Project SummaryEnteric bacteria and most opportunistic pathogens transmitted through soil and fresh water show exceptionaladaptability to a range of environments. Part of their adaptive potential is the ability to survive drastic osmolaritychanges. Upon a sudden dilution of external medium, such as in the rain, bacteria evade mechanical ruptureby engaging tension-activated channels that act as osmolyte release valves. The low-threshold MscS and high-threshold MscL, the two channel species that mediate the bulk of osmolyte exchange in E. coli, have beenextensively studied in terms of their structure and gating mechanisms. Yet, despite the progress in biophysicalstudies of these individual mechanosensitive channels, little is known about the actual release process thattakes place in the cell upon abrupt osmotic downshift. There is almost no data on the extent and rate of swelling,the kinetics of osmolyte release, the molecules that escape through specific channels, when and how thetransient permeability ceases, and finally, how all these parameters are linked to osmotic fitness. Our currentanalysis of the mechanism strongly suggests two key aspects: (i) the channels must release osmolytes fastenough to outpace the osmotic water influx and curb cell swelling; on the other hand (ii) the massive osmolytedissipation must be firmly terminated by inactivation of the low-threshold channel to facilitate recovery. Theproposed project aims at a self-consistent kinetic/physical model of the rescuing process based on acomprehensive phenomenological description of osmotically-induced solute exchange in live cultures of E.coli, cell envelope mechanics, and on spatial and thermodynamic properties of channel gating. (1) We willdetermine identities and availabilities of major osmolytes leaving cells through specific channels during osmoticshock using modern metabolomics. We will study the effects of major permeable and impermeable osmolyteson MscS and MscL gating and visualize permeation and interactions which may affect state distributions in MDsimulations. (2) To address several remaining questions about the mechanism of MscS opening andinactivation, we will determine crystal structures of mutants with stabilized resting and open states. Thetransition pathways between the states will then be reconstructed in simulations. (3) We will employ thestopped-flow technique to record the kinetics of light scattering in live cultures and assess permeabilities of thecell envelope to water and osmolytes; we will correlate the exchange rates with osmotic cell viability. Usingfluidics and videomicroscopy we will to determine the elasticity of the cell wall and the amount of membranereservoir inside the stretchable peptidoglycan. Parallel electrophysiological analysis will provide channeldensities and parameters for gating and inactivation. The detailed picture of the concerted action of two non-redundant channels in the course of osmotic permeability response and a set of ?vital? parameters will providegrounds for a quantitative model which would predict whether a particular magnitude and speed of osmoticdownshift will be tolerable or lethal.