ABSTRACT: Comparative proteomics of Bacteroides thetaiotaomicron samples comparing the total membrane (TM) and outer membrane vesicles (OMV) of WT B. thetaiotaomicron and delta 4364
Project description:Levoglucosan is produced in the pyrolysis of cellulose and starch, including from bushfires or the burning of biofuels, and is deposited from the atmosphere across the surface of the earth. We describe two levoglucosan degrading Paenarthrobacter spp. (Paenarthrobacter nitrojuajacolis LG01 and Paenarthrobacter histidinolovorans LG02) that were isolated by metabolic enrichment on levoglucosan as sole carbon source. Genome sequencing and proteomics analysis revealed expression of a series of gene clusters encoding known levoglucosan degrading enzymes, levoglucosan dehydrogenase (LGDH, LgdA), 3-keto-levoglucosan b-eliminase (LgdB1) and glucose 3-dehydrogenase (LgdC), along with an ABC transporter cassette and associated solute binding protein. However, no homologues of 3-ketoglucose dehydratase (LgdB2) were evident. The expressed gene clusters contained a range of putative sugar phosphate isomerase/xylose isomerases with weak similarity to LgdB2. Sequence similarity network analysis of genome neighbors revealed that homologues of LgdA, LgdB1 and LgdC are generally conserved in a range of bacteria in the phyla Firmicutes, Actinobacteria and Proteobacteria. One sugar phosphate isomerase/xylose isomerase cluster (LgdB3) was identified with limited distribution mutually exclusive with LgdB2. LgdB1, LgdB2 and LgdB3 adopt similar predicted 3D folds suggesting overlapping function in processing intermediates in LG metabolism. Our findings highlight the diversity within the LGDH pathway through which bacteria utilize levoglucosan as a nutrient source.
Project description:This project examines the impact of different growth media on the protein compositions of membranes of Bacteroides thetaiotaomicron.
Project description:Polysialic acid (polySia) is a linear polymer of α2,8-linked sialic acid residues that is of fundamental biological interest due to its pivotal roles in the regulation of the nervous, immune, and reproductive systems in healthy human adults. PolySia is also dysregulated in several chronic diseases, including cancers and mental health disorders. However, the mechanisms underpinning polySia biology in health and disease remain largely unknown, in part due to the lack of tools with which to study the glycan. The polySia-specific hydrolase, endoneuraminidase NF (EndoN), and the catalytically inactive polySia lectin EndoNDM, have been extensively used for studying polySia. However, EndoN is heat stable and remains associated with cells after washing. When studying polySia in systems with multiple polysialylated species, the residual EndoN that cannot be removed confounds data interpretation. We developed a strategy for site-specific immobilization of EndoN and EndoNDM on streptavidin-coated magnetic or agarose beads. We showed that immobilizing EndoN improves enzyme usefulness by allowing for effective removal of the enzyme from samples, while retaining hydrolase activity. Additionally, immobilization of EndoNDM allowed for the enrichment of polysialylated proteins from complex mixtures for their identification via mass spectrometry. We identified QSOX2 as a novel polysialylated protein secreted from MCF-7 cells. This method of site-specific immobilization can be utilized for other enzymes and lectins to yield insight into glycobiology.