David J. McNally

Silicon Enhances the Accumulation of Diterpenoid Phytoalexins in Rice: A Potential Mechanism for Blast Resistance

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.

Functional characterization of dehydratase/aminotransferase pairs from Helicobacter and Campylobacter: enzymes distinguishing the pseudaminic acid and bacillosamine 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-α-d-glucopyranose, a derivative of bacillosamine. To further define the Pse and bacillosamine biosynthetic pathways, we have undertaken functional characterization of UDP-α-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 His6-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-β-l-arabino-4-hexulose and UDP-2-acetamido-2,6-dideoxy-α-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-α-d-xylo-4-hexulose. Substrate-specificity studies demonstrated that the flagellar aminotransferases HP0366 and Cj1294 utilize only UDP-2-acetamido-2,6-dideoxy-β-l-arabino-4-hexulose as substrate producing UDP-4-amino-4,6-dideoxy-β-l-AltNAc, a precursor in the Pse biosynthetic pathway. In contrast, the Pgl aminotransferase Cj1121c (PglE) utilizes only UDP-2-acetamido-2,6-dideoxy-α-d-xylo-4-hexulose producing UDP-4-amino-4,6-dideoxy-α-d-GlcNAc (UDP-2-acetamido-4-amino-2,4,6-trideoxy-α-d-glucopyranose), a precursor used in the production of the Pgl glycan component 2,4-diacetamido-2,4,6-trideoxy-α-d-glucopyranose.

Functional Characterization of the Flagellar Glycosylation Locus in Campylobacter jejuni 81–176 Using a Focused Metabolomics Approach

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-α-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-α-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.

Targeted metabolomics analysis of Campylobacter coli VC167 reveals legionaminic acid derivatives as novel flagellar glycans.

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.

Commonality and Biosynthesis of the O-Methyl Phosphoramidate Capsule Modification in Campylobacter jejuni

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 β-d-GalfNAc residue, whereas Cj1422 transfers the MeOPN to C-4 of d-glycero-α-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.

Flagellar glycosylation in Clostridium botulinum

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‐α‐d‐galacto‐nonulosonic acid, (αLeg5GluNMe7Ac). 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.

Insertional mutagenesis of a fungal biocontrol agent led to discovery of a rare cellobiose lipid with antifungal activity.

ABSTRACT 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.

The HS:1 serostrain of Campylobacter jejuni has a complex teichoic acid-like capsular polysaccharide with nonstoichiometric fructofuranose branches and O-methyl phosphoramidate groups

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)‐α‐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 β‐d‐fructofuranoses substituted at C‐3 with CH3OP(O)(NH2)(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 31P 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.

Study of free oligosaccharides derived from the bacterial N-glycosylation pathway

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.

Suppression of induced resistance in cucumber through disruption of the flavonoid pathway

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.