Light regulation of bacterial conjugation by phytochromes through modulation of protein conformation, protein dynamics and protein-protein interactions in Agrobacterium fabrum

• Applicants: Dr. Norbert Krauß; Professor Dr. Tilman Lamparter
• Subject Area: Biophysics ; Biochemistry ; Plant Biochemistry and Biophysics
• Term: from 2018 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 409240911

Two phytochromes, Agp1 and Agp2, control conjugation (DNA transfer) in the soil bacterium Agrobacterium fabrum. Although much is known about 3D structures of bacterial phytochromes, their signal transduction pathways remain obscure. We want to analyse the early steps of signal transduction from phytochromes to conjugation in Agrobacterium fabrum by testing for possible light induced protein conformational changes of Agp1, analyse protein interaction between both Agrobacterium phytochromes and analyse a possible interaction between phytochromes and the relaxase TraA (or Atu6127). Different in vitro and in vivo methods will be combined in order to study protein interaction. The in vitro interaction between both phytochromes has already been shown by spectral changes, dark reversion, phosphorylation and "fluorescence resonance energy transfer" (FRET) between phytochromes labelled with fluorescent Atto dyes. We want to expand these FRET studies by including TraA and the response regulator of Agp1, AgRR1. TraA catalyses three reactions: nicking of the plasmid DNA at the sequence of oriT (origin of transfer), formation of a covalent bond with the DNA and unwinding of the double strand (helicase). We want to establish in vitro assays for all functions and then test whether Agp1 or Agp2 modulate one of the activities. For studying in vivo interaction we want to establish "bimolecular fluorescence complementation" based on "split YFP". We want to start with recombinant expression of fusion proteins and in vitro measurements before we express pairs of proteins (Agp1, Agp2 or TraA) each with the appropriate split-YFP tag in Agrobacterium. In this way we can find out which of the proteins interact in the living cell. And we plan to disentangle the specific roles of the three TraA proteins of Agrobacterium in conjugation by knockout studies. We believe that we will gain better understanding of light induced conformational changes of a phytochrome and in the early steps of light regulation of conjugation.

• DFG Programme: Research Grants