SynDLP: a new bacterial dynamin-like protein and its potential involvement in thylakoid membrane biogenesis
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Abstract
Billion years ago, the photosynthetic light reaction was a very successful invention of the nature. Primordial cyanobacteria were the first organism, which were able to cover their energy needs with almost endless resources (water, CO2 and sun light). During the earth history, the photosynthetic system has become a very successful way to provide energy not only for cyanobacteria. Some early eukaryotes engulfed cyanobacteria in an endosymbiotic event that led to the development of algae and plants. However, the photosynthesis light reaction is localized within a special membrane system, the thylakoid membrane. In this thesis, a protein was studied, which is potentially involved in the biogenesis of thylakoid membranes. Dynamin-like proteins (DLPs) from eukaryotes or bacterial DLPs (BDLPs) are mechanochemical large GTPase, which share membrane remodeling or membrane protection properties. They are involved in several processes affecting membranes, like fission, fusion and division of cell organelles as well as viral protection and membrane stress response. With the help of bioinformatic analysis, five potential BDLPs were identified in the genome of the model organism Synechocystis sp. PCC 6803. These five candidates were investigated in order to figure out if they are BDLPs. Moreover, the influence of the phenotype was studied by creating Synechocystis deletion or depletion strains, lacking the corresponding genes. Out of the five candidates only one was confirmed being a BDLP and subsequently be named SynDLP. During this project, expression and purification of this new BDLP was established and it has been shown that SynDLP is an asymmetric disulfide-linked dimer, in vitro. With the purified protein, a continuous, regenerative coupled GTPase assay confirmed the GTPase activity. In addition, membrane interaction studies with Laurdan fluorescence showed, that SynDLP interacts with negatively charged membranes. Moreover, SFG spectroscopy, TEM micrographs and a sedimentation assay revealed a nucleotide depending change in the conformation and in the oligomeric state. In addition, a SynDLP mutant was created, which does not form dimers and was compared to the WT. Besides the in vitro experiments, a Synechocystis deletion mutant was generated, lacking in the SynDLP gene. In vivo analysis suggested that the phenotype is slightly different, especially under high light growth conditions, compared to the WT strain.