Antigenic polysaccharides of bacteria: 40. The structures of O-specific polysaccharides from <Emphasis Type="Italic">Shigella dysenteriae</Emphasis> types 3 and 9 and <Emphasis Type="Italic">S. boydii</Emphasis> type 4 revised by NMR spectroscopy

The reported structures of O-specific polysaccharides from three type strains of Shigella bacteria were corrected by modern NMR techniques. The revisions concerned the configuration of the O-glycoside linkage (S. dysenteriae type 3, structure 1), the positions of monosaccharide residue glycosylation and acetalation by pyruvic acid (S. dysenteriae type 9, structure 2), and the attachment position of the side monosaccharide chain (S. boydii type 4, structure 3).

Open image in new window

     

Bacterial antigen polysaccharides. 43. Structure for the O-specific polysaccharide of <Emphasis Type="Italic">Providencia alcalifaciens</Emphasis> O46

Acidic O-specific polysaccharide containing D-glucose, D-glucuronic acid, L-fucose, and 2-acetamido-2-deoxy-D-glucose was obtained by mild acid degradation of lipopolysaccharide from Providencia alcalifaciens O46. The following structure of the hexasaccharide repeating unit of the O-specific polysaccharide was established using methylation analysis along with ¹H and ¹³C NMR spectroscopy, including 2D ¹H, ¹H-COSY, TOCSY, ROESY, ¹H, ¹³C-HSQC, and HMQC-TOCSY experiments:

Open image in new window

     

Antigenic polysaccharides of bacteria: 42. Structures of O-polysaccharides from two Shigella dysenteriae type 8 strains

The structure of the O-polysaccharide (O-antigen) from Shigella dysenteriae type 8 bacteria (strain 599) was corrected using modern NMR techniques (structure 1). The revisions concerned the position of the Glc residue (in the main, but not the side chain), the site of its substitution, and the configuration of the O-glycoside linkage of the GlcNAc residue. The S. dysenteriae type 8 bacterium (strain G1221), the second investigated representative, was found to produce another structural variant of the O-polysaccharide. It contains GlcNAc instead of the Glc residue in the main chain (structure 2). This data may lead to approval of division of S. dysenteriae type 8 into two subtypes:      

A completed structure of the O-polysaccharide from <Emphasis Type="Italic">Shigella dysenteriae</Emphasis> type 10

The structure of the O-specific polysaccharide from Shigella dysenteriae type 10, which has been reported previously in Bioorganic chemistry (1977, vol.3, pp. 1219–1225), is refined: →2)-β-D-Manp-(1→3)-α-D-ManpNAc-(1→3)-β-L-Rhap-(1→4)-α-D-GlcpNAc-(1→.     

Heterologous expression of <Emphasis Type="Italic">Shigella dysenteriae</Emphasis> serotype 1 O-antigen analog in <Emphasis Type="Italic">Escherichia coli</Emphasis> K-12 W3110 by transferring a minimum number of genes involved in O-polysaccharide biosynthesis

Objective

To heterologously produce the Shigella dysenteriae serotype 1 O-polysaccharide (O-PS, O-antigen) in Escherichia coli by transferring the minimum number of genes instead of the entire O-PS gene cluster.

Results

The three glycosyltransferase genes (rfbR, rfbQ and rfp) responsible for the formation of the O-repeat unit were introduced into E. coli K-12 W3110 to synthesize S. dysenteriae 1 O-PS. The specific O-antigen ladder type with different chain lengths of O-repeat units was observed in the recombinant E. coli strain by SDS-PAGE silver staining and western blotting using S. dysenteriae 1 lipopolysaccharide antiserum. Analysis by mass spectrometry and ion chromatography suggested generation of the specific S. dysenteriae 1 O-repeat unit structure with an extra glucose residue attached.

Conclusions

Recombinant E. coli expressing specific glycosyltransferase genes can generate the O-PS of S. dysenteriae 1 and might be able to synthesize heterologous O-antigens of various pathogenic bacteria for vaccine preparation.

     

Conformational analysis of cyclic dipeptides: I. , , , and

Conformational analysis of two pairs of synthetic cyclodipeptides formed by interaction of both side chain functional groups ( , and ) and of the main and side chains ( , and ) was achieved by the method of molecular mechanics. The energetically optimal conformational states of the molecules under study were determined. It was shown that the conformational motility of cyclic system of the compounds under study depends on the relative arrangement of the amide groups and the number of atoms in the cycle.     

Structure of acidic o-specific polysaccharide from the marine bacterium <Emphasis Type="Italic">Cellulophaga baltica</Emphasis>

The structure of an acidic O-specific polysaccharide from the marine bacterium Cellulophaga baltica was established by chemical methods and NMR spectroscopy. The polysaccharide was shown to consist of repeating tetrasaccharide units containing two mannose residues, one N-acetyl-D-glucosamine residue, and one D-glucuronic acid residue. An O-acetyl group was also found in the polysaccharide in nonstoichiometric amount. The polysaccharide had the following structure:

Open image in new window

     

The structure of the glycerophosphate-containing O-specific polysaccharide from <Emphasis Type="Italic">Escherichia coli</Emphasis> O130

A phosphorylated O-specific polysaccharide was obtained by mild acidic degradation of the lipopolysaccharide from the enteric bacterium Escherichia coli O130 and characterized by the methods of chemical analysis, including dephosphorylation and ¹H and ¹³C NMR spectroscopy. The polysaccharide was shown to be composed of branched tetrasaccharide repeating units containing two N-acetyl-D-galactosamine residues, D-galactose, D-glucose, and glycerophosphate residues (one of each). The polysaccharide has the following structure, which is unique among the known bacterial polysaccharides:

Open image in new window

     

Pancreatic lipase inhibitory activity of monogalactosyldiacylglycerols isolated from the freshwater microalga <Emphasis Type="Italic">Chlorella sorokiniana</Emphasis>

Chemical investigation of the freshwater microalga Chlorella sorokiniana led to the isolation of a monogalactosyldiacylglycerol (MGDG)-rich fraction possessing dose-dependent inhibitory activity against pancreatic lipase activity. The MGDG-rich fraction contains 12 MGDGs identified by LC/HRMS analysis. Among them, three MGDGs were new compounds, namely, (2S)-1-O-(7Z,10Z-hexadecadienoyl)-2-O-(7Z,10Z,13Z-hexadecatrienoyl)-3-O-β-D-galactopyranosylglycerol (1), (2S)-1-O-linoleoyl-2-O-(7Z,10Z-hexadecadienoyl)-3-O-β-D-galactopyranosylglycerol (6), and (2S)-1-O-oleoyl-2-O-(7Z,10Z-hexadecadienoyl)-3-O-β-D-galactopyranosylglycerol (8). The major galactolipids were isolated by semipreparative HPLC and tested for their effect toward pancreatic lipase inhibitory activity. All the tested MGDGs showed significant reduction of pancreatic lipase activity indicating possible beneficial use for management of lipase-related disorders such as obesity.     

Increase of 1-hydroxy-2-naphthoic acid concentration as a cause of temporary cessation of growth for <Emphasis Type="Italic">Arthrobacter</Emphasis> sp. K3: Kinetic analysis

Consumption of 1-hydroxy-2-naphthoic acid by strain Arthrobacter sp. K3 was investigated. Drastic increase in the substrate concentration in flow culture was shown to induce the lag phase of growth in case the initial substrate concentration in the medium was not saturating; the culture originally saturated with the substrate (S

Open image in new window

K _S ) was resistant to the concentration increase. In accordance with the constructed kinetic model, lag phase results from an accumulation of intermediates in the metabolic system.

     

Structural relationships between genetically closely related O-antigens of <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Shigella</Emphasis> spp.

Gene clusters for biosynthesis of 24 of 34 basic O-antigen forms of Shigella spp. are identical or similar to those of the genetically closely related bacterium Escherichia coli. For 18 of these relatedness was confirmed chemically by elucidation of the O-antigen (O-polysaccharide) structures. In this work, structures of the six remaining O-antigens of E. coli O32, O53, O79, O105, O183 (all related to S. boydii serotypes), and O38 (related to S. dysenteriae type 8) were established using ¹H and ¹³C NMR spectroscopy. They were found to be identical to the Shigella counterparts, except for the O32- and O38-polysaccharides, which differ in the presence of O-acetyl groups. The structure of the E. coli O105-related O-polysaccharide of S. boydii type 11 proposed earlier is revised. The contents of the O-antigen gene clusters of the related strains of E. coli and Shigella spp. and different mechanisms of O-antigen diversification in these bacteria are discussed in view of the O-polysaccharide structures established. These data illustrate the value of the O-antigen chemistry and genetics for elucidation of evolutionary relationships of bacteria.     

Mutational analysis of substrate specificity in a <Emphasis Type="Italic">Citrus paradisi</Emphasis> flavonol 3-<Emphasis Type="Italic">O</Emphasis>-glucosyltransferase

Citrus paradisi 3-O-glucosyltransferase (Cp3GT, Genbank Protein ID: ACS15351) and Citrus sinensis 3-O-glucosyltransferase (Cs3GT, Genbank Protein ID: AAS00612.2) share 95% amino acid sequence identity. Cp3GT was previously established as a flavonol-specific 3-O-glucosyltransferase by direct enzymatic analysis. Cs3GT is annotated as a flavonoid-3-O-glucosyltransferase and predicted to use anthocyanidins as substrates based on gene expression analysis correlated with the accumulation of anthocyanins in C. sinensis cv. Tarocco, a blood orange variety. Mutant enzymes in which amino acids found in Cs3GT were substituted for position equivalent residues in Cp3GT were generated, heterologously expressed in yeast, and characterized for substrate specificity. Structure–function relationships were investigated for wild type and mutant glucosyltransferases by homology modelling using a crystallized Vitis vinifera anthocyanidin/flavonol 3-O-GT (PDB: 2C9Z) as template and subsequent substrate docking. All enzymes showed similar patterns for optimal temperature, pH, and UDP/metal ion inhibition with differences observed in kinetic parameters. Although changes in the activity of the mutant proteins as compared to wild type were observed, cyanidin was never efficiently accepted as a substrate.     

Synthesis of oligosaccharide fragments of mannan from <Emphasis Type="Italic">Candida albicans</Emphasis> cell wall and their BSA conjugates

3-Aminopropyl glycosides of α-D-mannopyranosyl-(1→2)-α-D-mannopyranosyl-(1→2)-α-D-mannopyranosyl-(1→2)-α-D-mannopyranose, α-D-mannopyranosyl-(1→3)-α-D-mannopyranosyl-(1→2)-α-D-mannopyranosyl-(1→2)-α-D-mannopyranose, and α-D-mannopyranosyl-(1→2)-[α-D-mannopyranosyl-(1→3)]-α-D-mannopyranosyl-(1→2)-α-D-mannopyranosyl-(1→2)-α-D-mannopyranose were efficiently synthesized starting from ethyl 2-O-acetyl(benzoyl)-3,4,6-tri-O-benzyl-1-thio-α-D-mannopyranoside, ethyl 4,6-di-O-benzyl-2-O-benzoyl-1-thio-α-D-mannopyranoside, ethyl 4,6-di-O-benzyl-2,3-di-O-benzoyl-1-thio-α-D-mannopyranoside, and 2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl bromide. The oligosaccharide chains synthesized correspond to the three structural types of side chains of mannan from Candida albicans cell wall. A conjugate of the third pentasaccharide with bovine serum albumin was prepared using the squarate method.     

Two new steroid glycosides from the Far East starfish <Emphasis Type="Italic">Hippasteria kurilensis</Emphasis>

Two new steroid glycosides were isolated from the Far East starfish Hippasteria kurilensis collected in the Sea of Okhotsk. They were characterized as (22E,24R)-3-O-(2-O-methyl-β-D-xylopyranosyl)-24-O-[2-O-methyl-β-D-xylopyranosyl-(1→5)-α-L-arabinofuranosyl]-5α-cholest-22-ene-3β,4β,6α,7α,8,15β,24-heptaol (kurilensoside I) and (24S)-3-O-(2-O-methyl-β-D-xylopyranosyl)-24-O-(α-L-arabinofuranosyl)-5α-cholestane-3β,4β,6β,15α,24-pentaol (kurilensoside J). In addition, the earlier known glycosides linkosides F and L1, leviusculoside G, forbeside L, desulfated echinasteroside, and granulatoside A were isolated and identified. The structures of the new compounds were established with the help of two-dimentional NMR spectroscopy and mass- spectrometry.     

Effects of drought stress on the antioxidant system,osmolytes and secondary metabolites of <Emphasis Type="Italic">Saposhnikovia divaricata</Emphasis> seedlings

Saposhnikovia divaricata (Turcz.) Schischk is a traditional herb of East Asia. Bioactive chromones and volatile components in its roots are known to exhibit pharmacological functions. However, limited information is available on the drought resistance of this herb. In this study, potted Saposhnikovia divaricata seedlings were subjected to a progressive drought stress of 20 days by withholding water followed by twice rehydration, which resulted in some physiological, biochemical and secondary metabolite responses as well as drought acclimatization. A decline in leaf water content but increase in electrolyte leakage, malondialdehyde (MDA), hydrogen peroxide (H₂O₂), glutathione (GSH), proline, soluble sugar, prim-O-glucosylcimifugin and 4′-O-β-d-glucosyl-5-O-methylvisamminol content was observed. After rehydration, some of the indices recovered except proline, soluble sugar, prim-O-glucosylcimifugin and 4′-O-β-d-glucosyl-5-O-methylvisamminol content. Moreover, mild (day 8), moderate (days 12–16) and severe (day 20) drought phases were identified. A total of 18 volatile components were identified by GC–MS under different drought phases, of which aromatic alcohols (42.02%) and sesquiterpenes (37.35%) were the major components. The characteristic component named falcarinol was decreased by severe drought stress. This study demonstrated that Saposhnikovia divaricata had strong drought acclimatization, and resisted drought by activating the antioxidant system and accumulating osmolytes. In addition, moderate and severe drought stress promoted bioactive secondary metabolites prim-O-glucosylcimifugin and 4′-O-β-d-glucosyl-5-O-methylvisamminol accumulation. Severe drought stress reduced falcarinol relative content, which provided an insight for improving the quantity of Saposhnikovia divaricata bioactive components.     

Viral metagenomics analysis of feces from coronary heart disease patients reveals the genetic diversity of the <Emphasis Type="Italic">Microviridae</Emphasis>

Recent studies have declared that members of the ssDNA virus family Microviridae play an important role in multiple environments, as they have been found taking a dominant position in the human gut. The aim of this study was to analyze the overall composition of the gut virome in coronary heart disease (CHD) patients, and try to discover the potential link between the human gut virome and CHD. Viral metagenomics methods were performed to detect the viral sequences in fecal samples collected from CHD inpatients and healthy persons as controls. We present the analysis of the virome composition in these CHD patients and controls. Our data shows that the virome composition may be linked to daily living habits and the medical therapy of CHD. Virgaviridae and Microviridae were the two dominant types of viruses found in the enteric virome of CHD patients. Fourteen divergent viruses belonging to the family Microviridae were found, twelve of which were grouped into the subfamily Gokushovirinae, while the remaining two strains might represent two new subfamilies within Microviridae, according to the phylogenetic analysis. In addition, the genomic organization of these viruses has been characterized.

Open image in new window

     

Identification and functional characterization of DzS3GT,a cytoplasmic glycosyltransferase catalyzing biosynthesis of diosgenin 3-<Emphasis Type="Italic">O</Emphasis>-glucoside in <Emphasis Type="Italic">Dioscorea zingiberensis</Emphasis>

Dioscorea plants produce pharmaceutical diosgenin, which usually exists in plants in the form of saponins and has been a starting material for the production of steroids over seven decades. The first step of steroidal saponin biosynthesis from the corresponding aglycone is glycosylation by 3-O-sterol glycosyltransferase (S3GT), transferring the glycosyl from a sugar donor to the 3-OH position of the aglycone. In this study, a DzS3GT gene from Dioscorea zingiberensis was cloned and expressed in Escherichia coli, and the recombinant DzS3GT protein showed 3-O-sterol glycosyltransferase activity in vitro. Subcellular localization analysis revealed that the DzS3GT protein is located in the cytoplasm in rice protoplasts. The tissue profiles of DzS3GT differ from those reported SGT genes. DzS3GT is expressed strongly in leaves and very weakly in stems. The diosgenin 3-O-glucoside (trillin) content is much higher in the leaves than in other organs. The specificity of gene expression and saponins accumulation suggest that the biosynthesis of trillin may occur mainly in the leaves of D. zingiberensis. This is the first report of the cloning and biochemical characterization of a glycosyltransferase gene involved in the biosynthesis of diosgenin 3-O-glucoside in Dioscorea plants. In addition, the study provides a potential relevance to the biosynthesis and transport mechanism of steroidal saponins in Dioscorea plants.     

Molecular cloning and expression analysis of two <Emphasis Type="Italic">FAD2</Emphasis> genes from chia (<Emphasis Type="Italic">Salvia hispanica</Emphasis>)

FATTY ACID DESATURASE 2 (FAD2, EC 1.3.1.35), also known as delta-12 oleate desaturase, is a key enzyme for linoleic acid and α-linolenic acid biosynthesis. Chia (Salvia hispanica) seeds contain the highest known proportion of α-linolenic acid in any plant sources. In this study, two full-length FAD2 genes, named as ShFAD2-1 and ShFAD2-2, were isolated from S. hispanica based on RACE method. Both ShFAD2-1 and ShFAD2-2 proteins possess strong transmembrane helices, three histidine motifs and a C-terminal ER-located signal (YNNKL). Phylogenetic analysis showed that both ShFAD2-1 and ShFAD2-2 are grouped with constitutive plant FAD2s. Heterologous expression in Saccharomyces cerevisiae indicated that ShFAD2-1 and ShFAD2-2 genes both encode a bio-functional delta-12 oleate desaturase. ShFAD2-2 was mainly expressed in flowers and early-stage seeds while ShFAD2-1 expression was almost constitutive in different organs. qRT-PCR results demonstrated that ShFAD2-1 and ShFAD2-2 show a cold-induced and heat-repressed expression pattern, whereas they also were differentially up-regulated or repressed by other abiotic stresses. This is the first cloning and function characterization of FAD2 from S. hispanica, which can provide insights into molecular mechanism of high ALA traits of S. hispanica and enrich our understanding of the roles of FAD2 genes in various abiotic stresses.