Dr. Stefan Schulze Department of Biology University of Pennsylvania Thursday, April 1, 2021 - 11:10am Online via Zoom Special Information: Please contact Nancy Perkins at firstname.lastname@example.org for Zoom link and passcode Type of Event: Department Seminars Abstract: Protein glycosylation, one of the most complex post-translational modifications, plays central roles in a variety of cellular processes in prokaryotes. Elucidating its involvement in biofilm formation is crucial for a detailed understanding of antibiotic resistance and pathogenicity mechanisms. Yet the complexity and variability of glycoproteins in prokaryotes have made their system-wide analysis, thus far, virtually unachievable. Here, I will present an interdisciplinary approach that combines bioinformatics, comprehensive glycoproteomics and phenotypic characterizations for the functional analysis of prokaryotic glycosylation. This includes the development of universally applicable bioinformatic tools that are suitable to analyze the plethora of glycan compositions existing in prokaryotes, as well as the initiation of the Archaeal Proteome Project, a community-effort that combines proteomics datasets across a broad range of experimental conditions to harvest the wealth of information buried within them. Together with an in-depth proteomic analysis of glycosylation pathway mutants from the model archaeon Haloferax volcanii, this has led to the identification of the largest archaeal glycoproteome described so far. It also revealed the concurrence of two independent N-glycosylation pathways that can modify the same glycosylation sites. A variety of phenotypic assays of mutants defective in N-glycosylation pathways or glycosylated proteins were further used to show the involvement of glycosylation in crucial cellular processes such as biofilm formation and cell shape. The establishment of these approaches for H. volcanii not only provided new insights into the extent, complexity, and roles of glycosylation in archaea, but their applicability to a multitude of prokaryotes also paves the way for functional glycoproteomics in biofilm forming pathogens such as Pseudomonas aeruginosa.