Chemical characterization of pH-dependent structural epitopes of lipopolysaccharides from Rhizobium leguminosarum biovar phaseoli.

Lipopolysaccharide (LPS) was isolated from free-living Rhizobium leguminosarum bv. phaseoli CE3 cells grown at pH 4.8 (antigenically similar to bacteroid LPS) and compared with that from cells grown at pH 7.2 (free-living bacteria). Composition analysis revealed that pH 7.2 LPS differs from pH 4.8 LPS in that 2,3,4-tri-O-methylfucose is replaced by 2,3-di-O-methylfucose. The amount of 2-O-methylrhamnose is greater in the pH 4.8 LPS than in the pH 7.2 LPS. Analysis of the structural components of LPS (O-chain polysaccharide, core oligosaccharides, and the lipid A) revealed that all the composition differences in the various LPSs occur in the O-chain polysaccharide. These structural variations between pH 4.8 and pH 7.2 LPSs provide a chemical basis for the observed lack of cross-reactivity with pH 4.8 LPS of two monoclonal antibodies, JIM28 and JIM29, raised against free-living bacteria grown at pH 7.2. An LPS preparation isolated from bacteroids contained both 2,3,4-tri-O- and 2,3-di-O-methylfucose residues. This result is consistent with the finding that the two monoclonal antibodies react weakly with bacteroid LPS. It is concluded that methylation changes occur on the LPS O-chain of R. leguminosarum bv. phaseoli when the bacteria are grown at low pH and during nodule development.     

Identification of an ATP-binding cassette transporter for export of the O-antigen across the inner membrane in Rhizobium etli based on the genetic, functional, and structural analysis of an lps mutant deficient in O-antigen

For O-antigen lipopolysaccharide (LPS) synthesis in bacteria, transmembrane migration of undecaprenyl pyrophosphate-bound O-antigen oligosaccharide subunits or polysaccharide occurs before ligation to the core region of the LPS molecule. In this study, we identified by mutagenesis an ATP-binding cassette transporter in Rhizobium etli CE3 that is likely responsible for the translocation of the O-antigen across the inner plasma membrane. Mutant FAJ1200 LPS lacks largely the O-antigen, as shown by SDS-polyacrylamide gel electrophoresis and confirmed by immunoblot analysis. Furthermore, LPS isolated from FAJ1200 is totally devoid of any O-chain glycosyl residues and contains only those glycosyl residues that can be expected for the inner core region. The membrane component and the cytoplasmic ATP-binding component of the ATP-binding cassette transporter are encoded by wzm and wzt, respectively. The Tn5 transposon in mutant FAJ1200 is inserted in the wzm gene. This mutation resulted in an Inf- phenotype in bean plants.     

Structural characterization of the O-antigenic polysaccharide of the lipopolysaccharide from Rhizobium etli strain CE3. A unique O-acetylated glycan of discrete size, containing 3-O-methyl-6-deoxy-L-talose and 2,3,4-tri-O-,methyl-l fucose

The O-antigenic polysaccharide of the Rhizobium etli CE3 lipopolysaccharide (LPS) was structurally characterized using chemical degradations (Smith degradation and beta-elimination of uronosyl residues) in combination with alkylation analysis, electrospray, and matrix-assisted laser desorption ionization-time of flight mass spectrometry, tandem mass spectrometry, and (1)H COSY and TOCSY nuclear magnetic resonance spectroscopy analyses of the native polysaccharide and the derived oligosaccharides. The polysaccharide was found to be a unique, relatively low molecular weight glycan having a fairly discrete size, with surprisingly little variation in the number of repeating units (degree of polymerization = 5). The polysaccharide is O-acetylated and contains a variety of O-methylated glycosyl residues, rendering the native glycan somewhat hydrophobic. The molecular mass of the major de-O-acetylated species, including the reducing end 3-deoxy-d-manno-2-octulosonic acid (Kdo) residue, is 3330 Da. The polysaccharide is comprised of a trisaccharide repeating unit having the structure -->4)-alpha-d-GlcpA-(1-->4)-[alpha-3-O-Me-6-deoxy-Talp-(1--> 3)]-alpha -l-Fucp-(1-->. The nonreducing end of the glycan is terminated with the capping sequence alpha-2,3, 4-tri-O-Me-Fucp-(1-->4)-alpha-d-GlcpA-(1-->, and the reducing end of the molecule consists of the non-repeating sequence -->3)-alpha-l-Fucp-(1-->3)-beta-d-Manp-(1-->3)-beta-QuiNA cp-(1-->4)-a lpha-Kdop-(2-->, where QuiNAc is N-acetylquinovosamine (2-N-acetamido-2,6-dideoxyglucose). The reducing end Kdo residue links the O-chain polysaccharide to the core region oligosaccharide, resulting in a unique location for a Kdo residue in LPS, removed four residues distally from the lipid A moiety. Structural heterogeneity in the O-chain arises mainly from the O-acetyl and O-methyl substitution. Methylation analysis using trideuteriomethyl iodide indicates that a portion of the 2,3,4-tri-O-methylfucosyl capping residues, typically 15%, are replaced with 2-O-methyl- and/or 2,3-di-O-methylfucosyl residues. In addition, approximately 25% of the 3,4-linked branching fucosyl residues and 10% of the 3-linked fucosyl residues are 2-O-methylated. A majority of the glucuronosyl residues are methyl-esterified at C-6. These unique structural features may be significant in the infection process.     

The complete structure of the core of the LPS from Plesiomonas shigelloides 302-73 and the identification of its O-antigen biological repeating unit

Plesiomonas shigelloides is a Gram-negative opportunistic pathogen associated with gastrointestinal and extraintestinal infections, which especially invades immunocompromised patients and neonates. The lipopolysaccharides are one of the major virulence determinants in Gram-negative bacteria and are structurally composed of three different domains: the lipid A, the core oligosaccharide and the O-antigen polysaccharide.In the last few years we elucidated the structures of the O-chain and the core oligosaccharide from the P. shigelloides strain 302-73. In this paper we now report the characterization of the linkage between the core and the O-chain. The LPS obtained after PCP extraction contained a small number of O-chain repeating units. The product obtained by hydrazinolysis was analysed by FTICR-ESIMS and suggested the presence of an additional Kdo in the core oligosaccharide. Furthermore, the LPS was hydrolysed under mild acid conditions and a fraction that contained one O-chain repeating unit linked to a Kdo residue was isolated and characterized by FTICR-ESIMS and NMR spectroscopy. Moreover, after an alkaline reductive hydrolysis, a disaccharide α-Kdo-(2→6)-GlcNol was isolated and characterized. The data obtained proved the presence of an α-Kdo in the outer core and allowed the identification of the O-antigen biological repeating unit as well as its linkage with the core oligosaccharide.     

Lipid A and O-chain modifications cause Rhizobium lipopolysaccharides to become hydrophobic during bacteroid development

Modifications to the lipopolysaccharide (LPS) structure caused by three different growth conditions were investigated in the pea-nodulating strain Rhizobium leguminosarum 3841. The LPSs extracted by hot phenol-water from cultured cells fractionated into hydrophilic water and/or hydrophobic phenol phases. Most of the LPSs from cells grown under standard conditions extracted into the water phase, but a greater proportion of LPSs were extracted into the phenol phase from cells grown under acidic or reduced-oxygen conditions, or when isolated from root nodules as bacteroids. Compared with the water-extracted LPSs, the phenol-extracted LPSs contained greater degrees of glycosyl methylation and O-acetylation, increased levels of xylose, glucose and mannose and increased amounts of long-chain fatty acids attached to the lipid A moiety. The water- and phenol-phase LPSs also differed in their reactivity with monoclonal antibodies and in their polyacrylamide gel electrophoretic banding patterns. Phenol-extracted LPSs from rhizobia grown under reduced-oxygen conditions closely resembled the bulk of LPSs isolated from pea nodule bacteria (i.e. mainly bacteroids) in their chemical properties, reactivities with monoclonal antibodies and extraction behaviour. This finding suggests that, during symbiotic bacteroid development, reduced oxygen tension induces structural modifications in LPSs that cause a switch from predominantly hydrophilic to predominantly hydrophobic molecular forms. Increased hydrophobicity of LPSs was also positively correlated with an increase in the surface hydrophobicity of whole cells, as shown by the high degree of adhesion to hydrocarbons of bacterial cells isolated from nodules or from cultures grown under low-oxygen conditions. The implications of these LPS modifications are discussed for rhizobial survival and function in different soil and in planta habitats.     

Genetic locus and structural characterization of the biochemical defect in the O-antigenic polysaccharide of the symbiotically deficient Rhizobium etli mutant, CE166. Replacement of N-acetylquinovosamine with its hexosyl-4-ulose precursor

The O-antigen polysaccharide (OPS) of Rhizobium etli CE3 lipopolysaccharide (LPS) is linked to the core oligosaccharide via an N-acetylquinovosaminosyl (QuiNAc) residue. A mutant of CE3, CE166, produces LPS with reduced amounts of OPS, and a suppressed mutant, CE166 alpha, produces LPS with nearly normal OPS levels. Both mutants are deficient in QuiNAc production. Characterization of OPS from CE166 and CE166 alpha showed that QuiNAc was replaced by its 4-keto derivative, 2-acetamido-2,6-dideoxyhexosyl-4-ulose. The identity of this residue was determined by NMR and mass spectrometry, and by gas chromatography-mass spectrometry analysis of its 2-acetamido-4-deutero-2,6-dideoxyhexosyl derivatives produced by reduction of the 4-keto group using borodeuteride. Mass spectrometric and methylation analyses showed that the 2-acetamido-2,6-dideoxyhexosyl-4-ulosyl residue was 3-linked and attached to the core-region external Kdo III residue of the LPS, the same position as that of QuiNAc in the CE3 LPS. DNA sequencing revealed that the transposon insertion in strain CE166 was located in an open reading frame whose predicted translation product, LpsQ, falls within a large family of predicted open reading frames, which includes biochemically characterized members that are sugar epimerases and/or reductases. A hypothesis to be tested in future work is that lpsQ encodes UDP-2-acetamido-2,6-dideoxyhexosyl-4-ulose reductase, the second step in the synthesis of UDP-QuiNAc from UDP-GlcNAc.     

Structural characterization of the lipid A component of Sinorhizobium sp. NGR234 rough and smooth form lipopolysaccharide. Demonstration that the distal amide-linked acyloxyacyl residue containing the long chain fatty acid is conserved in rhizobium and Sinorhizobium sp

A broad-host-range endosymbiont, Sinorhizobium sp. NGR234 is a component of several legume-symbiont model systems; however, there is little structural information on the cell surface glycoconjugates. NGR234 cells in free-living culture produce a major rough lipopolysaccharide (LPS, lacking O-chain) and a minor smooth LPS (containing O-chain), and the structure of the lipid A components was investigated by chemical analyses, mass spectrometry, and NMR spectroscopy of the underivatized lipids A. The lipid A from rough LPS is heterogeneous and consists of six major bisphosphorylated species that differ in acylation. Pentaacyl species (52%) are acylated at positions 2, 3, 2', and 3', and tetraacyl species (46%) lack an acyl group at C-3 of the proximal glucosamine. In contrast to Rhizobium etli and Rhizobium leguminosarum, the NGR234 lipid A contains a bisphosphorylated beta-(1' --> 6)-glucosamine disaccharide, typical of enterobacterial lipid A. However, NGR234 lipid A retains the unusual acylation pattern of R. etli lipid A, including the presence of a distal, amide-linked acyloxyacyl residue containing a long chain fatty acid (LCFA) (e.g. 29-hydroxytriacontanoate) attached as the secondary fatty acid. As in R. etli, a 4-carbon fatty acid, beta-hydroxybutyrate, is esterified to (omega - 1) of the LCFA forming an acyloxyacyl residue at that location. The NGR234 lipid A lacks all other ester-linked acyloxyacyl residues and shows extensive heterogeneity of the amide-linked fatty acids. The N-acyl heterogeneity, including unsaturation, is localized mainly to the proximal glucosamine. The lipid A from smooth LPS contains unique triacyl species (20%) that lack ester-linked fatty acids but retain bisphosphorylation and the LCFA-acyloxyacyl moiety. The unusual structural features shared with R. etli/R. leguminosarum lipid A may be essential for symbiosis.     

Three genes encoding for putative methyl- and acetyltransferases map adjacent to the wzm and wzt genes and are essential for O-antigen biosynthesis in Rhizobium etli CE3

The elucidation of the structure of the O-antigen of Rhizobium etli CE3 predicts that the R. etli CE3 genome must contain genes encoding acetyl- and methyltransferases to confer the corresponding modifications to the O-antigen. We identified three open reading frames (ORFs) upstream of wzm, encoding the membrane component of the O-antigen transporter and located in the lps alpha-region of R. etli CE3. The ORFs encode two putative acetyltransferases with similarity to the CysE-LacA-LpxA-NodL family of acetyltransferases and one putative methyltransferase with sequence motifs common to a wide range of S-adenosyl-L-methionine-dependent methyltransferases. Mutational analysis of the ORFs encoding the putative acetyltransferases and methyltransferase revealed that the acetyl and methyl decorations mediated by these specific enzymes are essential for O-antigen synthesis. Composition analysis and high performance anion exchange chromatography analysis of the lipopolysaccharides (LPSs) of the mutants show that all of these LPSs contain an intact core region and lack the O-antigen polysaccharide. The possible role of these transferases in the decoration of the O-antigen of R. etli is discussed.     

Characterization of the lipopolysaccharide from a Rhizobium phaseoli mutant that is defective in infection thread development.

The lipopolysaccharide (LPS) from a Rhizobium phaseoli mutant, CE109, was isolated and compared with that of its wild-type parent, CE3. A previous report has shown that the mutant is defective in infection thread development, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that it has an altered LPS (K. D. Noel, K. A. VandenBosch, and B. Kulpaca, J. Bacteriol. 168:1392-1462, 1986). Mild acid hydrolysis of the CE3 LPS released a polysaccharide and an oligosaccharide, PS1 and PS2, respectively. Mild acid hydrolysis of CE109 LPS released only an oligosaccharide. Chemical and immunochemical analyses showed that CE3-PS1 is the antigenic O chain of this strain and that CE109 LPS does not contain any of the major sugar components of CE3-PS1. CE109 oligosaccharide was identical in composition to CE3-PS2. The lipid A's from both strains were very similar in composition, with only minor quantitative variations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of CE3 and CE109 LPSs showed that CE3 LPS separated into two bands, LPS I and LPS II, while CE109 had two bands which migrated to positions similar to that of LPS II. Immunoblotting with anti-CE3 antiserum showed that LPS I contains the antigenic O chain of CE3, PS1. Anti-CE109 antiserum interacted strongly with both CE109 LPS bands and CE3 LPS II and interacted weakly with CE3 LPS I. Mild-acid hydrolysis of CE3 LPS I, extracted from the polyacrylamide gel, showed that it contained both PS1 and PS2. The results in this report showed that CE109 LPS consists of only the lipid A core and is missing the antigenic O chain.     

Protoporphyrin formation in Rhizobium japonicum.

The obligately aerobic soybean root nodule bacterium Rhizobium japonicum produces large amounts of heme (iron protoporphyrin) only under low oxygen tensions, such as exist in the symbiotic root nodule. Aerobically incubated suspensions of both laboratory-cultured and symbiotic bacteria (bacteroids) metabolize delta-aminolevulinic acid to uroporphyrin, coproporphyrin, and protoporphyrin. Under anaerobic conditions, suspensions of laboratory-cultured bacteria form greatly reduced amounts of protoporphyrin from delta-aminolevulinic acid, whereas protoporphyrin formation by bacteroid suspensions is unaffected by anaerobiosis, suggesting that bacteroids form protoporphyrin under anaerobic conditions more readily than do free-living bacteria. Oxygen is the major terminal electron acceptor for coproporphyrinogen oxidation in cell-free extracts of both bacteroids and free-living bacteria. In the absence of oxygen, ATP, NADP, Mg2+, and L-methionine are required for protoporphyrin formation in vitro. In the presence of these supplements, coproporphyrinogenase activity under anaerobic conditions is 5 to 10% of that observed under aerobic conditions. Two mechanisms for coproporphyrinogen oxidation exist in R. japonicum: an oxygen-dependent process and an anaerobic oxidation in which electrons are transferred to NADP. The significance of these findings with regard to heme biosynthesis in the microaerophilic soybean root nodule is discussed.     

O-chain structure from the lipopolysaccharide of the human pathogen Halomonas stevensii strain S18214

Halomonas stevensii is a Gram-negative, pathogenic, moderately halophilic bacterium isolated from the blood of a renal care patient. It optimally grows at 30–35 °C at pH 8–9 and at a sea salt concentration ranging from 3.0% to 7.5%. Gram-negative bacterial infections are closely associated with the presence of the lipopolysaccharides (LPSs) on the outer membrane. These molecules consist of three regions covalently linked: the glycolipid (lipid A), the oligosaccharide region (core region), and the O-specific polysaccharide (O-chain, O-antigen). O-antigen seems to play an important role in the colonization step (adherence) and the ability to bypass host defense mechanisms. For this reason the structure elucidation of the O-chain repeating unit is important to improve knowledge about the role of LPS in the host-pathogen interaction. In this paper, we report the complete structure of the O-chain from the LPS of H. stevensii. The bacterial cells were cultivated and LPS was extracted by the PCP (phenol–chloroform–petroleum ether) method. After mild acid hydrolysis, the lipid A was removed by centrifugation and the obtained polysaccharide was analyzed by means of chemical analysis and one- and two-dimensional NMR spectroscopy giving the following structure:     

The structure of the carbohydrate backbone of the LPS from Myxococcus xanthus strain DK1622

Gram-negative rod shaped bacterium Myxococcus xanthus DK1622 produces a smooth-type LPS. The structure of the polysaccharide O-chain and the core-lipid A region of the LPS has been determined by chemical and spectroscopic methods. The O-chain was built up of disaccharide repeating units having the following structure: -->6)-alpha-D-Glcp-(1-->4)-alpha-D-GalpNAc6oMe*-(1--> with partially methylated GalNAc residue. The core region consisted of a phosphorylated hexasaccharide, containing one Kdo residue, unsubstituted at O-4, and no heptose residues. The lipid A component consisted of beta-GlcN-(1-->6)-alpha-GlcN1P disaccharide, N-acylated with 13-methyl-C14-3OH (iso-C15-3OH), C16-3OH, and 15-methyl-C16-3OH (iso-C17-3OH) acids. The lipid portion contained O-linked iso-C16 acid.     

Structural characterization of the outer core and the O-chain linkage region of lipopolysaccharide from Pseudomonas aeruginosa serotype O5.

The point of attachment of the O-chain in the outer core region of Pseudomonas aeruginosa serotype O5 lipopolysaccharide (LPS) was determined following a detailed analysis of the extended core oligosaccharide, containing one trisaccharide O-chain repeating unit, present in both the wild-type strain PAO1 and O-chain deficient mutant strains AK1401 and PAO-rfc. The structure of the extended core oligosaccharide was determined by various mass spectrometric methods as well as one-dimensional and two-dimensional NMR spectroscopy. Furthermore, the one-dimensional analogues of NOESY and TOCSY experiments were applied to confirm the structure of the outer core region in the O-chain polysaccharide. In both the extended core oligosaccharide and the core of the smooth LPS, a loss of one of the beta-glucosyl residues and the translocation of the alpha-rhamnosyl residue, followed by the attachment of the first O-chain repeating unit was observed. This process is complicated and could involve two distinct rhamnosyltransferases, one with alpha-1, 6-linkage specificity and another with alpha-1,3-linkage specificity. It is also plausible that an alpha-1,3 rhamnosyltransferase facilitates the addition of the 'new' alpha-rhamnosyl residue that will act as a receptor for the attachment of the single O-antigen repeating unit in the LPS of the semi-rough mutant. The 2-amino-2-deoxy-fucosyl residue of the first O-chain repeating unit directly attached to the core was found to have a beta-anomeric configuration instead of an alpha configuration, characteristic for this residue as a component of the O-chain polysaccharide. The results of this study provide the first example of the mechanistic implications of the structure of the outer core region in a fully assembled O-chain containing LPS, differing from the O-chain deficient rough LPS.     

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.     

Effective symbiosis between Rhizobium etli and Phaseolus vulgaris requires the alarmone ppGpp

The symbiotic interaction between Rhizobium etli and Phaseolus vulgaris, the common bean plant, ultimately results in the formation of nitrogen-fixing nodules. Many aspects of the intermediate and late stages of this interaction are still poorly understood. The R. etli relA gene was identified through a genome-wide screening for R. etli symbiotic mutants. RelA has a pivotal role in cellular physiology, as it catalyzes the synthesis of (p)ppGpp, which mediates the stringent response in bacteria. The synthesis of ppGpp was abolished in an R. etli relA mutant strain under conditions of amino acid starvation. Plants nodulated by an R. etli relA mutant had a strongly reduced nitrogen fixation activity (75% reduction). Also, at the microscopic level, bacteroid morphology was altered, with the size of relA mutant bacteroids being increased compared to that of wild-type bacteroids. The expression of the sigma(N)-dependent nitrogen fixation genes rpoN2 and iscN was considerably reduced in the relA mutant. In addition, the expression of the relA gene was negatively regulated by RpoN2, the symbiosis-specific sigma(N) copy of R. etli. Therefore, an autoregulatory loop controlling the expression of relA and rpoN2 seems operative in bacteroids. The production of long- and short-chain acyl-homoserine-lactones by the cinIR and raiIR systems was decreased in an R. etli relA mutant. Our results suggest that relA may play an important role in the regulation of gene expression in R. etli bacteroids and in the adaptation of bacteroid physiology.     

2-O-methylation of fucosyl residues of a rhizobial lipopolysaccharide is increased in response to host exudate and is eliminated in a symbiotically defective mutant

When Rhizobium etli CE3 was grown in the presence of Phaseolus vulgaris seed extracts containing anthocyanins, its lipopolysaccharide (LPS) sugar composition was changed in two ways: greatly decreased content of what is normally the terminal residue of the LPS, di-O-methylfucose, and a doubling of the 2-O-methylation of other fucose residues in the LPS O antigen. R. etli strain CE395 was isolated after Tn5 mutagenesis of strain CE3 by screening for mutant colonies that did not change antigenically in the presence of seed extract. The LPS of this strain completely lacked 2-O-methylfucose, regardless of whether anthocyanins were present during growth. The mutant gave only pseudonodules in association with P. vulgaris. Interpretation of this phenotype was complicated by a second LPS defect exhibited by the mutant: its LPS population had only about 50% of the normal amount of O-antigen-containing LPS (LPS I). The latter defect could be suppressed genetically such that the resulting strain (CE395 alpha 395) synthesized the normal amount of an LPS I that still lacked 2-O-methylfucose residues. Strain CE395 alpha 395 did not elicit pseudonodules but resulted in significantly slower nodule development, fewer nodules, and less nitrogenase activity than lps(+) strains. The relative symbiotic deficiency was more severe when seeds were planted and inoculated with bacteria before they germinated. These results support previous conclusions that the relative amount of LPS I on the bacterial surface is crucial in symbiosis, but LPS structural features, such as 2-O-methylation of fucose, also may facilitate symbiotic interactions.     

Structural characterization of the O-chain polysaccharide isolated from Bordetella avium ATCC 5086: variation on a theme(1)

The O-chain polysaccharide (O-PS) of Bordetella avium was isolated from the lipopolysaccharide by mild acid hydrolysis to remove the lipid A, followed by hydrofluorolysis to remove the lipopolysaccharide core oligosaccharide leaving a residual O-PS for structural analysis. High resolution (1)H and (13)C NMR and MALDI studies showed the O-chain to be a polymer composed of 1,4-linked 2-acetamidino-3-[3-hydroxybutanamido]-2,3-dideoxy-beta-D-glucopyranosyluronic acid residues.     

Stability of short and long O-chain lipopolysaccharide types in Rhizobium galegae and their correlation with symbiotic properties and growth conditions, tolerance of low pH, aluminum and salt in the growth medium

We recently showed that Rhizobium galegae strains had two different lipopolysaccharide types, short and long O-chain. In the present study we observed that the lipopolysaccharide type was a stable feature of the strain. Both types persisted in cells at all phases of the growth cycle, during differentiation of bacteria into bacteroids and when the cells were grown under environmental stress. When R. galegae strains were grown at low pH or in medium containing aluminum or salt, simulating conditions in acid soils of temperate regions and osmotic stress, respectively, the tolerance of low pH was associated with the long O-chain lipopolysaccharide and abundant acidic polysaccharide production.