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Journal of Bacteriology, September 2003, p. 5240-5247, Vol. 185, No. 17
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.17.5240-5247.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Yeast Two-Hybrid Studies on Interaction of Proteins Involved in Regulation of Nitrogen Fixation in the Phototrophic Bacterium Rhodobacter capsulatus

Alice Pawlowski, Kai-Uwe Riedel, Werner Klipp, Petra Dreiskemper, Silke Groß, Holger Bierhoff, Thomas Drepper, and Bernd Masepohl*

Lehrstuhl für Biologie der Mikroorganismen, Fakultät für Biologie, Ruhr-Universität Bochum, D-44780 Bochum, Germany

Received 4 April 2003/ Accepted 11 June 2003

Rhodobacter capsulatus contains two PII-like proteins, GlnB and GlnK, which play central roles in controlling the synthesis and activity of nitrogenase in response to ammonium availability. Here we used the yeast two-hybrid system to probe interactions between these PII-like proteins and proteins known to be involved in regulating nitrogen fixation. Analysis of defined protein pairs demonstrated the following interactions: GlnB-NtrB, GlnB-NifA1, GlnB-NifA2, GlnB-DraT, GlnK-NifA1, GlnK-NifA2, and GlnK-DraT. These results corroborate earlier genetic data and in addition show that PII-dependent ammonium regulation of nitrogen fixation in R. capsulatus does not require additional proteins, like NifL in Klebsiella pneumoniae. In addition, we found interactions for the protein pairs GlnB-GlnB, GlnB-GlnK, NifA1-NifA1, NifA2-NifA2, and NifA1-NifA2, suggesting that fine tuning of the nitrogen fixation process in R. capsulatus may involve the formation of GlnB-GlnK heterotrimers as well as NifA1-NifA2 heterodimers. In order to identify new proteins that interact with GlnB and GlnK, we constructed an R. capsulatus genomic library for use in yeast two-hybrid studies. Screening of this library identified the ATP-dependent helicase PcrA as a new putative protein that interacts with GlnB and the Ras-like protein Era as a new protein that interacts with GlnK.

* Corresponding author. Mailing address: Ruhr-Universität Bochum, Fakultät für Biologie, Lehrstuhl für Biologie der Mikroorganismen, D-44780 Bochum, Germany. Phone: 49 (234) 32 25632. Fax: 49 (234) 32 14620. E-mail: bernd.masepohl{at}ruhr-uni-bochum.de.

Journal of Bacteriology, September 2003, p. 5240-5247, Vol. 185, No. 17
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.17.5240-5247.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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