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Journal of Bacteriology, April 2007, p. 2743-2749, Vol. 189, No. 7
0021-9193/07/$08.00+0     doi:10.1128/JB.01865-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Characterization of the Signaling Domain of the NO-Responsive Regulator NorR from Ralstonia eutropha H16 by Site-Directed Mutagenesis{triangledown}

Andrea Klink, Bettina Elsner, Katja Strube, and Rainer Cramm*

Institut für Biologie/Mikrobiologie, Humboldt-Universität zu Berlin, Berlin, Germany

Received 12 December 2006/ Accepted 23 January 2007

In Ralstonia eutropha H16, the nitric oxide (NO)-responsive transcriptional activator NorR controls the expression of a dicistronic operon that encodes a membrane-bound NO reductase, NorB, and a protein of unknown function, NorA. The N-terminal domain (NTD) of NorR is responsible for perception of the signal molecule, nitric oxide. Thirteen out of 29 conserved residues of the NTD were exchanged by site-directed mutagenesis. Replacement of R63, R72, D93, D96, C112, D130, or F137 strongly decreased NorR-dependent promoter activation, while the exchange of Y95 or H110 led to an increase in promoter activity compared to that of the wild type. A purified truncated NorR comprising only the NTD (NorR-NTD) contained one iron atom per molecule and was able to bind NO in the as-isolated state. Based on the iron content of NorR-NTD proteins with single amino acid replacements, residues R72, D93, D96, C112, and D130 are likely candidates for iron ligands. Residues R63, Y95, and H110 appear not to be involved in NO binding but may take part in subsequent steps of the signal transduction mechanism of NorR.

* Corresponding author. Mailing address: Institut für Biologie/Mikrobiologie, Humboldt-Universität zu Berlin, Chausseestrasse 117, 10115 Berlin, Germany. Phone: (49) 30 2093 8111. Fax: (49) 30 2093 8102. E-mail: rainer.cramm{at}rz.hu-berlin.de.

{triangledown} Published ahead of print on 2 February 2007.

Journal of Bacteriology, April 2007, p. 2743-2749, Vol. 189, No. 7
0021-9193/07/$08.00+0     doi:10.1128/JB.01865-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Tucker, N. P., D'Autreaux, B., Yousafzai, F. K., Fairhurst, S. A., Spiro, S., Dixon, R. (2008). Analysis of the Nitric Oxide-sensing Non-heme Iron Center in the NorR Regulatory Protein. J. Biol. Chem. 283: 908-918 [Abstract] [Full Text]  
  • Strube, K., de Vries, S., Cramm, R. (2007). Formation of a Dinitrosyl Iron Complex by NorA, a Nitric Oxide-binding Di-iron Protein from Ralstonia eutropha H16. J. Biol. Chem. 282: 20292-20300 [Abstract] [Full Text]  


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