Complex O-acetylation in non-typeable Haemophilus influenzae lipopolysaccharide: evidence for a novel site of O-acetylation

The structure of the lipopolysaccharide (LPS) of non-typeable Haemophilus influenzae strain 723 has been elucidated using NMR spectroscopy and electrospray ionization mass spectrometry (ESI-MS) on O-deacylated LPS and core oligosaccharide material (OS), as well as ESI-MSn on permethylated dephosphorylated OS. It was found that the LPS contains the common structural element of H. influenzae, l-alpha-D-Hepp-(1-->2)-[PEtn-->6]-l-alpha-D-Hepp-(1-->3)-[beta-D-Glcp-(1-->4)]-l-alpha-D-Hepp-(1-->5)-[PPEtn-->4]-alpha-Kdo-(2-->6)-Lipid A, in which the beta-D-Glcp residue (GlcI) is substituted by phosphocholine at O-6 and the distal heptose residue (HepIII) by PEtn at O-3, respectively. In a subpopulation of glycoforms O-2 of HepIII was substituted by beta-D-Galp-(1-->4)-beta-D-Glcp-(1--> or beta-D-Glcp-(1-->. Considerable heterogeneity of the LPS was due to the extent of substitution by O-acetyl groups (Ac) and ester-linked glycine of the core oligosaccharide. The location for glycine was found to be at Kdo. Prominent acetylation sites were found to be at GlcI, HepIII, and the proximal heptose (HepI) residue of the triheptosyl moiety. Moreover, GlcI was acetylated at O-3 and/or O-4 and HepI was acetylated at O-2 as evidenced by capillary electrophoresis ESI-MSn in combination with NMR analyses. This is the first study to show that an acetyl group can substitute HepI of the inner-core region of H. influenzae LPS.     

The structure of the core region of the lipopolysaccharide from Geobacter sulfurreducens

The structure of the core part of the LPS from Geobacter sulfurreducens was analysed. The LPS contained no O-specific polysaccharide (O-side chain) and upon mild hydrolysis gave a core oligosaccharide, which was isolated by gel chromatography. It was studied by chemical methods, NMR and mass spectrometry, and the following structure was proposed. [carbohydrate structure: see text] where Q = 3-O-Me-alpha-L-QuiNAc-(1-->or H (approximately 3:2).     

Structure of the O-polysaccharide of Idiomarina zobellii KMM 231T containing two unusual amino sugars with the free amino group, 4-amino-4,6-dideoxy-D-glucose and 2-amino-2-deoxy-L-guluronic acid

Mild acid degradation of the lipopolysaccharide of the bacterium Idiomarina zobellii, type strain KMM 231T, with aq 2% HOAc at 100 degrees C, yielded an oligosaccharide, which represents one repeating unit of the O-polysaccharide. A polysaccharide was obtained by mild base degradation of the lipopolysaccharide. The following structure of the O-polysaccharide was elucidated by 1H and 13C NMR spectroscopy of the oligosaccharide and base-degraded lipopolysaccharide, including COSY, TOCSY, ROESY, 1H, 13C HSQC, HSQC-TOCSY and HMBC experiments: [-->3)-alpha-D-Quip4N-(1-->4)-alpha-D-GlcpA-(1-->6)-alpha-D-GlcpNAc-(1-->4)-alpha-L-GulpNA-(1-->3)-beta-D-FucpNAc-(1-->] The O-polysaccharide is distinguished by the presence of two unusual amino sugars, 4-amino-4,6-dideoxy-D-glucose (D-Qui4N) and 2-amino-2-deoxy-L-guluronic acid (L-GulNA), both having the free amino group. The unexpectedly high acid lability of the glycosidic linkage of 2-acetamido-2,6-dideoxy-D-galactose (D-FucNAc) could be associated with the presence of a free amino group adjacent to the site of attachment of FucNAc to Qui4N.     

Structural analysis of the lipopolysaccharide from nontypeable Haemophilus influenzae strain R2846

We here report the lipopolysaccharide (LPS) structures expressed by nontypeable Haemophilus influenzae R2846, a strain whose complete genome sequence has recently been obtained. Results were obtained by using NMR techniques and ESI-MS on O-deacylated LPS and core oligosaccharide material (OS) as well as ESI-MS (n) on permethylated dephosphorylated OS. A beta- d-Glc p-(1-->4)- d-alpha- d-Hep p-(1-->6)-beta- d-Glc p-(1-->4) unit was found linked to the proximal heptose (HepI) of the conserved triheptosyl inner-core moiety, l-alpha- d-Hep p-(1-->2)-[ PEtn-->6]- l-alpha- d-Hep p-(1-->3)- l-alpha- d-Hep p-(1-->5)-[ PPEtn-->4]-alpha-Kdo-(2-->6)-lipid A. The beta- d-Glc p (GlcI) linked to HepI was also branched with oligosaccharide extensions from O-4 and O-6. O-4 of GlcI was substituted with sialyllacto- N-neotetraose [alpha-Neu5Ac-(2-->3)-beta- d-Gal p-(1-->4)-beta- d-Glc pNAc-(1-->3)-beta- d-Gal p-(1-->4)-beta- d-Glc p-(1-->] and the related structure [( PEtn-->6)-alpha- d-Gal pNAc-(1-->6)-beta- d-Gal p-(1-->4)-beta- d-Glc pNAc-(1-->3)-beta- d-Gal p-(1-->4)-beta- d-Glc p-(1-->]. The distal heptose (HepIII) was substituted at O-2 by beta- d-Gal. Phosphate, phosphoethanolamine, phosphocholine, acetate, and glycine were found to substitute the core oligosaccharide. Two heptosyltransferase genes, losB1 and losB2, have been identified from the R2846 genome sequence and are candidates to add the noncore heptose to the LPS. Mutant strain R2846 losB1 did not show dd-heptose in the extension from HepI but still contained minor quantities of ld-heptose at the same position, indicating that the losB1 gene is required to add dd-heptose to GlcI. The LPS from strain R2846 losB1/ losB2 expressed no noncore heptose, consistent with losB2 directing the addition of ld-heptose.     

Structural determination of a neutral exopolysaccharide produced by Lactobacillus delbrueckii ssp. bulgaricus LBB.B332

The neutral exopolysaccharide produced by Lactobacillus delbrueckii ssp. bulgaricus LBB.B332 in skimmed milk was found to be composed of d-glucose, d-galactose, and l-rhamnose in a molar ratio of 1:2:2. Linkage analysis and 1D/2D NMR (1H and 13C) studies carried out on the native polysaccharide as well as on an oligosaccharide generated by a periodate oxidation protocol, showed the polysaccharide to consist of linear pentasaccharide repeating units with the following structure: -->3-alpha-D-Glcp-(1-->3)-alpha-D-Galp-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->2)-alpha-D-Galp-(1-->.     

Oligosaccharide polyester and triterpenoid saponins from the roots of Polygala japonica

An oligosaccharide polyester, 1-O-(E)-p-coumaroyl-(3-O-benzoyl)-beta-D-fructofuranosyl-(2-->1)-[6-O-(E)-feruloyl-beta-D-glucopyranosyl-(1-->2)]-[6-O-acetyl-beta-D-glucopyranosyl-(1-->3)-(4-O-acetyl)-beta-D-glucopyranosyl-(1-->3)]-4-O-[4-O-alpha-L-rhamnopyranosyl-(E)-p-coumaroyl]-alpha-D-glucopyranoside (polygalajaponicose I), and four triterpenoid saponins, 3beta, 23, 27-trihydroxy-29-O-beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranosyl-olean-12-en-28-oic acid (polygalasaponin XLVII), 3-O-beta-D-glucopyranosyl presenegenin 28-O-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-fucopyranosyl ester (polygalasaponin XLVIII), 3-O-beta-D-glucopyranosyl presenegenin 28-O-beta-D-galactopyranosyl-(1-->5)-beta-D-apiofuranosyl-(1-->4)-beta-D-xylopyranosyl-(1-->4)-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-glucopyranosyl ester (polygalasaponin XLIX) and 2beta, 27-dihydroxy-3-O-beta-D-glucopyranosyl 11-oxo-olean-12-en-23, 28-dioic acid 28-O-beta-D-galactopyranosyl-(1-->5)-beta-D-apiofuranosyl-(1-->4)-beta-D-xylopyranosyl-(1-->4)-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-fucopyranosyl ester (polygalasaponin L), in addition to five known compounds have been isolated from the roots of Polygala japonica.     

Structural analysis of lipopolysaccharide oligosaccharide epitopes expressed by non-typeable Haemophilus influenzae strain 176

The structure of the lipopolysaccharide (LPS) from non-typeable Haemophilus influenzae strain 176 has been investigated. Electrospray ionization-mass spectrometry (ESIMS) on O-deacylated LPS (LPS-OH) and core oligosaccharide (OS) samples obtained after mild-acid hydrolysis of LPS provided information on the composition and relative abundance of the glycoforms. ESIMS tandem-mass spectrometry on LPS-OH confirmed the presence of minor sialylated and disialylated glycoforms. Oligosaccharide samples were studied in detail using high-field NMR techniques. It was found that the LPS contains the common inner-core element of H. influenzae, L-alpha-D-Hepp-(1-->2)-[PEtn-->6]-L-alpha-D-Hepp-(1-->3)-[beta-D-Glcp-(1-->4)]-L-alpha-D-Hepp-(1-->5)-[PPEtn-->4]-alpha-Kdop-(2-->6)-Lipid A having glycosyl substitution at the O-3 position of the terminal heptose as recently observed for non-typeable H. influenzae strain 486 [M?nsson, M.; Bauer, S. H. J.; Hood, D. W.; Richards, J. C.; Moxon, E. R.; Schweda, E. K. H., Eur. J. Biochem. 2001, 268, 2148--2159]. The following LPS structures were identified as the major glycoforms, the most significant being indicated with an asterisk (*) (glycoforms are partly substituted with Gly at the terminal Hep):     

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). Comparative CE-MS analysis of A. salmonicida subsp. salmonicida core oligosaccharides from strains A449, 80204-1 and an in vivo rough isolate confirmed that the structure of the core oligosaccharide was conserved among different isolates of A. salmonicida.     

The structure of the phosphorylated carbohydrate backbone of the lipopolysaccharide of the phytopathogen bacterium Pseudomonas tolaasii

A novel core-lipid A backbone oligosaccharide was isolated and identified from the lipopolysaccharide fraction of the mushrooms pathogen bacterium Pseudomonas tolaasii. The oligosaccharide was obtained by alkaline treatment of the lipopolysaccharide fraction. Since the repeating unit of the O-antigen contained one residue of -->4)-alpha-l-GulpNAcAN, the hydrolysis was accompanied by beta-elimination on this residue and following depolymerization, producing a mixture of oligosaccharides. The complete structural elucidation showed the presence of a single core glycoform and was achieved by chemical analysis and by (1)H, (31)P, and (13)C NMR spectroscopy applying various 1D and 2D experiments. [structure: see text]. All sugars are alpha-d-pyranoses, if not stated otherwise. Hep is l-glycero-d-manno-heptose, Kdo is 3-deoxy-d-manno-oct-2-ulosonic acid, P is phosphate. QuiN and DeltaGulNA are present in nonstoichiometric amount.     

Structural characterization of a novel branching pattern in the lipopolysaccharide from nontypeable Haemophilus influenzae.

Structural analysis of the lipopolysaccharide (LPS) from nontypeable Haemophilus influenzae strain 981 has been achieved using NMR spectroscopy and ESI-MS on O-deacylated LPS and core oligosaccharide (OS) material as well as by ESI-MSn on permethylated dephosphorylated OS. A heterogeneous glycoform population was identified, resulting from the variable length of the OS branches attached to the glucose residue in the common structural element of H. influenzae LPS, l-alpha-d-Hepp-(1-->2)-[PEtn-->6]-l-alpha-d-Hepp-(1-->3)-[beta-d-Glcxp-(1-->4)]-l-alpha-d-Hepp-(1-->5)-[PPEtn-->4]-alpha-Kdop-(2-->6)-Lipid A. Notably, the O-6 position of the beta-d-Glcp residue was either substituted by PCho or the disaccharide branch beta-d-Galp-(1-->4)-d-alpha-d-Hepp, while the O-4 position was substituted by the globotetraose unit, beta-d-GalpNAc-(1-->3)-alpha-d-Galp-(1-->4)-beta-d-Galp-(1-->4)-beta-d-Glcp, or sequentially truncated versions thereof. This is the first time a branching sugar residue has been reported in the outer-core region of H. influenzae LPS. Additionally, a PEtn group was identified at O-3 of the distal heptose residue in the inner-core.     

Structural elucidation of the major Hex4 lipopolysaccharide glycoform from the lgtC mutant of Haemophilus influenzae strain Eagan

Lipopolysaccharide (LPS) oligosaccharide epitopes are major virulence factors of Haemophilus influenzae. The structure of LPS glycoforms of H. influenzae type b strain Eagan containing a mutation in the gene lgtC is investigated. LgtC is involved in the biosynthesis of globoside trisaccharide [alpha-D-Galp-(1-->4)-beta-d-Galp-(1-->4)-beta-D-Glcp-(1-->], an LPS epitope implicated in the virulence of this organism. Glycose and methylation analyses provided information on the composition while electrospray ionization mass spectrometry (ESI-MS) on O-deacylated LPS (LPS-OH) indicated the major glycoform to contain 4 hexoses attached to the common H. influenzae triheptosyl inner-core unit. The structure of the Hex4 glycoform in LPS-OH and core oligosaccharide samples was determined by NMR. It consists of an l-alpha-D-HepIIIp-(1-->2)-[PEtn-->6]-l-alpha-D-HepIIp-(1-->3)-l-alpha-D-HepIp-(1-->5)-[P-->4]-alpha-D-Kdop-(2--> to which a beta-D-Glcp-(1-->4)-alpha-D-Glcp disaccharide unit is extended from HepII at the C-3 position, while HepI and HepIII are substituted at the C-4 and C-2 positions with beta-D-Glcp and beta-D-Galp, respectively. This structure corresponds to that expressed as a subpopulation in the parent strain. 31P NMR studies permitted the identification of subpopulations of LPS containing Kdo substituted at the C-4 position with monophosphate or pyrophosphoethanolamine (PPEtn). HepIII was found to be substituted with either phosphate at the C-4 position or acetate at the C-3 position, but not both of them together in the same subpopulation. The subpopulations containing phosphate and acetate at HepIII and their location have not previously been reported.     

Structure of a lactic acid ether-containing and glycerol phosphate-containing O-polysaccharide from Proteus mirabilis O40

An O-polysaccharide was isolated by mild acid hydrolysis from the lipopolysaccharide of Proteus mirabilis O40 and studied by NMR spectroscopy, including 2D 1H, 1H COSY, TOCSY, ROESY, and 1H, 13C HMQC experiments, along with chemical methods. The polysaccharide was found to contain an ether of GlcNAc with lactic acid and glycerol phosphate in the main chain and to have the following structure: --> 3)-beta-D-GlcpNAc4(R-Lac)-(1 --> 3)-alpha-D-Galp-(1 --> 3)-D-Gro-1-P-(O --> 3)-beta-D-GlcpNAc-(1 --> where D-GlcpNAc4(R-Lac) stands for 2-acetamido-4-O-[(R)-1-carboxyethyl]-2-deoxy-D-glucose. This structure is unique among the known structures of the Proteus O-polysaccharides, which is in agreement with the classification of the strain studied into a separate O-serogroup. A serological relatedness of P. mirabilis O40 with some other Proteus strains was revealed and discussed in view of the O-polysaccharide structures.     

Structural analysis of lipooligosaccharide produced by Neisseria gonorrhoeae, strain MS11mk (variant A): a precursor for a gonococcal lipooligosaccharide associated with virulence.

We studied the structure of the lipooligosaccharide (LOS) that is produced by a variant A of strain MS11mk. This variant produces a single LOS that is recognized by monoclonal antibody (MAb) 2-1-L8. In a recent study of the pathogenesis of Neisseria gonorrhoeae in male volunteers, variant A gave rise to other phase variants that produce higher molecular weight LOSs, and these LOS were associated with virulence. Definition of the structure of the variant A LOS is important to understand the biosynthesis of LOS and its expression in vivo. The dephosphorylated oligosaccharide (OS) structure derived from the variant A LOS was analyzed by two-dimensional NMR and methylation analysis. The OS structure was found to be a truncated form of the LOS produced by strain F62 [Yamasaki et al. (1991) Biochemistry 30, 10566-10575]; the variant A OS is a hexamer, a beta-lactosyl residue linked to a tetrasaccharide: Gal beta 1-->4Glc beta 1-->4[GlcNAc alpha 1-->2Hep alpha 1-->3]Hep alpha 1-->KDO. We determined that the variant A LOS is a precursor for the synthesis of higher MW LOS. We also studied expression of the MAb 2-1-L8-defined epitope present on the variant A LOS. Our data indicate that the MAb-defined epitope is not a linear beta-lactosyl residue but its specificity is directed toward the phosphorylated GlcNAc-Hep-Hep residue. Since this MAb binds to gonococci, at least part of the phosphorylated diheptose area is exposed on the gonococcal surface.     

Structural analysis and biosynthesis gene cluster of an antigenic glycopeptidolipid from Mycobacterium intracellulare

Mycobacterium avium-Mycobacterium intracellulare complex (MAC) is the most common isolate of nontuberculous mycobacteria and causes pulmonary and extrapulmonary diseases. MAC species can be grouped into 31 serotypes by the epitopic oligosaccharide structure of the species-specific glycopeptidolipid (GPL) antigen. The GPL consists of a serotype-common fatty acyl peptide core with 3,4-di-O-methyl-rhamnose at the terminal alaninol and a 6-deoxy-talose at the allo-threonine and serotype-specific oligosaccharides extending from the 6-deoxy-talose. Although the complete structures of 15 serotype-specific GPLs have been defined, the serotype 16-specific GPL structure has not yet been elucidated. In this study, the chemical structure of the serotype 16 GPL derived from M. intracellulare was determined by using chromatography, mass spectrometry, and nuclear magnetic resonance analyses. The result indicates that the terminal carbohydrate epitope of the oligosaccharide is a novel N-acyl-dideoxy-hexose. By the combined linkage analysis, the oligosaccharide structure of serotype 16 GPL was determined to be 3-2'-methyl-3'-hydroxy-4'-methoxy-pentanoyl-amido-3,6-dideoxy-beta-hexose-(1-->3)-4-O-methyl-alpha-L-rhamnose-(1-->3)-alpha-L-rhamnose-(1-->3)-alpha-L-rhamnose-(1-->2)-6-deoxy-alpha-L-talose. Next, the 22.9-kb serotype 16-specific gene cluster involved in the glycosylation of oligosaccharide was isolated and sequenced. The cluster contained 17 open reading frames (ORFs). Based on the similarity of the deduced amino acid sequences, it was assumed that the ORF functions include encoding three glycosyltransferases, an acyltransferase, an aminotransferase, and a methyltransferase. An M. avium serotype 1 strain was transformed with cosmid clone no. 253 containing gtfB-drrC of M. intracellulare serotype 16, and the transformant produced serotype 16 GPL. Together, the ORFs of this serotype 16-specific gene cluster are responsible for the biosynthesis of serotype 16 GPL.     

Structure of a D-glycero -D-manno-heptan from the lipopolysaccharide of Helicobacter pylori

A lipopolysaccharide (LPS) was isolated by hot phenol-water extraction from Helicobacter pylori strain D4 and found to contain no fucosylated poly-N-acetyllactosamine chain typical of most H. pylori strains studied but a homopolymer of D-glycero-D-manno-heptose (DD-Hep). The heptan attached to a core oligosaccharide was released by mild acid degradation of the LPS, and the following structure of the trisaccharide-repeating unit was established by chemical methods and 1H and 13C NMR spectroscopy: --> 2)-D-alpha-D-Hepp-(1 --> 3)-D-alpha-D-Hepp-(1 --> 3)-D-alpha-D-Hepp-(1 -->. 1H NMR spectroscopy performed on small amounts of the intact LPS revealed the presence of the same polysaccharide in LPS of H. pylori strains D2 and D5, but not strain D10.     

The biofilm matrix of Pseudomonas sp. OX1 grown on phenol is mainly constituted by alginate oligosaccharides

The structure of the major constituent of the biofilm matrix produced by Pseudomonas sp. OX1, when grown on phenol as the sole carbon source is described. This investigation, carried out by chemical analysis, NMR spectroscopy and MALDI-TOF MS spectrometry, showed the presence of an oligosaccharide blend with the typical alginate structure, namely (1-->4) substituted beta-D-mannuronic (ManA) and alpha-L-guluronic acid (GulA). GulA residues were non-acetylated whereas ManA was always O-acetylated at C-2 or C-3.     

Synthesis of the O-linked pentasaccharide in glycoproteins of Trypanosoma cruzi and selective sialylation by recombinant trans-sialidase

The mucin-like glycoproteins of Trypanosoma cruzi have novel O-linked oligosaccharides that are acceptors of sialic acid in the trans-sialidase (TcTS) reaction. The transference of sialic acid from host glycoconjugates to the mucins is involved in infection and pathogenesis. The synthesis of the pentasaccharide, beta-D-Galp-(1-->2)-[beta-D-Galp-(1-->3)]-beta-D-Galp-(1-->6)-[beta-D-Galf-(1-->4)]-D-GlcpNAc and the corresponding alditol, previously isolated by reductive beta-elimination of the mucins, is described. The key step was the 6-O-glycosylation of a easily accessible derivative of beta-D-Galf-(1-->4)-D-GlcpNAc with a beta-D-Galp-(1-->2)-[beta-D-Galp-(1-->3)]-D-Galp donor using the trichloroacetimidate method. The beta-linkage was diastereoselectively obtained by the nitrile effect. The pentasaccharide is the major oligosaccharide in the mucins of T. cruzi, G strain and presents two terminal beta-D-Galp residues for possible sialylation by TcTS. A preparative sialylation reaction was performed with its benzyl glycoside and the sialylated product was isolated and characterized. NMR spectroscopic analysis showed that selective monosialylation occurred at the terminal (1-->3) linked galactopyranose.     

The structure of the O-specific polysaccharide chain of the Shewanella algae BrY lipopolysaccharide

An acidic O-specific polysaccharide was obtained by mild acid degradation of the Shewanella algae strain BrY lipopolysaccharide and was found to contain L-rhamnose, 2-acetamido-4-[D-3-hydroxybutyramido)]-2,4,6-trideoxy-D-glucose (D-BacNAc4NHbu), and 2-amino-2,6-dideoxy-L-galactose, N-acylated by the 4-carboxyl group of L-malic acid (L-malyl-(4-->2)-alpha-L-FucN) in the ratio 2:1:1. 1H and 13C NMR spectroscopy was applied to the intact polysaccharide, and the following structure of the repeating unit was established:-3)-alpha-D-BacNAc4NHbu-(1-->3)-alpha-L-Rha-(1-->2)-alpha-L-Rha-(1-->2)-L-malyl-(4-->2)-alpha-L-FucN-(1-. The repeating unit includes linkage via the residue of malic acid, reported here for the first time as a component of bacterial polysaccharides.     

Structural analysis of lipopolysaccharides from Haemophilus influenzae serotype f. Structural diversity observed in three strains.

Structural elucidation of the lipopolysaccharide (LPS) from three serotype f Haemophilus influenzae clinical isolates RM6255, RM7290 and RM6252 has been achieved using NMR spectroscopy techniques and ESI-MS on O-deacylated LPS and core oligosaccharide material (OS) as well as ESI-MSn on permethylated dephosphorylated OS. This is the first study to report structural details on LPS from serotype f strains. We found that the LPSs of all strains were highly heterogeneous mixtures of glycoforms expressing the common H. influenzae structural element l-alpha-d-Hepp-(1-->2)-[PEtn-->6]-l-alpha-d-Hepp-(1-->3)-[beta-d-Glcp-(1-->4)]-l-alpha-d-Hepp-(1-->5)-[PPEtn-->4]-alpha-Kdo-(2-->6)-lipid A with variable length of OS chains linked to each of the heptoses. The terminal heptose (HepIII) in RM6255 is substituted at the O-3 position by a beta-d-Glcp residue whereas HepIII in strains RM7290 and RM6252 is substituted at O-2 by the globoside unit (alpha-d-Galp-(1-->4)-beta-d-Galp-(1-->4)-beta-d-Glc) or truncated versions thereof. The central heptose (HepII) is substituted by an alpha-d-Galp-(1-->4)-beta-d-Galp-(1-->4)-beta-d-Glcp-(1-->4)-alpha-d-Glcp unit in RM7290 and RM6252 or truncated versions thereof. Strain RM6255 does not express galactose in its LPS and only shows a cellobiose unit elongating from HepII (beta-d-Glcp-(1-->4)-alpha-d-Glcp). ESI-MSn on dephosphorylated and permethylated OS provided information on the existence of additional minor isomeric glycoforms.     

Structural analysis of the lipooligosaccharide-derived oligosaccharide of Histophilus somni (Haemophilus somnus) strain 8025

Previous structural studies in our laboratory on lipooligosaccharide (LOS) inner core oligosaccharide (OS) had identified structures from several strains of Histophilus (Haemophilus) somni (738, 2336, 1P, 129Pt). Recently a type strain 8025 was proposed for this species and we therefore sought to determine the core OS structure of this H. somni strain. Core OS was isolated by standard methods from Westphal purified LOS. Structural information was established by a combination of monosaccharide and methylation analyses, NMR spectroscopy and mass spectrometry. The following structure for the core OS was determined on the basis of the combined data from these experiments: [carbohydrates: see text]. The structure determined contains aspects of other Histophilus somni core OS structures, such as the beta-Gal attached at the 2-position of Hep II (2336), PEtn only at the 6-position of Hep II (738, 129Pt) and a lactose extension from Hep I (1P). Since genetic manipulation has been achieved with this strain, the identification of the core OS structure will enable experiments designed to identify the role of glycosyltransferases involved in LOS biosynthesis.