiiron Protein Affords Protection against Nitrosative Stress In Vivo

“…However, two putative consensus sequences for a NorR-binding box [GT-(N 7 )-AC] were found upstream of the rd-roo coding region. According to genome database analyses, this inverted repeat is found in several bacteria upstream of gene loci encoding proteins involved in nitric oxide metabolism (6), which is in agreement with our results regarding the involvement of ROO in NO detoxification in D. gigas.…”

“…Total RNA was treated with DNaseI RNase-free (Roche), and Transcriptor Reverse Transcriptase (Roche) was used to synthesize cDNA with the random primer p(dN) 6 , according to the manufacturer's instructions. To rule out the presence of DNA in the RNA preparation, a control reaction was performed without the RT (no-RT control).…”

Dissimilatory Metabolism of Nitrogen Oxides in Bacteria: Comparative Reconstruction of Transcriptional Networks

“…NorR is a r 54 -dependent transcriptional activator that regulates expression of the NO reductase operons, norVW in E. coli and norAB in R. eutropha [21,22]. NorR binds to three tandem operator regions, inverted repeats with degenerate consensus GT-(N 7 )-AC, which are localized upstream of the r 54 promoter site [23]. By applying the motif detection procedure to the hcp promoter regions from four Vibrionales genomes, we identified two tandem palindromic sites with consensus GATGT-(N 7 )-ACATC (Figure S4).…”

“…Three tandem upstream activator sites with the core consensus GT-(N 7 )-AC were identified as NorR-binding sites observed in both promoter regions. Analysis of the adjacent regions of additional norR orthologs in bacterial genomes revealed similar tandem NorRbinding sites upstream of the norA and norB genes in Ralstonia species, norVW in Salmonella species, hmp in Vibrio cholerae and Pseudomonas aeruginosa, and hcp in V. vulnificus [23].…”

Characterization of the Signaling Domain of the NO-Responsive Regulator NorR from Ralstonia eutropha H16 by Site-Directed Mutagenesis

“…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).…”