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

Carlson, Russell W.; Kalembasa, S.; Turowski, D A; Pachori, Purnima; Noel, K. Dale

doi:10.1128/jb.169.11.4923-4928.1987

Cited by 139 publications

(130 citation statements)

References 32 publications

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“…Bacterial Strains-R. etli CE3 (the parent strain) and the R. etli mutants CE358 and CE359 were grown in a tryptone/yeast extract supplemented with Ca 2ϩ as described previously (25). Bacteria were harvested by centrifugation at late log/early stationary phase.…”

Section: Methodsmentioning

confidence: 99%

“…Polyacrylamide gel electrophoresis (PAGE) in the presence of deoxycholate (DOC) (27,28) of the water and phenol fractions indicated that LPS was recovered predominantly in the water layer in the case of strain CE3, while the two mutant strains yielded LPS in both the phenol and water layers. Water and phenol layers containing LPS were dialyzed and treated sequentially with ribonuclease, deoxyribonuclease, and proteinase K and then dialyzed and subjected to further purification by either gel filtration chromatography on Sepharose 4B (25,29) or by affinity chromatography on columns of polymyxin-agarose (Detoxi-Gel, Pierce). For the latter procedure, the crude extracts were treated with enzymes as described above and then dialyzed against 50 mM NH 4 HCO 3 , pH 8.0.…”

Section: Methodsmentioning

confidence: 99%

“…Lipopolysaccharide Purification-The parent strain, CE3, and the R. etli mutant strains CE358 and CE359, were extracted using the hot phenol-water procedure as described previously (25,26). Polyacrylamide gel electrophoresis (PAGE) in the presence of deoxycholate (DOC) (27,28) of the water and phenol fractions indicated that LPS was recovered predominantly in the water layer in the case of strain CE3, while the two mutant strains yielded LPS in both the phenol and water layers.…”

Section: Methodsmentioning

confidence: 99%

“…8. Furthermore, isolation of the intact O-chain released by mild acid hydrolysis from the parent strain LPS shows that the single 3-linked N-acetylquinovosaminosyl residue is an O-chain component, located distally to the reducing end Kdo residue (25). Further work is in progress to determine if this proposed location is correct.…”

Section: Analysis Of R Etli Mutant Ce358 Lps-the Glycosyl Compositiomentioning

confidence: 99%

“…The parent strain, CE3, was extracted with the hot phenol/ water procedure, and the LPSs were purified by the standard technique of size exclusion chromatography on Sepharose 4B (25,26,29). DOC-PAGE analysis showed the presence of typical high molecular weight LPS (LPS I, containing O-chain polysaccharide) and low molecular weight LPS (LPS II, which lacks the O-chain, and various LPSs that carry a truncated O-chain) (12,17).…”

Section: Purification Of R Etli Lpss and Initial Characterization-mentioning

confidence: 99%

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The Structures of the Lipopolysaccharides from Rhizobium etli Strains CE358 and CE359

Forsberg

Carlson

1998

Journal of Biological Chemistry

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The structural arrangement of oligosaccharides comprising the core region of Rhizobium etli CE3 lipopolysaccharide (LPS) has been elucidated through the characterization of the LPSs from two R. etli mutants. One mutant, CE358, completely lacks the O-chain polysaccharide, while the second mutant, CE359, contains a truncated portion of this polysaccharide. This structural arrangement of the core oligosaccharides in these LPSs was determined using electrospray ionization mass spectrometry, tandem mass spectrometry, and methylation analysis. Mild acid hydrolysis of the CE359 LPS produces two major core oligosaccharides: a tetrasaccharide (1) with the structure ␣-D-Galp- (136) Bacteria belonging to the family Rhizobiaceae are Gram-negative and are able to form nitrogen-fixing symbiotic relationships with legume plants. The surface polysaccharides, including the lipopolysaccharides (LPSs), 1 are involved in the normal infection process. Mutants that lack the O-chain polysaccharide portion of their LPSs are symbiotically defective in that they are unable to form normal infection threads (1, 2), and/or they cannot invade the root nodule cells (3-5). In addition, it has been shown that structural changes in the LPS occur during symbiotic infection and that most of these changes appear to take place in the O-chain polysaccharide portion of the LPS (6 -14). These structural adaptations in response to the environment of the host plant are likely to be important in order for the symbiont bacterium to induce a nitrogen-fixing nodule.In addition to the importance of determining the symbiotic "virulence" of these bacteria, rhizobial LPSs can have unique structural features compared with the LPSs from enteric bacteria. The most studied LPSs, the topic of this report, are those from Rhizobium etli and Rhizobium leguminosarum. All of the LPSs examined from wild-type or parent strains of these species are devoid of phosphate. Their lipid A region has a trisaccharide backbone consisting of one each of galacturonosyl (GalA), GlcN, and 2-aminogluconosyl (GlcN-onate) residues, the latter two residues being O-and N-acylated with ␤-hydroxy fatty acids and one very long chain fatty acid, 27-hydroxyoctacosanoic acid (15). This lipid A also appears not to carry any acyloxylacyl substituents. The core region has been found to consist of two oligosaccharides (16, 17) as shown below.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Analysis Of R Etli Mutant Ce358 Lps-the Glycosyl Compositiomentioning

confidence: 99%

Section: Purification Of R Etli Lpss and Initial Characterization-mentioning

confidence: 99%

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The Structures of the Lipopolysaccharides from Rhizobium etli Strains CE358 and CE359

Forsberg

Carlson

1998

Journal of Biological Chemistry

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Physical and biochemical characterization of a competition and nodulation defective mutant of Azorhizobium caulinodans ORS571

Kishore

Natarajan

Babu

1996

J. Basic Microbiol.

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The competitive ability of Azorhizobium caulinodans strain ORS57 1 was assessed using induced mutagenesis, which resulted in isolation of two mutants that were either defective in symbiotic properties or in competition for nodulation of Sesbania roscrata. One of the mutants was non-nodulating whereas the other one exhibited reduced competitiveness when inoculated along with the parent strain. Further characterization revealed that the non-nodulating mutant exhibited altered protein and lipopolysaccharide (LPS) profiles and the absence of flagella.One of the limitations in the practical realisation of legume-Rhizobium symbiotic nitrogen fixation technology is the Rhizobium competition problem -the failure of superior inoculant to occupy nodules in soils with a large population of indigenous Rhizobium. A number of strategies have been suggested to overcome the Rhizobium competition problem ranging from inoculation of plants with large numbers of Rhizobium (MEADE et al. 1985) over a long period to the stable transfer of genes involved in nodulation competitiveness to superior strains of Rhizobium (TRIPLET 1990).However, the basis of competitiveness among Rhizobium strain is largely unknown, and attempts to modify it by genetic manipulation have had varied results. Some Tn5 insertions in R. trijolii that reduce competitiveness are correlated with loss of motility or lack of trifoliotoxin production (TRIPLET and BATRA 1987). Chromosomal deletions or insertions in cryptic plasmids of R. meliloti have been shown to reduce strain competitiveness (HAHN and HENNECKE 1988). Similarly, competition defective mutants of R. fredii have been generated by Tn5 mutagenesis (MCLOUGHLIN et al. 1987). In R. loti, however, removal of a cryptic plasmid has been shown to increase competitiveness (PANKHURST et al. 1986). It has also been correlated with exopolysaccharide (EPS) production in R. fredii where induced mutants having reduced EPS production were competitively superior to the wild type of Bradyrhizobiurn (ZDOR and PUEPPKE 199 1).For the development of efficient inoculants, the most important issue is the selection and identification of a competitively superior inoculum strain. A number of tagging markers used for strain identification includes cultural characteristics, serological properties, intrinsic antibiotic resistance, phage typing, probes ("*P-labelled genomic DNA probes) of n8fi.x and nod genes; enzyme polymorphism, and SDS-PAGE protein and LPS patterns. All these techniques employed for determining nodule occupancy have one or the other practical limitation and the Rhizobium competition problem is still a major obstacle in the development of superior inoculants.Tn5 (carrying gusA (P-glucuronidase) as a reporter gene) has been used as a marker for Rhizobium in a wide range of studies (WILLSON et al. 1991). In the present study we describe the use of a Tn5 gusA marker system for assessing the competitive ability of Azorhizobium caulinoduns strain ORS57 1 with respect to other Rhizobium strains belonging to the genus Sesb...

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Structure of the Lipopolysaccharide from the Bradyrhizobium sp. ORS285 rfaL Mutant Strain

et al. 2017

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The importance of the outer membrane and of its main constituent, lipopolysaccharide, in the symbiosis between rhizobia and leguminous host plants has been well studied. Here, the first complete structural characterization of the entire lipopolysaccharide from an O‐chain‐deficient Bradyrhizobium ORS285 rfaL mutant is achieved by a combination of chemical analysis, NMR spectroscopy, MALDI MS and MS/MS. The lipid A structure is shown to be consistent with previously reported Bradyrhizobium lipid A, that is, a heterogeneous blend of penta‐ to hepta‐acylated species carrying a nonstoichiometric hopanoid unit and possessing very‐long‐chain fatty acids ranging from 26:0(25‐OH) to 32:0(31‐OH). The structure of the core oligosaccharide region, fully characterized for the first time here, is revealed to be a nonphosphorylated linear chain with methylated sugar residues, with a heptose residue exclusively present in the outer core region, and with the presence of two singly substituted 3‐deoxy‐d‐manno‐oct‐2‐ulosonic acid (Kdo) residues, one of which is located in the outer core region. The lipid A moiety is linked to the core moiety through an uncommon 4‐substituted Kdo unit.

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

Cited by 139 publications

References 32 publications

The Structures of the Lipopolysaccharides from Rhizobium etli Strains CE358 and CE359

The Structures of the Lipopolysaccharides from Rhizobium etli Strains CE358 and CE359

Physical and biochemical characterization of a competition and nodulation defective mutant of Azorhizobium caulinodans ORS571

Structure of the Lipopolysaccharide from the Bradyrhizobium sp. ORS285 rfaL Mutant Strain

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