Transcriptional regulation of the nos genes for nitrous oxide reductase in Pseudomonas aeruginosa

Arai, Hiroyuki; Mizutani, Masayuki; Igarashi, Yasuo

doi:10.1099/mic.0.25936-0

Cited by 91 publications

(113 citation statements)

References 45 publications

(36 reference statements)

Supporting

Mentioning

105

Contrasting

Order By: Relevance

“…Previous studies, carried out in P. aeruginosa PAO1 with the nirS, the norC and the nosZ promoters fused to the lacZ reporter gene, showed that the DNR transcription factor responds in vivo to N-oxides (Arai et al, 1999(Arai et al, , 2003. A similar response to NO in vivo has been reported for the DNR homologue NNR in Paracoccus denitrificans (Van Spanning et al, 1999) and, for the same regulator, also in E. coli using the FNR-dependent E. coli melR promoter (Hutchings et al, 2000).…”

Section: Introductionsupporting

confidence: 58%

“…In P. aeruginosa the denitrification pathway is regulated by redox signalling, through a cascade of transcription factors; in particular, the global oxygen-sensing regulator ANR (anaerobic regulation of arginine deaminase and nitrate reduction) (Galimand et al, 1991), a homologue of the Escherichia coli oxygen sensor FNR protein, activates, under anaerobic conditions, the gene coding for the transcription factor DNR (dissimilatory nitrate respiration regulator), which, in the presence of N-oxides, promotes the expression of the nir, the nor and the nos genes (Arai et al, 1995(Arai et al, , 1997(Arai et al, , 1999(Arai et al, , 2003 The DNR transcription factor belongs to the CRP/FNR (cAMP receptor protein/fumarate and nitrate reductase regulator) superfamily of regulators (Zumft, 2002;Körner et al, 2003). The members of this superfamily are usually homodimers, each monomer being formed by three domains (McKay & Steitz, 1981): (i) an N-terminal sensing domain (also referred to as the effector domain) with the typical fold of the cAMP-binding domain of CRP; (ii) a long dimerization a-helix recruited to form the dimer interface; and (iii) a C-terminal DNA-binding domain that contains a helix-turn-helix (HTH) motif.…”

Section: Introductionmentioning

confidence: 99%

“…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 cascade of transcription factors; in particular, the global oxygen-sensing regulator ANR (anaerobic regulation of arginine deaminase and nitrate reduction) (Galimand et al, 1991), a homologue of the Escherichia coli oxygen sensor FNR protein, activates, under anaerobic conditions, the gene coding for the transcription factor DNR (dissimilatory nitrate respiration regulator), which, in the presence of N-oxides, promotes the expression of the nir, the nor and the nos genes (Arai et al, 1995, 1997, 1999, 2003 Previous studies, carried out in P. aeruginosa PAO1 with the nirS, the norC and the nosZ promoters fused to the lacZ reporter gene, showed that the DNR transcription factor responds in vivo to N-oxides (Arai et al, 1999(Arai et al, , 2003. A similar response to NO in vivo has been reported for the DNR homologue NNR in Paracoccus denitrificans (Van Spanning et al, 1999) and, for the same regulator, also in E. coli using the FNR-dependent E. coli melR promoter (Hutchings et al, 2000).…”

mentioning

confidence: 99%

See 2 more Smart Citations

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

et al. 2009

View full text Add to dashboard Cite

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...

show abstract

Section: Introductionsupporting

confidence: 58%

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

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

et al. 2009

View full text Add to dashboard Cite

show abstract

“…The operon is under the control of Dnr, which is also a DnrD homologue. Although NosR is not necessary in this organism for nosZ transcription, a disruption of nosR halves the promoter activity of the operon (1). Sequence and topological similarities to NosR were also found in CprC, although this protein has a smaller periplasmic domain and lacks the polyferredoxin signature (40).…”

mentioning

confidence: 88%

“…Unfortunately, the mechanism of DnrD activation is still obscure. We can sum up the current situation only thus far: nosR transcription acts positively on its own promoter and activates downstream genes without an operon structure (1,13,21,38,45).…”

Section: Discussionmentioning

confidence: 99%

Functional Domains of NosR, a Novel Transmembrane Iron-Sulfur Flavoprotein Necessary for Nitrous Oxide Respiration

Wunsch

Zumft

2005

J Bacteriol

View full text Add to dashboard Cite

Bacterial nitrous oxide (N 2 O) respiration depends on the polytopic membrane protein NosR for the expression of N 2 O reductase from the nosZ gene. We constructed His-tagged NosR and purified it from detergentsolubilized membranes of Pseudomonas stutzeri ATCC 14405. NosR is an iron-sulfur flavoprotein with redox centers positioned at opposite sides of the cytoplasmic membrane. The flavin cofactor is presumably bound covalently to an invariant threonine residue of the periplasmic domain. NosR also features conserved CX 3 CP motifs, located C-terminally of the transmembrane helices TM4 and TM6. We genetically manipulated nosR with respect to these different domains and putative functional centers and expressed recombinant derivatives in a nosR null mutant, MK418nosR::Tn5. NosR's function was studied by its effects on N 2 O respiration, NosZ synthesis, and the properties of purified NosZ proteins. Although all recombinant NosR proteins allowed the synthesis of NosZ, a loss of N 2 O respiration was observed upon deletion of most of the periplasmic domain or of the C-terminal parts beyond TM2 or upon modification of the cysteine residues in a highly conserved motif, CGWLCP, following TM4. Nonetheless, NosZ purified from the recombinant NosR background exhibited in vitro catalytic activity. Certain NosR derivatives caused an increase in NosZ of the spectral contribution from a modified catalytic Cu site. In addition to its role in nosZ expression, NosR supports in vivo N 2 O respiration. We also discuss its putative functions in electron donation and redox activation.

show abstract

Assembly ofCuZandCuAin Nitrous Oxide Reductase

Pauleta

Moura

2017

Encyclopedia of Inorganic and Bioinorganic Chemistry

View full text Add to dashboard Cite

Nitrous oxide (N 2 O) reductase is a copper enzyme that catalyzes the reduction of N 2 O to dinitrogen, the last step of the denitrification pathway. This enzyme has two copper centers: a CuA center that is the electron transfer center and the “CuZ center” where the catalysis occurs. This enzyme has been the center of several studies over the last 20 years, and many of its spectroscopic and catalytic properties have been determined, as well as its structure in different oxidation states. These studies have also revealed that the CuA center is similar to the one present in cytochrome c oxidase, being a binuclear copper center, while the CuZ center is unique in biology, being a tetranuclear copper center bridged by a sulfur atom. Moreover, these studies have also identified that the CuZ center can exist in two forms, CuZ*(4Cu1S) and CuZ(4Cu2S). The first has a high turnover number in the fully reduced state, while the CuZ(4Cu2S) form of CuZ center has a very small turnover number and cannot explain the high capacity of the whole cells in reducing N 2 O, from which the enzyme is isolated with CuZ center mainly in that form. This fact envisages an activation mechanism still to be unraveled that might involve one or more enzymes/proteins encoded by the nos genes and a putative sulfur‐displacement mechanism. The biogenesis of these two centers has not been extensively studied, and at least with respect to the copper insertion, it has been postulated that there are alternative routes. The apo‐N 2 OR is synthesized in the cytoplasm in the apo form and is transported to the periplasmic space by either the Sec (unfolded state) or Tat system (dimer folded state), where the centers are assembled. Thus, CuA assembly has been proposed to be dependent on a copper chaperone, pCu A C, and Sco, a thiol‐disulfide reductase that reduces the disulfide bridge between the two cysteines in the apo‐CuA center. Relative to the CuZ center, the delivery of copper atoms is by NosL, an outer‐membrane protein, while the sulfur atoms are transported by the ABC transporter encoded by nosDFY genes. The other accessory proteins NosR and NosX have been assigned broader functions. NosR has been implicated in nosZ expression, as well as in maintaining the activity of N 2 OR, a function that has been shared by NosX. In this chapter the main biochemical properties of N 2 OR, as well as the biogenesis of its two copper centers, will be reviewed.

show abstract

Transcriptional regulation of the nos genes for nitrous oxide reductase in Pseudomonas aeruginosa

Cited by 91 publications

References 45 publications

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

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

Functional Domains of NosR, a Novel Transmembrane Iron-Sulfur Flavoprotein Necessary for Nitrous Oxide Respiration

Assembly ofCuZandCuAin Nitrous Oxide Reductase

Contact Info

Product

Resources

About