Methylation protection footprinting of wild type and mutant norR-norV intergenic region constructs with NorRΔGAF. Footprinting reactions contained ³²P-labelled 362-bp DNA fragments from pNPTfusV series plasmids (Table 1) spanning the norR-norV intergenic region 5′ end-labelled at the EcoRI end. Fragments encoded either the wild type promoter (A) or promoter constructs with NorR-binding site mutations at site 1 (B), site 2 (C) or site 3 (D). NorRΔGAF-DNA-binding reactions were treated with dimethyl sulphate. In each case, reactions carried out in the absence of protein are labelled ‘DNA only’. Other reactions contained between 88 and 700 nM NorRΔGAF as indicated. Residues are numbered relative to the norV transcript start site. Protected G residues are marked with lollipops on the left-hand side of each series. Arrows denote enhanced methylation at G residues.

ATPase activity and gel filtration profile of NorRΔGAF. (A) Plot of ATP turnover versus protein concentration, measured at a fixed concentration of ATP as substrate (1 mM). (B) Log of relative ATP hydrolysis rate V (compared to maximum rate Vmax at 47 μM) was plotted against the log of NorRΔGAF concentration. The slope of the linear regression slope was used to determine the Hill coefficient (1.98). (C) Different concentrations of NorRΔGAF were chromatographed at 4°C (upper) or preincubated with 1 mM ATP and chromatographed in the presence of 1 mM ATP at 4°C (lower). Corresponding molecular weight of standard globular proteins were indicated at their elution volume.

The ATPase activity and oligomerisation state of NorRΔGAF in the presence of the 266 bp dsDNA that contains all three enhancer sites. (A) Plot of ATP turnover versus NorRΔGAF concentration in the presence of 266 bp dsDNA, containing all three enhancer sites. Activity curves are also included for comparison of ATPase turnover at low concentrations in the presence and absence of shorter DNA fragments. (B) Gel filtration chromatography of 9 µM NorRΔGAF in complex with 0.75 µM 266 bp dsDNA (a molar ratio of 12 : 1 monomer: DNA) performed at 4°C using a Superose 6 column. The dotted line below the 9.3 ml elution peak represents the fractions analysed by negative-stain electron microscopy.

Negative-stain electron microscopy of NorRΔGAF in complex with 266 bp dsDNA, containing all three enhancer sites. (A) Raw micrographs of NorRΔGAF in complex with 21 bp NorR1 (right panel) or 66 bp NorR123 (left panel) DNA fragments. Scale bar 90 nm. (B) Top, raw micrograph of NorRΔGAF in complex with 266 bp dsDNA, with arrows showing some of the higher order oligomers. Scale bar 90 nm. Bottom, gallery of picked particles. Scale bar 15 nm. (C) Selected class averages of 7–10 particles per class generated from 5000 particles show ring-shaped particles with a diameter of 126 Å. Scale bar 11 nm.

Effect of NorR-binding sites on activation of a norV-lacZ reporter. Strains MC100V, MC101V, MC102V and MC103V (Table 1) were grown under either aerobic or anaerobic conditions and induced with potassium nitrite (4 mM). norV-lacZ expression was then determined by measuring β-galactosidase activity. β-galactosidase activity was minimal in MC1000 lacking a promoter fusion construct.

ATPase and open complex stimulating activities of NorRΔGAF associated with wild type and mutant promoter DNA constructs. (A) Rates of ATP hydrolysis by NorRΔGAF were monitored at 340 nm for 20 min at 37°C (filled bars) after which 5 nM of the wild type norR-norVW fragment was added, and the rates were monitored for a further 20 min at 37°C (empty bars). ATPase activities of NorRΔGAF in the presence of DNA constructs carrying a mutant NorR-binding site were compared with the wild type construct. ATPase activities are expressed as specific activity relative to protein concentration in mmol ATP min⁻¹ mol NorR⁻¹. The bar representation is a sum of the values obtained without and with DNA. (B) Each open complex assay reaction contained 1 nM of a ³²P-labelled DNA fragment encoding either the wild type or mutant norR-norVW intergenic region. All lanes contained the components required for open complex formation except lanes 1, 6, 11 and 16, which contained DNA only and lanes 2, 7, 12 and 17, which contained 115 nM NorRΔGAF but lacked σ⁵⁴. NorRΔGAF concentrations were 115 nM (lanes 3, 8 13, 18), 230 nM (lanes 4, 9, 14, 19) and 460 nM (lanes 5, 10, 15, 20). Free DNA, NorRΔGAF/DNA and open complexes are indicated by the arrows to the left of the figure. (C) Heparin resistant open complex species were quantified using a Fujix BAS 1000 phosphorimager. Bands were quantified by their intensity relative to the open complex band formed with the wild type DNA construct at 460 nM NorRΔGAF, which was assumed to be 100%.

Effect of individual enhancer sites on ATPase activity and oligomerisation states of NorRΔGAF. (A) Comparison of the ATPase activity of NorRΔGAF in the absence (cf. Fig 4A) and presence of NorR1 or NorR123 dsDNA. A molar ratio of 1: 1 and 3: 1 activator monomer: DNA was maintained for complexes formed with NorR1 and NorR123 fragments, respectively. (B) Gel filtration studies of NorRΔGAF (20 μM) in the absence or presence of dsDNA as in A. Complexes were also chromatographed in the presence of 1 mM ATP, with no significant effect observed on the elution profile. (C) left: ATPase activities of 26.5 μM NorRΔGAF and NorR_178–452 AAA+ domain compared. The assays were performed at 37°C, and turnover in min⁻¹ was calculated when [α-³²P]ADP formed was ∼20 % of total radiolabeled nucleotide. Right: the elution profile of NorR AAA+ domain (100 µM) gel filtered through a Superdex 200 column in the presence and absence of 1 mM ATP at 4°C. Standard globular proteins were used for calibration: thyroglobulin (669 kDa), β-amylase (200 kDa), bovine serum albumin (66 kDa) and carbonic anhydrase (29 kDa).

NorRΔGAF has a higher affinity for the 266 bp DNA fragment than for the 66 bp DNA fragment NorR123. NorRΔGAF concentrations between 0 and 1000 nM (as indicated above the gels) were incubated with either the 266 bp DNA fragment (A) or the 66 bp DNA fragment (B). The percentage of fully shifted DNA was quantified using a Fujix BAS 1000 phosphorimager (C).