Structural studies of the O-specific polysaccharide of Hafnia alvei strain PCM 1207 lipopolysaccharide.

The structure of the O-specific side-chain of the Hafnia alvei strain PCM 1207 lipopolysaccharide (LPS) has been investigated. Methylation analysis, partial acid hydrolysis, matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) MS, fast atom bombardment (FAB)-MS/MS and 1H- and 13C-NMR spectroscopy were the principal methods used. Glycerol phosphate was identified as a constituent in the polysaccharide and the following structure of a pentasaccharide repeating unit was established: The polysaccharide is partially (approximately 10%) substituted with O-acetyl groups. The lipopolysaccharide was also subjected to high resolution magic angle spinning (HR-MAS) NMR analysis, which showed both the signals of the O-specific polysaccharide as well as several signals from unsubstituted core oligosaccharides. This confirmed the presence of the described structure in the native LPS.     

Structural and serological studies on Hafnia alvei O-specific polysaccharide of alpha-D-mannan type isolated from the lipopolysaccharide of strain PCM 1223

On the basis of chemical and methylation analyses, one- and two-dimensional (1)H- and (13)C-NMR spectroscopy, including COSY, TOCSY, NOESY and (1)H, (13)C HSQC experiments, a neutral O-specific polysaccharide isolated from Hafnia alvei strain PCM 1223 lipopolysaccharide (LPS) was found to be an alpha-mannan composed of pentasaccharide repeating units having the following structure:-->3)-alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->. Immunoblotting showed a strong cross-reactivity between anti-H. alvei PCM 1223 serum and LPSs of Escherichia coli O9 and Klebsiella pneumoniae O3. The serological relationship of the LPSs of these bacteria is due to the structural identity of their O-specific polysaccharides, though the LPSs differ in their core regions.     

Structure of the O-polysaccharide from the lipopolysaccharide of Hafnia alvei strain PCM 1529

The following structure of the pentasaccharide repeating unit of an acidic O-polysaccharide of Hafnia alvei PCM 1529 was established by sugar and methylation analyses along with 1D and 2D 1H and 13C NMR spectroscopy: [Carbohydrate structure: see text].     

Structure of the O-polysaccharide from the lipopolysaccharide of Hafnia alvei strain PCM 1546

An acidic O-polysaccharide isolated by mild acid hydrolysis from the lipopolysaccharide of Hafnia alvei PCM 1546 is composed of D-Gal, D-Glc, D-GlcA, D-GalNAc and O-acetyl groups in the ratios 1:1:1:2:1.6. On the basis of sugar and methylation analyses along with 1D and 2D 1H and 13C NMR spectroscopy, the following structure of the pentasaccharide repeating unit of the polysaccharide was established: [see equation in text].     

Non-typical lipopolysaccharide core regions of some Hafnia alvei strains: structural and serological studies

The lipopolysaccharides of Hafnia alvei strains 23, 1222 and 39 were found to have non-typical core region. On the basis of sugar and methylation analyses, 1H-nuclear magnetic resonance spectra and matrix-assisted laser-desorption ionization-time of flight mass spectrometry, it was concluded that the core oligosaccharide of strains 23 and 1222 has the same structure as Escherichia coli R4 core region, and the core oligosaccharide of strain 39 has the structure of Salmonella Ra core. Using the serological methods (passive hemagglutination, enzyme-linked immunosorbent assay and immunoblotting) and the anti-conjugate sera directed against E. coli R4 and Salmonella Ra core oligosaccharides we have confirmed the structural results presented above.     

O-specific polysaccharide of Hafnia alvei lipopolysaccharide isolated from strain 1211. Structural study using chemical methods, gas-liquid chromatography/mass spectrometry and NMR spectroscopy.

The Hafnia alvei strain 1211 O-specific polysaccharide is composed of 3-amino-N-(D-3'-hydroxybutyryl)-3,6-dideoxy-D-galactose, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine and D-glucose (1:1:2:2). On the basis of sugar and methylation analyses, Smith degradation, and one- and two-dimensional 1H- and 13C-NMR spectroscopy, the polysaccharide was shown to be an O-acetylated polymer of the repeating hexasaccharide unit, ----2D(4-OAc)Fucp3NAcyl beta 1----6DGlcpNAc alpha 1---- (DGlcp beta 1----3)4DGalpNAc alpha 1----3DGlcpNAc beta 1----2DGlcp beta 1----, where DFucp3NAcyl = 3-amino-N-(D-3'-hydroxybutyryl)-3,6-dideoxy-D- galactopyranose. The O-specific polysaccharide showed some microheterogeneity due to incomplete substitution by terminal glucose.     

Structure of the lipopolysaccharide core region of Hafnia alvei strains 1185 and 1204

Sugar and methylation analyses using gas chromatography/mass spectrometry and NMR spectroscopy proved that the core oligosaccharides of Hafnia alvei strains 1185 and 1204 have the following formula: carbohydrate sequence [see text] where Kdo = 3-deoxy-oct-2-ulosonic acid and P-PEtN = diphosphorylethanolamine. The structure shown above is a slight modification of the typical core region of H. alvei lipopolysaccharides. The difference refers to one sugar only: terminal galactose is present in the core of strains of 1185 and 1204, while terminal glucose in the typical core.     

Structural studies of the lipopolysaccharide from Haemophilus parainfluenzae strain 20

Haemophilus parainfluenzae is a Gram-negative bacterium that colonizes the upper respiratory tract of humans and is a part of normal flora. In this study, we investigated the lipopolysaccharide (LPS) expressed by H. parainfluenzae strain 20. Using NMR and MS techniques on LPS, oligosaccharide samples and lipid A, the structures for O-antigen, core oligosaccharide and lipid A could be established. It was found that the biological repeating unit of the O-antigen is →4)-α-d-GalpNAc-(1→P→6)-β-d-Glcp-(1→3)-α-d-FucpNAc4N-(1→, in which d-FucpNAc4N is 2-acetamido-4-amino-2,4,6-trideoxy-d-galactose. This sugar is in β-configuration when linked to O-4 of the glucose residue of β-d-Galp-(1→2)-l-α-d-Hepp-(1→2)-[PEtn→6]-l-α-d-Hepp-(1→3)-[β-d-Glcp-(1→4)]-l-α-d-Hepp-(1→5)-[PPEtn→4]-α-Kdo-(2→6)-lipid A. LPS from a wbaP mutant of H. parainfluenzae strain 20 did not contain an O-antigen, consistent with the wbaP gene product being required for expression of O-antigen in fully extended LPS.     

Structural analysis of a novel sialic-acid-containing trisaccharide from Rhodobacter capsulatus 37b4 lipopolysaccharide.

Sialic-acid-containing lipopolysaccharides from Rhodobacter capsulatus 37b4 (S-form lipopolysaccharide), KB-1 (R-type lipopolysaccharide) and Sp 18 (deep R-type lipopolysaccharide) were investigated for the linkage and substitution of sialic acids. Methylation analysis and behaviour towards acid and enzymic hydrolysis indicated a non-reducing terminal location of sialic acids in the R-type lipopolysaccharide of strain Sp 18, whereas an internal, chain-linked location of sialic acids was found in the lipopolysaccharides of strains 37b4 and KB-1. For these latter strains, methylation analysis revealed a substitution of sialic acids by other sugars at position 7 for strain 37b4 and positions 4 and 7 for strain KB-1. In accordance with the chain-linked position of sialic acids, mild hydrolysis of R. capsulatus 37b4 lipopolysaccharide with acetic acid released a trisaccharide with sialic acid at the reducing terminus. Structural investigation of this trisaccharide by methylation analysis, 1H- and 13C-NMR spectroscopy revealed the presence of the disaccharide Gal1-6Glc at the non-reducing end, probably with an alpha-anomeric configuration of the galactose residue, i.e. melibiose, beta-glycosidically linked to position 7 of sialic acid. Therefore the structure Gal alpha 1-6Glc beta 1-7Neu5Ac is proposed for this core oligosaccharide from R. capsulatus 37b4 lipopolysaccharide.     

Structural analysis of the lipid A isolated from Hafnia alvei 32 and PCM 1192 lipopolysaccharides

Hafnia alvei, a Gram-negative bacterium, is an opportunistic pathogen associated with mixed hospital infections, bacteremia, septicemia, and respiratory diseases. The majority of clinical symptoms of diseases caused by this bacterium have a lipopolysaccharide (LPS, endotoxin)-related origin. The lipid A structure affects the biological activity of endotoxins predominantly. Thus, the structure of H. alvei lipid A was analyzed for the first time. The major form, asymmetrically hexa-acylated lipid A built of β-d-GlcpN4P-(1→6)-α-d-GlcpN1P substituted with (R)-14:0(3-OH) at N-2 and O-3, 14:0(3-(R)-O-12:0) at N-2′, and 14:0(3-(R)-O-14:0) at O-3′, was identified by ESI-MSⁿ and MALDI-time-of-flight (TOF) MS. Comparative analysis performed by MS suggested that LPSs of H. alvei 32, PCM 1192, PCM 1206, and PCM 1207 share the identified structure of lipid A. LPSs of H. alvei are yet another example of enterobacterial endotoxins having the Escherichia coli-type structure of lipid A. The presence of hepta-acylated forms of H. alvei lipid A resulted from the addition of palmitate (16:0) substituting 14:0(3-OH) at N-2 of the α-GlcpN residue. All the studied strains of H. alvei have an ability to modify their lipid A structure by palmitoylation.     

Structural studies of the lipopolysaccharide of Moritella viscosa strain M2-226

The structure of the O-specific side chain of the lipopolysaccharide from the Gram-negative psychrophilic bacterium Moritella viscosa strain M2-226, responsible for the winter ulcer in Atlantic salmon, has been determined. Monosaccharide analysis and (1)H and (13)C NMR spectroscopy were employed to elucidate the structure. It was concluded that the polysaccharide is composed of a trisaccharide repeating unit with the following structure: →3)-β-D-GlcpNAc-(1→4)-[α-D-GlcpA-(1→3)]-α-L-Fucp-(1→ .     

The structure of the O-chain of the lipopolysaccharide of a prototypal diarrheagenic strain of Hafnia alvei that has characteristics of a new species under the genus Escherichia.

The structure of the O-polysaccharide of the lipopolysaccharide from a diarrheal strain isolated in Bangladesh was studied with sugar, and methylation analysis, NMR spectroscopy, mass spectrometry and partial acid hydrolysis. The strain was first designated as Hafnia alvei, but later found to be a possible new species in the genus Escherichia. Two different polysaccharides were detected, a major and a minor one. The structure of the major polysaccharide is given below, while the structure of the minor one was not investigated. The structure of the repeating unit was established as The structure does not resemble any of the previously investigated lipopolysaccharide O-chains from Escherichia coli or H. alvei, but could fit in either group based on types of sugar residues and acidity. Phenotypic microbiological studies cannot definitely assign it to either species of the two genera. Genetic hybridization studies indicate that the Bangladeshi isolates may require a new species designation under the genus Escherichia.     

响应面法优化赖氨酸脱羧酶产酶培养基

目的:对蜂房哈夫尼菌产L-赖氨酸脱羧酶培养基进行优化.方法:采用响应面优化的方法.首先单因素实验得到最适培养基成分为:葡萄糖2%,酵母膏2%,MsSO40.03%,KH2PO40.01%,NaCl 0.3%,L-赖氨酸0.5%,维生素B6 0.1%,玉米浆4%,酶活达到180.85U/mL.在此基础上,用PB试验筛选出对酶活影响显著的3个因素(葡萄糖、酵母膏、玉米浆),再通过Box-behnken实验对这三个因素进行优化.结果:得到产酶最佳培养基为葡萄糖1.84%,酵母膏2.20%,玉米浆3.66%,MgSO40.03%,K2HPO4 0.01%,NaCl 0.3%,L-赖氨酸0.5%,维生素B60.1%.结论:响应面优化的方法使酶活达到203.14U/mL,比优化前的比酶活(7.03U/ML)提高28.9倍,在单因素的基础上提高了11.3%.     

Structural analysis of the lipopolysaccharide from Chlamydophila psittaci strain 6BC.

The lipopolysaccaride of Chlamydophila psittaci 6BC was isolated from tissue culture-grown elementary bodies using a modified phenol/water procedure followed by extraction with phenol/chloroform/light petroleum. Compositional analyses indicated the presence of 3-deoxy-Dmanno-oct-2-ulosonic acid, GlcN, organic bound phosphate and fatty acids in a molar ratio of approximately 3. 3 : 2 : 1.8 : 4.6. Deacylated lipopolysaccharide was obtained after successive microscale treatment with hydrazine and potassium hydroxide, and was then separated by high performance anion-exchange chromatography into two major fractions, the structures of which were determined by 600 MHz NMR spectroscopy as alpha-Kdo-(2-->8)-alpha-Kdo-(2-->4)-alpha-Kdo-(2-->6)-beta-D-GlcpN -(1 -->6)-alpha-D-GlcpN 1,4'-bisphosphate and alpha-Kdo-(2-->4)-[alpha-Kdo-(2-->8)]-alpha-Kdo-(2-->4)-alpha-Kdo-(2- ->6)-beta-D-GlcpN-(1-->6)-alpha-D-GlcpN 1,4'-bisphosphate. The distribution of fatty acids in lipid A was determined by compositional analyses and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry experiments on lipid A and de-O-acylated lipid A. It was shown that the carbohydrate backbone of lipid A is replaced by a complex mixture of fatty acids, including long-chain and branched (R)-configured 3-hydroxy fatty acids, the latter being exclusively present in an amide linkage.     

Structural analysis of the lipopolysaccharide from nontypeable Haemophilus influenzae strain 1003.

Structural analysis of the lipopolysaccharide (LPS) of nontypeable Haemophilus influenzae strain 1003 has been achieved by the application of high-field NMR techniques, ESI-MS, capillary electrophoresis coupled to ESI-MS, composition and linkage analyses on O-deacylated LPS and core oligosaccharide material. 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)-[PP Etn-->4]-alpha-Kdop-(2-->6)-Lipid A, in which the beta-D-Glcp residue is substituted by phosphocholine at O-6 and an acetyl group at O-4. A second acetyl group is located at O-3 of the distal heptose residue (HepIII). HepIII is chain elongated at O-2 by either a beta-D-Glcp residue (major), lactose or sialyllactose (minor, i.e. alpha-Neu5Ac-(2-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp), where a third minor acetylation site was identified at the glucose residue. Disialylated species were also detected. In addition, a minor substitution of ester-linked glycine at HepIII and Kdo was observed.     

Structure of the O-specific, sialic acid containing polysaccharide chain and its linkage to the core region in lipopolysaccharide from Hafnia alvei strain 2 as elucidated by chemical methods, gas-liquid chromatography/mass spectrometry, and 1H NMR spectroscopy

Mild acid hydrolysis of Hafnia alvei strain 2 lipopolysaccharide released no O-specific polysaccharide but instead gave a monomeric octasaccharide repeating unit with N-acetylneuraminic acid as the reducing terminus. In addition, a dimer of the octasaccharide repeating unit, and also a decasaccharide composed of a fragment of the O-specific polysaccharide chain and the core region, were obtained in minute amounts. On the basis of the sugar and methylation analyses, periodate oxidation, and 1H NMR spectroscopy of the lipopolysaccharide hydrolytic products, the biological repeating unit of the O-specific polysaccharide was shown to be a branched octasaccharide: (Formula; see text) The linkage between the O-specific polysaccharide chain and core region has also been determined and has yield strong evidence that N-acetylneuraminic acid is an inherent lipopolysaccharide component. The lipopolysaccharide of H. alvei strain 2 is the first lipopolysaccharide reported to contain 4-substituted neuraminic acid in its O-specific polysaccharide region.     

Structural analysis of the lipopolysaccharide from the nontypable Haemophilus influenzae strain SB 33.

The structure of the core region of the lipopolysaccharide (LPS) from the nontypable Haemophilus influenzae strain SB 33 was elucidated. The LPS was subjected to a variety of degradative procedures. The structures of the derived oligosaccharide products were established by monosaccharide and methylation analyses, NMR spectroscopy and mass spectrometry. These analyses revealed a series of related phosphocholine (PCho) containing structures differing in the number of hexose residues. The results pointed to each species containing a conserved phosphoethanolamine (PEtn) substituted heptose-containing trisaccharide inner-core moiety. The major LPS glycoforms were identified as 2-Hex, 3-Hex and 4-Hex species according to the number of hexose residues present.     

Structural studies of the O-polysaccharide from the Escherichia coli O77 lipopolysaccharide.

The structure of the O-antigen polysaccharide (PS) from Escherichia coli O77 has been determined. Sugar and methylation analysis together with 1H and 13C NMR spectroscopy were the main methods used. The PS is composed of tetrasaccharide repeating units with the following structure:-->2)-alpha-D-Manp-(1-->2)-beta-D-Manp-(1-->3)-alpha-D-GlcpNAc-(1-->6)-alpha-D-Manp-(1-->