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Efforts to provide active research context to introductory courses in basic sciences are likely to better engage learners and provide a framework for relevant concepts. A simple teaching and learning experiment was conducted to use concepts in organic chemistry to solve problems in the life sciences. Bryant University is a liberal arts university with new programs in biological and environmental sciences. This laboratory exercise was developed for introductory organic chemistry and designed to highlight its applicability to the biological sciences. Students participated in an ongoing project to identify antigens on the surface of "Candida albicans" hyphae by testing a variety of lipid extraction procedures and characterizing the fatty acid and carbohydrate composition of their extractions by thin-layer chromatography and gas chromatography-mass spectrometry. Student extracts were also tested in an immunoblot assay with a specific antibody fragment (scFv) that recognizes an unknown component of the cell surface of "C. albicans" hyphae. In order to build communication skills, students were required to prepare and present posters detailing the project and their findings.

In this study, intact flagellin proteins were purified from strains of Clostridium difficile and analyzed using quadrupole time of flight and linear ion trap mass spectrometers. Top-down studies showed the flagellin proteins to have a mass greater than that predicted from the corresponding gene sequence. These top-down studies revealed marker ions characteristic of glycan modifications. Additionally, diversity in the observed masses of glycan modifications was seen between strains. Electron transfer dissociation mass spectrometry was used to demonstrate that the glycan was attached to the flagellin protein backbone in O linkage via a HexNAc residue in all strains examined. Bioinformatic analysis of C. difficile genomes revealed diversity with respect to glycan biosynthesis gene content within the flagellar biosynthesis locus, likely reflected by the observed flagellar glycan diversity. In C. difficile strain 630, insertional inactivation of a glycosyltransferase gene (CD0240) present in all sequenced genomes resulted in an inability to produce flagellar filaments at the cell surface and only minor amounts of unmodified flagellin protein. doi: 10.1128/JB.00861-09

N-Glycosylation of proteins is recognized as one of the most common post-translational modifications. It was believed that N-glycosylation occurred exclusively in eukaryotes until the recent discovery, of the general protein glycosylafion pathway (Pgl) in Campylobacter jejuni, which has similarities to the eukaryotic system and adds proteins en bloc from a lipid carrier to a protein acceptor. In addition to N-linked glycans, a number of pathogenic bacteria such as Pseudomonas aeruginosa and Neisseria species have been shown to O-glycosylate their proteins through polyisoprene-linked intermediates. To date, most techniques to analyze lipid-linked oligosaccharides (LLOs) of these pathways involve the use of radiolabels and chromatographic separation. With the increasing frequency of reports of bacterial protein glycosylation that proceed through lipid-mediated steps, there is a need for technologies capable of characterizing these newly described bacterial systems as well as eukaryotic pathways from biologically relevant samples in an accurate, rapid, and cost-effective manner. In this paper, a new glycomics strategy based on porous graphite carbon (PGC) liquid chromatography mass spectrometry (LC-MS) was devised and validated on the C. jejuni N-glycan pathway, lipid-linked oligosaccharide intermediates of the Pgl pathway from crude lipid extracts were separated using online chromatography on a capillary PGC column with a chloroform gradient. By exploiting the retention properties of hydrophobic and polar analytes on PGC, baseline separation of LLOs with minor changes in oligosaccharide structure and polyisoprene chain length was obtained. This method is capable of analyzing low levels of LLOs (from approximately [10.sup.6] bacterial cells) and distinguishing the LLOs that differ by as little as one monosaccharide or polyisoprene unit. Furthermore, we have demonstrated for the first time that oligosaccharides of the C. jejuni Pgl pathway are assembled on different polyisoprenes, e. g. [C.sub.45], [C.sub.60], and apparent hydroxylated forms, in addition to those previously reported (i.e., [C.sub.50] and [C.sub.55]). The hydroxylated forms of the LLOs are believed to be an intermediate in the degradation of accumulated LLOs for polyisoprene carrier recycling. 10.1021/ac9013622

N-Glycosylation of proteins is recognized as one of the most common post-translational modifications. Until recently it was believed that N-glycosylation occurred exclusively in eukaryotes before the discovery of the general protein glycosylation pathway (Pgl) in Campylobacter jejuni. To date, most techniques to analyze lipid-linked oligosaccharides (LLOs) of these pathways involve the use of radiolabels and chromatographic separation. Technologies capable of characterizing eukaryotic and the newly described bacterial N-glycosylation systems from biologically relevant samples in a quick, accurate, and cost-effective manner are needed. In this paper a new glycomics strategy based on lectin-affinity capture was devised and validated on the C. jejuni N-glycan pathway and the engineered Escherichia coli strains expressing the functional C. jejuni pathway. The lipid-linked oligosaccharide intermediates of the Pgl pathway were then enriched using SBA-agarose affinity-capture and examined by capillary electrophoresis--mass spectrometry (CE--MS). We demonstrate that this method is capable of detecting low levels of LLOs, the sugars are indeed assembled on undecaprenylpyrophosphate, and structural information for expected and unexpected LLOs can be obtained without further sample manipulation. Furthermore, CE--MS analyses of C. jejuni and the E. coli 'glyco-factories' showed striking differences in the assembly and control of N-glycan biosynthesis.

The characterization of a soluble derivative of the family 3 lytic transglycosylase (LT) MltB (termed sMltB) from Pseudomonas acruginosa is done by investigating the role of several residues predicted to exist at the -1 subsite of sMltB and participate in substrate binding. The replacement of Gln 100, a residue that would appear to interact with the N-acetyl group on N-acetylmuramic acid (MurNAc), did not show any changes in substrate affinity or activity.

The importance and specificity of polyisoprenol in the Campylobacter jejuni N-linked glycosylation pathway are discussed.

The SUPREX method and difference spectroscopy is applied for the determination of protein-ligand interactions in an engineered soluble form of Pseudomonas aeruginosa membrane-bound lytic transglycosylase B (MltB1) (sMltB). The data showed that N-acetylglucosamine (GlcNAc) alone does not bind to MltB with any measurable affinity but does contribute to the binding of GlcNAc-N-acetylmuramic acid-dipeptide.

The β-hexosaminidase inhibitor and structural analog of the putative oxazolium reaction intermediate of lytic transglycosylases, N-acetylglucosamine thiazoline (NAG–thiazoline), was synthesized in 46% overall yield and tested as an inhibitor of Escherichia coli growth. NAG–thiazoline, at concentrations up to 1 mg/ml, was not found to affect the viability of E. coli DH5α. However, the compound did induce morphological changes to the cells. Growth of cells in the presence of NAG–thiazoline caused an apparent inhibition of the biosynthesis of the cylindrical regions of the cells such that they became much shorter in length. The surface of these shorter cells was found to be much less hydrophobic compared to untreated cells as determined by the bacterial adhesion to hydrocarbon (BATH) assay. In addition, the co-administration of NAG–thiazoline with 1.7 × MIC concentrations of ampicillin prevented cell lysis suggesting that the compound inhibited autolytic enzymes, in particular the lytic transglycosylases. [Copyright &y& Elsevier]

N- Glycosylation of proteins is recognized as one of the most common post-translational modifications. Until recently it was believed that N-glycosylation occurred exclusively in eukaryotes until the discovery of the general protein glycosylation pathway (Pgl) in Campylobacter jejuni. We have developed a new glycomics strategy based on lectin-affinity capture of lipid-linked oligosaccharides (LLOs) coupled to capillary electrophoresis mass spectrometry. The LLO intermediates of the C. jejuni Pgl pathway were used to validate the methodology and to better characterize the bacterial model system for protein N-glycosylation. This method provides a rapid, non-radioactive approach for the characterization of intermediates in polysaccharide biosynthesis and is a useful tool for glycoengineering efforts in bacteria.