Abstract
The structures of Rhizobium leguminosarum and Rhizobium etli lipid A are distinct from those found in other Gram-negative bacteria. Whereas the more typical Escherichia coli lipid A is a hexa-acylated disaccharide of glucosamine that is phosphorylated at positions 1 and 4', R. etli and R. leguminosarum lipid A consists of a mixture of structurally related species (designated A-E) that lack phosphate. A conserved distal unit, comprised of a diacylated glucosamine moiety with galacturonic acid residue at position 4' and a secondary 27-hydroxyoctacosanoyl (27-OH-C28) as part of a 2' acyloxyacyl moiety, is present in all five components. The proximal end is heterogeneous, differing in the number and lengths of acyl chains and in the identity of the sugar itself. A proximal glucosamine unit is present in B and C, but an unusual 2-amino-2-deoxy-gluconate moiety is found in D-1 and E. We now demonstrate that membranes of R. leguminosarum and R. etli can convert B to D-1 in a reaction that requires added detergent and is inhibited by EDTA. Membranes of Sinorhizobium meliloti and E. coli lack this activity. Mass spectrometry demonstrates that B is oxidized in vitro to a substance that is 16 atomic mass units larger, consistent with the formation of D-1. The oxidation of the lipid A proximal unit is also demonstrated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry in the positive and negative modes using the model substrate, 1-dephospho-lipid IV(A). With this material, an additional intermediate (or by product) is detected that is tentatively identified as a lactone derivative of 1-dephospho-lipid IV(A). The enzyme, presumed to be an oxidase, is located exclusively in the outer membrane of R. leguminosarum as judged by sucrose gradient analysis. To our knowledge, an oxidase associated with the outer membranes of Gram-negative bacteria has not been reported previously.
Highlights
The structures of Rhizobium leguminosarum and Rhizobium etli lipid A are distinct from those found in other Gram-negative bacteria
Whereas the more typical Escherichia coli lipid A is a hexa-acylated disaccharide of glucosamine that is phosphorylated at positions 1 and 4, R. etli and R. leguminosarum lipid A consists of a mixture of structurally related species that lack phosphate
We demonstrate that membranes of R. leguminosarum and R. etli can convert B to D-1 in a reaction that requires added detergent and is inhibited by EDTA
Summary
Materials—Glass-backed 0.25-mm Silica Gel 60 thin layer chromatography plates were from Merck. The pellet containing the membranes was washed by homogenization in the same volume of 50 mM HEPES, pH 7.5. The washed membranes were homogenized in 50 mM HEPES, pH 7.5, at a protein concentration of ϳ5–15 mg/ml. The cell pellet was resuspended in 7.8 ml of 50 mM HEPES, pH 7.5, containing 0.5 mM EDTA, and the cells were lysed by passage through a French pressure cell at 10,000 p.s.i. Crude cell-free extract was prepared by removal of unbroken cells and large debris by centrifugation at 12,100 ϫ g for 10 min at 4 °C, and the supernatant was recovered. The washed membranes were homogenized with a 25-gauge 1/2 syringe needle in a total volume of 2.5 ml of 50 mM HEPES, pH 7.5, containing 0.5 mM EDTA. The activity for each fraction was calculated as a percentage of the total activity throughout the entire gradient
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