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Abstract

Riboswitches are RNA elements that modulate the expression of genes involved in essential metabolic pathways and occur mainly in the bacterial world. Binding of a specific small ligand to the aptamer domain of the riboswitch typically results in repression of the downstream genes, either by transcriptional termination or inhibition of translation. As riboswitches constitute widespread genetic control elements, they are auspicious novel targets for antibiotic substances. The fact that hardly any new, effective antibiotic groups have been discovered in recent decades poses a significant problem, especially regarding the spread of multi-resistant pathogens, such as those from the so called ESKAPE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species). In the first part of this study, thiamine pyrophosphate (TPP) riboswitches from relevant pathogenic strains were identified using a newly adapted dual-luciferase reporter gene assay. TPP riboswitches from the ESKAPE pathogens Enterobacter spp., A. baumannii, P. aeruginosa, K. pneumoniae and E. faecium as well as from the pathogens Streptococcus pneumoniae and Mammaliicoccus sciuri were experimentally validated. The experimental outcome shows that the functionality of some of these riboswitches is dependent on the upstream promoter region. In addition to the affirmation of their function as regulators of gene expression, the riboswitches were characterized by their mode of action using translational and transcriptional reporter gene fusions. The thiE TPP riboswitch from M. sciuri and the thiBPQ riboswitch from Enterobacter spp. exhibit translational regulation, while the thiC riboswitches from P. aeruginosa and Enterobacter spp. mainly operate via termination of transcription. For the thiC riboswitches from A. baumannii, E. faecium and K. pneumoniae a combined function of translation inhibition and transcriptional termination was found. Interestingly, none of the riboswitches examined in this study were affected by the antibiotic pyrithiamine, which is known to block the E. coli thiC riboswitch. The study of the K. pneumoniae thiC riboswitch identified several key nucleotides in the extended P3 stem region of the aptamer that are responsible for the non-responsiveness of the riboswitch to pyrithiamine pyrophosphate. The second chapter of this study addresses the riboswitch-binding protein RibR. This protein was first described in Bacillus subtilis as a regulatory element of flavin mononucleotide (FMN) riboswitches and has two different functional parts. While the N-terminal part has flavokinase activity, the RNA-binding activity could be assigned to the C-terminal part of the protein. Expression of ribR in B. subtilis is induced by the presence of the sulfur sources methionine and taurine. The RibR protein of B. subtilis (RibRsub) counteracts the effect of the ligand FMN on FMN riboswitch activity, allowing expression of the downstream genes involved in riboflavin biosynthesis even in the presence of high FMN levels. In Bacillus amyloliquefaciens, a truncated RibR-variant (RibRamy) has been identified, which only consists of a putative RNA-binding domain. Electrophoretic mobility shift assays showed that RibRamy, just like RibRsub, binds both ribD FMN riboswitch aptamers from B. subtilis and B. amyloliquefaciens. CRISPR-Cas9 genome editing was used to generate B. subtilis ribR mutants expressing different versions of ribRsub and ribRamy. Enzyme assays with cell-free extracts from these strains which allowed monitoring of the FMN riboswitch activity confirmed that RibRamy derepressed the FMN riboswitch even in the presence of FMN. This effect depends on two conserved arginine residues in the primary structures of the B. subtilis and B. amyloliquefaciens RibR proteins as shown by site directed mutagenesis experiments. Further results suggest a link between RibRamy and the response to oxidative stress in B. amyloliquefaciens.