Nitric oxide is an intermediate of denitrification, and is one of the radical species deployed by macrophages against invading pathogens, therefore bacterial responses to NO are of considerable importance. The Escherichia coli flavorubredoxin and its associated oxidoreductase reduce NO to nitrous oxide under anaerobic conditions, and are encoded by the norVW transcription unit. Expression of norVW requires the NO sensing regulatory protein NorR and is dependent on RNA polymerase containing the alternative sigma factor, σ54. We have purified NorR and shown that it binds to three sites in the norVW promoter region, located 75–140 bp upstream of the experimentally verified transcription start site. We have also identified two binding sites for the integration host factor, one between the NorR sites and the σ54-RNA polymerase binding site, and a second downstream of the norVW transcription start site. Comparison of the norVW promoters of enteric bacteria along with known and putative NorR-regulated promoters from Vibrio, Ralstonia and Pseudomonas species suggests that NorR binding sites contain an invariant GT(N7)AC motif flanking an AT-rich central region. The identification of a consensus for NorR binding sites will help to elucidate additional members of the NorR regulon.
- DNA footprinting
- NO sensor
- σ54-dependent activator
In denitrifying bacteria, NO is produced endogenously by the stepwise reduction of nitrate and nitrite . Pathogenic bacteria also encounter NO produced as an antimicrobial defence by phagocytic cells such as macrophages . Escherichia coli is known to have at least two proteins capable of detoxifying NO under different conditions. Flavohaemoglobin (encoded by hmp) is believed to be responsible for the aerobic and micro-aerobic detoxification of NO . Flavorubredoxin and its redox partner encoded by the norVW genes have been reported to have an NO reductase activity under anaerobic and micro-aerobic conditions in E. coli [4–6].
NorR is a σ54-dependent transcriptional activator required for the expression of norVW in response to NO, nitrate, nitrite and nitroprusside under both aerobic and anaerobic conditions [4,6–9]. The norVW and nor genes are divergently transcribed and are separated by a 187 bp intergenic region (Figure 1A). NorR activates transcription under both aerobic and anaerobic conditions, suggesting there may be additional NorR targets, since the flavorubredoxin is only active in the absence of oxygen [6,9].
Similar to other σ54-dependent transcriptional activators, NorR has a modular domain architecture . It contains a central AAA+ (ATPase associated with various cellular activities) domain required for the interaction with σ54 and coupling of ATP hydrolysis to open promoter complex formation , a C-terminal DNA binding domain implicated in DNA sequence recognition and an N-terminal GAF (cGMP specific and stimulated phosphodiesterases, Anabaena adenylate cyclases and Escherichia coli FhlA) domain which is probably responsible for detection of the NO signal [8,12].
Purified NorR binds co-operatively to three sites upstream of the norVW promoter
We overexpressed NorR using the pET21a expression vector in E. coli BL21(DE3) (Novagen, Madison, WI, U.S.A.) and purified the protein by heparin agarose and gel filtration chromatographies. NorR bound co-operatively to multiple sites in the norR/norVW intergenic region in gel retardation assays. DNase I footprinting and methylation protection experiments identified three NorR binding sites located 75–140 bp upstream of the norV transcription start site  (Figure 1B). The norV-proximal site appeared to have a higher affinity for NorR than the other sites suggesting a mechanism for co-operative binding of NorR. From these footprinting experiments, we have been able to deduce a consensus for NorR binding sites, which is GT(N7)AC (Figure 2A). Knowledge of the NorR consensus binding site will assist in the future identification of other members of the NorR regulon. An alignment of known NorR-dependent promoters from diverse bacteria demonstrates that the E. coli NorR binding site is well conserved amongst other proteobacteria (Figure 2B).
Characterization of the σ54-dependent promoter and two IHF (integration host factor) binding sites in the intergenic region
Methylation protection experiments with purified σ54 and core RNA polymerase (Epicentre) have confirmed the predicted binding site for σ54-RNA polymerase holoenzyme in the norVW promoter [10,13]. The holoenzyme protected guanine residues at −24 and −12 (Figure 1B), consistent with the pattern observed with other σ54-dependent promoters and the conserved nature of these residues in the σ54 consensus recognition sequence [14–16]. The norVW transcription start site was identified by primer extension and is located in the expected position downstream of the σ54-RNA polymerase recognition sequence (Figure 1B). Transcription from this promoter is not detectable in untreated cultures, but is activated in cells treated with nitrite or nitroprusside [6,13].
IHF is a small heterodimeric protein capable of bending DNA and consequently affecting transcriptional activation. For example, IHF can induce the bending of DNA to allow an activator bound upstream to interact with downstream–bound σ54-RNA polymerase [17,18]. We identified two IHF binding sites in the norR/norVW intergenic region by DNase I footprinting  (Figure 1B). IHF site 1 is located between the σ54 promoter and the norV-proximal NorR binding site (NorR1) consistent with a role in facilitating an interaction between DNA-bound NorR and σ54-RNA polymerase. IHF site 2 appears to have a lower affinity than site 1 and is located between the norV transcriptional start and the norV start codon, a scenario previously observed with csgD , where it is believed to play a role in oxygen-dependent gene regulation.
The architecture of the norR-norVW intergenic region is complex with three NorR and two IHF binding sites. Owing to the distance between the σ54-dependent norVW promoter and the NorR sites, some bending of the DNA is presumably required to allow contact between NorR and σ54-RNA polymerase. We suggest that IHF site 1 is ideally located to allow this interaction to take place to facilitate transcriptional activation of the norVW promoter. Interestingly, we observed enhanced methylation at guanine residues adjacent to NorR binding sites in methylation protection experiments, suggesting that NorR is capable of inducing some degree of DNA curvature independent of IHF. Although the high affinity NorR 1 site is suitably positioned to act as an upstream activator sequence for norVW, it is possible that co-operative binding of NorR to all three sites is necessary for maximal promoter activation. In addition, NorR sites 2 and 3 may play a role in autogenous regulation of the divergently transcribed norR promoter.
It is not currently known whether NO interacts directly or indirectly with the GAF domain of NorR. One hypothesis is that the GAF domain has an inhibitory effect on the AAA+ domain in the ‘off’ state, and that the reception of the signal (directly or otherwise) releases this inhibition , enabling the mechanochemical function of the AAA+ domain to drive isomerization of σ54-RNA polymerase from the closed to the open promoter complex, thus initiating transcription of norVW.
Identification of the consensus NorR binding site will facilitate the search for other potential members of the NorR regulon in diverse bacteria. Intriguingly, our sequence analysis suggests that NorR activates a σ54-dependent promoter upstream of the hmp gene in Pseudomonas aeruginosa and Vibrio cholerae. This implies that, in some cases, NorR has a role in regulating the expression of other NO detoxifying enzymes.
This work was funded by a BBSRC grant (83/P18536) to R.D. and S.S.
The 10th Nitrogen Cycle Meeting 2004: Focused Meeting held at the University of East Anglia, Norwich, U.K., 2–4 September 2004. Edited by C.S. Butler (Newcastle upon Tyne, U.K.) and D.J. Richardson (Norwich, U.K.). Sponsored by the COST (European Cooperation in the field of Scientific and Technical Research) Office and the ESF (European Science Foundation).
Abbreviations: AAA+, ATPase associated with various cellular activities; GAF, cGMP specific and stimulated phosphodiesterases, Anabaena adenylate cyclases and Escherichia coli FhlA; IHF, integration host factor
- © 2005 The Biochemical Society