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Biomedical subjects

O Holst

Publications and source records attributed to O Holst.

At least 55 records · Page 3Linked to original sources
PubMed1998

Identification of a novel core type in Salmonella lipopolysaccharide. Complete structural analysis of the core region of the lipopolysaccharide from Salmonella enterica sv. Arizonae O62.

For the first time, the complete structure of a lipopolysaccharide (LPS) core region from Salmonella enterica has been identified that is different from the Ra core type generally thought to be present in all Salmonella LPS. The LPSs from two rough mutants and the smooth form of S. enterica sv. Arizonae IIIa O62, which all failed to react with an Ra core type-specific monoclonal antibody and were resistant to phage FO1, were analyzed after chemical modification using monosaccharide analysis, mass spectrometry, and NMR spectroscopy. In the novel core type, the terminal D-GlcNAc residue present in the Ra core type, is replaced by a D-Glc residue. The O-specific polysaccharide is alpha1-->4-linked to the second distal Glc residue of the core. Furthermore, phosphoryl substituents attached to O-4 of L-glycero-D-manno-heptose (Hep) I and II were identified as 2-aminoethyl diphosphate (on Hep I) and phosphate (Hep II). [structure: see text] Abbreviations in Structure I are as follows: Hepp, L-glycero-D-manno-heptopyranose; Kdo, 3-deoxy-D-manno-oct-2-ulopyranosonic acid; PPEA, 2-aminoethyl diphosphate; R, O-specific polysaccharide. The presence of this novel core type in LPS of S. enterica should be taken into account in the development of a general antibody-based diagnostic system for Salmonella.

Carbohydrate Conformation↗
PubMed1998

Enzymatic specificity and hydrolysis pattern of the catalytic domain of the xylanase Xynl from Rhodothermus marinus.

The catalytic domain of a xylanase from Rhodothermus marinus was produced in Escherichia coli. The catalytic domain belongs to glycosyl hydrolase family 10. The produced protein has a 22-amino acid leader peptide followed by a 411-amino acid truncated xylanase. The molecular mass was 48 kDa and the recombinant xylanase had a pI of 4.9. The pH and temperature optima for activity were determined to be 7.5 and 80 degrees C, respectively. At that temperature the enzyme had a half-life of 1 h 40 min. An addition of 1 mM calcium stabilized the activity of the enzyme at 80 degrees C. The xylanase had its highest specific activity on oat spelt xylan but was active also on other xylans and to a limited extent on some other polysaccharides (soluble glucans). No exo- or endo-cellulase activity was observed. Hydrolysis of xylo-oligomers and oat spelt xylan was studied and the predominant products of hydrolysis were xylobiose and xylotriose. The enzyme was inactive on xylobiose, xylotriose and on the soluble fraction from oat spelt xylan. The R. marinus xylanase is shown to have a strong preference for internal linkages and is therefore classified as an endo-xylanase.

Amino Acid Sequence↗
PubMed1998

Structural studies of the O-antigen isolated from the phenol-soluble lipopolysaccharide of Acinetobacter baumannii (DNA group 2) strain 9.

A polysaccharide containing D-GalNAc, D-Glc and 4-acetamido-4,6-dideoxy-D-glucose (Qui4NAc) was isolated from the phenol-soluble lipopolysaccharide originating from Acinetobacter baumannii strain 9. The structure of the repeating unit was shown by means of monosaccharide analyses, Smith-degradation, partial acid hydrolysis, mass spectrometry, and NMR spectroscopy to be a branched pentasaccharide, in which the tetrasaccharide backbone is built from amino sugars only. [structure: see text] The polysaccharide was identified by serological and western blot analyses as the O-antigen of the lipopolysaccharide.

Acinetobacter↗
PubMed1998

Cloning, sequencing and overexpression of a Rhodothermus marinus gene encoding a thermostable cellulase of glycosyl hydrolase family 12.

A gene library from the thermophilic eubacterium Rhodothermus marinus, strain ITI 378, was constructed in pUC18 and transformed into Escherichia coli. Of 5400 transformants, 3 were active on carboxymethylcellulose. Three plasmids conferring cellulase activity were purified and were all found to contain the same cellulase gene, celA. The open reading frame for the celA gene is 780 base pairs and encodes a protein of 260 amino acids with a calculated molecular mass of 28.8 kDa. The amino acid sequence shows homology with cellulases in glycosyl hydrolase family 12. The celA gene was overexpressed in E. coli when the pET23, T7 phage RNA polymerase system was used. The enzyme showed activity on carboxymethylcellulose and lichenan, but not on birch xylan or laminarin. The expressed enzyme had six terminal histidine residues and was purified by using a nickel nitrilotriacetate column. The enzyme had a pH optimum of 6-7 and its highest measured initial activity at 100 degrees C. The heat stability of the enzyme was increased by removal of the histidine residues. It then retained 75% of its activity after 8 h at 90 degrees C.

Bacterial Proteins↗
PubMed1998

The structure of the lipopolysaccharide from Klebsiella oxytoca rough mutant R29 (O1-/K29-).

The lipopolysaccharide from Klebsiella oxytoca rough mutant R29 (O1-/K29-) has been isolated and its complete structure has been elucidated by compositional analyses, NMR spectroscopy, and laser-desorption mass spectrometry. The carbohydrate backbone has the structure [formula: see text] of which the GlcN residues (the lipid A backbone) are acylated by 14:(3-OH) (amide-linked) and 12:0, 14:0(3-OH)(ester-linked) fatty acids.

Carbohydrate Sequence↗
PubMed1998

Biotechnological possibilities for waste tyre-rubber treatment.

Every year large amounts of spent rubber material, mainly from rubber tyres, are discarded. Of the annual total global production of rubber material, which amounts to 16-17 million tonnes, approximately 65% is used for the production of tyres. About 250 millions spent car tyres are generated yearly in USA only. This huge amount of waste rubber material is an environmental problem of great concern. Various ways to remediate the problem have been proposed. Among these are road fillings and combustion in kilns. Spent tyres, however, comprise valuable material that could be recycled if a proper technique can be developed. One way of recycling old tyres is to blend ground spent rubber with virgin material followed by vulcanization. The main obstacle to this recycling is bad adhesion between the crumb and matrix of virgin rubber material due to little formation of interfacial sulphur crosslinks. Micro-organisms able to break sulphur-sulphur and sulphur-carbon bonds can be used to devulcanize waste rubber in order to make polymer chains on the surface more flexible and facilitate increased binding upon vulcanization. Several species belonging to both Bacteria and Archaea have this ability. Mainly sulphur oxidizing species, such as different species of the genus Thiobacillus and thermoacidophiles of the order of Sulfolobales, have been studied in this context. The present paper will give a background to the problem and an overview of the biotechnological possibilities for solutions of waste rubber as an environmental problem, focusing on microbial desulphurization.

Bacteria↗
PubMed1997

Cloning and sequence of a thermostable multidomain xylanase from the bacterium Rhodothermus marinus.

The gene (xyn1) encoding a Rhodothermus marinus xylanase has been cloned and expressed in Escherichia coli. The gene comprises 5 different domains in an unusual combination. The cellulose binding domains (CBDs) encoded by xyn1 are repeated in tandem at the N-terminus and show similarity with the CBD family IV. The xyn1-gene is the first example encoding a CBD family IV in combination with a xylan hydrolyzing catalytic domain of the glycosyl hydrolase family 10.

Amino Acid Sequence↗
PubMed1997

Structural studies of the O-antigenic polysaccharide of the lipopolysaccharide from Acinetobacter (DNA group 11) strain 94 containing 3-amino-3,6-dideoxy-D-galactose substituted by the previously unknown amide-linked L-2-acetoxypropionic acid or L-2-hydroxypropionic acid.

A polysaccharide containing D-Gal, D-GalNAc, 3-(L-2-acetoxypropionamido)-3,6-dideoxy-D-galactose (approximately 80%) and 3-(L-2-hydroxypropionamido)-3,6-dideoxy-D-galactose (approximately 20%) was isolated by mild acid hydrolysis, followed by gel-permeation chromatography, from the phenol-soluble lipopolysaccharide (phenol/water extracted) derived from Acinetobacter strain 94. The polysaccharide, characterised by means of monosaccharide analyses, partial acid hydrolysis, and NMR studies, consisted of a branched tetrasaccharide repeating unit, as depicted below, in which Fucp3Nacyl represents 3-(L-2-hydroxypropionamido)-3,6-dideoxy-D-galactose, in which approximately 80% of the acyl residues are O-acetylated. These Fucp3N derivatives and an O-acetylated acyl group are therefore constituents of bacterial LPS, but to our knowledge are not present in any other natural carbohydrates. [sturcture: see text]

Acinetobacter↗
PubMed1997

Deletion of the heptosyltransferase genes rfaC and rfaF in Escherichia coli K-12 results in an Re-type lipopolysaccharide with a high degree of 2-aminoethanol phosphate substitution.

The chromosomal genes rfaC and rfaF of Escherichia coli W3110 were inactivated by allelic-replacement mutagenesis to generate a defined strain lacking both heptosyltransferases which catalyze in lipopolysaccharide (LPS) biosynthesis the transfer of the first two L-glycero-D-manno-heptose (Hep) residues to 3-deoxy-D-manno-2-octulosonic acid (Kdo). The LPS of the mutant was isolated and its chemical structure was investigated by compositional analysis and nuclear magnetic resonance spectroscopy of isolated, deacylated oligosaccharide phosphates. The basic structure was a tetrasaccharide alpha-Kdo-(2-->4)-alpha-Kdo-(2-->6)-beta-D-GlcN4P-(1-->6)-alpha-D- GlcN1P which in LPS was substituted at position 07 of Kdo II by 2-aminoethanol phosphate in non-stoichiometric amounts. 2-Aminoethanol was cleaved during deacylation of the LPS by successive hydrazinolysis and KOH treatment and, in addition, phosphate migration from 07 to 08 of Kdo II occurred. Thus, the oligosaccharides alpha-Kdo7P-(2-->4)-alpha-Kdo-(2-->6)-beta-D-GlcN4P-(1-->6)- alpha-D-GlcN1P and alpha-Kdo8P-(2-->4)-alpha-Kdo-(2-->6)-beta-D-GlcN4P-(1-->6)- alpha-D-GlcN1P could be isolated. KOH treatment of the two trisphosphates and authentic methyl 3-deoxy-D-manno-octulopyranoside 7-(2-acetamidoethyl phosphate) proved that phosphate migration only took place when the phosphate group was substituted with 2-aminoethanol. Complementation studies with plasmid-encoded rfaC and rfaF genes revealed that the mutant strain can be used in combination with LPS-specific antibodies for the cloning and characterization of heptosytransferases which glycosylate Kdo residues of the inner core region of LPS.

Carbohydrate Conformation↗
PubMed1997

Structural investigation of the lipopolysaccharide from Acinetobacter haemolyticus strain NCTC 10305 (ATCC 17906, DNA group 4).

The structure of the lipopolysaccharide (LPS) from Acinetobacter haemolyticus strain NCTC 10305 (DNA group 4) was elucidated by means of analytical chemistry, NMR spectroscopy and fast-atom-bombardment mass spectrometry. Several oligosaccharides were obtained after deacylation or successive de-O-acylation, dephosphorylation, reduction, and de-N-acylation of LPS. In the major fraction of the LPS, the core is attached to the lipid A through D-glycero-D-talo-2-octulopyranosonic acid (Ko), whereas in a minor fraction (<20%) Ko is replaced by 3-deoxy-D-manno-octulopyranosonic acid (Kdo). The structures of the phosphorylated carbohydrate backbones of these LPS fractions are [structure: see text] with Dha = 3-deoxy-D-lyxo-2-heptulosaric acid, Sug = sugar and is Ko in a major fraction and Kdo in a minor fraction. All sugar residues have the D-configuration and are present in the pyranose form. Mass spectrometry of de-O-acylated LPS revealed the presence of an additional hexose residue in minor amounts, the position and nature of which could not be identified.

Acinetobacter↗
PubMed1997

Structural and serological characterisation of the O-antigenic polysaccharide of the lipopolysaccharide from Acinetobacter strain 90 belonging to DNA group 10.

Water-soluble lipopolysaccharide (phenol/water extraction) isolated from Acinetobacter strain 90, which belongs to DNA group 10, was hydrolysed with 1% acetic acid, ultracentrifuged, and water-soluble products finally eluted from a Sephadex G-50 column. The major fraction, a polysaccharide, contained D-Gal, D-GlcNAc, D-GalNAc, and 4,6-dideoxy-4-[(R)-3-hydroxybutyramido]-D-galactose (Fuc4NBuOH). The polysaccharide was characterised by means of monosaccharide analyses, Smith-degradation, N-deacetylation/deamination, and NMR studies, and was shown to have a branched pentasaccharide repeating unit. [structure in text] This structure was specifically recognised in western blots and enzyme immunoassays by polyclonal rabbit antisera.

Acinetobacter↗
PubMed1997

Structural and serological characterisation of the O-antigenic polysaccharide of the lipopolysaccharide from Acinetobacter junii strain 65.

A polysaccharide containing rhamnose (Rha) and Gal was isolated by acetic acid hydrolysis, followed by gel-permeation chromatography, from the water-soluble lipopolysaccharide (phenol/water extracted) from Acinetobacter junii strain 65. The polysaccharide was characterised by means of monosaccharide analyses, Smith degradation, and NMR studies, and was shown to have a linear pentasaccharide repeating unit, as depicted below. This structure was specifically recognised in western blots and enzyme immunoassays by polyclonal rabbit antisera. [structure in text]

Acinetobacter↗
PubMed1997

Structural and serological characterisation of the O-antigenic polysaccharide of the lipopolysaccharide from Acinetobacter haemolyticus strain ATCC 17906.

A polysaccharide containing 2-acetamido-2-deoxy-D-galacturonic acid (GalNAcA), 2.4-diacetamido-2,4,6-trideoxy-D-glucose (QuiNAc4NAc), and D-alanine (Ala) was isolated from the water-soluble lipopolysaccharide (LPS) originating from the reference strain for Acinetobacter haemolyticus (DNA group 4) strain ATCC 17906. The polysaccharide, characterised by means of monosaccharide analyses and NMR studies, was shown to be based on a linear trisaccharide repeating unit, as shown below, with the alanine group amide-bound to position 6 of one GalNAcA residue. It was specifically recognised in western blots by polyclonal rabbit antisera. [structure: see text]

Acinetobacter↗
PubMed1997

The structure of the carbohydrate backbone of the lipopolysaccharide from Acinetobacter strain ATCC 17905.

The structure of the carbohydrate backbone of the lipopolysaccharide from Acinetobacter strain ATCC 17905 was studied. After deacylation of the lipopolysaccharide, a mixture of two compounds (ratio approximately 2:1) was isolated by high-performance anion-exchange chromatography, the structures of which were determined by NMR spectroscopy and electrospray-mass spectrometry as [STRUCUTRE IN TEXT] [Sug, 3-deoxy-D-manno-2-octulopyranosonic acid (Kdo) in oligosaccharide 1 (major portion) and D-glycero-D-talo-2-octulopyranosonic acid (Ko) in oligosaccharide 2 (minor portion)]. All monosaccharide residues also possess the D-configuration and are present in the pyranose form.

Acinetobacter↗
PubMed1997

Structural and serological characterisation of the O-specific polysaccharide from lipopolysaccharide of Acinetobacter calcoaceticus strain 7 (DNA group 1).

S-form lipopolysaccharide was isolated by phenol/water extraction from a strain of Acinetobacter calcoaceticus (DNA group 1 ). The structure of the O-antigenic polysaccharide was determined by compositional analysis and NMR spectroscopy of the de-O-acylated lipopolysaccharide. The isolated polysaccharide obtained after hydrolysis of lipopolysaccharide in 0.01 M trifluoroacetic acid has the following structure: [STRUCTURE IN TEXT] in which Pyr is pyruvate. The O-acetyl substitution of D-Gal was non-stoichiometric. The O-antigen was specifically recognised in western blots by polyclonal rabbit antisera.

Acinetobacter calcoaceticus↗
PubMed1996

Structural and serological characterisation of two O-specific polysaccharides of Acinetobacter.

Extraction of dry bacteria of Acinetobacter strain 34 (DNA group 2) or Acinetobacter strain 108 (DNA group 13) by phenol/water yielded a polymer that was identified by means of serological studies and fatty acid analysis as S-form lipopolysaccharide. Degradation of the lipopolysaccharides of strains 34 and 108 in 1% acetic acid and 5% acetic acid, respectively, and gel-permeation chromatography gave the respective O-antigenic polysaccharides, the structures of which were determined, by compositional analysis and NMR spectroscopy of the polysaccharide, as [Sequence: see text] for strain 108, where D-Fucp3NBuOH represents 3-[(R)-3-hydroxybutyramido] -3,6-dideoxy-D-galactose and D-GalpANAc represents 2-acetamido-2-deoxy-D-galacturonic acid. Both structures were specifically recognised in Western blots by polyclonal rabbit antisera and there was no cross-reaction between these two structures.

Acinetobacter↗