Yeast two-hybrid studies on interaction of proteins involved in regulation of nitrogen fixation in the phototrophic bacterium Rhodobacter capsulatus

Rhodobacter capsulatus contains two I'll-like proteins, GInB and GInK, 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 PH-like proteins and proteins known to be involved in regulating nitrogen fixation. Analysis of defined protein pairs demonstrated the following interactions: GInB-NtrB, GInB-NifA1, GInB-NifA2, GInB-DraT, GInK-NifA1, GInK-NifA2, and GInK-DraT. These results corroborate earlier genetic data and in addition show that I'll-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 GInB-GInB, GInB-GInK, NifA1-NiIA1, NifA2-NifA2, and NifA1-NifA2, suggesting that fine tuning of the nitrogen fixation process in R. capsulatus may involve the formation of GInB-GInK heterotrimers as well as NifA1-NifA2 heterodimers. In order to identify new proteins that interact with GInB 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 GInB and the Ras-like protein Era as a new protein that interacts with GlnK.