Dersch

Colonization of the intestinal tract and dissemination to subepithelial tissues by the enteric pathogen Yersinia pseudotuberculosis is initiated by a set of early-stage virulence factors, such as PsaA pili and the cell invasion factor InvA, which are induced by the pivotal virulence regulator RovA. RovA is a dimeric MarR-type transcriptional regulator. Its synthesis is strictly autoregulated by a positive and a negative (threshold valve) feedback loop and it is controlled in response to temperature, growth phase and nutrient composition of the medium. Recently, we could demonstrate that RovA is a protein thermometer harbouring an intrinsic thermosensor. A temperature shift between 25°C and 37°C induces reversible con­formational changes. This reduces the DNA-binding capacity of RovA and renders the regula­tory protein more susceptible to degradation by the Lon protease. Modelling of rovA regulation predicted bimodal expression and analysis using rovA-gfp fusions confirmed phenotypic heterogeneity of rovA expression. As no thermally controlled bistable system has been identified so far, we want to analyze the molecular basis and underlying drivers for temperature-dependent cell-to-cell variation by time-lapse fluorescence microscopy and flow cytometry. This will allow us to follow gene expression at certain temperatures in subpopulations over multiple generations for lineage recon­struction and permits us to address stochastics, bistability and hysteresis of the system. Furthermore, effect of rovA mutants that abolish autoregulation, thermosensing, or proteolytic degradation and influence of nutrient-dependent control mechanisms on bistability of rovA will be investigated. Gained knowledge about the mechanisms determining rovA bistability will be incooperated into the mathe­matical model and used for further model-guided modulation of the system to validate and optimize the mathematical model. As experimental evidence for the benefits of phenotypic variations is limited, a major goal of this project is also to determine whether bistability of the crucial virulence factor contributes to a better overall fitness during infection and improves virulence.