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Abstract

Bacteria control import and export of compounds, adhesion to surfaces, cell-to-cell interactions and interactions with host via proteins they feature on their cell surface. Recent evidence has emerged that members of a category of proteins anchored to bacterial membranes through their lipidated tails, called lipoproteins, can be exposed at the cell surface of Gram-negative bacteria. Escherichia coli is thought to carry around 100 lipoproteins, but what fraction of them is surface exposed and whether this is a general phenomenon remains unknown. In my PhD project, I systematically investigated whether lipoproteins are surface exposed in E. coli. In this work, I developed a Surface Proteome Quantification (SPQ) method, which systematically and quantitatively assesses protein cell-surface exposure at a proteome-wide level, using surface biotin labelling combined with quantitative mass spectrometry. Thereby I identified at least 23 outer membrane (OM) lipoproteins to be significantly surface exposed (out of 62 quantified OM lipoproteins) to similar levels as the bona-fide surface exposed OM β-barrel proteins. Then, 5 surface identified lipoproteins, 3 OM lipoproteins and 2 inner membrane lipoproteins, were verified for their surface exposure using independent biochemical methods. Furthermore, using the SPQ method, I identified a strong dependency of the lipoprotein cell-surface exposure on the BAM (β-barrel assembly machinery) and explored the role of the different components of the BAM complex. In parallel, I generated the first proteome-wide quantitative examination of the E. coli membrane proteome after physical separation of the inner- and outer-membrane, revealing an additional complexity and some mis-annotations in protein localization within the bacterial cell envelope. Finally, I investigated the physiological consequences on the Rcs stress response system through a mislocalized model surface exposed lipoprotein, RcsF. Overall, the complexity and importance of protein localization in the bacterial cell envelope, with the focus on lipoproteins, is investigated and discussed in this thesis.