Bacterial mechanosensitive channels are major players in cells’ ability to cope with hypo-osmotic stress. Excess turgor pressure due to fast water influx is reduced as the channels, triggered by membrane tension, open and release osmolytes. In bacteria, the bulk release of ions and other osmolytes is mainly mediated by two families of mechanosensitive channels: MscS and MscL. The MscL family channels form large non-selective pathways in the membrane and gate near the lytic tension. In this way, they act as a final back up mechanism against osmotic downshock. MscS family channels require less tension to open and display great diversity in structure and functionality. Chapter 2 describes the first multifaceted phenomenological study of the emergency osmolyte release system in wild type Pseudomonas aeruginosa in comparison with E.coli. We recorded the kinetics of cell equilibration reported by light scattering responses to osmotic up- and down-shocks using the stopped-flow technique. We also performed the first electrophysiological characterization of the mechanosensitive ion channels in Pseudomonas aeruginosa. We presented a quantitative biophysical description of “osmotic fitness” which would be of interest to microbiologists, epidemiologists, ecologists and general environmental scientists. Chapter 3 presents the combined theoretical and experimental analysis of the full functional cycle of the bacterial channel MscS, which plays a major role in osmotic adjustments and environmental stability of most bacteria. We modeled MscS gating as a finite state continuous-time Markov chain and obtained analytical expressions for the steady state solution and the inactivated state area (which is experimentally hard to determine). In Chapter 4, we derived a general formalism to extract the free energy difference between the closed and open states of mechanosensitive ion channels (ΔF) from non-equilibrium work distributions associated with the channels’ gating. Our new approach bridges the gap between recent developments in non-equilibrium thermodynamics of small systems and ion channel biophysics. Our study also serves as an experimental verification of non-equilibrium work relations in a biological system. Therefore, the results in this thesis are sufficiently general and would be of interest to a broad community.