Project Summary Enteric bacteria and most opportunistic pathogens transmitted through soil and fresh water show exceptional adaptability to a range of environments. Part of their adaptive potential is the ability to survive drastic osmolarity changes. Upon a sudden dilution of external medium, such as in the rain, bacteria evade mechanical rupture by 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 been extensively studied in terms of their structure and gating mechanisms. Yet, despite the progress in biophysical studies of these individual mechanosensitive channels, little is known about the actual release process that takes 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 the transient permeability ceases, and finally, how all these parameters are linked to osmotic fitness. Our current analysis of the mechanism strongly suggests two key aspects: (i) the channels must release osmolytes fast enough to outpace the osmotic water influx and curb cell swelling; on the other hand (ii) the massive osmolyte dissipation must be firmly terminated by inactivation of the low-threshold channel to facilitate recovery. The proposed project aims at a self-consistent kinetic/physical model of the rescuing process based on a comprehensive 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 will determine identities and availabilities of major osmolytes leaving cells through specific channels during osmotic shock using modern metabolomics. We will study the effects of major permeable and impermeable osmolytes on MscS and MscL gating and visualize permeation and interactions which may affect state distributions in MD simulations. (2) To address several remaining questions about the mechanism of MscS opening and inactivation, we will determine crystal structures of mutants with stabilized resting and open states. The transition pathways between the states will then be reconstructed in simulations. (3) We will employ the stopped-flow technique to record the kinetics of light scattering in live cultures and assess permeabilities of the cell envelope to water and osmolytes; we will correlate the exchange rates with osmotic cell viability. Using fluidics and videomicroscopy we will to determine the elasticity of the cell wall and the amount of membrane reservoir inside the stretchable peptidoglycan. Parallel electrophysiological analysis will provide channel densities and parameters for gating and inactivation. The detailed picture of the concerted action of two non- redundant channels in the course of osmotic permeabilit...