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

两种光敏色素Agp1和Agp2控制着土壤细菌蚕豆农杆菌的接合(DNA转移)。虽然细菌光敏色素的三维结构已为人们所知，但它们的信号转导途径仍然不清楚。我们希望通过检测Agp1可能的光诱导蛋白构象变化来分析蚕豆农杆菌从光敏色素到接合信号转导的早期步骤，分析两种光敏色素之间的蛋白质相互作用，并分析光敏色素与松弛酶TraA(或Atu6127)之间可能的相互作用。为了研究蛋白质的相互作用，将采用不同的体外和体内方法相结合。这两种光敏色素之间的体外相互作用已经通过光谱变化、暗回复、磷酸化和荧光阿托染料标记的光敏色素之间的“荧光共振能量转移”(FRET)来显示。我们希望通过包括TraA和Agp1的反应调节因子AgRR1来扩大这些FRET的研究。TRAA催化三个反应：在ORIT(转移起点)序列上划破质粒DNA，与DNA形成共价键，以及解开双链(螺旋酶)。我们希望建立所有功能的体外检测，然后测试Agp1或Agp2是否调节其中一种活动。为了研究体内相互作用，我们希望建立基于分裂YFP的双分子荧光互补作用。我们希望在农杆菌中表达每对带有适当Split-YFP标签的蛋白质(Agp1、Agp2或TraA)之前，首先进行融合蛋白的重组表达和体外测量。通过这种方式，我们可以找出哪些蛋白质在活细胞中相互作用。我们计划通过基因敲除研究来解开农杆菌三种TraA蛋白在接合中的特定作用。我们相信，我们将更好地了解光诱导的光敏色素的构象变化，以及在光调控结合的早期步骤。

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.