| Summary: | Escherichia coli (E. coli) frequently experiences changes in environmental osmolarity thus requiring homeostatic adjustments for proper cell function. In a hypoosmotic shock the central players are the mechanosensitive channels (MS channels), considered the major routes for the release of cytoplasmic solutes to achieve rapid reduction of turgor pressure. In the present study a combination of in vivo and in vitro techniques were used to investigate three of the seven MS channels present in the genome of E. coli, these channels being MscL, MscS and MscK. The first chapter focused on three residues (R46, R59 and K60) in the transmembrane helices TM1 and TM2 of MscS. It has been hypothesized that these residues act as part of the tension sensor mechanism, interacting either with membrane phospholipids or ions within the pore of the channel. In addition to MscS, the second chapter included MscL and MscK and analyzed the precise localization and diffusion rate of these proteins in the cytoplasmic membrane by the use of a super resolution fluorescence microscopy technique. The same technique was used in the third chapter to quantify the two main MS channels, MscL and MscS. As well as providing a single cell census these analyses provided a correlation between abundance of channels and cells survival during a downshock, the situation in which these channels gate and protect cells. Quantification revealed a high abundance of these proteins, thus MS channels were also investigated for an alternative role, namely the release of solutes during hyperosmotic shocks. This last chapter focused specifically on glutamate excretion, which is part of the hyperosmotic response. In summary, the results presented in this thesis substantially increased our knowledge of MS channels, both from a functional and a quantitative perspective.
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