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Journal of Bacteriology, December 2002, p. 6777-6785, Vol. 184, No. 24
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.24.6777-6785.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Mutant Forms of the Azotobacter vinelandii Transcriptional Activator NifA Resistant to Inhibition by the NifL Regulatory Protein

Francisca Reyes-Ramirez,{dagger} Richard Little, and Ray Dixon*

Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, United Kingdom

Received 8 July 2002/ Accepted 19 September 2002

The Azotobacter vinelandii {sigma}54-dependent transcriptional activator protein NifA is regulated by the NifL protein in response to redox, carbon, and nitrogen status. Under conditions inappropriate for nitrogen fixation, NifL inhibits transcription activation by NifA through the formation of the NifL-NifA protein complex. NifL inhibits the ATPase activity of the central AAA+ domain of NifA required to drive open complex formation by {sigma}54-RNA polymerase and may also inhibit the activator-polymerase interaction. To analyze the mechanism of inhibition in greater detail, we isolated NifA mutants which are resistant to the inhibitory action of NifL. Mutations in both the amino-terminal GAF domain and the catalytic AAA+ domain of NifA were isolated. Several mutants blocked inhibition by NifL in response to both nitrogen and redox status, whereas some of the mutant NifA proteins were apparently able to discriminate between the forms of NifL present under different environmental conditions. One mutant protein, NifA-Y254N, was resistant to NifL under conditions of anaerobic nitrogen excess but was relatively sensitive to NifL under aerobic growth conditions. The properties of the purified mutant protein in vitro were consistent with the in vivo phenotype and indicate that NifA-Y254N is not responsive to the nitrogen signal conveyed by the interaction of NifL with A. vinelandii GlnK but is responsive to the oxidized form of NifL when ADP is present. Our observations suggest that different conformers of NifL may be generated in response to discrete signal transduction events and that both the GAF and AAA+ domains of NifA are involved in the response to NifL.

* Corresponding author. Mailing address: Department of Molecular Microbiology, John Innes Centre, Colney Ln., Norwich NR4 7UH, United Kingdom. Phone: 44 1603-450747. Fax: 44 1603-450778. E-mail: ray.dixon{at}bbsrc.ac.uk.

{dagger} Present address: School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom.

Journal of Bacteriology, December 2002, p. 6777-6785, Vol. 184, No. 24
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.24.6777-6785.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Little, R., Martinez-Argudo, I., Perry, S., Dixon, R. (2007). Role of the H Domain of the Histidine Kinase-like Protein NifL in Signal Transmission. J. Biol. Chem. 282: 13429-13437 [Abstract] [Full Text]  
  • Thummer, R., Klimmek, O., Schmitz, R. A. (2007). Biochemical Studies of Klebsiella pneumoniae NifL Reduction Using Reconstituted Partial Anaerobic Respiratory Chains of Wolinella succinogenes. J. Biol. Chem. 282: 12517-12526 [Abstract] [Full Text]  
  • Martinez-Argudo, I., Little, R., Dixon, R. (2004). A crucial arginine residue is required for a conformational switch in NifL to regulate nitrogen fixation in Azotobacter vinelandii. Proc. Natl. Acad. Sci. USA 101: 16316-16321 [Abstract] [Full Text]  
  • Martinez-Argudo, I., Little, R., Shearer, N., Johnson, P., Dixon, R. (2004). The NifL-NifA System: a Multidomain Transcriptional Regulatory Complex That Integrates Environmental Signals. J. Bacteriol. 186: 601-610 [Full Text]  
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