Gram-negative bacteria are characterized by a cell envelope consisting of an inner membrane (IM) and an outer membrane (OM), which are separated by the peptidoglycan-containing periplasm. While the integral IM proteins are alpha-helical, all but one known integral OM proteins (OMPs) are beta-barrels. All OMPs are synthesized in the cytosol with an N-terminal signal sequence that directs them to the Sec translocon for transport through the IM. Extensive genetic and biochemical research brought to light many aspects of protein translocation across the IM; however, details about protein assembly into the OM have remained obscure. The Omp85 protein was first described in Neisseria meningitdis as an essential protein and the key component of the OMP assembly machinery. Homologs of Omp85 are present in Gram-negative bacteria and in mitochondria. The Omp85 homolog in Escherichia coli, known as BamA, was shown to be associated with four lipoproteins only one of which is essential. Several chaperones have been identified that play a role in OMP biogenesis during transit of the periplasm. Among them, Skp and SurA appear to be the most prominent ones in E. coli. So far, most of the research concerning OMP assembly in Gram-negative bacteria has been performed in E. coli. However, with the release of more and more genome sequences of Gram-negative bacteria, it becomes clear that the OMP assembly process may not be entirely similar in all Gram-negatives, since some of the components implicated in the assembly process cannot always be found in each genome. Furthermore, Gram-negatives other than E. coli were shown to demonstrate different phenotypes when genes for OM assembly components are inactivated, and their analysis may yield novel insights into the biogenesis of the OM. The goal of this work was to characterize the OMP assembly process in N. meningitidis to further our understanding of OMP assembly in Gram-negative bacteria in general. To that end, we characterized the Omp85-containing beta-barrel assembly complex in N. meningitidis. Several components were found to be similar to those found in E. coli, but one was lacking and a new component was also identified. We addressed the roles of the major periplasmic chaperones that are generally implicated in OMP assembly, i.e. SurA and Skp, in N. meningitidis and found considerable differences compared to E. coli, particularly with respect to the role of SurA. Emanating from the observation that E. coli BamA did not interact with a neisserial OMP in vitro, we addressed the species specificity of Omp85 by analyzing the functionality of Omp85 homologs from other proteobacteria in E. coli and in N. meningitidis. Further, we purified E. coli and N. meningitidis Omp85 proteins and some of their subdomains and studied properties, such as secondary structure, pore formation and substrate recognition in vitro. Altogether this thesis provides broad study of the OMP assembly in N. meningitidis
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