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The importance of RNA in molecular and cell biology has long been underestimated. Besides transmitting genetic information, studies of recent years have revealed crucial tasks of RNA especially in gene regulation. Riboswitches are natural RNA-based genetic switches and known only for ten years. They directly sense small-molecule metabolites and regulate in response the expression of the corresponding metabolic genes. Within recent years, artificial riboswitches have been developed that operate according to user-defined demands. Hence, they represent powerful tools for synthetic biology.
This study focused on the development of engineered catalytic riboswitches for conditional gene expression in eukaryotes. A self-cleaving hammerhead ribozyme was linked to a tetracycline binding aptamer in order to regulate ribozyme cleavage allosterically with tetracycline. By integrating such a hybrid molecule into a gene of interest, mRNA cleavage and thereby gene expression is controllable in a ligand dependent manner. The linking domain between ribozyme and aptamer was randomised. Tetracycline inducible ribozymes were isolated after eleven cycles of in vitro selection (SELEX). 80% of the analysed ribozymes show cleavage that strongly depends on tetracycline. In the presence of 1 μM tetracycline, their cleavage rates are comparable to that of the parental hammerhead ribozyme. In the absence of tetracycline, cleavage rates are inhibited up to 333-fold. The allosteric ribozymes bind tetracycline with similar affinity and specificity as the parental aptamer. Ribozyme cleavage is fully induced within minutes after addition of tetracycline. Interestingly, the isolated linker domains exhibit structural consensus motives rather than consensus sequences.
When transferred to yeast, three switches reduced reporter gene expression by 30 - 60% in the presence of tetracycline; none of them controlled gene expression in mammalian cells. In vitro selected molecules do not necessarily retain their characteristics when applied in a cellular context. Therefore, high throughput screening and selection systems have been developed in mammalian cells. The screening system is based on two fluorescent reporter proteins (GFP and mCherry). 1152 individual constructs of the selected ribozyme pool were tested, but none of them reduced reporter gene expression significantly in the presence of tetracycline. The selection system employs a fusion peptide encoding two selection markers (Hygromycin B phosphotransferase and HSV thymidine kinase) facilitating both negative and positive selection. 6.5 x 104 individual constructs of the selected ribozyme pool are currently under investigation.
Myxobacteria are on order of Gram-negative, soil dwelling bacteria that feature an impressive number of properties: they can glide on solid surfaces by using two different motility motors, subsist by preying on other microorganisms, are often producers of multiple natural products, and upon adverse environmental conditions, they are able to form multicellular structures called “fruiting bodies”. The process, in which these macroscopically visible structures arise from independent single cells, has been the predominant subject of myxobacterial research for many decades. More precisely, researchers have strived for the discovery of genes, proteins and small molecules that act as signals, receivers or modulators of this complex process. In this regard, the species Myxococcus xanthus has evolved into the model organism due to its relatively simple and reliable handling in a laboratory environment. The research underlying this thesis focused on the identification and biosynthesis of lipids that may act as intercellular signaling molecules during the course of fruiting body formation of the myxobacterium Myxococcus xanthus as part of the “E-signal” system. In general, lipids containing branched-chain fatty acids with an uneven number of carbon atoms were found to be important players in this particular process. Nevertheless, their exact roles remain largely unknown as of this day. The first publication that is part of this thesis deals with an aspect that even strengthened the importance of role of iso-branched compounds in myxobacteria: myxobacterial metabolism is able to transform precursors of iso-lipids to isoprenoids. It addresses the question whether isoprenoids in general are important for fruiting body formation. Phenotypic analysis of mutants impaired in the biosynthesis of the central isoprenoid precursor 3-hydroxymethylglutaryl-Coenzyme A (3-HMG-CoA) from acetate and/or branched chain keto acids and their genetic and metabolic complementation clearly showed that isoprenoids are essential for fruiting body formation and confirmed that leucine derived isovalerate is an important source for isoprenoid precursors in myxobacteria. The second, and by far and away most tedious and sophisticated study, addressed the question as to how myxobacteria form fatty acid derived iso-branched ether lipids and to what extent they are important for fruiting body formation and sporulation. In a previous study, those unusual lipids were identified as specific biomarkers for myxobacterial development. No biochemical pathways to ether lipids specific for prokaryotes were known by then. In this study, a putative candidate gene that may be in involved in ether lipid biosynthesis was investigated. A combination of gene disruption and complementation experiments, phenotypic analysis and monitoring of ether lipid formation by means of GC-MS demonstrated its involvement in myxobacterial ether lipid biosynthesis and the importance of these lipids for the developmental process. Heterologous expression and biochemical testing of this gene together with in-silico sequence analysis and docking experiments confirmed the functions of its predicted domains. The discussion section provides an additional suggestion on how the ether bond formation is performed. Furthermore and most importantly, iso-branched ether lipids were found to be essential for sporulation but not for fruiting body formation. In summary, one or several molecules derived from an iso-branched alkylglycerol seem to play a role during sporulation in M. xanthus and a multidomain enzyme unique for myxobacteria is involved in their biosynthesis. The last manuscript addresses the complexity of lipid metabolism in myxobacteria. Prior to this work, there was limited knowledge about the exact composition of the myxobacterial lipidome and no method was available to monitor putative changes in the myxobacterial lipidome down to the single molecular species for studying lipid biosynthesis or regulation. An ultra-performance liquid chromatography coupled with mass spectrometry based method with electrospray ionization (UPLC-ESI-MS) utilizing standard equipment and a water/acetonitrile/isopropanol based eluent system proved to be geared for the construction of lipid profiles for wild type and mutant cells of M. xanthus and to show their differences. Fragmentation spectra based structure elucidation of lipid molecular species resulted in the identification of 99 molecular species comprising glycerophosphoethanolamines, glycerophosphoglycerols, glycerolipids, ceramides and ceramide phosphoinositols. The latter have never been described for any prokaryotes before. Three dimensional plots were created from the relative intensity differences of the single molecular ion species between the different samples to provide an efficient and versatile visualization of the data and enable the researcher to quickly detect differences.