Yeast two-hybrid studies on interaction of proteins involved in regulation of nitrogen fixation in the phototrophic bacterium Rhodobacter capsulatus.
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.
Pawlowski
Alice
A
Lehrstuhl für Biologie der Mikroorganismen, Fakultät für Biologie, Ruhr-Universität Bochum, D-44780 Bochum, Germany.
Riedel
Kai-Uwe
KU
Klipp
Werner
W
Dreiskemper
Petra
P
Gross
Silke
S
Bierhoff
Holger
H
Drepper
Thomas
T
Masepohl
Bernd
B
D001419:Bacteria / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D008982:Molybdenum / Q000378:metabolism; D009586:Nitrogen Fixation; D009591:Nitrogenase / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D016354:Rhodobacter capsulatus / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D020798:Two-Hybrid System Techniques
D001426:Bacterial Proteins; D000644:Quaternary Ammonium Compounds; D008982:Molybdenum; D009591:Nitrogenase