The transcription factor DNR from Pseudomonas aeruginosa specifically requires nitric oxide and haem for the activation of a target promoter in Escherichia coli

Abstract

Pseudomonas aeruginosa is a well-known pathogen in chronic respiratory diseases such as cystic fibrosis. Infectivity of P. aeruginosa is related to the ability to grow under oxygen-limited conditions using the anaerobic metabolism of denitrification, in which nitrate is reduced to dinitrogen via nitric oxide (NO). Denitrification is activated by a cascade of redox-sensitive transcription factors, among which is the DNR regulator, sensitive to nitrogen oxides. To gain further insight into the mechanism of NO-sensing by DNR, we have developed an Escherichia coli-based reporter system to investigate different aspects of DNR activity. In E. coli DNR responds to NO, as shown by its ability to transactivate the P. aeruginosa norCB promoter. The direct binding of DNR to the target DNA is required, since mutations in the helix-turn-helix domain of DNR and specific nucleotide substitutions in the consensus sequence of the norCB promoter abolish the transcriptional activity. Using an E. coli strain deficient in haem biosynthesis, we have also confirmed that haem is required in vivo for the NO-dependent DNR activity, in agreement with the property of DNR to bind haem in vitro. Finally, we have shown, we believe for the first time, that DNR is able to discriminate in vivo between different diatomic signal molecules, NO and CO, both ligands of the reduced haem iron in vitro, suggesting that DNR responds specifically to NO. INTRODUCTIONPseudomonas aeruginosa is one of the most important opportunistic pathogens; it is a Gram-negative bacterium which colonizes the inflamed lungs of cystic fibrosis patients, causing persistent infections (Yoon et al., 2006). P. aeruginosa is able to grow in the absence of oxygen, through anaerobic metabolism, which is important for infectivity and for the formation of biofilm (Hassett et al., 2002; Barraud et al., 2006), a surface-associated antibioticresistant microbial community (Singh et al., 2000). The molecular race between host and pathogen thus includes strategies that are centred on the ability of P. aeruginosa to survive under oxygen-limited conditions; cells lying near the edge of the mucous layer rapidly deplete oxygen (O 2 ), creating a gradient where O 2 drops to very low levels. Under these microaerobic conditions P. aeruginosa grows in thick biofilms probably employing both microaerobic respiration and the denitrifying redox chain Alvarez-Ortega & Harwood, 2007;Platt et al., 2008). The complete denitrification pathway involves four enzymes: nitrate reductase, nitrite reductase, nitric oxide reductase (NOR) and nitrous oxide reductase, operating sequentially to reduce nitrate to dinitrogen gas via nitrite (NO { 2 ), nitric oxide (NO) and nitrous oxide (N 2 O) (Zumft, 1997). The expression and the activity of the NIR and NOR enzymes are tightly controlled because it is mandatory for the bacteria to keep the concentration of intracellular NO below cytotoxic levels, to limit nitrosative stress.In P. aeruginosa the denitrification pathway is regulated by redox signalling, through a cas...

Global regulator Anr represses PlcH phospholipase activity in Pseudomonas aeruginosa when oxygen is limiting

“…nitric oxide, which binds to the haem cofactor present on Dnr and induces a conformational change in concert with carbon dioxide, allowing the transcription factor to bind to DNA (Arai et al, 1999;Castiglione et al, 2009;Giardina et al, 2009). Despite the fact that the Anr-and Dnrbinding sites are indistinguishable from one another Rodionov et al, 2005), we did not detect higher plcH levels in the dnr mutant indicating that Dnr was not playing a role in plcH repression under our conditions (Fig.…”

Unusual Heme-Binding PAS Domain from YybT Family Proteins

“…The binding of CO and CN Ϫ resulted in spectra that are consistent with a hexacoordinated iron heme; whereas the characteristic 398-nm Soret band for the Fe(II)-NO complex is indicative of a pentacoordinated heme that often is observed for the NO sensors, such as H-NOX, DNR, and E75 (5,9,18,32). The transition from hexa-to pentacoordinated heme iron upon NO binding suggests that NO triggers the dissociation of the unidentified proximal ligand from the iron center.…”

“…The transition from hexa-to pentacoordinated heme iron upon NO binding suggests that NO triggers the dissociation of the unidentified proximal ligand from the iron center. Such change in coordination number has been reported for NO sensor proteins as well as proteins such as CooA (5,8,34 (5,12,25,26). The other examples include the sensor domain of the Drosophila nuclear receptor E75 and the N-terminal domain of the hemeregulated eukaryotic initiation factor 2␣ kinase (HRI-ND) (18,32,43).…”

Origin and Impact of Nitric Oxide in Pseudomonas aeruginosa Biofilms

“…The expression of the denitrification genes is controlled by the activity of the DNR transcription factor (52), which is also highly expressed under biofilm dispersal conditions (12) and may play a role in this process. DNR is a heme-based gas sensor of the cAMP receptor protein (CRP)-FNR superfamily, which positively responds to NO (48,49,51).…”

“…In this metabolic pathway, nitrate is reduced to NO and then to N 2 in four reaction steps, each catalyzed by a specific reductase ( Fig. 1) whose expression is tightly controlled, mainly by the arginine nitrate regulation (ANR) and dissimilative nitrate respiration regulator (DNR) transcription factors (48)(49)(50)(51)(52)(53). Two putative nitrate reductases are present in P. aeruginosa, i.e., the inner membranebound nitrate reductase NarGHI ( Fig.…”