All kingdoms possess a large fraction of RNA-based regulation. We identified several small non-coding regulatory RNAs (ncRNAs) in the human bacterial pathogen Listeria monocytogenes that controlled virulence by a direct RNA:RNA interaction. My group have also identified several 5´-untranslated RNAs (5´-UTRs) known to control expression of their downstream mRNA by a switch mechanism triggered by certain metabolites, specific compartments of the host or by different temperatures.In the suggested project, we will analyze the mechanism by how various RNA-species function on a molecular level by biochemical and genetic approaches. By constructing mutations (deletion and base-substitutions), the role of the regulatory RNAs and their targets during pathogenesis will be pin-pointed using different virulence model organisms. For 5´-UTRs binding specific metabolites, we will add non-metabolic analogs to examine if such molecules can block the function of the 5´-UTRs and hence infection. The core structure of one identified ncRNA will be used as a scaffold to develop an RNA interference system in bacteria.At least one RNA-helicase has been shown to be essential for bacterial motility and growth at 4°C. It is being purified to test its in vitro properties at mRNA targets and at different temperatures. Its in vivo role will be analyzed by genetic techniques.Bacterial resistance against different antibiotics is an increasing problem worldwide. We have identified one pyridine molecule specifically targeting listerial virulence gene expression and its mechanism of action will be revealed by genetic and biochemical techniques. A diffusible, although yet unknown molecule, with bacteriostatic activity was observed and its nature and mechanism will be revealed mainly by biochemical experiments.Our work will give important knowledge of how the bacterium uses RNA to sense its surroundings, but will also identifiy new types of antibacterial agents.