ABSTRACT: Comparsion of multiple BC k56-2 muants to assess protoeme chnages. seven strain comparsion between WT vs delta pglL vs delta 1086 vs delta 2974 vs delta CepI vs delta CepR vs delta pglL complemented AmrAB::S7-pglL-his. LFQ based quantification
Project description:Characterisation of the effect of multiple pglL muants and complement within Burkholderia cenocepacia k56-2 to confirm glycosylation phenotype. Six strain comparsion (WT vs delta pglL 1 vs delta pglL 2 vs delta pglL 1 complement native pglL vs delta pglL 1 complement S7-pglL-his vs delta pglL 2 complement native pglL
Project description:Characterisation of the effect of glycosylation disruption within Burkholderia cenocepacia k56-2. Five strain comparsion (WT vs delta pglL vs delta OGC vs Delta pglL OGC vs Delta pglL complemented S7-pglL-his)
Project description:Comparsion of DNA interacting proteome of BC muants to assess chnages in transcriptional proteins. Four strain comparsion between WT vs delta pglL vs delta ogc vs delta pglL complemented AmrAB::S7-pglL-his. LFQ based quantification
Project description:Comparsion of the endogenous peptide pool within Burkholderia Cenocepacia strains to identify evidence for glycoprotein degradation in the absent of glycosylation. WT vs delta pglL vs pglL complement were compared (4 biological of each)
Project description:Comparsion of proteomes of Campylobacter fetus subsp. fetus to compare protein level via iBAQ analysis, expression (by LFQ) and coverage between Campylobacter fetus subsp. fetus strain82-40 vs Campylobacter fetus subsp. fetus strain ATCC 27374
Project description:Neisseria meningitidis PglL belongs to a novel family of bacterial oligosaccharyltransferases (OTases) responsible for O-glycosylation of type IV pilins. Although members of this family are widespread among pathogenic bacteria, there is little known about their mechanism. Understanding the O-glycosylation process may uncover potential targets for therapeutic intervention, and can open new avenues for the exploitation of these pathways for biotechnological purposes. In this work, we demonstrate that PglL is able to transfer virtually any glycan from the undecaprenyl pyrophosphate (UndPP) carrier to pilin in engineered Escherichia coli and Salmonella cells. Surprisingly, PglL was also able to interfere with the peptidoglycan biosynthetic machinery and transfer peptidoglycan subunits to pilin. This represents a previously unknown post-translational modification in bacteria. Given the wide range of glycans transferred by PglL, we reasoned that substrate specificity of PglL lies in the lipid carrier. To test this hypothesis we developed an in vitro glycosylation system that employed purified PglL, pilin, and the lipid farnesyl pyrophosphate (FarPP) carrying a pentasaccharide that had been synthesized by successive chemical and enzymatic steps. Although FarPP has different stereochemistry and a significantly shorter aliphatic chain than the natural lipid substrate, the pentasaccharide was still transferred to pilin in our system. We propose that the primary roles of the lipid carrier during O-glycosylation are the translocation of the glycan into the periplasm, and the positioning of the pyrophosphate linker and glycan adjacent to PglL. The unique characteristics of PglL make this enzyme a promising tool for glycoengineering novel glycan-based vaccines and therapeutics.
Project description:O-glycosylation of proteins in Neisseria meningitidis is catalyzed by PglL, which belongs to a protein family including WaaL O-antigen ligases. We developed two hidden Markov models that identify 31 novel candidate PglL homologs in diverse bacterial species, and describe several conserved sequence and structural features. Most of these genes are adjacent to possible novel target proteins for glycosylation. We show that in the general glycosylation system of N. meningitidis, efficient glycosylation of additional protein substrates requires local structural similarity to the pilin acceptor site. For some Neisserial PglL substrates identified by sensitive analytical approaches, only a small fraction of the total protein pool is modified in the native organism, whereas others are completely glycosylated. Our results show that bacterial protein O-glycosylation is common, and that substrate selection in the general Neisserial system is dominated by recognition of structural homology.
Project description:Outer membrane vesicles (OMV) are spherical structures derived from the outer membrane (OM) of Gram-negative bacteria. OMVs from the predominant gut microbiota members, Bacteroides, are proposed to play key roles in gut homeostasis. OMV biogenesis in Bacteroides is a poorly understood process. Here, we revisited the protein composition of B. theta OMVs by mass spectrometry. We confirmed that OMVs produced by this organism contain large quantities of glycosidases and proteases, with most of them being lipoproteins. We found that many of these OMV-enriched lipoproteins are encoded by polysaccharide utilization loci (PULs), such as the sus operon. We examined the subcellular localization of the components of the sus operon and found that the alpha-amylase SusG is highly enriched in OMVs while the oligosaccharide importer SusC remains mostly in the OM. We show that all OMV-enriched lipoproteins possess a lipoprotein export sequence (LES) that mediates translocation of SusG from the periplasmic face of the OM towards the extracellular milieu and is required for SusG to localize preferentially to OMVs. We also show that surface-exposed SusG in OMVs is active and can rescue growth of bacterial cells incapable of growing on starch as only carbon source. Our results support the role of OMVs as "public goods" that can be utilized by other organisms with different metabolic capabilities.