Alginate is a viscous extracellular polymer produced by mucoid strains of Pseudomonas aeruginosa that cause chronic pulmonary infections in patients with cystic fibrosis. Alginate is polymerized from GDP-mannuronate to a linear polymer of ,B-1-4-linked residues of D-mannuronate and its C5-epimer, L-guluronate. We previously identified a gene called algG in the alginate biosynthetic operon that is required for incorporation of L-guluronate residues into alginate. In this study, we tested the hypothesis that the product of algG is a C5-epimerase that directly converts D-mannuronate to L-guluronate. The DNA sequence of algG was determined, and an open reading frame encoding a protein (AlgG) of approximately 60 kDa was identified. The inferred amino terminus of AlgG protein contained a putative signal sequence of 35 amino acids. Expression of aIgG in Escherichia coli demonstrated both 60-kDa pre-AIgG and 55-kDa mature AlgG proteins, the latter of which was localized to the periplasm. An N-terminal analysis of AIgG showed that the signal sequence was removed in the mature form. Pulse-chase experiments in both E. coli and P. aeruginosa provided evidence for conversion of the 60-to the 55-kDa size in vivo. Expression of algG from a plasmid in an algG (i.e., polymannuronate-producing) mutant of P. aeruginosa restored production of an alginate containing Lguluronate residues. The observation that AlgG is apparently processed and exported from the cytoplasm suggested that it may act as a polymer-level mannuronan C5-epimerase. An in vitro assay for mannuronan C5 epimerization was developed wherein extracts of E. coli expressing high levels of AlgG were incubated with polymannuronate. Epimerization of D-mannuronate to L-guluronate residues in the polymer was detected enzymatically, using a L-guluronate-specific alginate lyase of Klebsieila aerogenes. Epimerization was also detected in the in vitro reaction between recombinant AlgG and poly-D-mannuronate, using high-performance anion-exchange chromatography. The epimerization reaction was detected only when acetyl groups were removed from the poly-D-mannuronate substrate, suggesting that AIgG epimerization activity in vivo may be sensitive to acetylation of the D-mannuronan residues. These results demonstrate that AlgG has polymer-level mannuronan C5-epimerase activity.Alginate is an unbranched polysaccharide produced by Pseudomonas species (10, 13), Azotobacter vinelandii (31), and several species of brown seaweed (22). Alginate is composed of D-mannuronate and its C5-epimer, L-guluronate, which are linked by 13-1-4 glycosidic bonds (13). The L-guluronate is probably derived from D-mannuronate by the action of a C5-epimerase (Fig. 1). In bacteria, alginate is modified by the addition of O-acetyl groups on some D-mannuronate residues (8, 42). The sugar residues of alginate do not show repeating subunits characteristic of other bacterial exopolysaccharides (46). Mucoid strains of Pseudomonas aeruginosa produce alginate as a capsule-like exopolysaccharide and are responsible f...
Two long-chain fatty acids, 27-oxo-octacosanoic acid (28:0(27-oxo)) and heptacosane-1,27-dioic acid (27:0-dioic) were identified for the first time in phenol-chloroform-petroleum ether extracts of Legionella pneumophila, indicating that they are constituents of lipopolysaccharide. The fatty acids were characterised by combined gas-liquid chromatography/mass spectrometry and proton nuclear magnetic resonance spectroscopy. Moreover, minor amounts of 29-oxo-triacontanoic (30:0(29-oxo)) acid and nonacosane-1,29-dioic acid (29:0-dioic) as well as 27-hydroxy-octacosanoic acid (28:0(27-OH)) were present in the phenol-chloroform-petroleum ether extract.
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.
The lipopolysaccharide (endotoxin) content in airborne dust samples from three different poultry slaughterhouses was determined with both the chromogenic Limulus amebocyte lysate assay and gas chromatographymass spectrometry analysis of lipopolysaccharide-derived 3-hydroxy fatty acids. Gram-negative cell walls were also measured by using two-dimensional gas chromatography/electron-capture analysis of diaminopimelic acid originating from the peptidoglycan. The correlation between the results of the Limulus assay and those of gas chromatography-mass spectrometry for determination of the lipopolysaccharide content in the dust samples was poor, whereas a good correlation was obtained between lipopolysaccharide and diaminopimelic acid concentrations with the gas chromatographic methods. The results suggest that it is predominantly cellwall-dissociated lipopolysaccharides that are measured with the Limulus assay, whereas the gas chromatographic methods allow determination of total concentrations of lipopolysaccharide, including Limulus-inactive lipopolysaccharide, gram-negative cells, and cellular debris.
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