Studies of the light-induced signal transduction in Agrobacterium fabrum

Organisms regardless of their origin or type, monitor their environment and respond differently to any environmental changes that occur. These changes not only can alter biochemical reactions that are taking part in the signal transmission, but also the properties of the proteins involved. Therefore, biochemical, or biophysical analyses are a useful approach to understand bacterial cellular mechanisms such as the ones in the bacterium Agrobacterium fabrum (former Agrobacterium tumefaciens) and its phytochrome system. The biliprotein photoreceptors Agp1 and Agp2, found in A. fabrum, exhibit opposite spectral characteristics regarding light-induced and thermal conversion between the red absorbing Pr form and the far-red absorbing Pfr form and could control various cellular mechanisms. These phytochromes have been shown to be able to act together, regulating various mechanisms or processes, such as bacterial conjugation, plant infection and cell growth, in addition to the phosphorylation, and their photoconversion ability. Understanding a signal transduction pathway requires the understanding of the function of proteins, which in turn requires a detailed knowledge of their structure, dynamics, and the different conformational states they can adopt. Therefore, structures studies by X-ray crystallography of the photosensory core modules of various phytochromes were analysed, revealing insights into their conformational changes upon photoconversion. Various hypothesises were already published, on how the structure of phytochromes varies upon photoconversion and in transducing the light signal. Nonetheless, it is still not clear how this signal transduction pathway occurs and how phytochromes are involved in. For this reason, it is aimed in this research to understand the signal transduction pathways of a bacterial phytochrome. These pathways are protein-driven processes, involving photoisomerization, light induced protein conformational changes and possible protein-protein interactions. Within this work, in vitro studies using Agp1 mutants were performed. These mutants were prepared to introduce a single cysteine residue at defined positions, enabling targeted labelling with a maleimide fluorophore. First, the influence of mutations in Agp1 on the autophosphorylation was examined, to determine that these single mutations did not significantly affect the protein function, serving thus as a control experiment. The autophosphorylation results of Agp1 mutants revealed a similar behaviour as for Agp1 wild type, exhibiting a higher autophosphorylation activity in the Pr form than in the Pfr form. Subsequently, these Agp1 mutants were then investigated by time resolved fluorescence anisotropy, to gain insight into the dynamics of the protein. Results have indeed shown that the dynamics of the phytochrome at the corresponding (sub-) domain, changes between the Pr and Pfr form for both the PCM and HK modules, where mainly the dynamics in the Pfr form is lower than in the Pr form. Thus, emphasizing the conformational changes of the phytochrome upon photoconversion that are involved in the transmission of the light signal. Moreover, interaction studies were also carried out via FRET, where Agp1 and Agp2 were labelled with different fluorophores. Results have indeed shown an interaction between both phytochromes, suggesting that the PAS-GAF bidomain of Agp2 interacts with the histidine kinase module of Agp1 and that the FRET efficiency is light-regulated. A different approach for the study of the signal transduction in A. fabrum was realised through in vivo analysis of the bacterial conjugal transfer, since it was proved to be affected by the red light sensitive phytochromes and by their histidine kinases. The conjugation assay in this work revealed the importance of the TraA gene, which is supposed to be the first protein involved in the conjugation cascade of Agp1. Three TraA homologues are presented in A. fabrum and are encoded by the Ti plasmid (Atu6127), the linear chromosome and the At plasmid. Results showed that the knockout of the TraA encoded by the pTi, inhibited the conjugal transfer in A. fabrum and that phytochromes regulate the expression of the other TraA genes encoded by the linear chromosome and the pAt