DNA binding activity of the Escherichia coli nitric oxide sensor NorR suggests a conserved target sequence in diverse proteobacteria

Abstract

The Escherichia coli nitric oxide sensor NorR was shown to bind to the promoter region of the norVW transcription unit, forming at least two distinct complexes detectable by gel retardation. Three binding sites for NorR and two integration host factor binding sites were identified in the norR-norV intergenic region. The derived consensus sequence for NorR binding sites was used to search for novel members of the E. coli NorR regulon and to show that NorR binding sites are partially conserved in other members of the proteobacteria.

FIG. 1.

Binding of NorR to the norR-norV intergenic region. (A) Gel mobility shift assays contained a ³²P-labeled 362-bp EcoRI-BamHI fragment spanning the norR-norV intergenic region. NorR-retarded species are labeled A and B. Concentrations (nanomolar) of NorR were 0 (lane 2), 1 (lane 3), 20 (lane 4), 40 (lane 5), 60 (lane 6), 80 (lane 7), 100 (lane 8), 120 (lane 9), 140 (lane 10), 160 (lane 11), 180 (lane 12), 200 (lane 13), and 500 (lane 14). Lane 1 contained 1 μM IHF; the IHF-bound species is labeled C. A 360-bp fragment containing the nifH promoter (lane 15) was not bound by 500 nM NorR (lane 16). (B) Three independent gel mobility shift experiments (including the example shown in panel A) were quantified with a Fujix BAS 1000 phosphorimager. The total amount of retarded DNA is plotted as a percentage of the radioactivity present in each lane.

FIG. 2.

DNase I footprinting of NorR with the template strand of the norVW promoter. The DNA fragment was the 362-bp EcoRI-BamHI fragment, 5′ end-labeled at the EcoRI site. G+A sequencing tracks prepared with the Maxam and Gilbert method are in lanes 1 and 15. Lane 14 contained no NorR. Binding reactions shown in lanes 3 to 14 contained increasing concentrations of NorR identical to those shown in Fig. 1. Regions of NorR protection were deduced from three independent experiments and are denoted by the solid lines to the right of the footprint, labeled 1 for the high-affinity site and 2 and 3 for the low-affinity sites.

FIG. 3.

Methylation protection of the norR-norV intergenic region by NorR (A) and σ⁵⁴ RNA polymerase (B). Binding reaction mixtures contained the 362-bp EcoRI-BamHI promoter fragment 5′ end-labeled at either the EcoRI end (lanes 1 and 2) or the BamHI end (lanes 3 and 4) and were treated with dimethyl sulfate. In each case, lanes 1 and 3 contained DNA only and lanes 2 and 4 contained either 500 nM NorR (A) or 1 μM σ⁵⁴ plus 1.5 μM RNA polymerase (B). Residues are numbered with respect to the norV transcription start site, designated +1 (see Fig. 4 and 5). Protected G residues are marked with lollipops, and enhanced methylation at G residues is denoted by arrows.

FIG. 4.

Localization of the norVW mRNA start site by primer extension. Anaerobic cultures of E. coli DH10B grown in LB medium supplemented with 0.1% glucose were grown to exponential phase (optical density at 650 nm, 0.45) and were treated with 4 mM NaNO₂ (lane 2), 40 mM NaNO₃ (lane 3), or 100 μM sodium nitroprusside (lane 4). After 20 min, total RNA was extracted and 20 μg was used for each reaction. Lane 1 contains the untreated control. Dideoxy sequencing reactions (lanes G, A, T, and C) were prepared with the same primer. The arrow marks the major norVW transcriptional start site.

FIG. 5.

DNase I footprinting of IHF with the coding strand of the norVW promoter. The DNA fragment was the 362-bp EcoRI-BamHI fragment, 5′ end-labeled at the BamHI site. IHF site 1 (A) and IHF site 2 (B) are denoted by solid lines. G+A sequencing tracks prepared with the Maxam and Gilbert method are in lane 1 in each case. IHF concentrations in binding reaction mixtures (micromolar) were 0 (lanes 2), 1 (lanes 3), 0.1 (lanes 4), 0.03 (lanes 5), and 0.02 (lanes 6).

FIG. 6.

Protein binding sites in the norR-norV intergenic region. (A) The approximate extent of protection by NorR in DNase I footprinting experiments is indicated by underlining. Nucleotides contacted by NorR or σ⁵⁴ RNA polymerase are shaded, those protected from methylation by NorR are marked by filled circles; enhancement of methylation is indicated by open circles. The predicted σ⁵⁴ promoter is boxed. The high-affinity NorR binding site is labeled 1, the low-affinity sites are labeled 2 and 3. The major norV mRNA initiates at a cytidine residue indicated by an arrow. Start codons for norR and norV are shown in bold. IHF binding sites 1 and 2 are labeled and are indicated by wavy lines. (B) Conservation of NorR binding sites among selected bacterial species (Sty, S. enterica serovar Typhimurium; Sfl, S. flexneri; Pae, P. aeruginosa; Reu1, R. eutropha megaplasmid genes; Reu2, R. eutropha chromosomal genes). The three E. coli NorR binding sites are labeled 1, 2, and 3 as in panel A, and norR start codons are shaded. Conserved sequence elements in predicted NorR binding sites and σ⁵⁴ promoters are highlighted. The promoter is located upstream of genes for flavorubredoxin (E. coli, S. enterica serovar Typhimurium, and S. flexneri), flavohemoglobin (P. aeruginosa), and NO reductase (R. eutropha).