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With many ongoing clinical trials utilizing adeno-associated virus (AAV) gene therapy, it is necessary to find scalable and serotype-independent primary capture and recovery methods to allow for efficient and robust manufacturing processes. Here, we demonstrate the ability of a hydrophobic interaction chromatography membrane to capture and recover AAV1, AAV5, AAV8, and AAV "Mutant C" (a novel serotype incorporating elements of AAV3B and AAV8) particles from cell culture media and cell lysate with recoveries of 76%-100% of loaded material, depending on serotype. A simple, novel technique that integrates release and recovery of cell-associated AAV capsids is demonstrated. We show that by the addition of lyotropic salts to AAV-containing cell suspensions, AAV is released at an equivalent efficiency to mechanical lysis. The addition of the lyotropic salt also promotes a phase separation, which allows physical removal of large amounts of DNA and insoluble cellular debris from the AAV-containing aqueous fraction. The AAV is then captured and eluted from a hydrophobic interaction chromatography membrane. This integrated lysis and primary capture and recovery technique facilitates substantial removal of host-cell DNA and host-cell protein impurities., (© 2020 The Author(s).)

The ability of an anion exchange membrane to purify a γ-retrovirus was assessed and optimised with respect to different loading and wash buffers. Recoveries of infectious virus greater than 50% were consistently obtained, while specific titre was increased up to one thousand fold when compared to the material loaded. Specific proteins removed and retained by this optimised process were identified by mass spectrometry. It was possible to successfully bind and elute the equivalent of 1.27 × 10(8) Ifu/ml of ion exchange membrane. This could then be highly concentrated, with infectious virus concentrated to a maximum of 420-fold compared to the load., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

A new disposable adsorbent material for fast anion-exchange capture of nano-complexes without prefiltering, clarification or pre-processing of samples was developed based on plastic microcapillary films (MCFs). An MCF containing 19 parallel microcapillaries, each with a mean internal diameter of 142 ± 10 μm, was prepared using a melt extrusion process from an ethylene-vinyl alcohol copolymer (EVOH). The MCF internal surfaces were functionalised using branched chain chemistries to attach quaternary amine groups producing an anion-exchange adsorbent. The purification of nano-complexes using this newly fabricated MCF-EVOH-Q was successfully demonstrated with the capture of lentivirus from pre-filtered culture harvest. This 5m chromatographic substrate was found to bind and elute ∼40% of bound lentivirus or 2.5 × 10(6)infectious units (ifu). The unique properties of this chromatographic substrate that allow the passage of large particulates was further demonstrated with the capture of lentiviral particles from unfiltered un-processed culture media containing cells and cell debris. Using this approach, 56% or 1 × 10(7)ifu of captured lentivirus was eluted. A device based on this new material might be used at an early stage in clinical lentiviral production to harvest lentiviral particles, directly from bioreactors., (Copyright © 2012. Published by Elsevier B.V.)

Large quantities of soil organic carbon in Arctic permafrost zones are becoming increasingly unstable due to a warming climate. High temperatures and substantial rainfall in July 2007 in the Canadian High Arctic resulted in permafrost active layer detachments (ALDs) that redistributed soils throughout a small watershed in Nunavut, Canada. Molecular biomarkers and NMR spectroscopy were used to measure how ALDs may lead to microbial activity and decomposition of previously unavailable soil organic matter (SOM). Increased concentrations of extracted bacterial phospholipid fatty acids (PLFAs) and large contributions from bacterial protein/peptides in the NMR spectra at recent ALDs suggest increased microbial activity. PLFAs were appreciably depleted in a soil sample where ALDs occurred prior to 2003. However an enrichment of bacterial derived peptidoglycan was observed by (1)H-(13)C heteronuclear multiple quantum coherence (HMQC) and (1)H diffusion edited (DE) NMR and enhanced SOM degradation was observed by (13)C solid-state NMR. These data suggest that a previous rise in microbial activity, as is currently underway at the recent ALD site, led to degradation and depletion of labile SOM components. Therefore, this study indicates that ALDs may amplify climate change due to the release of labile SOM substrates from thawing High Arctic permafrost.

N-Linked protein glycosylation is conserved throughout the three domains of life and influences protein function, stability, and protein complex formation. N-Linked glycosylation is an essential process in Eukaryotes; however, although N-glycosylation affects multiple cellular processes in Archaea and Bacteria, it is not needed for cell survival. Methods for the analyses of N-glycosylation in eukaryotes are well established, but comparable techniques for the analyses of the pathways in Bacteria and Archaea are needed. In this chapter we describe new methods for the detection and analyses of N-linked, and the recently discovered free oligosaccharides (fOS), from whole cell lysates of Campylobacter jejuni using non-specific pronase E digestion and permethylation followed by mass spectrometry. We also describe the expression and immunodetection of the model N-glycoprotein, AcrA, fused to a hexa-histidine tag to follow protein glycosylation in C. jejuni. This chapter concludes with the recent demonstration that high-resolution magic angle spinning NMR of intact bacterial cells provides a rapid, non-invasive method for analyzing fOS in C. jejuni in vivo. This combination of techniques provides a powerful tool for the exploration, quantification, and structural analyses of N-linked and free oligosaccharides in the bacterial system.

Glycomics which is the study of saccharides and genes responsible for their formation requires the continuous development of rapid and sensitive methods for the identification of glycan structures. It involves glycoanalysis which relies upon the development of methods for determining the structure and interactions of carbohydrates. For the application of functional glycomics to microbial virulence, carbohydrates and their associated metabolic and carbohydrate processing enzymes and respective genes can be identified and exploited as targets for drug discovery, glyco-engineering, vaccine design, and detection and diagnosis of diseases. Glycomics also encompasses the detailed understanding of carbohydrate-protein interactions and this knowledge can be applied to research efforts focused toward the development of vaccines and immunological therapies to alleviate infectious diseases.

The food-borne pathogen Campylobacter jejuni is one of the leading causes of bacterial gastroenteritis worldwide and the most frequent antecedent in neuropathies such as the Guillain-Barré and Miller Fisher syndromes. C. jejuni was demonstrated to possess an N-linked protein glycosylation pathway that adds a conserved heptasaccharide to >40 periplasmic and membrane proteins. Recently, we showed that C. jejuni also produces free heptasaccharides derived from the N-glycan pathway reminiscent of the free oligosaccharides (fOS) produced by eukaryotes. Herein, we demonstrate that C. jejuni fOS are produced in response to changes in the osmolarity of the environment and bacterial growth phase. We provide evidence showing the conserved WWDYG motif of the oligosaccharyltransferase, PglB, is necessary for fOS release into the periplasm. This report demonstrates that fOS from an N-glycosylation pathway in bacteria are potentially equivalent to osmoregulated periplasmic glucans in other Gram-negative organisms.

The lipopolysaccharide of Pseudomonas aeruginosa PAO1 contains an unusual sugar, 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid (d-ManNAc3NAcA). wbpB, wbpE, and wbpD are thought to encode oxidase, transaminase, and N-acetyltransferase enzymes. To characterize their functions, recombinant proteins were overexpressed and purified from heterologous hosts. Activities of His(6)-WbpB and His(6)-WbpE were detected only when both proteins were combined in the same reaction. Using a direct MALDI-TOF mass spectrometry approach, we identified ions that corresponded to the predicted products of WbpB (UDP-3-keto-d-GlcNAcA) and WbpE (UDP-d-GlcNAc3NA) in the coupled enzyme-substrate reaction. Additionally, in reactions involving WbpB, WbpE, and WbpD, an ion consistent with the expected product of WbpD (UDP-d-GlcNAc3NAcA) was identified. Preparative quantities of UDP-d-GlcNAc3NA and UDP-d-GlcNAc3NAcA were enzymatically synthesized. These compounds were purified by high-performance liquid chromatography, and their structures were elucidated by NMR spectroscopy. This is the first report of the functional characterization of these proteins, and the enzymatic synthesis of UDP-d-GlcNAc3NA and UDP-d-GlcNAc3NAcA.

The rare 6-deoxysugar D-rhamnose is a component of bacterial cell surface glycans, including the D-rhamnose homopolymer produced by Pseudomonas aeruginosa, called A-band O polysaccharide. GDP-D-rhamnose synthesis from GDP-D-mannose is catalyzed by two enzymes. The first is a GDP-D-mannose-4,6-dehydratase (GMD). The second enzyme, RMD, reduces the GMD product (GDP-6-deoxy-D-lyxo-hexos-4-ulose) to GDP-d-rhamnose. Genes encoding GMD and RMD are present in P. aeruginosa, and genetic evidence indicates they act in A-band O-polysaccharide biosynthesis. Details of their enzyme functions have not, however, been previously elucidated. We aimed to characterize these enzymes biochemically, and to determine the structure of RMD to better understand what determines substrate specificity and catalytic activity in these enzymes. We used capillary electrophoresis and NMR analysis of reaction products to precisely define P. aeruginosa GMD and RMD functions. P. aeruginosa GMD is bifunctional, and can catalyze both GDP-d-mannose 4,6-dehydration and the subsequent reduction reaction to produce GDP-D-rhamnose. RMD catalyzes the stereospecific reduction of GDP-6-deoxy-D-lyxo-hexos-4-ulose, as predicted. Reconstitution of GDP-D-rhamnose biosynthesis in vitro revealed that the P. aeruginosa pathway may be regulated by feedback inhibition in the cell. We determined the structure of RMD from Aneurinibacillus thermoaerophilus at 1.8 A resolution. The structure of A. thermoaerophilus RMD is remarkably similar to that of P. aeruginosa GMD, which explains why P. aeruginosa GMD is also able to catalyze the RMD reaction. Comparison of the active sites and amino acid sequences suggests that a conserved amino acid side chain (Arg185 in P. aeruginosa GMD) may be crucial for orienting substrate and cofactor in GMD enzymes.

Flagellins from Clostridium botulinum were shown to be post-translationally modified with novel glycan moieties by top-down MS analysis of purified flagellin protein from strains of various toxin serotypes. Detailed analyses of flagellin from two strains of C. botulinum demonstrated that the protein is modified by a novel glycan moiety of mass 417 Da in O-linkage. Bioinformatic analysis of available C. botulinum genomes identified a flagellar glycosylation island containing homologs of genes recently identified in Campylobacter coli that have been shown to be responsible for the biosynthesis of legionaminic acid derivatives. Structural characterization of the carbohydrate moiety was completed utilizing both MS and NMR spectroscopy, and it was shown to be a novel legionaminic acid derivative, 7-acetamido-5-(N-methyl-glutam-4-yl)-amino-3,5,7,9-tetradeoxy-D-glycero-alpha-D-galacto-nonulosonic acid, (alphaLeg5GluNMe7Ac). Electron transfer dissociation MS with and without collision-activated dissociation was utilized to map seven sites of O-linked glycosylation, eliminating the need for chemical derivatization of tryptic peptides prior to analysis. Marker ions for novel glycans, as well as a unique C-terminal flagellin peptide marker ion, were identified in a top-down analysis of the intact protein. These ions have the potential for use in for rapid detection and discrimination of C. botulinum cells, indicating botulinum neurotoxin contamination. This is the first report of glycosylation of Gram-positive flagellar proteins by the 'sialic acid-like' nonulosonate sugar, legionaminic acid.

Pseudomonas aeruginosa PAK (serotype O6) produces a single polar, glycosylated flagellum composed of a-type flagellin. To determine whether or not flagellin glycosylation in this serotype requires O-antigen genes, flagellin was isolated from the wild type, three O-antigen-deficient mutants wbpL, wbpO, and wbpP, and a wbpO mutant complemented with a plasmid containing a wild-type copy of wbpO. Flagellin from the wbpO mutant was smaller (42 kDa) than that of the wild type (45 kDa), or other mutants strains, and exhibited an altered isoelectric point (pI 4.8) when compared with PAK flagellin (pI 4.6). These differences were because of the truncation of the glycan moiety in the wbpO-flagellin. Thus, flagellin glycosylation in P. aeruginosa PAK apparently requires a functional WbpO but not WbpP. Because WbpP was previously proposed to catalyze a metabolic step in the biosynthesis of B-band O-antigen that precedes the action of WbpO, these results prompted us to reevaluate the two-step pathway catalyzed by WbpO and WbpP. Results from WbpO-WbpP-coupled enzymatic assays showed that either WbpO or WbpP is capable of initiating the two-step pathway; however, the kinetic parameters favored the WbpO reaction to occur first, converting UDP-N-acetyl-D-glucosamine to UDP-N-acetyl-D-glucuronic acid prior to the conversion to UDP-N-acetyl-D-galacturonic acid by WbpP. This is the first report to show that a C4 epimerase could utilize UDP-N-acetylhexuronic acid as a substrate.

In this study we investigated the commonality and biosynthesis of the O-methyl phosphoramidate (MeOPN) group found on the capsular polysaccharide (CPS) of Campylobacter jejuni. High resolution magic angle spinning NMR spectroscopy was used as a rapid, high throughput means to examine multiple isolates, analyze the cecal contents of colonized chickens, and screen a library of CPS mutants for the presence of MeOPN. Sixty eight percent of C. jejuni strains were found to express the MeOPN with a high prevalence among isolates from enteritis, Guillain Barré, and Miller-Fisher syndrome patients. In contrast, MeOPN was not observed for any of the Campylobacter coli strains examined. The MeOPN was detected on C. jejuni retrieved from cecal contents of colonized chickens demonstrating that the modification is expressed by bacteria inhabiting the avian gastrointestinal tract. In C. jejuni 11168H, the cj1415-cj1418 cluster was shown to be involved in the biosynthesis of MeOPN. Genetic complementation studies and NMR/mass spectrometric analyses of CPS from this strain also revealed that cj1421 and cj1422 encode MeOPN transferases. Cj1421 adds the MeOPN to C-3 of the beta-d-GalfNAc residue, whereas Cj1422 transfers the MeOPN to C-4 of D-glycero-alpha-L-gluco-heptopyranose. CPS produced by the 11168H strain was found to be extensively modified with variable MeOPN, methyl, ethanolamine, and N-glycerol groups. These findings establish the importance of the MeOPN as a diagnostic marker and therapeutic target for C. jejuni and set the groundwork for future studies aimed at the detailed elucidation of the MeOPN biosynthetic pathway.

The heteropolymeric O-antigen of the lipopolysaccharide from Pseudomonas aeruginosa serogroup O5 as well as the band-A trisaccharide from Bordetella pertussis contain the di-N-acetylated mannosaminuronic acid derivative, beta-D-ManNAc3NAcA (2,3-diacetamido-2,3-dideoxy-beta-D-mannuronic acid). The biosynthesis of the precursor for this sugar is proposed to require five steps, through which UDP-alpha-D-GlcNAc (UDP-N-acetyl-alpha-D-glucosamine) is converted via four steps into UDP-alpha-D-GlcNAc3NAcA (UDP-2,3-diacetamido-2,3-dideoxy-alpha-D-glucuronic acid), and this intermediate compound is then epimerized by WbpI (P. aeruginosa), or by its orthologue, WlbD (B. pertussis), to form UDP-alpha-D-ManNAc3NAcA (UDP-2,3-diacetamido-2,3-dideoxy-alpha-D-mannuronic acid). UDP-alpha-D-GlcNAc3NAcA, the proposed substrate for WbpI and WlbD, was obtained through chemical synthesis. His6-WbpI and His6-WlbD were overexpressed and then purified by affinity chromatography using FPLC. Capillary electrophoresis was used to analyse reactions with each enzyme, and revealed that both enzymes used UDP-alpha-D-GlcNAc3NAcA as a substrate, and reacted optimally in sodium phosphate buffer (pH 6.0). Neither enzyme utilized UDP-alpha-D-GlcNAc, UDP-alpha-D-GlcNAcA (UDP-2-acetamido-2,3-dideoxy-alpha-D-glucuronic acid) or UDP-alpha-D-GlcNAc3NAc (UDP-2,3-diacetamido-2,3-dideoxy-alpha-D-glucose) as substrates. His6-WbpI or His6-WlbD reactions with UDP-alpha-D-GlcNAc3NAcA produce a novel peak with an identical retention time, as shown by capillary electrophoresis. To unambiguously characterize the reaction product, enzyme-substrate reactions were allowed to proceed directly in the NMR tube and conversion of substrate into product was monitored over time through the acquisition of a proton spectrum at regular intervals. Data collected from one- and two-dimensional NMR experiments showed that His6-WbpI catalysed the 2-epimerization of UDP-alpha-D-GlcNAc3NAcA, converting it into UDP-alpha-D-ManNAc3NAcA. Collectively, these results provide evidence that WbpI and WlbD are UDP-2,3-diacetamido-2,3-dideoxy-alpha-D-glucuronic acid 2-epimerases.

Glycosylation of Campylobacter flagellin is required for the biogenesis of a functional flagella filament. Recently, we used a targeted metabolomics approach using mass spectrometry and NMR to identify changes in the metabolic profile of wild type and mutants in the flagellar glycosylation locus, characterize novel metabolites, and assign function to genes to define the pseudaminic acid biosynthetic pathway in Campylobacter jejuni 81-176 (McNally, D. J., Hui, J. P., Aubry, A. J., Mui, K. K., Guerry, P., Brisson, J. R., Logan, S. M., and Soo, E. C. (2006) J. Biol. Chem. 281, 18489-18498). In this study, we use a similar approach to further define the glycome and metabolomic complement of nucleotide-activated sugars in Campylobacter coli VC167. Herein we demonstrate that, in addition to CMP-pseudaminic acid, C. coli VC167 also produces two structurally distinct nucleotide-activated nonulosonate sugars that were observed as negative ions at m/z 637 and m/z 651 (CMP-315 and CMP-329). Hydrophilic interaction liquid chromatography-mass spectrometry yielded suitable amounts of the pure sugar nucleotides for NMR spectroscopy using a cold probe. Structural analysis in conjunction with molecular modeling identified the sugar moieties as acetamidino and N-methylacetimidoyl derivatives of legionaminic acid (Leg5Am7Ac and Leg5AmNMe7Ac). Targeted metabolomic analyses of isogenic mutants established a role for the ptmA-F genes and defined two new ptm genes in this locus as legionaminic acid biosynthetic enzymes. This is the first report of legionaminic acid in Campylobacter sp. and the first report of legionaminic acid derivatives as modifications on a protein.

A recent study that examined multiple strains of Campylobacter jejuni reported that HS:19, a serostrain that has been associated with the onset of Guillain-Barré syndrome, had unidentified labile, capsular polysaccharide (CPS) structures. In this study, we expand on this observation by using current glyco-analytical technologies to characterize these unknown groups. Capillary electrophoresis electrospray ionization MS and NMR analysis with a cryogenically cooled probe (cold probe) of CPS purified using a gentle enzymatic method revealed a hyaluronic acid-type [-4)-beta-D-GlcA6NGro-(1-3)-beta-D-GlcNAc-(1-]n repeating unit, where NGro is 2-aminoglycerol. A labile alpha-sorbofuranose branch located at C2 of GlcA was determined to have the L configuration using a novel pyranose oxidase assay and is the first report of this sugar in a bacterial glycan. A labile O-methyl phosphoramidate group, CH3OP(O)(NH2)(OR) (MeOPN), was found at C4 of GlcNAc. Structural heterogeneity of the CPS was due to nonstoichiometric glycosylation with sorbose at C2 of GlcA and the nonstoichiometric, variably methylated phosphoramidate group. Examination of whole bacterial cells using high-resolution magic angle spinning NMR revealed that the MeOPN group is a prominent feature on the cell surface for this serostrain. These results are reminiscent of those in the 11168 and HS:1 strains and suggest that decoration of CPS with nonstoichiometric elements such as keto sugars and the phosphoramidate is a common mechanism used by this bacterium to produce a structurally complex surface glycan from a limited number of genes. The findings of this work with the HS:19 serostrain now present a means to explore the role of CPS as a virulence factor in C. jejuni.

Flagellin glycosylation is a necessary modification allowing flagellar assembly, bacterial motility, colonization, and hence virulence for the gastrointestinal pathogen Helicobacter pylori [Josenhans, C., Vossebein, L., Friedrich, S., and Suerbaum, S. (2002) FEMS Microbiol. Lett., 210, 165-172; Schirm, M., Schoenhofen, I.C., Logan, S.M., Waldron, K.C., and Thibault, P. (2005) Anal. Chem., 77, 7774-7782]. A causative agent of gastric and duodenal ulcers, H. pylori, heavily modifies its flagellin with the sialic acid-like sugar 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-alpha-l-manno-nonulosonic acid (pseudaminic acid). Because this sugar is unique to bacteria, its biosynthetic pathway offers potential as a novel therapeutic target. We have identified six H. pylori enzymes, which reconstitute the complete biosynthesis of pseudaminic acid, and its nucleotide-activated form CMP-pseudaminic acid, from UDP-N-acetylglucosamine (UDP-GlcNAc). The pathway intermediates and final product were identified from monitoring sequential reactions with nuclear magnetic resonance (NMR) spectroscopy, thereby confirming the function of each biosynthetic enzyme. Remarkably, the conversion of UDP-GlcNAc to CMP-pseudaminic acid was achieved in a single reaction combining six enzymes. This represents the first complete in vitro enzymatic synthesis of a sialic acid-like sugar and sets the groundwork for future small molecule inhibitor screening and design. Moreover, this study provides a strategy for efficient large-scale synthesis of novel medically relevant bacterial sugars that has not been attainable by chemical methods alone.

N-Glycosylation of proteins is recognized as one of the most common posttranslational modifications in eukaryotes. To date, most glycomics techniques are limited to examining eukaryotic pathways. Technologies capable of characterizing newly described N-linked glycosylation systems in bacteria from biologically relevant samples in an accurate, rapid, and cost-effective manner are needed. In this paper, a new glycomics strategy, based on the combination of nonspecific proteolytic digestion and permethylation, was devised that can be used for both eukaryotic and bacterial glycoproteins. Eukaryotic glycoproteins were digested with Pronase E with a higher ratio (enzyme/protein, 2:1-3:1) and a longer reaction time (48-72 h). The Asn-glycans were then purified using porous graphitic carbon cartridges followed by permethylation. The mass spectrometric data indicated that hydroxyl groups were methylated, while the amino group in asparagine underwent beta-elimination. Both modifications were evident from an increase of 111 Da in the molecular masses of permethylated Asn-glycans compared to the corresponding free oligosaccharides. NMR spectroscopy corroborated these results by showing that the mass difference resulted from beta-elimination of the free amino group in the asparagine residue. The method was validated with the characterization of the N-linked glycoproteins in total protein extracts from the bacterium Campylobacter jejuni 11168H. In addition to detecting the Asn-linked bacterial heptasaccharide, we also observed an unexpected free heptasaccharide intermediate that required a functional glycosylation pathway. These results demonstrate the usefulness of this method for screening N-linked glycans expressed by eukaryotes and bacteria and for detecting novel intermediates of N-linked glycosylation pathways.

High-speed counter-current chromatography (HSCCC) methods were developed for the study of induced defense metabolites in wheat (Triticum aestivum) against powdery mildew (Blumeria graminis f. sp. tritici). A single HSCCC purification step afforded extraction of mg-quantities of an induced compound with antifungal activity. Subsequent LC-MS and NMR analyses have led to the characterization of 5,6-O-methyl trans-aconitic acid, the first such report of this compound in a plant species. The inducible nature of aconitic acid was evidenced by comparing the metabolite profiles of leaf extracts from plants treated or not with soluble silicon and infected or not with powdery mildew. In a second step, dual-mode HSCCC was used to enhance the separation of other forms of aconitic acid in wheat. Based on these results, it was concluded that 5,6-O-methyl trans-aconitic acid plays an important role as a defense molecule in wheat plants and that HSCCC is a powerful separation method for purifying such compounds from complex plant-pathogen interactions.

Bacterial genome sequencing has provided a wealth of genetic data. However, the definitive functional characterization of hypothetical open reading frames and novel biosynthetic genes remains challenging. This is particularly true for genes involved in protein glycosylation because the isolation of their glycan moieties is often problematic. We have developed a focused metabolomics approach to define the function of flagellin glycosylation genes in Campylobacter jejuni 81-176. A capillary electrophoresis-electrospray mass spectrometry and precursor ion scanning method was used to examine cell lysates of C. jejuni 81-176 for sugar nucleotides. Novel nucleotide-activated intermediates of the pseudaminic acid (Pse5NAc7NAc) pathway and its acetamidino derivative (PseAm) were found to accumulate within select isogenic mutants, and use of a hydrophilic interaction liquid chromatography-mass spectrometry method permitted large scale purifications of the intermediates. NMR with cryo probe (cold probe) technology was utilized to complete the structural characterization of microgram quantities of CMP-5-acetamido-7-acetamidino-3,5,7,9-tetradeoxy-L-glycero-alpha-L-manno-nonulosonic acid (CMP-Pse5NAc7Am), which is the first report of Pse modified at C7 with an acetamidino group in Campylobacter, and UDP-2,4-diacetamido-2,4,6-trideoxy-alpha-D-glucopyranose, which is a bacillosamine derivative found in the N-linked proteinglycan. Using this focused metabolomics approach, pseB, pseC, pseF, pseI, and for the first time pseA, pseG, and pseH were found to be directly involved in either the biosynthesis of CMP-Pse5NAc7NAc or CMP-Pse5NAc7Am. In contrast, it was shown that pseD, pseE, Cj1314c, Cj1315c, Cjb1301, Cj1334, Cj1341c, and Cj1342c have no role in the CMP-Pse5NAc7NAc or CMP-Pse5NAc7Am pathways. These results demonstrate the usefulness of this approach for targeting compounds within the bacterial metabolome to assign function to genes, identify metabolic intermediates, and elucidate novel biosynthetic pathways.

Helicobacter pylori and Campylobacter jejuni have been shown to modify their flagellins with pseudaminic acid (Pse), via O-linkage, while C. jejuni also possesses a general protein glycosylation pathway (Pgl) responsible for the N-linked modification of at least 30 proteins with a heptasaccharide containing 2,4-diacetamido-2,4,6-trideoxy-alpha-D-glucopyranose, a derivative of bacillosamine. To further define the Pse and bacillosamine biosynthetic pathways, we have undertaken functional characterization of UDP-alpha-D-GlcNAc modifying dehydratase/aminotransferase pairs, in particular the H. pylori and C. jejuni flagellar pairs HP0840/HP0366 and Cj1293/Cj1294, as well as the C. jejuni Pgl pair Cj1120c/Cj1121c using His(6)-tagged purified derivatives. The metabolites produced by these enzymes were identified using NMR spectroscopy at 500 and/or 600 MHz with a cryogenically cooled probe for optimal sensitivity. The metabolites of Cj1293 (PseB) and HP0840 (FlaA1) were found to be labile and could only be characterized by NMR analysis directly in aqueous reaction buffer. The Cj1293 and HP0840 enzymes exhibited C6 dehydratase as well as a newly identified C5 epimerase activity that resulted in the production of both UDP-2-acetamido-2,6-dideoxy-beta-L-arabino-4-hexulose and UDP-2-acetamido-2,6-dideoxy-alpha-D-xylo-4-hexulose. In contrast, the Pgl dehydratase Cj1120c (PglF) was found to possess only C6 dehydratase activity generating UDP-2-acetamido-2,6-dideoxy-alpha-D-xylo-4-hexulose. Substrate-specificity studies demonstrated that the flagellar aminotransferases HP0366 and Cj1294 utilize only UDP-2-acetamido-2,6-dideoxy-beta-L-arabino-4-hexulose as substrate producing UDP-4-amino-4,6-dideoxy-beta-L-AltNAc, a precursor in the Pse biosynthetic pathway. In contrast, the Pgl aminotransferase Cj1121c (PglE) utilizes only UDP-2-acetamido-2,6-dideoxy-alpha-D-xylo-4-hexulose producing UDP-4-amino-4,6-dideoxy-alpha-D-GlcNAc (UDP-2-acetamido-4-amino-2,4,6-trideoxy-alpha-D-glucopyranose), a precursor used in the production of the Pgl glycan component 2,4-diacetamido-2,4,6-trideoxy-alpha-D-glucopyranose.

Recently, the CPS biosynthetic loci for several strains of Campylobacter jejuni were sequenced and revealed evidence for multiple mechanisms of structural variation. In this study, the CPS structure for the HS:1 serostrain of C. jejuni was determined using mass spectrometry and NMR at 600 MHz equipped with an ultra-sensitive cryogenically cooled probe. Analysis of CPS purified using a mild enzymatic method revealed a teichoic acid-like [-4)-alpha-d-Galp-(1-2)-(R)-Gro-(1-P](n), repeating unit, where Gro is glycerol. Two branches at C-2 and C-3 of galactose were identified as beta-d-fructofuranoses substituted at C-3 with CH(3)OP(O)(NH(2))(OR) groups. Structural heterogeneity was due to nonstoichiometric glycosylation at C-3 of galactose and variable phosphoramidate groups. Identical structural features were found for cell-bound CPS on intact cells using proton homonuclear and (31)P heteronuclear two-dimensional HR-MAS NMR at 500 MHz. In contrast, spectroscopic data acquired for hot water/phenol purified CPS was complicated by the hydrolysis and subsequent loss of labile groups during extraction. Collectively, the results of this study established the importance of using sensitive isolation techniques and HR-MAS NMR to examine CPS structures in vivo when labile groups are present. This study uncovered how incorporation of variable O-methyl phosphoramidate groups on nonstoichiometric fructose branches is used in C. jejuni HS:1 as a strategy to produce a highly complex polysaccharide from its small CPS biosynthetic locus and a limited number of sugars.

UDP-N-acetyl-L-fucosamine is a precursor to l-fucosamine in the lipopolysaccharide of Pseudomonas aeruginosa serotype O11 and the capsule of Staphylococcus aureus type 5. We have demonstrated previously the involvement of three enzymes, WbjB, WbjC, and WbjD, in the biosynthesis of UDP-2-acetamido-2,6-dideoxy-L-galactose or UDP-N-acetyl-L-fucosamine (UDP-l-FucNAc). An intermediate compound from the coupled-reaction of WbjB-WbjC with the initial substrate UDP-2-acetamido-2-deoxy-alpha-D-glucose or UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) was purified, and the structure was determined by NMR spectroscopy to be UDP-2-acetamido-2,6-dideoxy-L-talose (UDP-L-PneNAc). WbjD could then convert this intermediate into a new product with the same mass, consistent with a C-2 epimerization reaction. Those results led us to propose a pathway for the biosynthesis of UDP-L-FucNAc; however, the exact enzymatic activity of each of these proteins has not been defined. Here, we describe a fast protein liquid chromatography (FPLC)-based anion-exchange procedure, which allowed the separation and purification of the products of C-2 epimerization due to WbjD. Also, the application of a cryogenically cooled probe in NMR spectrometry offers the greatest sensitivity for determining the structures of minute quantities of materials, allowing the identification of the final product of the pathway. Our results showed that WbjB is bifunctional, catalyzing firstly C-4, C-6 dehydration and secondly C-5 epimerization in the reaction with the substrate UDP-D-GlcNAc, producing two intermediates. WbjC is also bifunctional, catalyzing C-3 epimerization of the second intermediate followed by reduction at C-4. The FPLC-based procedure provided good resolution of the final product of WbjD reaction from its epimer/substrate UDP-l-PneNAc, and the use of the cryogenically cooled probe in NMR revealed unequivocally that the final product is UDP-L-FucNAc.

ABSTRACT In this study, cucumber plants (Cucumis sativus) expressing induced resistance against powdery mildew (caused by Podosphaera xanthii) were infiltrated with inhibitors of cinnamate 4-hydroxylase, 4-coumarate:CoA ligase (4CL), and chalcone synthase (CHS) to evaluate the role of flavonoid phytoalexin production in induced disease resistance. Light and transmission electron microscopy demonstrated ultrastructural changes in inhibited plants, and biochemical analyses determined levels of CHS and beta-glucosidase enzyme activity and 4CL protein accumulation. Our results showed that elicited plants displayed a high level of induced resistance. In contrast, down regulation of CHS, a key enzyme of the flavonoid pathway, resulted in nearly complete suppression of induced resistance, and microscopy confirmed the development of healthy fungal haustoria within these plants. Inhibition of 4CL ligase, an enzyme largely responsible for channeling phenylpropanoid metabolites into the lignin pathway, had little effect on induced disease resistance. Biochemical analyses revealed similar levels of 4CL protein accumulation for all treatments, suggesting no alterations of nontargeted functions within inhibited plants. Collectively, the results of this study support the idea that induced resistance in cucumber is largely correlated with rapid de novo biosynthesis of flavonoid phytoalexin compounds.

ABSTRACT Although several reports underscore the importance of silicon (Si) in controlling Magnaporthe grisea on rice, no study has associated this beneficial effect with specific mechanisms of host defense responses against this fungal attack. In this study, however, we provide evidence that higher levels of momilactone phytoalexins were found in leaf extracts from plants inoculated with M. grisea and amended with silicon (Si(+)) than in leaf extracts from inoculated plants not amended with silicon (Si(-) ) or noninoculated Si(+) and Si(-) plants. On this basis, the more efficient stimulation of the terpenoid pathway in Si(+) plants and, consequently, the increase in the levels of momilactones appears to be a factor contributing to enhanced rice resistance to blast. This may explain the lower level of blast severity observed on leaves of Si(+) plants at 96 h after inoculation with M. grisea. The results of this study strongly suggest that Si plays an active role in the resistance of rice to blast rather than the formation of a physical barrier to penetration by M. grisea.

Extraction of cucumber leaf tissue expressing induced resistance against powdery mildew fungi revealed the presence of two new major C-glycosyl flavonoid products: vitexin-6-(4-hydroxy-1-ethylbenzene) (cucumerin A, 1) and isovitexin-8-(4-hydroxy-1-ethylbenzene) (cucumerin B, 2). In addition, the known C-glycosyl flavonoids apigenin-8-C-beta-D-glucopyranoside (vitexin, 3), apigenin-6-C-beta-D-glucopyranoside (isovitexin, 4), luteolin-8-C-beta-D-glucopyranoside (orientin, 5), and luteolin-6-C-beta-D-glucopyranoside (isoorientin, 6), as well as 4-hydroxycinnamic acid (p-coumaric acid, 7) and its methyl ester (p-came, 8), were found in higher quantities within resistant plants. The structures of 1-8 were elucidated using spectroscopic methods and unambiguously confirmed for 3-8 using co-chromatography experiments with authentic standards. On the basis of the results of this study and the reported biological activities of C-glycosyl flavonoids, these compounds would play a vital role in the defense strategy of this species by acting as phytoalexins.

Insertional mutagenesis was applied for the first time to a fungal biocontrol agent, Pseudozyma flocculosa, in an attempt to obtain mutants with altered antagonistic properties. Transformants were obtained via DNA-mediated transformation. Molecular analyses of the transformants revealed that multiple copies of the plasmid were integrated in tandem at one to many chromosomal loci. The transformants were screened for their biocontrol properties using standard bioassays, and the 160 tested transformants were classified into four groups: group I mutants (22 transformants) showed a stronger antagonistic effect than the wild type (WT) while those of group II (107 transformants) had a comparable antagonistic effect; group III mutants (17 transformants) had a decreased antagonistic effect relative to WT and group IV mutants (14 transformants) had lost their biocontrol properties. Culture extracts of the mutants (group IV) and WT were analyzed and compared for the presence of active metabolites which were then separated by solid-phase extraction and purified using conventional methods. Nuclear magnetic resonance experiments and analytical studies on a metabolite specifically produced by the WT revealed the presence of 2-(2',4'-diacetoxy-5'-carboxy-pentanoyl) octadecyl cellobioside (flocculosin), a novel glycolipid with strong antifungal properties; the production of this compound would account for the biocontrol activity of P. flocculosa.