The Yersinia genus includes human and animal pathogens (plague, enterocolitis). The fine structures of the endotoxin lipids A of seven strains of Yersinia enterocolitica, Yersinia ruckeri and Yersinia pestis were determined and compared using mass spectrometry. These lipids differed in secondary acylation at C-2P P: this was dodecanoic acid (C 12 ) for two strains of Y. enterocolitica and Y. ruckeri, tetradecanoic acid (C 14 ) in two other Y. enterocolitica and hexadecenoic acid (C 16:1 ) in Y. pestis. The enterocolitica lipids having a mass identical to that of Escherichia coli were found to be structurally different. The results supported the idea of a relation between membrane fluidity and environmental adaptability in Yersinia.z 2000 Federation of European Biochemical Societies.
Membrane-derived oligosaccharides are branched, substituted fi-glucans localized in the periplasmic space of Escherichia coil and other Gram-negative bacteria.The biosynthesis of membrane-derived oligosaccharides and of analogous periplasmic oligosaccharides found in plant bacteria is of particular interest because it is subject to strict osmotic regulation [Miller, K. J., Kennedy, E. P., and Reinhold, V. N. (1986) Membrane-derived oligosaccharides (MDO) of Escherichia coli are periplasmic glucans variously substituted with sn-1-phosphoglycerol (derived from phosphatidylglycerol), phosphoethanolamine, and O-succinyl ester residues (1, 2). The synthesis of MDO and of analogous periplasmic glucans in other Gram-negative bacteria is subject to strict osmotic regulation (3,4). In E. coli, osmotic regulation is thought to be exerted at the level of the synthesis of the glucan chains. An enzyme system in E. coli that catalyzes the synthesis of (,B1-2)-linked glucan chains (the "backbone" of the MDO structure) from UDP-glucose was described by Weissborn and Kennedy (5). The system was found to require both a membrane enzyme fraction and a soluble protein termed transglucosylation factor. This enzyme system catalyzes the following reaction:[1]Here Glc-O-R is octyl P-D-glucopyranoside, a model substrate thought to function as a primer because of its resemblance to the postulated membrane-bound, nascent MDO chains. Genetic as well as biochemical evidence indicates that this enzyme system plays an essential role in the biosynthesis of MDO. Mutants in the mdoA locus are defective in the membrane component of the system catalyzing reaction 1 and are also blocked in the synthesis of MDO glucan chains in vivo (5, 6). We now report the purification of the transglucosylation factor to apparent homogeneity and the discovery that it is identical to acyl carrier protein (ACP), the well-characterized and intensively studied protein required for the biosynthesis of fatty acids and membrane phospholipids in E. coli (7). MATERIALS AND METHODSAssay of Transglucosylation Factor. The assay system contained the following components in the indicated final concentrations: 50 mM Tris HCl (pH 7.0); 5 mM MgSO4; 0.5 mM ATP; 5 mM 2-mercaptoethanol; 2 mM octyl P-Dglucopyranoside; and UDP-[3H]glucose (5) with a specific activity of 1600 cpm/nmol (1 mM). The final volume was usually 0.06 ml. Each tube contained membrane enzyme fraction (about 8 ,ug of protein) prepared from E. coli strain DF214 as described by Weissborn and Kennedy (5). Various amounts of transglucosylation factor or purified ACP were added. After incubation at 37°C for 90 min, the reaction was stopped by the addition of an equal volume of ethanol, and the conversion of labeled UDP-glucose to neutral glucan was measured as described by Weissborn and Kennedy (5). Significant activity (about 10% of that found with a saturating amount of transglucosylation factor) was found in the absence of added factor.ACP and Antibody Against ACP. ACP of E. coli and antibody directed again...
Structural studies of Bordetella endotoxins (LPSs) have revealed remarkable differences: (i) between their LPSs and those of other bacterial pathogens; (ii) among the LPSs of the seven identified Bordetella species; and (iii) among the LPSs of some Bordetella strains. The lipid As have the "classical" bisphosphorylated diglucosamine backbone but tend to have fewer and species-specific fatty acid components compared to those of other genera. Nevertheless, three strains of B. bronchiseptica have at least three different fatty acid distributions; however, the recently identified B. hinzii and B. trematum LPSs had identical lipid A structures. The B. pertussis core is a dodecasaccharide multi-branched structure bearing amino and carboxylic groups. Another unusual feature is the presence of free amino sugars in the central core region and a complex distal trisaccharide unit containing five amino groups of which four are acetylated and one is methylated. The B. pertussis LPS does not have O-chains and that of B. trematum had only a single O-unit, unlike the LPSs of all the other species of the smooth-type. The O-chain-free cores of non-B. pertussis LPSs were always built on the B. pertussis core model but most were species-specifically incomplete. The LPS structures of three B. bronchiseptica strains were found to be different from each other. The O-chains of B. bronchiseptica and B. parapertussis were almost identical and had some features in common with B. hinzii O-chain. Serological analyses are consistent with the determined LPS structures.
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