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Structure & Function of Bacterial Transport Machines in their Cellular Context https://doi.org/10.17863/CAM.105459 Kirykowicz, Angela Mary Luisi, Ben Zhang, Peijun Membranes confer cells with individual identity and capacity to regulate their response to their environment. A critical aspect of having a membranous partition is the ability to transport substances into and out of cells as part of life-sustaining functions. In pathogenic bacteria, transporters aid infection and survival in the host. Two such transporters in Gram-negative bacterial species are the MacA-MacB-TolC (MacAB-TolC) antibiotic efflux pump and the Type I Secretion System (T1SS), responsible respectively for antibiotic resistance and export of protein virulence factors. To pass the Gram-negative envelope in a one-step translocation process, both machines use a tripartite system, consisting of outer membrane protein TolC, a periplasmic adapter protein (MacA or haemolysin D (HlyD) in the T1SS), and an inner membrane protein (MacB or haemolysin B (HlyB) in the T1SS). Both use the power of ATP-hydrolysis to export their substrates. Here, I utilise computational and experimental approaches to elucidate the mechanism of function for both machines. I conduct molecular dynamics (MD) simulations of membrane embedded HlyB component of the T1SS with and without its haemolysin A (HlyA) substrate as *in silico* experiments. I also conduct MD simulations with and without substrate for a related peptidase. I show that substrate recognition is via conserved charge-charge interactions. I also show that HlyB has an asymmetric preferential interaction with cardiolipin when its substrate is present, which is not seen in the peptidase simulations. I propose that this preference is part of the mechanism of transport, with cardiolipin providing energy via the proton-motive force. I test this hypothesis through flow cytometry detection of labelled substrate trapped T1SS in a mixed population of cells, by comparing parental MG1655 *Escherichia coli* with a cardiolipin deficient MG1655 strain. I found that the cardiolipin deficient strain has reduced T1SS levels compared to its parent. To aid structural studies, I optimise the expression of the T1SS using a flow cytometry based sequential design strategy where conditions are iteratively tested via detection of substrate trapped T1SS and updated until no more improvement can be made. I also test purification strategies for single-particle cryo-electron microscopy studies. Finally, I apply further bioinformatic approaches and synthesise my computational and experimental results to propose a mechanism of transport and suggest future experimental tests. I conduct MD simulations of MacB in membrane with and without a trapped lipid. I show that this trapped lipid locks MacB into an open state, allowing for substrate entry into the pump. I contextualise the results by comparing MD simulations to MacB-like structures and propose a revised mechanism of transport as a function of its free-energy landscape. Lastly, I explore the use of cryo-electron tomography (cryo-ET) as a method to obtain *in vivo* structural insights. I show that the use of “ghost” partially lysed *E. coli* can produce high-contrast specimens for tomography. I collect a tomographic dataset of “ghost” MacAB-TolC containing cells and apply subtomogram averaging. Preliminary results suggest that MacAB-TolC forms an array in cells, and that MacB is structurally flexible, likely in its nucleotide-binding domain. Together, these studies of the MacAB-TolC efflux pump and the T1SS shed light on their function and suggest new avenues of research to explore in order to fulfil the goal of finding novel inhibitors. University of Cambridge 2023-09-29 Thesis Doctoral Doctor of Philosophy (PhD) eng Diamond Doctoral Scholarship (via United Kingdom Research Institute, UKRI) European Research Council https://www.repository.cam.ac.uk/handle/1810/363352 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/fada7826-f472-464c-87d9-ae6f9d2354e0/download bcae81d17891434e79046db2ffddc2b8 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/1338b757-a2eb-469a-940c-0d719d956090/download 87eda9de84448d1f82354d60eee3eb5f https://www.rioxx.net/licenses/all-rights-reserved/ antibiotic efflux pumps bacterial secretion systems cryo-electron microscopy cryo-electron tomography macab-tolc molecular dynamics type I secretion system