Anders Sonesson

Chemical composition of a lipopolysaccharide from Legionella pneumophila

Lipopolysaccharide isolated from Legionella pneumophila (Phil. 1) was examined for chemical composition. The polysaccharide split off by mild acid hydrolysis contained rhamnose, mannose, glucose, quinovosamine, glucosamine and 2-keto-3-deoxyoctonate, in molar proportions 1.6:1.8:1.0:1.5:4.1:2.7. Heptoses were absent and glucose was probably mainly phosphorylated. The carbohydrate backbone of the lipid A part consisted of glucosamine, quinovosamine and glycerol, in the molar ratios 3.9:1.0:3.4, with glycerol as a phosphorylated moiety. A complex fatty acid substitution pattern comprising eight O-ester-linked, exclusively nonhydroxylated acids, and nineteen amide-linked, exclusively 3-hydroxylated acids was revealed. Both straight- and branched (iso and anteiso) carbon chains occurred. The major hydroxy fatty acid was 3-hydroxy-12-methyltridecanoic acid and six others were of a chain-length above 20 carbon atoms, with 3-hydroxy-20-methyldocosanoic acid as the longest. Two dihydroxy fatty acids, 2,3-dihydroxy-12-methyltridecanoic and 2,3-dihydroxytetradecanoic acids, were also detected. These results suggest that L. pneumophila contains a rather complex and unusual lipopolysaccharide structure of considerable biological and chemotaxonomic interest.

Determination of environmental levels of peptidoglycan and lipopolysaccharide using gas chromatography with negative-ion chemical-ionization mass spectrometry utilizing bacterial amino acids and hydroxy fatty acids as biomarkers.

D-Alanine and diaminopimelic acid originating from bacterial peptidoglycans and hydroxy fatty acids from lipopolysaccharides (endotoxins) were analysed by gas chromatography using a chiral column (Chirasil-Val as stationary phase) and selected-ion monitoring detection with negative-ion chemical-ionization mass spectrometry. The amino acids were analysed as N-heptafluorobutyryl isobutyl esters after rapid hydrolysis of peptidoglycan followed by isolation of the amino acids with disposable ion-exchange columns. Racemization of amino acid enantiomers was controlled by using deuterium chloride in the hydrolysis. The hydroxy acids were analysed as O-pentafluorobenzoyl methyl esters. Most of the bacteria present in airborne dust from a poultry confinement building were found to be Gram-positive according to the analytical chemical method whereas the Limulus amoebocyte lysate test suggested the presence of appreciable amounts of lipopolysaccharides of Gram-negative bacteria. Further studies are required to compare the utility of these two methods for determining endotoxins in complex environments.

Composition of 2,3-dihydroxy fatty acid-containing lipopolysaccharides from Legionella israelensis, Legionella maceachernii and Legionella micdadei

Lipopolysaccharides (LPS) from Legionella israelensis, L. maceachernii and L. micdadei were analysed for chemical composition. The main sugar of the lipid A fractions was in each case 2,3-diamino-2,3-dideoxy-D-glucose. Lipid A of L. israelensis also contained a substantial amount of D-glucosamine. In each lipid A fraction a complex fatty acid pattern was detected. This comprised at least 19 different 3-hydroxy fatty acids (amide-linked), three 2,3-dihydroxy fatty acids (amide-linked), non-hydroxy fatty acids (ester-linked) as well as long-chain (omega-1)-oxo, (omega-1)-hydroxy and (1,omega)-dioic fatty acids (ester-linked). In addition, L. maceachernii and L. micdadei contained alpha-hydroxylated long-chain (omega-1)-oxo and (1,omega)-dioic fatty acids. The polysaccharide parts of L. maceachernii and L. micdadei LPS were similar and contained mainly L-rhamnose, L-fucose, D-mannose, D-glucose, L-fucosamine, D-glucosamine, 2-keto-3-deoxy-octonic acid (Kdo) as well as the rare octose yersiniose A. The corresponding composition of L. israelensis LPS was simpler and consisted mainly of L-rhamnose and 3-amino-3,6-dideoxy-D-mannose. LPS of L. israelensis and L. micdadei contained, in addition, 2-keto-octonic acid linked to Kdo. Phosphorylated sugar constituents were detected in all three LPS, whereas ethanolamine was found only in LPS from L. maceachernii. The SDS-PAGE band pattern of L. micdadei differed from the two others in a higher proportion of the low molecular mass constituents.

Compulsory higher education teacher training in Sweden: development of a national standards framework based in the scholarship of teaching and learning

Today visible proofs of excellence in teaching and learning are increasingly important aspects of institutional branding in higher education (HE). Teaching competence is brought forward as a central aspect of the quality of programmes. Still, the induction of new university teachers is managed in many different ways. Approaches may vary according to how teaching competence is perceived; as growing from practice only, requiring formal courses or, for instance, outlined in the Scholarship of Teaching and Learning (SoTL) movement. In Sweden, the HE Ordinance from 2002 states that to get permanent positions, lecturers should have completed Compulsory HE Teacher Training (CHETT). The size and organisation of the courses were not regulated in the Ordinance and institutional practices varied. In a three-year project intended learning outcomes for CHETT was suggested. These outcomes are based on SoTL and linked to an estimated workload of 10 weeks. Based on a national survey in 2006, institutional responses to the proposals are analysed.

Cytoskeleton components of inside-out and right-side-out plasma membrane vesicles from plants

SummaryIsolated plasma membrane vesicles purified by aqueous polymer two-phase partitioning were used as a model system for studies on the membrane-associated (cortical) cytoskeleton in plants. Actin, as identified by immunoblotting, was found to be specifically attached to plasma membrane vesicles from cauliflower (Brassica oleracea L.). The actin was not washed off as the vesicles were turned inside-out, indicative of a fairly strong attachment. Triton X-100 extraction of plasma membrane vesicles resulted in an insoluble and hence pelletable fraction where actin could be found together with several other proteins. Our results show that the cortical cytoskeleton is to some extent co-purified with the plasma membrane, and we believe that well defined, inside-out and right-side-out plasma membrane vesicles can be used to study the structure and dynamics of the plant cortical cytoskeleton.

Chemical characterization of lipopolysaccharides from Legionella feeleii, Legionella hackeliae and Legionella jordanis

Lipopolysaccharides (LPS) from Legionella feeleii serogroup 1, L. hackeliae serogroup 1 and L. jordanis were subjected to chemical analysis. All three LPS contained D-mannose, D-glucose, D-glucosamine, L-glycero-D-manno-heptose, 2-keto-3-deoxyoctonic acid and glycerol. In addition the LPS of L. feeleii was characterized by L-quinovose (tentatively identified) and L-fucosamine, L. hackeliae LPS by D-quinovosamine, D-galactosamine and D-galacturonic acid, and L. jordanis LPS by D-quinovosamine. Phosphorylated sugars were detected in all three LPS. The backbone sugar of the lipid A part was in each case 2,3-diamino-2,3-dideoxy-D-glucose substituted with a complex pattern of fatty acid, including 20-22 different amide-linked (non-branched and methyl-branched) 3-hydroxy fatty acids of chain-length ranging from 12 to 23 carbon atoms. The fatty acid patterns included also ester-linked nonhydroxylated entities and the uncommon 27-oxo-octacosanoic acid and 29-oxotriacontanoic acid. The LPS of L. hackeliae and L. jordanis also contained heptacosane-1,27-dioic and nonacosane-1,29-dioic acid, and their 2-hydroxy analogues were characteristic of L. jordanis LPS. SDS-PAGE patterns of the three LPS were distinctly different. Both L. feeleii and L. jordanis produced smooth-form LPS with characteristic ladder patterns, whereas L. hackeliae LPS were of more rough-type character.

The Characterization of Plasma Membrane-Bound Tubulin of Cauliflower Using Triton X-114 Fractionation

The cortical microtubules determine how cellulose microfibrils are deposited in the plant cell wall and are thus important for the control of cell expansion. To understand how microtubules can control microfibril deposition, the components that link the microtubules to the plasma membrane (PM) of plant cells must be isolated. To obtain information on the properties of the tubulin-membrane associations, cauliflower (Brassica oleracea) PM was subjected to Triton X-114 fractionation, and the distribution of [alpha]- and [beta]-tubulin was analyzed using immunoblotting. Approximately one-half of the PM-associated tubulin was solubilized by Triton X-114 and 10 to 15% of both [alpha]- and [beta]-tubulin was recovered in the detergent phase (indicative of hydrophobic properties) and 30 to 40% was recovered in the aqueous phase. The hydrophobic tubulin could be released from the membrane by high pH extraction with preserved hydrophobicity. A large part of the PM-associated tubulin was found in the Triton-insoluble fraction. When this insoluble material was extracted a second time, a substantial amount of hydrophobic tubulin was released if the salt concentration was increased, suggesting that the hydrophobic tubulin was linked to a high-salt-sensitive protein aggregate that probably includes other components of the cytoskeleton. The hydrophobicity of a fraction of PM-associated tubulin could reflect a direct or indirect interaction of this tubulin with the lipid bilayer or with an integral membrane protein and may represent the anchoring of the cortical microtubules to the PM, a key element in the regulation of cell expansion.

The branched-chain octose yersiniose A is a lipopolysaccharide constituent of Legionella micdadei and Legionella maceachernii

Abstract Lipopolysaccharides (LPS) were isolated from the two closely related species Legionella maceachernii and L. micdadei . The chemical composition of their polysaccharide parts have been have analysed and were found to be qualitatively similar. Hydrolysates included the sugars 2-keto-3-deoxyoctanoic acid, rhamnose, fucose, mannose, fucosaine, glucosamine, glucose and glycerol. The two latter constituents were present both phosporylated and unphosphorylated. In addition both LPS contained an uncommon sugar. Mass spectra of this compound as alditol acetate and methylglycoside (trifluoracetylated) were identical to those of yersiniose A ( 3,6- dideoxy -4-C-[1-( S )- hydroxyethyl ]- d -xylo- hexose ) isolated from Yersinia frederiksenii O:16.29. This identity was further substantiated by retention characteristics from anion-exchange chromatography, and gas chromatography both as alditol acetate and trifluoroacetylated methylglycoside. Thus, yersiniose A is a constituent of L. maceachernii and L. micdadei LPS and may prove useful as a marker in Legionella diagnostics.

Gas chromatographic determination of (phosphorylated) 2-keto-3-deoxyoctonic acid, heptoses and glucosamine in bacterial lipopolysaccharides after treatment with hydrofluoric acid, methanolysis and trifluoroacetylation

Quantification of phosphorylated sugar constituents of lipopolysaccharides has been performed by the following sequence: dephosphorylation by treatment with hydrofluoric acid, cleavage to monomeric constituents by methanolysis and analysis of the released sugars by capillary gas chromatography. Lipopolysaccharides of Salmonella minnesota Rd1P+, Bordetella pertussis NIH 114 and Vibrio cholerae, NAG and 95R strains, were used as model substances. Comparison of the chromatographic data obtained from hydrofluoric acid-treated and untreated lipopolysaccharide preparations indicated that all lipopolysaccharides examined contained one moiety of glucosamine bound to phosphate in a stable linkage. 2-Keto-3-deoxyoctonic acid appeared phosphorylated to a variable extent. Lipopolysaccharides of the two V. cholerae strains contained one moiety of fully phosphorylated 2-keto-3-deoxyoctonic acid, whereas in that of S. minnesota Rd1P+ only one of the three moieties was phosphorylated. Lipopolysaccharide of B. pertussis had one moiety of 2-keto-3-deoxyoctonic acid, ca. 70% phosphorylated. All four of the preparations examined contained L-glycero-D-manno-heptose in amounts varying from 2.6 to 5.2 moieties. In the lipopolysaccharides of B. pertussis and strain 95R of V. cholerae this sugar was unphosphorylated, whereas the two remaining strains contained one phosphorylated moiety of this sugar. Phosphorylated lipopolysaccharide constituents can be analysed by this approach on a 50-100 micrograms scale.

Determination of endotoxins by gas chromatography: evaluation of electron-capture and negative-ion chemical-ionization mass spectrometric detection of halogenated derivatives of β-hydroxymyristic acid

The sensitivity and selectivity of gas chromatography for analysing several halogenated ester derivatives of beta-hydroxymyristic acid were studied using both selected-ion monitoring detection with negative-ion chemical-ionization mass spectrometry and electron-capture detection. Six different derivatization methods were compared with respect to yield, chemical stability and formation of by-products. Procedures for removal of excess reagents using disposable silica columns and thin-layer chromatography were elaborated. The 3-O-pentafluorobenzoyl-methyl ester was the preferred derivative since it provided high sensitivity and had the molecular ion as the base peak in the mass spectrum. The detection limit was 0.5 pg with electron-capture detection and 0.3 pg with the mass spectrometric system. Using beta-hydroxymyristic acid as a chemical marker it was possible to detect Escherichia coli endotoxin in aqueous solution at a level of 1 ng/ml.