The Isolation and Partial Characterization of the Lipopolysaccharides from Several <i>Rhizobium trifolii</i> Mutants Affected in Root Hair Infection

Carlson, Russell W.; Shatters, Robert G.; Duh, Jauh‐Lin; Turnbull, E. A.; Hanley, Brian A.; Rolfe, Barry G.; Djordjevic, Michael A.

doi:10.1104/pp.84.2.421

Cited by 38 publications

(28 citation statements)

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“…phaseoli and R. leguminosarum bv. trifolii, it was shown that this high-molecular-weight fraction corresponds to LPSI containing the 0-antigen side chain (7,8). To decide whether the mutants described here completely lack the LPSI or produce very reduced quantities, a more sensitive immunoblotting assay would be necessary.…”

Section: Discussionmentioning

confidence: 99%

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Genes involved in lipopolysaccharide production and symbiosis are clustered on the chromosome of Rhizobium leguminosarum biovar viciae VF39

1989

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Four mutants of Rhizobium leguminosarum biovar viciae VF39 altered in lipopolysaccharide (LPS) synthesis were isolated upon random TnS mutagenesis. These mutants produced matt colonies on TY medium and showed autoagglutination and loss of motility. On sodium dodecyl sulfate-polyacrylamide gels, they lacked a slow-migrating carbohydrate band, corresponding to the complete LPS (LPSI). All four mutants formed small white nodules on Vicia hirsuta. These nodules were infected but showed no nitrogen-fixing activity and senesced prematurely. Three of the mutants were complemented by a wild-type cosmid to synthesis of normal LPS and induction of nitrogen-fixing nodules. By hybridization and in vivo cloning experiments, the mutations were mapped within different EcoRI fragments which could be localized on the VF39 chromosome. Crosscomplementation analyses revealed that the three mutants were affected in different transcriptional units. The results indicate that a cluster of genes necessary for LPSI production and symbiotic efficiency is located within a defined region of 20 kilobases on the R. keguminosarum bv. viciae chromosome.The establishment of nitrogen-fixing nodules in the Rhizobium-legume symbiosis is a complex multistep interaction between microsymbiont and its specific host plant (for a review, see reference 2). Early events involve recognition and deformation or curling of root hair cells. Cortical cell division, leading to the formation of a unique organ, the nodule, is induced probably by diffusible bacterial substances. Invasion of the nodule by the bacteria is initiated by penetration of the root hair cell wall and the formation of an infection thread, in which the bacteria are carried towards the dividing root cortex cells. Bacteria are eventually released from the infection thread into the plant cells. They remain separated from the host cytoplasm by peribacteroid membranes and differentiate into bacteroids able to reduce atmospheric nitrogen.This complex developmental process obviously requires continuous signal exchange between plant and bacterial cells. Undoubtedly, bacterial cell surface components play an important role in this specific interaction; particularly extracellular polysaccharides (EPSs) and lipopolysaccharides (LPSs) have been hypothesized to be involved in the symbiotic process (for reviews see references 5 and 15).The LPS of gram-negative bacteria consists of lipid A, a core oligosaccharide and an 0-antigenic side chain. The Rhizobium LPSs are very heterogenous molecules and vary greatly among different species as among strains of a single species (6, 49). Therefore, rhizobial LPS has been suggested to be involved in specific recognition and attachment of bacteria to the root hair cells of compatible host plants, mediated by the binding of lectins (47). While recent work (12) supports an essential function of lectins in host-plant specificity, a role of LPS in host-specific nodulation remains unclear.Differences in LPS composition were described between nodulating and nonnodulating str...

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Section: Discussionmentioning

confidence: 99%

“…trifolii (previously called R. trifolit) (8,34,35). Similarly, quantitative differences in the LPS of a Nodmutant of a fast-growing Rhizobium japonicum strain were observed (9).…”

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Genes involved in lipopolysaccharide production and symbiosis are clustered on the chromosome of Rhizobium leguminosarum biovar viciae VF39

1989

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“…The surface carbohydrates of rhizobia, which include the lipopolysaccharide (LPS), extracellular polysaccharide, and capsular polysaccharide, have all been hypothesized to play a role in symbiosis (9,11,18,20,22,23). The LPSs of Rhizobium species resemble their enterobacterial counterparts in having structurally distinct regions; the 0-chain polysaccharide, the core oligosaccharide, and the hydrophobic lipid A (10,(13)(14)(15)(16)(17); however, there are many differences in the structural details of Rhizobium LPSs compared with enterobacterial LPSs (1-3, 12, 25-27).…”

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

Bhat

Carlson

1992

J Bacteriol

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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-0-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 (0-chain polysaccharide, core oligosaccharides, and the lipid A) revealed that all the composition differences in the various LPSs occur in the 0-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 0-chain of R. leguminosarum bv. phaseoli when the bacteria are grown at low pH and during nodule development.Rhizobia are gram-negative soil bacteria that form nitrogen-fixing symbiotic associations with leguminous plants. In terms of host selection, the bacteria exhibit a great deal of specificity. The surface carbohydrates of rhizobia, which include the lipopolysaccharide (LPS), extracellular polysaccharide, and capsular polysaccharide, have all been hypothesized to play a role in symbiosis (9,11,18,20,22,23). The LPSs of Rhizobium species resemble their enterobacterial counterparts in having structurally distinct regions; the 0-chain polysaccharide, the core oligosaccharide, and the hydrophobic lipid A (10, 13-17); however, there are many differences in the structural details of Rhizobium LPSs compared with enterobacterial LPSs (1-3, 12, 25-27).Earlier reports on the role of LPSs in symbiosis were directed toward the possibility that LPSs were involved in the specific attachment of the symbiont to the host plant (8,9,30). Recent studies indicate involvement of LPSs at a later stage in the nodule development. Mutants which do not have the 0-chain region either fail to form normal infection threads or are not released properly from the infection thread into the host root cells (7,13,19,33,34,37).Using monoclonal antibodies to Rhizobium leguminosarum bv. viciae bacteroids, it has been shown that there are changes in LPS epitopes which occur during differentiation of bacteria into bacteroids (6,35,39,42). Some of these changes can be produced ex planta, by growing the bacteria at low pH or low 02 tension, conditions which mimic, in part, the microenvironment of the root nodule (28). Similar results have been obtained for another strain of R. leguminosarum bv. viciae and for R leguminosarum bv. phaseoli (21, 38). These epitope changes occur only ...

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“…trifolii (16,50,51) and Rhizobium fredii (17) after the loss of whole plasmids. Various mutants with alterations in LPS were defective in the symbiotic association at different stages of development.…”

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confidence: 99%

“…By a different approach, in an attempt to characterize the participation of LPS in symbiosis, changes in the composition of LPS were found among nodulating and nonnodulating strains of Rhizobium leguminosarum bv. trifolii (16,50,51) and Rhizobium fredii (17) after the loss of whole plasmids. Various mutants with alterations in LPS were defective in the symbiotic association at different stages of development.…”

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A Rhizobium meliloti lipopolysaccharide mutant altered in competitiveness for nodulation of alfalfa

et al. 1992

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A transposon Tn5-induced mutant of Rhizobium meliloti Rm2011, designated Rm6963, showed a rough colony morphology on rich and minimal media and an altered lipopolysaccharide (LPS). Major differences from the wild-type LPS were observed in (i) hexose and 2-keto-3-deoxyoctonate elution profiles of crude phenol extracts chromatographed in Sepharose CL-4B, (ii) silver-stained sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis patterns of crude and purified LPS fractions, and (iii) immunoreactivities otherwise present in purified LPS of the parental strain Rm2011. In addition, Rm6963 lost the ability to grow in Luria-Bertani medium containing the hydrophobic compounds sodium deoxycholate or SDS and showed a decrease in survival in TY medium supplemented with high calcium concentrations. The mutant also had altered symbiotic properties. Rm6963 formed nodules that fixed nitrogen but showed a delayed or even reduced ability to nodulate the primary root of alfalfa without showing changes in the position of nodule distribution profiles along the roots. Furthermore, 2 to 3 weeks after inoculation, plants nodulated by Rm6963 were smaller than control plants inoculated with wild-type bacteria in correlation with a transient decrease in nitrogen fixation. In most experiments, the plants recovered later by expressing a full nitrogen-fixing phenotype and developing an abnormally high number of small nodules in lateral roots after 1 month. Rm6963 was also deficient in the ability to compete for nodulation. In coinoculation experiments with equal bacterial numbers of both mutant and wild-type rhizobia, only the parent was recovered from the uppermost root nodules. A strain ratio of approximately 100 to 1 favoring the mutant was necessary to obtain an equal ratio (1:1) of nodule occupancy.These results show that alterations in Rm6963 which include LPS changes lead to an altered symbiotic phenotype during the association with alfalfa that affects the timing of nodule emergence, the progress of nitrogen fixation, and the strain competitiveness for nodulation.Bacteria of the genera Rhizobium and Bradyrhizobium have the ability to infect roots of leguminous plants to form nitrogen-fixing nodules (1, 43). The resulting symbiosis, highly specific with respect to the involved partners, develops along an ordered multistage interaction, with participation of plant and rhizobial components and specific soluble signals (26, 33). Thus, a complex, developmentally regulated association takes place under the coordinated expression of several symbiotic genes in the plant and the bacteria (44, 49).Several bacterial surface components have been investigated and proved to be necessary for a compatible interaction of the bacteria with the host plant. Glucans (31), exopolysaccharides (5,20,21,42,45), and lipopolysaccharides (LPS) (6, 13, 19, 47) all appear to play a role during symbiotic interaction, especially during plant recognition and infection (1, 11).The symbiotic role of LPS-and also exopolysaccharides-was at first investigated by...

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The Isolation and Partial Characterization of the Lipopolysaccharides from Several Rhizobium trifolii Mutants Affected in Root Hair Infection

Cited by 38 publications

References 28 publications

Genes involved in lipopolysaccharide production and symbiosis are clustered on the chromosome of Rhizobium leguminosarum biovar viciae VF39

Genes involved in lipopolysaccharide production and symbiosis are clustered on the chromosome of Rhizobium leguminosarum biovar viciae VF39

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

A Rhizobium meliloti lipopolysaccharide mutant altered in competitiveness for nodulation of alfalfa

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