This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hutchings, M. I.
Right arrow Articles by Spiro, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hutchings, M. I.
Right arrow Articles by Spiro, S.

 Previous Article  |  Next Article 

Journal of Bacteriology, November 2000, p. 6434-6439, Vol. 182, No. 22
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Heterologous NNR-Mediated Nitric Oxide Signaling in Escherichia coli

Matthew I. Hutchings,1 Neil Shearer,1 Sarah Wastell,1 Rob J. M. van Spanning,2 and Stephen Spiro1,*

School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom,1 and Department of Molecular Cell Physiology, Faculty of Biology, BioCentrum Amsterdam, Vrije Universiteit, NL-1081 HV Amsterdam, The Netherlands2

Received 11 July 2000/Accepted 5 September 2000

The transcription factor NNR from Paracoccus denitrificans was expressed in a strain of Escherichia coli carrying a plasmid-borne fusion of the melR promoter to lacZ, with a consensus FNR-binding site 41.5 bp upstream of the transcription start site. This promoter was activated by NNR under anaerobic growth conditions in media containing nitrate, nitrite, or the NO+ donor sodium nitroprusside. Activation by nitrate was abolished by a mutation in the molybdenum cofactor biosynthesis pathway, indicating a requirement for nitrate reductase activity. Activation by nitrate was modulated by the inclusion of reduced hemoglobin in culture media, because of the ability of hemoglobin to sequester nitric oxide and nitrite. The ability of nitrate and nitrite to activate NNR is likely due to the formation of NO (or related species) during nitrate and nitrite respiration. Amino acids potentially involved in NNR activity were replaced by site-directed mutagenesis, and the activities of NNR derivatives were tested in the E. coli reporter system. Substitutions at Cys-103 and Tyr-35 significantly reduced NNR activity but did not abolish the response to reactive nitrogen species. Substitutions at Phe-82 and Tyr-93 severely impaired NNR activity, but the altered proteins retained the ability to repress an FNR-repressible promoter, so these mutations have a "positive control" phenotype. It is suggested that Phe-82 and Tyr-93 identify an activating region of NNR that is involved in an interaction with RNA polymerase. Replacement of Ser-96 with alanine abolished NNR activity, and the protein was undetectable in cell extracts. In contrast, NNR in which Ser-96 was replaced with threonine retained full activity.

* Corresponding author. Mailing address: School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom. Phone: 44 1603 593222. Fax: 44 1603 592250. E-mail: s.spiro{at}uea.ac.uk.

Journal of Bacteriology, November 2000, p. 6434-6439, Vol. 182, No. 22
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Castiglione, N., Rinaldo, S., Giardina, G., Cutruzzola, F. (2009). The transcription factor DNR from Pseudomonas aeruginosa specifically requires nitric oxide and haem for the activation of a target promoter in Escherichia coli. Microbiology 155: 2838-2844 [Abstract] [Full Text]  
  • Hodson, C. T. C., Lewin, A., Hederstedt, L., Le Brun, N. E. (2008). The Active-Site Cysteinyls and Hydrophobic Cavity Residues of ResA Are Important for Cytochrome c Maturation in Bacillus subtilis. J. Bacteriol. 190: 4697-4705 [Abstract] [Full Text]  
  • Bushart, T. J., Roux, S. J. (2007). Conserved Features of Germination and Polarized Cell Growth: A Few Insights from a Pollen-Fern Spore Comparison. ANN BOT (LOND) 99: 9-17 [Abstract] [Full Text]  
  • Lewin, A., Crow, A., Oubrie, A., Le Brun, N. E. (2006). Molecular Basis for Specificity of the Extracytoplasmic Thioredoxin ResA. J. Biol. Chem. 281: 35467-35477 [Abstract] [Full Text]  
  • Hutchings, M. I., Hong, H.-J., Leibovitz, E., Sutcliffe, I. C., Buttner, M. J. (2006). The {sigma}E Cell Envelope Stress Response of Streptomyces coelicolor Is Influenced by a Novel Lipoprotein, CseA.. J. Bacteriol. 188: 7222-7229 [Abstract] [Full Text]  
  • Lee, Y.-Y., Shearer, N., Spiro, S. (2006). Transcription factor NNR from Paracoccus denitrificans is a sensor of both nitric oxide and oxygen: isolation of nnr* alleles encoding effector-independent proteins and evidence for a haem-based sensing mechanism.. Microbiology 152: 1461-1470 [Abstract] [Full Text]  
  • Clark, R. W., Lanz, N. D., Lee, A. J., Kerby, R. L., Roberts, G. P., Burstyn, J. N. (2006). Unexpected NO-dependent DNA binding by the CooA homolog from Carboxydothermus hydrogenoformans. Proc. Natl. Acad. Sci. USA 103: 891-896 [Abstract] [Full Text]  
  • Mazoch, J., Kunak, M., Kucera, I., van Spanning, R. J. M. (2003). Fine-tuned regulation by oxygen and nitric oxide of the activity of a semi-synthetic FNR-dependent promoter and expression of denitrification enzymes in Paracoccus denitrificans. Microbiology 149: 3405-3412 [Abstract] [Full Text]  
  • Corker, H., Poole, R. K. (2003). Nitric Oxide Formation by Escherichia coli: DEPENDENCE ON NITRITE REDUCTASE, THE NO-SENSING REGULATOR Fnr, AND FLAVOHEMOGLOBIN Hmp. J. Biol. Chem. 278: 31584-31592 [Abstract] [Full Text]  
  • Mesa, S., Bedmar, E. J., Chanfon, A., Hennecke, H., Fischer, H.-M. (2003). Bradyrhizobium japonicum NnrR, a Denitrification Regulator, Expands the FixLJ-FixK2 Regulatory Cascade. J. Bacteriol. 185: 3978-3982 [Abstract] [Full Text]  
  • Gardner, A. M., Gessner, C. R., Gardner, P. R. (2003). Regulation of the Nitric Oxide Reduction Operon (norRVW) in Escherichia coli. ROLE OF NorR AND sigma 54 IN THE NITRIC OXIDE STRESS RESPONSE. J. Biol. Chem. 278: 10081-10086 [Abstract] [Full Text]  
  • Van Doorslaer, S., Dewilde, S., Kiger, L., Nistor, S. V., Goovaerts, E., Marden, M. C., Moens, L. (2003). Nitric Oxide Binding Properties of Neuroglobin. A CHARACTERIZATION BY EPR AND FLASH PHOTOLYSIS. J. Biol. Chem. 278: 4919-4925 [Abstract] [Full Text]  
  • Arai, H., Mizutani, M., Igarashi, Y. (2003). Transcriptional regulation of the nos genes for nitrous oxide reductase in Pseudomonas aeruginosa. Microbiology 149: 29-36 [Abstract] [Full Text]  
  • Hutchings, M. I., Mandhana, N., Spiro, S. (2002). The NorR Protein of Escherichia coli Activates Expression of the Flavorubredoxin Gene norV in Response to Reactive Nitrogen Species. J. Bacteriol. 184: 4640-4643 [Abstract] [Full Text]  
  • Lewin, A. C., Doughty, P. A., Flegg, L., Moore, G. R., Spiro, S. (2002). The ferric uptake regulator of Pseudomonas aeruginosa has no essential cysteine residues and does not contain a structural zinc ion. Microbiology 148: 2449-2456 [Abstract] [Full Text]  
  • Busch, A., Friedrich, B., Cramm, R. (2002). Characterization of the norB Gene, Encoding Nitric Oxide Reductase, in the Nondenitrifying Cyanobacterium Synechocystis sp. Strain PCC6803. Appl. Environ. Microbiol. 68: 668-672 [Abstract] [Full Text]  
  • Hutchings, M. I., Crack, J. C., Shearer, N., Thompson, B. J., Thomson, A. J., Spiro, S. (2002). Transcription Factor FnrP from Paracoccus denitrificans Contains an Iron-Sulfur Cluster and Is Activated by Anoxia: Identification of Essential Cysteine Residues. J. Bacteriol. 184: 503-508 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»