| Čoahkkáigeassu: | The Tol-Pal system is a conserved machine in double-membraned bacteria that stabilises the Gram-negative bacterial outer membrane. In this thesis, I aim to elucidate the mechanism by which the Tol-Pal system dynamically stabilises this important protective layer. I examine how assembly of the <em>trans</em>-periplasmic motor-transducer complex recruits a membrane stabilising lipoprotein, Pal, to the division site. To contextualise the experimental aspects of this thesis, I first examine the physiological role of the Tol-Pal system, and its cellular function in <em>E. coli</em>. Through use of mutagenesis, fluorescence microscopy, and live cell assays, I demonstrate that system function can be modulated by altering the secondary structure of the periplasm spanning transducer protein, TolA. I also examine the cell-wide implications of reducing TolA function in such a manner, with respect to membrane stability and cell division (chapter three). Subsequent bioinformatic investigations into the phylogeny of the Tol-Pal system reveal its unexpectedly broad distribution throughout the kingdom of bacteria and hint at its evolutionary origin (chapter four). Further molecular insights were gleaned through <em>in vivo</em> crosslinking, where I probe the conformational rearrangements that occur within the TolQR motor in response to proton flux (chapter five). Additional modelling and bioinformatic data reveal the putative TolA binding site on the TolQR motor, and I demonstrate the biological relevance of a previously unknown interaction motif involved in TolA-TolR interactions and force transduction (chapter six). Finally, I describe the optimisation of electron cryotomography (cryo-ET) to structurally visualise the TolQR-TolA assembly in an energetically stalled state (chapter seven). Overall, this work reveals the function, distribution, conservation and organisation of bacterial Tol-Pal systems and their likely evolutionary origin. I also propose a model for the mechanism by which these protein machines use proton flux to stabilise the outer membrane in <em>E. coli</em>.
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