RESUMEN
The lipopolysaccharide (LPS) core of the Gram-negative bacterium Rhizobium leguminosarum is more amenable to enzymatic study than that of Escherichia coli because much of it is synthesized from readily available sugar nucleotides. The inner portion of the R. leguminosarum core contains mannose, galactose, and three 3-deoxy-D-manno-octulosonate (Kdo) residues, arranged in the order: lipid A-(Kdo)2-Man-Gal-Kdo-[O antigen]. A mannosyltransferase that uses GDP-mannose and the conserved precursor Kdo2-[4'-32P]lipid IVA (Kadrmas, J. L., Brozek, K. A., and Raetz, C. R. H. (1996) J. Biol. Chem. 271, 32119-32125) is proposed to represent a key early enzyme in R. leguminosarum core assembly. Conditions for demonstrating efficient galactosyl- and distal Kdo-transferase activities are now described using a coupled assay system that starts with GDP-mannose and Kdo2-[4'-32P]lipid IVA. As predicted, mannose incorporation precedes galactose addition, which in turn precedes distal Kdo transfer. LPS core mutants with Tn5 insertions in the genes encoding the putative galactosyltransferase (lpcA) and the distal Kdo-transferase (lpcB) are shown to be defective in the corresponding in vitro glycosylation of Kdo2-[4'-32P]lipid IVA. We have also discovered the new gene (lpcC) that encodes the mannosyltransferase. The gene is separated by several kilobase pairs from the lpcAB cluster. All three glycosyltransferases are carried on cosmid pIJ1848, which contains at least 20 kilobase pairs of R. leguminosarum DNA. Transfer of pIJ1848 into R. meliloti 1021 results in heterologous expression of all three enzymes, which are not normally present in strain 1021. Expression of the lpc genes individually behind the T7 promoter results in the production of each R. leguminosarum glycosyltransferase in E. coli membranes in a catalytically active form, demonstrating that lpcA, lpcB, and lpcC are structural genes.
Asunto(s)
Glicosiltransferasas/genética , Lipopolisacáridos/biosíntesis , Rhizobium leguminosarum/enzimología , Bacteriófago T7/genética , Secuencia de Bases , Secuencia de Carbohidratos , Clonación Molecular , Cósmidos , Cartilla de ADN , Glicosilación , Lipopolisacáridos/química , Datos de Secuencia Molecular , Mutagénesis InsercionalRESUMEN
Heptosyltransferase I, encoded by the rfaC(waaC) gene of Escherichia coli, is thought to add L-glycero-D-manno-heptose to the inner 3-deoxy-D-manno-octulosonic acid (Kdo) residue of the lipopolysaccharide core. Lipopolysaccharide isolated from mutants defective in rfaC lack heptose and all other sugars distal to heptose. The putative donor, ADP-L-glycero-D-manno-heptose, has never been fully characterized and is not readily available. In cell extracts, the analog ADP-mannose can serve as an alternative donor for RfaC-catalyzed glycosylation of the acceptor, Kdo2-lipid IVA. Using a T7 promoter construct that overexpresses RfaC approximately 15,000-fold, the enzyme has been purified to near homogeneity. NH2-terminal sequencing confirms that the purified enzyme is the rfaC gene product. The subunit molecular mass is 36 kDa. Enzymatic activity is dependent upon the presence of Triton X-100 and is maximal at pH 7.5. The apparent Km (determined at near saturating concentrations of the second substrate) is 1.5 mM for ADP-mannose and 4.5 microM for Kdo2-lipid IVA. Chemical hydrolysis of the RfaC reaction product at 100 degrees C in the presence of sodium acetate and 1% sodium dodecyl sulfate generates fragments consistent with the inner Kdo residue of Kdo2-lipid IVA as the site of mannosylation. The analog, Kdo-lipid IVA, functions as an acceptor, but is mannosylated at less than 1% the rate of Kdo2-lipid IVA. The purified enzyme displays no activity with ADP-glucose, GDP-mannose, UDP-glucose, or UDP-galactose. Mannosylation of Kdo2-lipid IVA catalyzed by RfaC proceeds in high yield and may be useful for the synthesis of lipopolysaccharide analogs. Pure RfaC can also be used together with Kdo2-[4'-32P]lipid IVA to assay for the physiological donor (presumably ADP-L-glycero-D-manno-heptose) in a crude, low molecular weight fraction isolated from wild type cells.
Asunto(s)
Escherichia coli/enzimología , Glicosiltransferasas/metabolismo , Lípido A/análogos & derivados , Lipopolisacáridos/biosíntesis , Secuencia de Aminoácidos , Secuencia de Carbohidratos , Glucolípidos/metabolismo , Glicosilación , Glicosiltransferasas/efectos de los fármacos , Glicosiltransferasas/genética , Glicosiltransferasas/aislamiento & purificación , Concentración de Iones de Hidrógeno , Lípido A/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Azúcares de Nucleósido Difosfato/metabolismo , Octoxinol/farmacología , Proteínas Recombinantes/metabolismo , Análisis de Secuencia , Especificidad por SustratoRESUMEN
The genes for seven of nine enzymes needed for the biosynthesis of Kdo2-lipid A (Re endotoxin) in Escherichia coli have been reported. We have now identified a novel gene encoding the lipid A 4'-kinase (the sixth step of the pathway). The 4'-kinase transfers the gamma-phosphate of ATP to the 4'-position of a tetraacyldisaccharide 1-phosphate intermediate (termed DS-1-P) to form tetraacyldisaccharide 1,4'-bis-phosphate (lipid IVA). The 4'-phosphate is required for the action of distal enzymes, such as Kdo transferase and also renders lipid A substructures active as endotoxin antagonists or mimetics. Lysates of E. coli generated using individual lambda clones from the ordered Kohara library were assayed for overproduction of 4'-kinase. Only one clone, [218]E1D1, which directed 2-2.5-fold overproduction, was identified. This construct contains 20 kilobase pairs of E. coli DNA from the vicinity of minute 21. Two genes related to the lipid A system map in this region: msbA, encoding a putative translocator, and kdsB, the structural gene for CMP-Kdo synthase. msbA forms an operon with a downstream, essential open reading frame of unknown function, designated orfE. orfE was cloned into a T7 expression system. Washed membranes from cells overexpressing orfE display approximately 2000-fold higher specific activity of 4'-kinase than membranes from cells with vector alone. Membranes containing recombinant, overexpressed 4'-kinase (but not membranes with wild-type kinase levels) efficiently phosphorylate three DS-1-P analogs: 3-aza-DS-1-P, base-treated DS-1-P, and base-treated 3-aza-DS-1-P. A synthetic hexaacylated DS-1-P analog, compound 505, can also be phosphorylated by membranes from the overproducer, yielding [4'-32P] lipid A (endotoxin). The overexpressed lipid A 4'-kinase is very useful for making new 4'-phosphorylated lipid A analogs with potential utility as endotoxin mimetics or antagonists. We suggest that orfE is the structural gene for the 4'-kinase and that it be redesignated lpxK.
Asunto(s)
Proteínas Bacterianas/genética , Escherichia coli/genética , Genes Bacterianos , Lípido A/análogos & derivados , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Mapeo Cromosómico , Clonación Molecular , Escherichia coli/enzimología , Expresión Génica , Biblioteca de Genes , Lípido A/metabolismo , Lipopolisacáridos/metabolismo , Sistemas de Lectura Abierta , Fosforilación , Proteínas RecombinantesRESUMEN
The lipopolysaccharide of Rhizobium leguminosarum differs from that of other Gram-negative organisms. R. leguminosarum lipid A lacks phosphate groups, but it contains a galacturonic acid residue at the 4'-position and an aminogluconate moiety in place of the usual glucosamine 1-phosphate unit. R. leguminosarum lipid A is esterified with a peculiar long chain fatty acid, 27-hydroxyoctacosanoate, not found in enteric Gram-negative bacteria, and the inner core of R. leguminosarum contains mannose and galactose in place of heptose. Despite these differences, the biosynthesis of R. leguminosarum lipid A is initiated by the same seven enzyme pathway as in Escherichia coli (Raetz, C. R. H. (1993) J. Bacteriol. 175, 5745-5753) to form the phosphorylated precursor, (Kdo)2-lipid IVA, which is then processed differently. We now describe several novel Rhizobium-specific enzymes that recognize and modify (Kdo)2-lipid IVA. The 1- and 4'-phosphatases were detected using (Kdo)2-[1-32P]-lipid IVA and (Kdo)2-[4'-32P]-lipid IVA, respectively, as shown by release of 32Pi. In the presence of GDP-mannose and/or UDP-galactose, membranes of R. leguminosarum first transferred mannose and then galactose to (Kdo)2-[4'-32P]-lipid IVA. In addition, at least two hydrophobic metabolites were generated from (Kdo)2-[4'-32P]-lipid IVA in a manner that was dependent upon both membranes and a cytosolic factor from R. leguminosarum. These compounds are attributed to novel acylations of (Kdo)2-[4'-32P]-lipid IVA. E. coli membranes and cytosol did not catalyze any of the unique reactions detected in R. leguminosarum extracts. Our findings establish the conservation and versatility of (Kdo)2-lipid IVA as a lipid A precursor in bacteria.
Asunto(s)
Aciltransferasas/metabolismo , Galactosiltransferasas/metabolismo , Lípido A/metabolismo , Lipopolisacáridos/biosíntesis , Manosiltransferasas/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Rhizobium leguminosarum/enzimología , Azúcares Ácidos/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Carbohidratos , Citosol/metabolismo , Escherichia coli , Glucolípidos/metabolismo , Guanosina Difosfato Manosa/metabolismo , Lípido A/análogos & derivados , Modelos Biológicos , Datos de Secuencia Molecular , Uridina Difosfato Galactosa/metabolismoRESUMEN
The lipopolysaccharide structure of the nitrogen-fixing bacterium Rhizobium leguminosarum differs from that of Escherichia coli in several ways, one of which is the sugar composition of the core. The E. coli inner core consists of 3-deoxy-D-manno-octulosonic acid (Kdo) and L-glycero-D-manno-heptose (heptose), while the inner core of R. leguminosarum contains 2-keto-3-deoxy-D-manno-octulosonic acid (Kdo), mannose, galactose, and galacturonic acid. The two Kdo residues and their linkages appear to be identical in both species. The linkages of heptose in E. coli and of mannose in R. leguminosarum to Kdo are both alpha1-5. We now characterize a membrane-associated glycosyl transferase in R. leguminosarum extracts that incorporates mannose into nascent lipopolysaccharide, using Kdo2-lipid IVA as the acceptor and GDP-mannose (or synthetic ADP-mannose) as the donor. The mannosyl transferase is associated with the inner membrane. The apparent Km values for GDP-mannose and Kdo2-lipid IVA are 4.3 microM and 7.1 microM, respectively, in the presence of excess co-substrate. Extracts of E. coli do not catalyze GDP-mannose-dependent glycosylation of Kdo2-lipid IVA, but they are active when ADP-mannose is substituted for GDP-mannose. Given the structural similarity of ADP-mannose to ADP-heptose, we examined the possibility that heptosyl transferase I of E. coli (the product of the rfaC gene) catalyzes mannose transfer from ADP-mannose to Kdo2-lipid IVA. Extracts of E. coli mutants defective in the rfaC gene are unable carry out ADP-mannose-dependent glycosylation of Kdo2-lipid IVA. Plasmids bearing rfaC+ not only restore the missing activity but also direct its overexpression. Our assay using ADP-mannose as a substitute for ADP-heptose (which is not readily available) should facilitate the purification and characterization of heptosyl transferase I of E. coli. The GDP-mannose-dependent enzyme of R. leguminosarum may represent a functional equivalent of E. coli RfaC.
Asunto(s)
Glucosiltransferasas/metabolismo , Lipopolisacáridos/metabolismo , Manosa/metabolismo , Manosiltransferasas/metabolismo , Rhizobium leguminosarum/metabolismo , Escherichia coli/enzimología , Glucolípidos/metabolismo , Glicosilación , Guanosina Difosfato Manosa/metabolismo , Cinética , Lípido A/análogos & derivados , Lípido A/metabolismo , Manosa/análogos & derivados , Fracciones Subcelulares/metabolismo , Azúcares ÁcidosRESUMEN
Lipid A from several strains of the N2-fixing bacterium Rhizobium leguminosarum displays significant structural differences from Escherichia coli lipid A, one of which is the complete absence of phosphate groups. However, the first seven enzymes of E. coli lipid A biosynthesis, leading from UDP-GlcNAc to the phosphorylated intermediate, 2-keto-3-deoxyoctulosonate (Kdo2)-lipid IVA, are present in R. leguminosarum. We now describe a membrane-bound phosphatase in R. leguminosarum extracts that removes the 4' phosphate of Kdo2-lipid IVA. The 4' phosphatase is selective for substrates containing the Kdo domain. It is present in extracts of R. leguminosarum biovars phaseoli, viciae, and trifolii but is not detectable in E. coli and Rhizobium meliloti. A nodulation-defective strain (24AR) of R. leguminosarum biovar trifolii, known to contain a 4' phosphatase residue on its lipid A, also lacks measurable 4' phosphatase activity. The Kdo-dependent 4' phosphatase appears to be a key reaction in a pathway for generating phosphate-deficient lipid A.
Asunto(s)
Lípido A/biosíntesis , Monoéster Fosfórico Hidrolasas/metabolismo , Rhizobium leguminosarum/metabolismo , Secuencia de Carbohidratos , Escherichia coli/metabolismo , Genes Bacterianos , Lípido A/química , Datos de Secuencia Molecular , Estructura Molecular , Mutación , Fosfatos/química , Monoéster Fosfórico Hidrolasas/genética , Rhizobium leguminosarum/genética , Sinorhizobium meliloti/metabolismo , Especificidad de la Especie , Fracciones Subcelulares/metabolismoRESUMEN
Competition binding and UV melting studies of a DNA model system consisting of three, four or five mutually complementary oligonucleotides demonstrate that unpaired bases at the branch point stabilize three- and five-way junction loops but destabilize four-way junctions. The inclusion of unpaired nucleotides permits the assembly of five-way DNA junction complexes (5WJ) having as few as seven basepairs per arm from five mutually complementary oligonucleotides. Previous work showed that 5WJ, having eight basepairs per arm but lacking unpaired bases, could not be assembled [Wang, Y.L., Mueller, J.E., Kemper, B. and Seeman, N.C. (1991) Biochemistry, 30, 5667-5674]. Competition binding experiments demonstrate that four-way junctions (4WJ) are more stable than three-way junctions (3WJ), when no unpaired bases are included at the branch point, but less stable when unpaired bases are present at the junction. 5WJ complexes are in all cases less stable than 4WJ or 3WJ complexes. UV melting curves confirm the relative stabilities of these junctions. These results provide qualitative guidelines for improving the way in which multi-helix junction loops are handled in secondary structure prediction programs, especially for single-stranded nucleic acids having primary sequences that can form alternative structures comprising different types of junctions.