Rhizobium fredii and Rhizobium meliloti produce 3-deoxy-D-manno-2-octulosonic acid-containing polysaccharides that are structurally analogous to group II K antigens (capsular polysaccharides) found in Escherichia coli.

The polysaccharide components from cultured cells of Rhizobium fredii USDA205 and Rhizobium meliloti AK631 were extracted with hot phenol-water and separated by repetitive gel filtration chromatography. Polyacrylamide gel electrophoresis, nuclear magnetic resonance spectrometry, and gas chromatography analyses showed that both of these bacterial species produce unique polysaccharides that contain a high proportion of 3-deoxy-D-manno-2-octulosonic acid (Kdo). These polysaccharides, which constituted a major portion of the extracted carbohydrate, are not excreted into the growth media (i.e., they are not extracellular polysaccharides) and are structurally distinct from the lipopolysaccharides. The primary structure of the preponderant polysaccharide from R. fredii USDA205 was determined by high-performance anion-exchange liquid chromatography, nuclear magnetic resonance spectrometry, fast atom bombardment-mass spectrometry, and gas chromatography-mass spectrometry; it consists of repeating units of [-->3)-alpha-D-Galp-(1-->5)-beta-D-Kdop-(2-->]n. This molecule is structurally analogous to the constituents of one subgroup of K antigens (capsular polysaccharides) produced by Escherichia coli. Polysaccharides of this type have not previously been identified as components of rhizobial cells. The Kdo-containing polysaccharide from R. meliloti, which has not been completely characterized, appears to be structurally related to that of R. fredii.     

A bacteriophage-associated glycanase cleaving beta-pyranosidic linkages of 3-deoxy-D-manno-2-octulosonic acid (KDO)

A bacteriophage growing on Escherichia coli K13, K20, and K23 strains carries a glycanase that catalyzes the hydrolytic cleavage of the beta-ketopyranosidic linkages of 3-deoxy-D-manno-2-octulosonic acid (KDO) in the respective capsular polysaccharides. The main cleavage product of the K23 polysaccharide has been identified by 1H- and 13C-n.m.r. spectroscopy as beta beta Ribfl----7 beta KDOp2----3-beta Ribfl----7KDO. Cleavage of polysaccharides containing alpha-pyranosidic, or 5-substituted beta-pyranosidic KDO is not catalyzed by the enzyme.     

Synthesis of trisaccharides containing 3-deoxy-D-manno-2-octulosonic acid residues related to the KDO-region of enterobacterial lipopolysaccharides

Glycosylation of methyl (allyl 7,8-O-carbonyl-3-deoxy-alpha-D-manno-2-octulo-pyranosid)o nate with an alpha-(2----4) linked per-O-acetylated KDO-disaccharide bromide derivative under Helferich conditions afforded a 2:1 mixture of the alpha- and beta-linked trisaccharide derivatives in 50% yield. Removal of the protecting groups gave sodium O-[sodium (3-deoxy-alpha-D-manno-2-octulopyranosyl)onate]-(2----4)-O-[ sodium (3-deoxy-alpha- and -beta-D-manno-2-octulopyranosyl)onate]-(2----4)-sodium (allyl 3-deoxy-alpha-D-manno-2-octulopyranosid)onate. Radical copolymerization of the allyl glycosides afforded artificial antigens, suitable for defining antibody specificities directed against the KDO-region of enterobacterial lipopolysaccharides.     

Hydrolytic release, and identification by g.l.c.-m.s., of 3-deoxy-D-manno-2-octulosonic acid in the lipopolysaccharides isolated from bacteria of the Vibrionaceae

The identification of the peracetylated methyl glycosides of 3-deoxy-D-manno-2-octulosonic acid (KDO) methyl esters was achieved by g.l.c.-m.s. These peracetylated methyl glycoside methyl esters were obtained from fully acetylated lipopolysaccharides and core oligosaccharides of representative strains of the Vibrionaceae family by the following sequence of mild reactions: acetolysis, methanolysis, and acetylation. KDO was shown to be present in all of the lipopolysaccharides (LPS), a result in direct contrast to the generally accepted view of the absence of this compound in LPS from this family of bacteria.     

Methylation analysis of the heptose/3-deoxy-D-manno-2-octulosonic acid region (inner core) of the lipopolysaccharide from Salmonella minnesota rough mutants

A modified methylation analysis is described which allows the elucidation of the structure of the inner core region [heptose/3-deoxy-D-manno-2-octulosonic acid (KDO)] of enterobacterial lipopolysaccharides (LPS) of Salmonella minnesota rough mutants (Re, strain R595; and Rd2P-, strain R4). Methylation, carboxyl-reduction, remethylation, hydrolysis, carbonyl-reduction, and acetylation of the Re-mutant LPS yielded the 2,6-di-O-acetyl and 2,4,6-tri-O-acetyl derivatives of partially methylated 3-deoxyoctitol in equimolar amounts, indicating the presence of a terminal and a 4-linked pyranosidic KDO residue. For Rd2P- LPS, the hydrolysis step involved 0.1M trifluoroacetic acid at 100 degrees for 1 h which cleaved ketosidic linkages, and the final products included the foregoing acetyl derivatives in the molar ratio of 1:02 and a partially methylated and acetylated 3-deoxyoctitol derivative which was substituted at O-5 by a methylated heptopyranosyl residue. Trideuteriomethylation of the latter product followed by methanolysis and acetylation gave 5-O-acetyl-3-deoxy-1,7,8-tri-O-methyl-2,4,6-tri-O-trideuteriomethyl++ +-D- glycero-D-talo/galacto-octitol and 1,5-di-O-acetyl-2,3,4,6,7-penta-O-methyl-L-glycero-D-manno-heptitol++ +. These results prove the presence of a (2----4)-linked KDO disaccharide in Re LPS and show that the core region of Rd2P- LPS contains a terminal alpha-L-glycero-D-manno-heptopyranosyl group and a non-substituted, a 4-O-, and a 4,5-di-O-substituted pyranosidic KDO residue in the molar ratios 1:1:0.2:1.     

Cloning and characterization of ce/8A gene from Rhizobium leguminosarum bv. trifolii 1536

AIMS: To isolate the cellulase gene from Rhizobium leguminosarum bv. trifolii 1536. METHODS AND RESULTS: By the shot-gun method a clone (cel8A) harbouring 3.1 kb genomic DNA fragment from R. leguminosarum bv. trifolii 1536 was obtained. The cel8A gene coded 348 amino acids and it belongs to the glycosyl hydrolase family 8. The molecular mass of Cel8A protein induced from Escherichia coli DH5alpha, appeared to be 35 kDa. The optimum pH and optimum temperature was 7.0, and about 30 degrees C for its enzymatic activity respectively. CONCLUSIONS: R. leguminosarum bv. trifolii 1536 had cel8A gene having an open reading frame of 1047 bp coded for the activity of hydrolyzation of carboxymethyl cellulose. SIGNIFICANCE AND IMPACT OF THE STUDY: The production of celluloytic enzyme by R. leguminosarum bv. trifolii was confirmed, which would play specific roles in rhizobia. Future study should focus on its role in the infection and nodulation phenomena.     

Origin of the 2-amino-2-deoxy-gluconate unit in Rhizobium leguminosarum lipid A. Expression cloning of the outer membrane oxidase LpxQ

An unusual feature of the lipid A from the plant endosymbionts Rhizobium etli and Rhizobium leguminosarum is the presence of a proximal sugar unit consisting of a 2-amino-2-deoxy-gluconate moiety in place of glucosamine. An outer membrane oxidase that generates the 2-amino-2-deoxy-gluconate unit from a glucosamine-containing precursor is present in membranes of R. leguminosarum and R. etli but not in S. meliloti or Escherichia coli. We now report the identification of a hybrid cosmid that directs the overexpression of this activity by screening 1800 lysates of individual colonies of a R. leguminosarum 3841 genomic DNA library in the host strain R. etli CE3. Two cosmids (p1S11D and p1U12G) were identified in this manner and transferred into S. meliloti, in which they also directed the expression of oxidase activity in the absence of any chromosomal background. Subcloning and sequencing of the oxidase gene on a 6.5-kb fragment derived from the approximately 20-kb insert in p1S11D revealed that the enzyme is encoded by a gene (lpxQ) that specifies a protein of 224 amino acid residues with a putative signal sequence cleavage site at position 28. Heterologous expression of lpxQ using the T7lac promoter system in E. coli resulted in the production of catalytically active oxidase that was localized in the outer membrane. A new outer membrane protein of the size expected for LpxQ was present in this construct and was subjected to microsequencing to confirm its identity and the site of signal peptide cleavage. LpxQ expressed in E. coli generates the same products as seen in R. leguminosarum membranes. LpxQ is dependent on O(2) for activity, as demonstrated by inhibition of the reaction under strictly anaerobic conditions. An ortholog of LpxQ is present in the genome of Agrobacterium tumefaciens, as shown by heterologous expression of oxidase activity in E. coli.     

The pea nodule environment restores the ability of a Rhizobium leguminosarum lipopolysaccharide acpXL mutant to add 27-hydroxyoctacosanoic acid to its lipid A

Members of the Rhizobiaceae contain 27-hydroxyoctacosanoic acid (27OHC(28:0)) in their lipid A. A Rhizobium leguminosarum 3841 acpXL mutant (named here Rlv22) lacking a functional specialized acyl carrier lacked 27OHC(28:0) in its lipid A, had altered growth and physiological properties (e.g., it was unable to grow in the presence of an elevated salt concentration [0.5% NaCl]), and formed irregularly shaped bacteroids, and the synchronous division of this mutant and the host plant-derived symbiosome membrane was disrupted. In spite of these defects, the mutant was able to persist within the root nodule cells and eventually form, albeit inefficiently, nitrogen-fixing bacteroids. This result suggested that while it is in a host root nodule, the mutant may have some mechanism by which it adapts to the loss of 27OHC(28:0) from its lipid A. In order to further define the function of this fatty acyl residue, it was necessary to examine the lipid A isolated from mutant bacteroids. In this report we show that addition of 27OHC(28:0) to the lipid A of Rlv22 lipopolysaccharides is partially restored in Rlv22 acpXL mutant bacteroids. We hypothesize that R. leguminosarum bv. viciae 3841 contains an alternate mechanism (e.g., another acp gene) for the synthesis of 27OHC(28:0), which is activated when the bacteria are in the nodule environment, and that it is this alternative mechanism which functionally replaces acpXL and is responsible for the synthesis of 27OHC(28:0)-containing lipid A in the Rlv22 acpXL bacteroids.     

Biosynthesis of lipopolysaccharide in Escherichia coli. Cytoplasmic enzymes that attach 3-deoxy-D-manno-octulosonic acid to lipid A

Previous studies in our laboratory led to the elucidation of the covalent structure of a tetraacyldisaccharide 1,4'-bisphosphate precursor of lipid A (designated lipid IVA), that accumulates at 42 degrees C in temperature-sensitive mutants defective in 3-deoxy-D-manno-octulosonic acid (KDO) biosynthesis (Raetz, C. R. H., Purcell, S., Meyer, M. V., Qureshi, N., and Takayama, K. (1985) J. Biol. Chem. 260, 16080-16088). Using [4'-32P]lipid IVA as the probe, we now demonstrate the existence of cytoplasmic KDO-transferases in Escherichia coli capable of attaching 2 KDO residues, derived from CMP-KDO, to lipid IVA. A partial purification has been developed to obtain a cytoplasmic subfraction that adds these 2 KDO residues with a 90% yield. The product is shown to have the stoichiometry of (KDO)2-IVA by fast atom bombardment mass spectrometry and NMR spectroscopy. The partially purified enzyme can utilize alternative lipid-disaccharide cosubstrates bearing five or six fatty acyl chains, but it has an absolute requirement for a monophosphate residue at position 4' of the lipid acceptor. When reincubated with a crude cytoplasmic fraction, a nucleoside triphosphate and Mg2+, (KDO)2-IVA is rapidly metabolized to more polar substances, the identity of which is unknown. The KDO-transferase(s) described in the present study should be very useful for the semisynthetic preparation of complex lipopolysaccharide substructures and analogs.     

Analysis of the lipid moiety of lipopolysaccharide from Rhizobium tropici CIAT899: identification of 29-hydroxytriacontanoic acid.

The lipid moieties of two lipid A's isolated from the phenolic and aqueous fractions of lipopolysaccharide from Rhizobium tropici CIAT899 have been studied. Several 3-hydroxy fatty acids and two long-chain hydroxy fatty acids, 27-hydroxyoctacosanoic acid, and 29-hydroxytriacontanoic acid were identified; the ratios of these acids are the same in both lipid A's. These results can be used for chemotaxonomic purposes.     

Acyl-acyl carrier protein specificity of UDP-GlcNAc acyltransferases from gram-negative bacteria: relationship to lipid A structure.

Lipid A, the component of lipopolysaccharide that provides the membrane anchor of the core and O-antigen sugars, is known to contain characteristic R-3-hydroxy fatty acids bound to the 2,2' (N-linked) and 3,3' (O-linked) positions of the glucosamine disaccharide in different gram-negative bacteria. The studies reported here show that it is the acyl-acyl carrier protein specificities of the enzymes UDP-GlcNAc-O-acyltransferase and UDP-3-O-[(R)-3-hydroxyacyl]-GlcN-N-acyltransferase that determine the nature of these fatty acids.     

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from structurally unrelated single carbon sources by newly isolated Pseudomonas sp. EL-2

Summary A Pseudomonas sp. EL-2 strain capable of synthesizing poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] was isolated from activated sludge. For simulation of P(3HB-co-3HV) production in the cells, deficiency of nutrients such as NH₄ ⁺, SO₄ ^2- and Mg²⁺ was crucial and the maximum content of P(3HB-co-3HV) could reach 46% on NH₄ ⁺-deficient medium. This organism synthesized P(3HB-co-3HV) with 3HV monomer in the range from 1.9 to 49.3 mol% from unrelated single carbon sources such as glucose, fructose, propionate, or sorbitol. P(3HB-co-3HV)s containing a higher fraction of 3HV were produced by adding propionic acid to glucose medium.     

Expression and characterization of the protein Rv1399c from Mycobacterium tuberculosis. A novel carboxyl esterase structurally related to the HSL family.

The Mycobacterium tuberculosis genome contains an unusually high number of proteins involved in the metabolism of lipids belonging to the Lip family, including various nonlipolytic and lipolytic hydrolases. Driven by a structural genomic approach, we have biochemically characterized the Rv1399c gene product, LipH, previously annotated as a putative lipase. Rv1399c was overexpressed in E. coli as inclusion bodies and refolded. Rv1399c efficiently hydrolyzes soluble triacylglycerols and vinyl esters. It is inactive against emulsified substrate and its catalytic activity is strongly inhibited by the diethyl paranitrophenyl phosphate (E600). These kinetic behaviors unambiguously classify Rv1399c as a nonlipolytic rather than a lipolytic hydrolase. Sequence alignment reveals that this enzyme belongs to the alpha/beta hydrolase fold family and shares 30-40% amino acid sequence identity with members of the hormone-sensitive lipase subfamily. A model of Rv1399c derived from homologous three-dimensional structures reveals a canonical catalytic triad (Ser162, His290 and Asp260) located at the bottom of a solvent accessible pocket lined by neutral or charged residues. Based on this model, kinetic data of the Arg213Ala mutant partially explain the role of the guanidinium moiety, located close to His290, to confer an unusual low pH shift of the catalytic histidine in the wild type enzyme. Overall, these data identify Rv1399c as a new nonlipolytic hydrolase from M. tuberculosis and we thus propose to reannotate its gene product as NLH-H.     

3-Deoxy-D-manno-oct-2-ulosonic acid (Kdo) transferase of Legionella pneumophila transfers two kdo residues to a structurally different lipid A precursor of Escherichia coli

The 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) transferase gene of Legionella pneumophila was cloned and sequenced. Despite remarkable structural differences in lipid A, the gene complemented a corresponding Escherichia coli mutant and was shown to encode a bifunctional enzyme which transferred 2 Kdo residues to a lipid A acceptor of E. coli.     

Biosynthesis of KDN (2-keto-3-deoxy-D-glycero-D-galacto-nononic acid). Identification and characterization of a KDN-9-phosphate synthetase activity from trout testis.

Although the deaminoneuraminic acid or KDN glycotope (2-keto-3-deoxy-D-glycero-D-galacto-nononic acid) is expressed in glycoconjugates that range in evolutionary diversity from bacteria to man, there is little information as to how this novel sugar is synthesized. Accordingly, biosynthetic studies were initiated in trout testis, an organ rich in KDN, to determine how this sialic acid is formed. These studies have shown that the pathway consists of the following three sequential reactions: 1) Man + ATP --> Man-6-P + ADP; 2) Man-6-P + PEP --> KDN-9-P + P(i); 3) KDN-9-P --> KDN + P(i). Reaction 1, catalyzed by a hexokinase, is the 6-O-phosphorylation of mannose to form D-mannose 6-phosphate (Man-6-P). Reaction 2, catalyzed by KDN-9-phosphate (KDN-9-P) synthetase, condenses Man-6-P and phosphoenolpyruvate (PEP) to form KDN-9-P. Reaction 3, catalyzed by a phosphatase, is the dephosphorylation of KDN-9-P to yield free KDN. It is not known if a kinase specific for Man (Reaction 1) and a phosphatase specific for KDN-9-P (Reaction 3) may exist in tissues actively synthesizing KDN. In this study, the KDN-9-P synthetase, an enzyme that has not been previously described, was identified as at least one key enzyme that is specific for the KDN biosynthetic pathway. This enzyme was purified 50-fold from rainbow trout testis and characterized. The molecular weight of the enzyme was estimated to be about 80,000, and activity was maximum at neutral pH in the presence of Mn(2+). N-Acetylneuraminic acid 9-phosphate (Neu5Ac-9-P) synthetase, which catalyzes the condensation of N-acetyl-D-mannosamine 6-phosphate and phosphoenol-pyruvate to produce Neu5Ac-9-P, was co-purified with the KDN-9-P synthetase. Substrate competition experiments revealed, however, that syntheses of KDN-9-P and Neu5Ac-9-P were catalyzed by two separate synthetase activities. The significance of these studies takes on added importance with the recent discovery that the level of free KDN is elevated in human fetal cord but not matched adult red blood cells and in ovarian cancer cells (Inoue, S., Lin, S-L., Chang, T., Wu, S-H., Yao, C-W., Chu, T-Y., Troy, F. A., II, and Inoue, Y. (1998) J. Biol. Chem. 273, 27199-27204). This unexpected finding emphasizes the need to understand more fully the role that free KDN and KDN-glycoconjugates may play in normal hematopoiesis and malignancy.     

Two-dimensional NMR spectroscopy and structures of six lipid A species from Rhizobium etli CE3. Detection of an acyloxyacyl residue in each component and origin of the aminogluconate moiety

The chemical structures of six lipid A species (A, B, C, D-1, D-2, and E) purified from Rhizobium etli CE3 were investigated by one- and two-dimensional NMR spectroscopy. The R. etli lipid A subtypes each contain an unusual acyloxyacyl residue at position 2' as part of a conserved distal glucosamine moiety but differ in their proximal units. All R. etli lipid A species lack phosphate groups. However, they are derivatized with an alpha-linked galacturonic acid group at position 4', as shown by nuclear Overhauser effect spectroscopy. Component B, which had been not been reported in previous studies, features a beta, 1'-6 linked disaccharide of glucosamine acylated at positions 2, 3, 2', and 3' in a pattern that is typical of lipid A found in other Gram-negative bacteria. D-1 contains an acylated aminogluconate unit in place of the proximal glucosamine residue of B. C and E lack ester-linked beta-hydroxyacyl chains at position 3, as judged by their H-3 chemical shifts, and may be synthesized from B and D-1, respectively, by the R. etli 3-O-deacylase. D-2 is an isomer of D-1 that forms nonenzymatically by acyl chain migration. A may be an elimination product derived from D-1 during hydrolysis at 100 degrees C (pH 4.5), a step needed to release lipid A from lipopolysaccharide. Based on these findings, we propose a biosynthetic scheme for R. etli lipid A in which B is generated first by a variation of the E. coli pathway. The aminogluconate unit of D-1 could then be made from B by enzymatic oxidation of the proximal glucosamine. As predicted by our hypothesis, enzyme(s) can be demonstrated in extracts of R. etli that convert (14)C-labeled B to D-1.     

Separation and characterization of six (1 leads to 3)-beta-glucanases from Saccharomyces cerevisiae.

Using a system of chromatography through columns of DEAE-Bio-Gel, HTP-Bio-Gel, and CM-Bio-Gel, we isolated and characterized six different (1 leads to 3)-beta-glucanases from cell wall autolysates and cell extracts of Saccharomyces cerevisiae haploid strain 2180B. These enzymes were designated glucanases I, II, IIIA, IIIB, IV, and V. The haploid mating type S. cerevisiae strain 2180A and the diploid strains S. cerevisiae 2180D and S. cerevisiae 595 contained the same complex of glucanases. Glucanases II and IIIA were exoenzymes, and glucanases I, IIIB, IV, and V were endoenzymes. The enzymes exhibited different molecular weights, kinetic properties, and activities on isolated yeast cell walls. The products of substrate (laminarin) hydrolysis were quantified by using high-pressure liquid chromatography and were significantly different for the four endoglucanases.     

Isolation and identification of novel ADP-ribosylated proteins from Streptomyces coelicolor A3(2)

An important role of protein ADP-ribosylation in bacterial morphogenesis has been proposed (J. Bacteriol. 178, 3785-3790; 178, 4935-4941). To clarify the detail of ADP-ribosylation, we identified a new kind of target protein for ADP-ribosylation in Streptomyces coelicolor A3(2) grown to the late growth phase. All four proteins (MalE, BldKB, a periplasmic protein for binding branched-chain amino-acids, and a periplasmic solute binding protein) were functionally similar and participated in the regulation of transport of metabolites or nutrients through the membrane. ADP-ribosylation was likely to occur on a cysteine residue, because the modification group was removed by mercuric chloride treatment. The modification site may be the site of lipoprotein modification necessary for protein export. This report is the first suggesting that certain proteins involved in membrane transport can be ADP-ribosylated.