Functional characterization of the initiation enzyme of S-layer glycoprotein glycan biosynthesis in Geobacillus stearothermophilus NRS 2004/3a.

The glycan chain of the S-layer glycoprotein of Geobacillus stearothermophilus NRS 2004/3a is composed of repeating units [-->2)-alpha-l-Rhap-(1-->3)-beta-l-Rhap-(1-->2)-alpha-l-Rhap-(1-->], with a 2-O-methyl modification of the terminal trisaccharide at the nonreducing end of the glycan chain, a core saccharide composed of two or three alpha-l-rhamnose residues, and a beta-d-galactose residue as a linker to the S-layer protein. In this study, we report the biochemical characterization of WsaP of the S-layer glycosylation gene cluster as a UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase that primes the S-layer glycoprotein glycan biosynthesis of Geobacillus stearothermophilus NRS 2004/3a. Our results demonstrate that the enzyme transfers in vitro a galactose-1-phosphate from UDP-galactose to endogenous phosphoryl-polyprenol and that the C-terminal half of WsaP carries the galactosyltransferase function, as already observed for the UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase WbaP from Salmonella enterica.

The structure of the core-O-chain linkage region of the lipopolysaccharide from Bordetella hinzii.

Linkage region between core and the O-chain of the lipopolysaccharide from Bordetella hinzii has been analyzed by NMR and MS analysis of the products, obtained by anhydrous HF treatment or consecutive ammonia and AcOH treatment of the LPS. The following structure of this region was deduced from the experimental results: [structure: see text] This structure is identical to the structure of the respective region of Bordetella parapertussis LPS. Polysaccharide part (PS) consists of not more than 15 2,3-diacetamido-2,3-dideoxyhexuronamides, methylated at the only hydroxyl group of the non-reducing terminal monosaccharide.

O-Acetylation in the O-specific polysaccharide isolated from Shigella flexneri serotype 2a.

Shigella flexneri causes diarrheal diseases especially in infants and children in developing countries. Modifications of the lipopolysaccharide (LPS) molecule, like bacteriophage-mediated glucosylation and acetylation of the O-specific chain (O-SP), are important for the LPS antigenicity and consequently for the immunogenicity of the polysaccharide-based vaccines against shigellosis. Here, we report the degree of O-acetylation and the localisation of O-acetyl groups and side-chain glucose substitution in the O-SP (scheme) in different preparations of S.

Structure and serological analysis of the Hafnia alvei 481-L O-specific polysaccharide containing phosphate in the backbone chain.

The lipopolysaccharide was extracted from cells of Hafnia alvei 481-L bacterial strain and, after mild acid hydrolysis, the O-specific polysaccharide was isolated and characterised. On the basis of chemical analyses and NMR spectroscopic studies of the polysaccharide and oligosaccharides obtained after Smith degradation, or hydrogen fluoride treatment, it was found that the repeating unit of the O-specific polysaccharide is a phosphorylated hexasaccharide: [see text]. The biological repeating unit of the H.

The HS:19 serostrain of Campylobacter jejuni has a hyaluronic acid-type capsular polysaccharide with a nonstoichiometric sorbose branch and O-methyl phosphoramidate group.

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.

Lipopolysaccharides from Serratia marcescens possess one or two 4-amino-4-deoxy-L-arabinopyranose 1-phosphate residues in the lipid A and D-glycero-D-talo-oct-2-ulopyranosonic acid in the inner core region.

The carbohydrate backbones of the core-lipid A region were characterized from the lipopolysaccharides (LPSs) of Serratia marcescens strains 111R (a rough mutant strain of serotype O29) and IFO 3735 (a smooth strain not serologically characterized but possessing the O-chain structure of serotype O19). The LPSs were degraded either by mild hydrazinolysis (de-O-acylation) and hot 4 M KOH (de-N-acylation), or by hydrolysis in 2 % aqueous acetic acid, or by deamination. Oligosaccharide phosphates were isolated by high-performance anion-exchange chromatography.

Characterization of the lipopolysaccharide and beta-glucan of the fish pathogen Francisella victoria.

Lipopolysaccharide (LPS) and beta-glucan from Francisella victoria, a fish pathogen and close relative of highly virulent mammal pathogen Francisella tularensis, have been analyzed using chemical and spectroscopy methods. The polysaccharide part of the LPS was found to contain a nonrepetitive sequence of 20 monosaccharides as well as alanine, 3-aminobutyric acid, and a novel branched amino acid, thus confirming F. victoria as a unique species.

Carbohydrate-containing components of biofilms produced in vitro by some staphylococcal strains related to orthopaedic prosthesis infections.

The capacity of coagulase-negative staphylococci to colonize implanted medical devices is generally attributed to their ability to produce biofilms. Biofilm of the model strain of Staphylococcus epidermidis RP62A was shown to contain two carbohydrate-containing moieties, a linear poly-beta-(1-->6)-N-acetyl-D-glucosamine (PNAG) and teichoic acid. In the present study, we investigated several biofilm-producing staphylococci isolated from infected orthopaedic implants and characterized the composition of the laboratory-grown biofilms using chemical analysis and 1H nuclear magnetic resonance spectroscopy.

Complete structures of Bordetella bronchiseptica and Bordetella parapertussis lipopolysaccharides.

The structures of the lipopolysaccharide (LPS) core and O antigen of Bordetella bronchiseptica and Bordetella parapertussis are known, but how these two regions are linked to each other had not been determined. We have studied LPS from several strains of these microorganisms to determine the complete carbohydrate structure of the LPS. LPS was analyzed using different chemical degradations, NMR spectroscopy, and mass spectrometry.

Structural elucidation of the extracellular and cell-wall teichoic acids of Staphylococcus aureus MN8m, a biofilm forming strain.

Extracellular teichoic acid, an essential constituent of the biofilm produced by Staphylococcus epidermidis strain RP62A, is also an important constituent of the extracellular matrix of another biofilm producing strain, Staphylococcus aureus MN8m. The structure of the extracellular and cell wall teichoic acids of the latter strain was studied by NMR spectroscopy and capillary electrophoresis-mass spectrometry. Both teichoic acids were found to be a mixture of two polymers, a (1-->5)-linked poly(ribitol phosphate), substituted at the 4-position of ribitol residues with beta-GlcNAc, and a (1-->3)-linked poly(glycerol phosphate), partially substituted with the D-Ala at 2-position of glycerol residue.

H2BC: a new technique for NMR analysis of complex carbohydrates.

It is demonstrated that the H2BC NMR pulse sequence (J. Am. Chem.

Structural studies of the core region of Aeromonas salmonicida subsp. salmonicida lipopolysaccharide.

The core oligosaccharide structure of the in vivo derived rough phenotype of Aeromonas salmonicida subsp. salmonicida was investigated by a combination of compositional, methylation, CE-MS and one- and two-dimensional NMR analyses and established as the following: [carbohydrate: see text] where R=alpha-D-Galp-(1-->4)-beta-D-GalpNAc-(1--> or alpha-D-Galp-(1--> (approx. ratio 4:3).

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-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.

The structure of a polysaccharide isolated from Inonotus levis P. Karst. mushroom (Heterobasidiomycetes).

Inonotus levis biomass was extracted with 5% NaOH containing NaBH4, the insoluble material was discarded and the solution dialyzed. It was further treated with proteinase and the polymeric fraction isolated by gel chromatography. It contained mostly a polysaccharide of the following structure: where a non-reducing terminal glucuronic acid residue was present in about half of the molecules, making thus some of the chains acidic and others neutral.

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.

Application of electrospray ionization with Fourier transform ion cyclotron resonance mass spectrometry for structural screening of core oligosaccharides from lipopolysaccharides of the bacteria Proteus.

Electrospray ionization with Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICRMS) was used for screening and structural elucidation of core oligosaccharides isolated from lipopolysaccharides of bacteria of the genus Proteus. Mass spectra allowed the determination of the molecular masses with high accuracy and the estimation of the chemical heterogeneity of the samples. They did not, however, provide sufficient information to identify structural details of the branched oligosaccharides.

High-resolution anatomic, diffusion tensor, and magnetization transfer magnetic resonance imaging of the optic chiasm at 3T.

Purpose: To evaluate techniques for anatomical and physiological imaging of the intracranial optic nerve (ON), optic chiasm (OC), and optic tract (OT) at 3T with the aim of visualizing axonal damage in multiple sclerosis (MS).

Materials And Methods: Imaging was performed on a 3T scanner employing a custom-designed head coil that consisted of a coil array with four coils (30 x 30 cm(2)). Oblique fast spin echo (FSE) images, magnetization transfer (MT)-enhanced 3D gradient-echo (GRE) time-of-flight (TOF) images, and line scan diffusion images (LSDI) were obtained.

On-resonance low B1 pulses for imaging of the effects of PARACEST agents.

Application of the exchange-sensitive, low-power RF pulses positioned on the bulk water resonance for imaging of the effects of PARACEST agents is proposed as an alternative to the standard CW off-resonance irradiation. Specifically, we applied a low-power WALTZ-16 RF train, with the 90 degrees pulse unit replaced by a pulse of the fixed length (WALTZ-16*). Using this sequence, the bulk water signal was found to be sensitive to exchange lifetimes with PARACEST complex bound protons, the transverse relaxation time of bulk water, and longitudinal relaxation time of bound protons.

The structure of the capsular polysaccharide of Shewanella oneidensis strain MR-4.

Capsular polysaccharides were extracted from Shewanella oneidensis strain MR-4, grown on two different culture media. The polysaccharides were analyzed using 1H and 13C NMR spectroscopy, and the following structure of the repeating unit was established: [structure: see text] where the residue of 4-amino-4,6-dideoxy-D-glucose (Qui4N) was substituted with different N-acyl groups depending on the growth media. All monosaccharides are present in the pyranose form.

The role of galacturonic acid in outer membrane stability in Klebsiella pneumoniae.

In most members of the Enterobacteriaceae, including Escherichia coli and Salmonella, the lipopolysaccharide core oligosaccharide backbone is modified by phosphoryl groups. The negative charges provided by these residues are important in maintaining the barrier function of the outer membrane. Mutants lacking the core heptose region and the phosphate residues display pleiotrophic defects collectively known as the deep-rough phenotype, characterized by changes in outer membrane structure and function.

Cold temperature-induced modifications to the composition and structure of the lipopolysaccharide of Yersinia pestis.

Following a report of variations in the lipopolysaccharide (LPS) structure of Yersinia pestis at mammalian (37 degrees C) and flea (25 degrees C) temperatures, a number of changes to the LPS structure were observed when the bacterium was cultivated at a temperature of winter-hibernating rodents (6 degrees C). In addition to one of the known Y. pestis LPS types, LPS of a new type was isolated from Y.

The structure of the antigenic O-polysaccharide of the lipopolysaccharide of Edwardsiella ictaluri strain MT104.

The structural characterization of the antigenic O-polysaccharide component of the lipopolysaccharide produced by the fish pathogenic bacterium Edwardsiella ictaluri MT104 was undertaken by the application of NMR spectroscopy and chemical analysis. The O-chain was found to be a linear polymer of a repeating tetrasaccharide unit composed of D-glucose, 2-acetamido-2-deoxy-D-galactose, and D-galactose in a 1:2:1 ratio having the structure: [carbohydrate structure]; see text.

Extracellular carbohydrate-containing polymers of a model biofilm-producing strain, Staphylococcus epidermidis RP62A.

Staphylococcus aureus and coagulase-negative staphylococci, primarily Staphylococcus epidermidis, are recognized as a major cause of nosocomial infections associated with the use of implanted medical devices. It has been established that clinical isolates often produce a biofilm, which is involved in adherence to biomaterials and provides enhanced resistance of bacteria against host defenses and antibiotic treatments. It has been thought that the staphylococcal biofilm contains two polysaccharides, one responsible for primary cell adherence to biomaterials (polysaccharide/adhesin [PS/A]) and an antigen that mediates bacterial aggregation (polysaccharide intercellular adhesin [PIA]).

Structural characterization of the antigenic capsular polysaccharide and lipopolysaccharide O-chain produced by Actinobacillus pleuropneumoniae serotype 15.

The specific capsular polysaccharide produced by Actinobacillus pleuropneumoniae serotype 15 was determined to be a high-molecular-mass polymer having [alpha]D + 69 degrees (water) and composed of a linear backbone of phosphate diester linked disaccharide units of 2-acetamido-2-deoxy-D-glucose (D-GlcNAc) and 2-acetamido-2-deoxy-D-galactose (D-GalNAc) residues (1:1). Thirty percent of the D-GalNAc residues were substituted at O-4 by beta-D-galactopyranose (beta-D-Galp) residues. Through the application of chemical and NMR methods, the capsule, which defines the serotype specificity of the bacterium, was found to have the structure [structure: see text].