Underlying principles of bistability in the expression of the pivotal virulence regulator RovA in Yersinia pseudotuberculosis and role for virulence

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In Yersinia pseudotuberculosis, important virulence-related traits are under the control of the transcriptional regulator RovA, which uses a built-in thermosensor to control its activity. Thermal shifts between 25°C and 37°C, encountered upon host entry and exit, induce reversible conformational changes in the RovA dimer that attenuate DNA-binding capacity of RovA and renders the regulatory protein more susceptible to degradation by the Lon protease. The synthesis of RovA is strictly autoregulated by a positive and a negative feedback loop, and besides thermal control, it is regulated in response to growth phase and nutrient composition of the medium. Expression analysis of rovA-gfp fusions at different temperatures over multiple generations using time-lapse fluorescence microscopy and flow cytometry revealed that two distinct subpopulations (ON and OFF) of Y. pseudotuberculosis emerge in a certain temperature range in response to a bimodal behavior of RovA. We elucidated the molecular mechanisms underlying the temperature-tunable bistable switch and developed deterministic and stochastic models, which allow us to explain and predict the influence of temperature and alterations of regulatory elements controlling RovA synthesis on phenotypic switching. The analysis of mutant strains that are characterized by a modified bimodal behavior of RovA in a mouse infection model further indicated that phenotypic heterogeneity of RovA is advantageous for pathogenesis and fitness during infection. However, which biological functions are influenced by the bistable behavior of RovA and which of them support a better fitness and/or improve pathogenicity of the bacteria are still unknown and will be part of our future study. To address these questions we will: (i) analyze the spatio-temporal distribution of the distinct subpopulation (ON and OFF) in infected host tissues to gain insight where, when and to what extent rovA is heterogenously expressed during the infection(ii) unravel the biological functions which are differently expressed in the RovA ON and OFF subpopulation in vitro and during the infection, and address their significance for bacterial fitness and pathogenesis(iii) investigate the role of nutrients (metabolites) for the generation of RovA bistability during the infection and determine their importance for persistence and bacterial pathogenicity. Gained knowledge about the influence of metabolites on the bistable phenotype of rovA will be incooperated into the mathematical models and used for model-guided modulation of the system to validate and optimize the models.This will provide valuable information about the existence, properties and role of the RovA-expressing and non-expressing cells within bacterial communities during the infection, and about how bistability of the system promote adaptation to rapid changes, e.g. when they encounter a different environment inside or outside their hosts.