Activation of a consensus FNR-dependent promoter by DNR of Pseudomonas aeruginosa in response to nitrite

Hasegawa, N.

doi:10.1016/s0378-1097(98)00334-6

Cited by 14 publications

(18 citation statements)

References 13 publications

(23 reference statements)

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“…We cannot exclude, however, that other features of the nor promoter may preclude productive interaction between FNR and the E. coli transcriptional machinery. Interestingly, although the E. coli FNR protein seems unable to activate the nor promoter (this work), the DNR protein is able to recognize in P. aeruginosa an FNR-dependent E. coli promoter (Hasegawa et al, 1998), making the scenario more complicated. …”

mentioning

confidence: 81%

“…N-oxide sensors belong to different subgroups of the CRP/ FNR superfamily, namely the DNR, NnrR and FNR subgroups (Rinaldo et al, 2006), which recognize the same consensus sequence in their target promoters, the FNR box (TTGATN 4 ATCAA) (Eiglmeier et al, 1989;Hoeren et al, 1993;Hasegawa et al, 1998); these proteins contain a highly conserved amino acid sequence motif Glu-SerArg (E-SR), directly involved in the interaction with the FNR box . Another transcription factor belonging to the E subgroup of the CRP/FNR superfamily, i.e.…”

Section: Introductionmentioning

confidence: 99%

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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

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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...

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confidence: 81%

Section: Introductionmentioning

confidence: 99%

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

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“…DNR does not carry the cysteine residues and is proposed to sense the existence of N-oxides, especially NO, rather than oxygen limitation (Arai et al, 1995b(Arai et al, , 1999b. The promoters regulated by FNR-like regulators have a sequence similar to the consensus FNRbinding motif (FNR box) (TTGAT----ATCAA), and both ANR and DNR recognize the consensus FNR box (Hasegawa et al, 1998). There are motifs similar to the FNR box in the promoter regions of the nir and nor operons, but the nir and nor promoters are activated only by DNR and not by ANR.…”

Section: Nitrate (Nomentioning

confidence: 99%

“…We have reported that both of the FNR-like regulators of Pseudomonas aeruginosa, ANR and DNR, can activate an artificial FNR-dependent promoter (Hasegawa et al, 1998). It is still unknown how ANR and DNR distinguish their target promoters.…”

Section: Effect Of Dnr On Nosr Promoter Activitymentioning

confidence: 99%

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

2003

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The genes for nitrous oxide (N 2 O) reduction, nosRZDFYL, are clustered on the chromosome of Pseudomonas aeruginosa. Promoter assays using transcriptional fusions to lacZ revealed that the structural gene for nitrous oxide reductase, nosZ, is transcribed with the upstream nosR gene. The nosR gene product is not required for the activity of the nosR promoter. A sequence similar to the consensus FNR-binding motif was found 41?5 bp upstream from the major transcriptional start point of nosR. Mutation of the motif significantly reduced the promoter activity. DNR, an FNR-related transcriptional regulator required for the expression of denitrification genes in P. aeruginosa, is necessary for the transcription of nosR, indicating that the motif is recognized by DNR. Nitrite (NO 2 2 ), nitric oxide (NO) and NO-generating reagents induced nosR promoter activity, but N 2 O did not. The NO 2 2 -induced nosR promoter activity was reduced by mutation of the NO 2 2 reductase gene. However, a low concentration of NO 2 2 induced the promoter activity in a NO reductase mutant. These results indicate that NO is the inducer molecule for transcription of the nos genes.

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“…For example, the secondary regulator Dnr regulates enzymes involved in the stepwise reduction of nitrate to nitrogen gas in the absence of oxygen during denitrification (Arai et al, 1997(Arai et al, , 1999Hasegawa et al, 1998). In the course of this process, nitrite is reduced to PlcH activity frees lipids and the choline-containing head group, which can be catabolized to choline phosphate (not shown), choline, GB and DMG by P. aeruginosa when oxygen is available (Massimelli et al, 2005, Wargo et al, 2008.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2014

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Haemolytic phospholipase C (PlcH) is a potent virulence and colonization factor that is expressed at high levels by Pseudomonas aeruginosa within the mammalian host. The phosphorylcholine liberated from phosphatidylcholine and sphingomyelin by PlcH is further catabolized into molecules that both support growth and further induce plcH expression. We have shown previously that the catabolism of PlcH-released choline leads to increased activity of Anr, a global transcriptional regulator that promotes biofilm formation and virulence. Here, we demonstrated the presence of a negative feedback loop in which Anr repressed plcH transcription and we proposed that this regulation allowed for PlcH levels to be maintained in a way that promotes productive host-pathogen interactions. Evidence for Anr-mediated regulation of PlcH came from data showing that growth at low oxygen (1 %) repressed PlcH abundance and plcH transcription in the WT, and that plcH transcription was enhanced in an Danr mutant. The plcH promoter featured an Anr consensus sequence that was conserved across all P. aeruginosa genomes and mutation of conserved nucleotides within the Anr consensus sequence increased plcH expression under hypoxic conditions. The Anr-regulated transcription factor Dnr was not required for this effect. The loss of Anr was not sufficient to completely derepress plcH transcription as GbdR, a positive regulator of plcH, was required for expression. Overexpression of Anr was sufficient to repress plcH transcription even at 21 % oxygen. Anr repressed plcH expression and phospholipase C activity in a cell culture model for P. aeruginosa-epithelial cell interactions. INTRODUCTIONPseudomonas aeruginosa squanders few opportunities to colonize a vulnerable human host. In addition to engendering a range of nosocomial infections, including keratitis (Green et al., 2008), burn wounds (de Macedo & Santos, 2005), endocarditis (Baddour et al., 2005) and implanted medical devices (Hidron et al., 2008), it also colonizes the lungs of 80 % of persons with the heritable disease cystic fibrosis (CF) (Rajan & Saiman, 2002;Rosenfeld et al., 2003). Colonization by P. aeruginosa contributes to the decline of lung function in this patient population (Rajan & Saiman, 2002), due in part to its wide array of virulence factors that promote its growth and persistence within the host. One such virulence factor is the well-characterized exotoxin haemolytic phospholipase C (PlcH), which is secreted by P. aeruginosa when in association with eukaryotic hosts. PlcH is essential for virulence in co-culture with fungal filaments, on Arabidopsis leaves, in Galleria mellonella larvae and in mammalian hosts (Hogan & Kolter, 2002;Jander et al., 2000;Rahme et al., 2000), highlighting its importance in diverse models of infection.PlcH degrades the abundant phospholipids phosphatidylcholine (PC) and sphingomyelin, which are found in eukaryotic membranes and in lung surfactant (Berka & Vasil, 1982;Vasil, 2006). PC degradation releases both fatty acids and choline-containing c...

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Activation of a consensus FNR-dependent promoter by DNR of Pseudomonas aeruginosa in response to nitrite

Cited by 14 publications

References 13 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

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

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

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