Bacterial polysaccharide which binds Rhizobium trifolii to clover root hairs.

Immunofluorescence, quantitative immunoprecipitation, and inhibition of bacterial agglutination and passive hemagglutination indicate that cross-reactive antigenic determinants are present on the surface of Rhizobium trifolii and clover roots. These determinants are immunochemically unique to this Rhizobium-legume cross-inoculation group. The multivalent lectin trifoliin and antibody to the clover root antigenic determinants bind competitively to two acidic heteropolysaccharides isolated from capsular material of R. Trifolii 0403. The major polysaccharide is an antigen which lacks heptose, 2-keto-3-deoxyoctulosonic acid, and endotoxic lipid A. The minor polysaccharide in the capsular material of R. Trifolii 0403 contains the same antigen in addition to heptose, 2-keto-3-deoxyoctonate, and lipid A. The acidic polysaccharides of two strains of R. trifolii share the clover r-ot cross-reactive antigenic determinant despite other differences in their carbohydrate composition. Studies with monovalent antigen-binding fragments of anti-clover root antibody and Azotobacter vinelandii hybrid transformants carrying the unique antigenic determinant suggest that these polysaccharides bind R. trifolii to the clover root hair tips which contain trifoliin.     

Alteration of the Trifoliin A-Binding Capsule of Rhizobium trifolii 0403 by Enzymes Released from Clover Roots

The effect of white clover root exudate on capsules of Rhizobium trifolii 0403 was examined. The clover lectin trifoliin A was detected in root exudate of two clover varieties by indirect immunofluorescence with antibody against this lectin purified from clover seed. Trifoliin A bound uniformly to encapsulated, heat-fixed cells during 1 h of incubation with root exudate. After 4 to 8 h of incubation, trifoliin A was only bound to one pole of the cells. Transmission electron microscopy showed that the capsule itself was altered. The disorganization of the acidic polymers of the capsule began in the equatorial center of the rod-shaped cell and then progressed toward the poles at unequal rates. Trifoliin A could no longer be detected on heat-fixed cells after 12 h of incubation with root exudate. However, trifoliin A was detected in situ on one pole of cells grown for 4 days in the clover root environment of Fahraeus slide cultures. Inhibition studies with the hapten 2-deoxy-d-glucose showed that trifoliin A in root exudate had a higher affinity for one of the cell poles. Immunoelectrophoresis was used to monitor the alteration of the extracellular polysaccharides from R. trifolii 0403 by concentrated root exudate. These polysaccharides were converted into products which eventually lost their ability to immunoprecipitate with homologous antibody. This progressive loss of antigenic reactivity proceeded more rapidly with root exudate from seedlings grown under nitrogen-free conditions than with root exudate from plants grown with 15 mM KNO(3). The root exudate, depleted of trifoliin A by immunoaffinity chromatography, was still able to alter the capsule of R. trifolii 0403. Reconstitution experiments showed that the substance(s) in root exudate which induced this alteration of the capsule was of a high molecular weight, heat labile, trypsin sensitive, and antigenically unrelated to trifoliin A. A variety of glycosidase activities were also detected in the fraction depleted of trifoliin A. These results suggest that enzymes in clover root exudate alter the trifoliin A-binding capsule in a way which would favor polar attachment of R. trifolii to clover root hairs.     

Transient appearance of lectin receptors onRhizobium trifolii

The appearance on the surface ofRhizobium trifolii 0403 of determinants important to both clover lectin (trifoliin) binding and adherence of the bacteria to clover root epithelial surfaces was studied by quantitative agglutination, immunofluorescence, and direct microscopic techniques. These unique determinants were found for only transient periods of time—as cells left lag phase and as they entered stationary phase of growth in broth. When present, these receptors were associated with a fibrillar polyanionic capsule surrounding the cells when grown on solid medium. These studies support earlier proposals that the architecture of the rhizobial cell surface is not constant in composition, but changes with the phase of growth.     

Specific phases of root hair attachment in the Rhizobium trifolii-clover symbiosis

The time course and orientation of attachment of Rhizobium trifolii 0403 to white clover root hairs was examined in slide cultures by light and electron microscopy. Inocula were grown for 5 days on defined BIII agar medium and represented the large subpopulation of fully encapsulated single cells which uniformly bind the clover lectin trifoliin A. When 10(7) cells or more were added per seedling, bacteria attached within minutes, forming randomly oriented clumps at the root hair tips. Several hours later, single cells attached polarly to the sides of the root hair. This sequence of attachment to clover root hairs was selective for R. trifolii at inoculum sizes of 10(7) to 4 X 10(8) per seedling, specifically inhibited if 2-deoxy-D-glucose, a hapten for trifoliin A, was present in the inoculum, and not observed when 4 X 10(8) cells were added to alfalfa seedling roots or to large clover root cell wall fragments which lacked trifoliin A but still had trifoliin A receptors. Once attached, R. trifolii 0403 became progressively less detachable with 2-deoxy-D-glucose. At smaller inoculum sizes (10(5) to 10(6) cells per seedling), there was no immediate clumping of R. trifolii at clover root hair tips, although polar binding of bacteria along the root hair surface was observed after 4 h. The interface between polarly attached bacteria and the root hair cell wall was shown to contain trifoliin A by immunofluorescence microscopy. Also, this interface was shown by transmission electron microscopy to contain electron-dense granules of host origin. Scanning electron microscopy revealed an accumulation of extracellular microfibrils associated with the lateral and polar surfaces of the attached bacteria, detectable after 12 h of incubation with seedling roots. At this same time, there was a significant reduction in the effectiveness of 2-deoxy-D-glucose in dislodging bacteria already attached to root hairs and an increase in firm attachment of bacteria to the root hair surface, which withstood the hydrodynamic shear forces of high-speed vortexing. These results are interpreted as a sequence of phases in attachment, beginning with specific reversible interactions between bacterial and plant surfaces (phase I attachment), followed by production of extracellular microfibrils which firmly anchor the bacterium to the root hair (phase 2 adhesion). Thus, attachment of R. trifolii to clover root hairs is a specific process requiring more than just the inherent adhesiveness of the bacteria to the plant cell wall.(ABSTRACT TRUNCATED AT 400 WORDS)     

Specific phases of root hair attachment in the Rhizobium trifolii-clover symbiosis.

The time course and orientation of attachment of Rhizobium trifolii 0403 to white clover root hairs was examined in slide cultures by light and electron microscopy. Inocula were grown for 5 days on defined BIII agar medium and represented the large subpopulation of fully encapsulated single cells which uniformly bind the clover lectin trifoliin A. When 10(7) cells or more were added per seedling, bacteria attached within minutes, forming randomly oriented clumps at the root hair tips. Several hours later, single cells attached polarly to the sides of the root hair. This sequence of attachment to clover root hairs was selective for R. trifolii at inoculum sizes of 10(7) to 4 X 10(8) per seedling, specifically inhibited if 2-deoxy-D-glucose, a hapten for trifoliin A, was present in the inoculum, and not observed when 4 X 10(8) cells were added to alfalfa seedling roots or to large clover root cell wall fragments which lacked trifoliin A but still had trifoliin A receptors. Once attached, R. trifolii 0403 became progressively less detachable with 2-deoxy-D-glucose. At smaller inoculum sizes (10(5) to 10(6) cells per seedling), there was no immediate clumping of R. trifolii at clover root hair tips, although polar binding of bacteria along the root hair surface was observed after 4 h. The interface between polarly attached bacteria and the root hair cell wall was shown to contain trifoliin A by immunofluorescence microscopy. Also, this interface was shown by transmission electron microscopy to contain electron-dense granules of host origin. Scanning electron microscopy revealed an accumulation of extracellular microfibrils associated with the lateral and polar surfaces of the attached bacteria, detectable after 12 h of incubation with seedling roots. At this same time, there was a significant reduction in the effectiveness of 2-deoxy-D-glucose in dislodging bacteria already attached to root hairs and an increase in firm attachment of bacteria to the root hair surface, which withstood the hydrodynamic shear forces of high-speed vortexing. These results are interpreted as a sequence of phases in attachment, beginning with specific reversible interactions between bacterial and plant surfaces (phase I attachment), followed by production of extracellular microfibrils which firmly anchor the bacterium to the root hair (phase 2 adhesion). Thus, attachment of R. trifolii to clover root hairs is a specific process requiring more than just the inherent adhesiveness of the bacteria to the plant cell wall.(ABSTRACT TRUNCATED AT 400 WORDS)     

Trifolin: a Rhizobium recognition protein from white clover

A protein agglutinin, trifoliin, was purified from white clover seeds and seedling roots. Trifoliin specifically agglutinates the symbiont of clover, Rhizobium trifolii, at concentrations as low as 0.2 microgram protein/ml, and binds to the surface of encapsulated R. trifolii 0403. This clover protein has a subunit with Mr approximately 50 000, an isoelectric point of 7.3, and contains carbohydrate. Antibody to purified trifoliin binds to the root hair region of 24-h-old clover seedlings, but does not bind to alfalfa, birdsfoot trefoil or joint vetch. The highest concentration of trifoliin on a clover root is present at sites where material in the capsule of R. trifolii binds. 2-Deoxy-D-glucose elutes trifoliin from intact clover-seedling roots, suggesting that this protein is anchored to root cell walls through its carbohydrate binding sites. We propose that trifoliin on the root hair surface plays an important role in the recognition of R. trifolii by clover.     

Presence of trifoliin A,a rhizobium-binding lectin,in clover root exudate

Trifoliin A, a Rhizobium-binding glycoprotein from white clover, was detected in sterile clover root exudate by a sensitive immunofluorescence assay employing encapsulated cells of Rhizobium trifolii 0403 heat-fixed to microscope slides. Its presence in root exudate was further examined by immunoaffinity chromatography. The binding of trifoliin A to cells was specifically inhibited by the hapten, 2-deoxyglucose. Significantly higher quantities of trifoliin A were detected in root exudate of seedlings grown hydroponically in nitrogen-free medium than in rooting medium containing 15 mM NO, a concentration which completely suppressed root hair infection by the nitrogen-fixing symbiont. The presence of trifoliin A in root exudate may make it possible for recognition processes to occur before the microsymbiont attaches to its plant host.     

Trifolin: A Rhizobium recognition protein from white clover

A protein agglutinin, trifoliin, was purified from white clover seeds and seedling roots. Trifoliin specifically agglutinates the symbiont of clover, Rhizobium trifolii, at concentrations as low as 0.2 μg protein/ml, and binds to the surface of encapsulated R. trifolii 0403. This clover protein has a subunit with M_r ≈ 50 000, an isoelectric point of 7.3, and contains carbohydrate. Antibody to purified trifoliin binds to the root hair region of 24-h-old clover seedlings, but does not bind to alfalfa, birdsfoot trefoil or joint vetch. The highest concentration of trifoliin on a clover root is present at sites where material in the capsule of R. trifolii binds. 2-Deoxy-d-glucose elutes trifoliin from intact clover-seedling roots, suggesting that this protein is anchored to root cell walls through its carbohydrate binding sites. We propose that trifoliin on the root hair surface plays an important role in the recognition of R. trifolii by clover.     

Regulation by fixed nitrogen of host-symbiont recognition in the Rhizobium-clover symbiosis

Either NO₃⁻ (16 millimolar) or NH₄⁺ (1 millimolar) completely inhibited infection and nodulation of white clover seedlings (Trifoliin repens) inoculated with Rhizobium trifolii. The binding of R. trifolii to root hairs and the immunologically detectable levels of the plant lectin, trifoliin, on the root hair surface had parallel declining slopes as the concentration of either NO₃⁻ or NH₄⁺ was increased in the rooting medium. This supports the role of trifoliin in binding R. trifolii to clover root hairs. Agglutination of R. trifolii by trifoliin from seeds was not inhibited by these levels of NO₃⁻ or NH₄⁺. The results suggest that these fixed N ions may play important roles in regulating an early recognition process in the Rhizobium-clover symbiosis, namely the accumulation of high numbers of infective R. trifolii cells on clover root hairs.     

Rhizobium lipopolysaccharide modulates infection thread development in white clover root hairs.

The interaction between Rhizobium lipopolysaccharide (LPS) and white clover roots was examined. The Limulus lysate assay indicated that Rhizobium leguminosarum bv. trifolii (hereafter called R. trifolii) released LPS into the external root environment of slide cultures. Immunofluorescence and immunoelectron microscopy showed that purified LPS from R. trifolii 0403 bound rapidly to root hair tips and infiltrated across the root hair wall. Infection thread formation in root hairs was promoted by preinoculation treatment of roots with R. trifolii LPS at a low dose (up to 5 micrograms per plant) but inhibited at a higher dose. This biological activity of LPS was restricted to the region of the root present at the time of exposure to LPS, higher with LPS from cells in the early stationary phase than in the mid-exponential phase, incubation time dependent, incapable of reversing inhibition of infection by NO3- or NH4+, and conserved among serologically distinct LPSs from several wild-type R. trifolii strains (0403, 2S-2, and ANU843). In contrast, infections were not increased by preinoculation treatment of roots with LPSs from R. leguminosarum bv. viciae strain 300, R. meliloti 102F28, or members of the family Enterobacteriaceae. Most infection threads developed successfully in root hairs pretreated with R. trifolii LPS, whereas many infections aborted near their origins and accumulated brown deposits if pretreated with LPS from R. meliloti 102F28. LPS from R. leguminosarum 300 also caused most infection threads to abort. Other specific responses of root hairs to infection-stimulating LPS from R. trifolii included acceleration of cytoplasmic streaming and production of novel proteins. Combined gas chromatography-mass spectroscopy and proton nuclear magnetic resonance analyses indicated that biologically active LPS from R. trifolii 0403 in the early stationary phase had less fucose but more 2-O-methylfucose, quinovosamine, 3,6-dideoxy-3-(methylamino)galactose, and noncarbohydrate substituents (O-methyl, N-methyl, and acetyl groups) on glycosyl components than did inactive LPS in the mid-exponential phase. We conclude that LPS-root hair interactions trigger metabolic events that have a significant impact on successful development of infection threads in this Rhizobium-legume symbiosis.     

Stimulation of clover root hair infection by lectin-binding oligosaccharides from the capsular and extracellular polysaccharides of Rhizobium trifolii

A polysaccharide depolymerase isolated from the phage lysate of Rhizobium trifolii 4S was used to fragment capsular polysaccharides (CPS) and extracellular polysaccharides (EPS) of R. trifolii 0403 into oligosaccharides. These products were analyzed for clover lectin (trifoliin A)-binding ability, effect on infection of white clover root hairs, and changes in glycosyl and noncarbohydrate composition with culture age. The oligosaccharides from CPS of cultures grown on agar plates for 3, 5, and 7 days exhibited lectin-binding ability at levels similar to those of the corresponding intact CPS. The intact EPS did not bind to clover lectin, although the oligosaccharide fragments from EPS did. In contrast, oligosaccharides from deacetylated CPS had less than half the lectin-binding ability of the native polysaccharide substrate. The CPS from 5-day-old cultures, its corresponding oligosaccharide fragments, and the oligosaccharide fragments of EPS from 5-day-old cultures, all at a concentration of 2.5 micrograms per seedling, stimulated infection thread formation in root hairs of clover seedlings inoculated with R. trifolii 0403. Thus, this bacteriophage-induced polysaccharide depolymerase converted the acidic CPS and EPS of R. trifolii 0403 into biologically active oligosaccharides capable of binding trifoliin A and stimulating root hair infection. The amount of the noncarbohydrate substitutions (pyruvate, acetate, and ether-linked 3-hydroxybutyrate) in the CPS oligosaccharides changed with culture age as shown by 1H-nuclear magnetic resonance spectroscopy. The binding of trifoliin A, therefore, appears to be sensitive to changes in the degree of substitution of noncarbohydrate substitutions in the CPS of R. trifolii 0403.     

Lack of a direct nitrate-trifoliin A interaction in the Rhizobium-clover symbiosis

Summary Nitrate added at critical concentrations to plant growth medium inhibits the infection of legume roots by Rhizobium. The direct interaction, of nitrate and trifoliin A, a Rhizobium-recognition lection from white clover (Trifolium repens L.), was examined as a possible basis for this regulation. Selective molecular ultrafiltration studies to detect ligand-protein interactions showed that radioactive¹³NO₃ ⁻ did not bind directly to trifoliin A when incubated at two molar ratios. Immunoprecipitation of trifoliin A by its homologous antibody was unaffected by 15 mM NO₃ ⁻. In addition, there was no apparent reduction in attachment ofR. trifolii 0403 to root hairs of clover seedings during 1 h of incubation in the presence of 15 mM NO₃ ⁻. These results show that nitrate inhibition of these early steps of the infection process is not due to a direct interaction of nitrate with trifoliin A or its glycosylated receptors.     

Development and trifoliin A-binding ability of the capsule of Rhizobium trifolii.

The age-dependent lectin-binding ability of Rhizobium trifolii 0403 capsular polysaccharide (CPS) was examined by following the development of the capsule and its ability to interact with the white clover lectin trifoliin A. Bacteria grown on agar plates for 3, 5, 7, 14, and 21 days were examined by electron microscopy and immunofluorescence microscopy with antibodies prepared against either R. trifolii 0403 CPS or trifoliin A after pretreatment with the lectin. The capsule began to develop at one pole by day 3 and completely surrounded the cells in cultures incubated for 5 days or longer. The capsular polysaccharide on cells cultured for 3 and 5 days was completely reactive with trifoliin A, became noticeably less reactive by day 7, and was only reactive with the lectin at one pole of a few cells after that time. The quantity and location of lectin receptors on bacteria of different ages directly correlated with their attachment in short-term clover root hair-binding studies. Cells from 3- or 21-day-old cultures attached almost exclusively in a polar fashion, whereas cells grown for 5 days attached to root hairs randomly and in the highest numbers. CPS isolated from a 5-day-old culture had higher lectin-binding ability than CPS from 3- and 7-day-old cultures, whereas the CPS from a 14-day-old culture had the lowest. Chemical analyses of the isolated CPS showed changes in the levels of uronic acids (as glucuronic acid), pyruvate, and O-acetyl substitutions with culture age, but the neutral sugar composition remained relatively constant. These results provide evidence that the age-dependent distribution of lectin receptors dictates the level and orientation of attachments of R. trifolii 0403 to clover root hairs.     

Characterization of the anomalous infection and nodulation of subterranean clover roots by Rhizobium leguminosarum 1020

Anomalous nodulation of Trifolium subterraneum (subterranean clover) roots by Rhizobium leguminosarum 1020 was examined as a model of modified host-specificity in a Rhizobium-legume symbiosis. Consistent with previous reports, these nodules (i) appeared most often at sites of secondary root emergence, (ii) were ineffective in nitrogen fixation and (iii) were as numerous as nodules formed by an effective Rhizobium trifolii strain. R. leguminosarum 1020, grown on agar plates or in the clover root environment, did not bind the white clover lectin, trifoliin A. This strain did not attach in high numbers, and did not induce shepherd's crooks or infection threads, in subterranean clover root hairs. However, R. leguminosarum 1020 did cause branching, moderate curling and other deformations of root hairs. The bacteria probably entered the clover root through breaks in the epidermis at sites of lateral root emergence. The anomalous nodulation was inhibited by nitrate. Only trace amounts of leghaemoglobin were detected in the nodules by Western blot analysis. The nodules were of the meristematic type and initially contained well-developed infection, bacteroid and senescent zones. Infection threads were readily found in the infection zone of the nodule. However, the bacteroid-containing tissue senesced more rapidly than in the effective symbiosis between subterranean clover and R. trifolii 0403. This anomalous nodulation of subterranean clover by R. leguminosarum 1020 suggests a naturally-occurring alternative route of infection that allows Rhizobium to enlarge its host range.     

Alteration of surface properties in a Tn5 mutant strain of Rhizobium trifolii 0403.

A symbiotically defective mutant strain of Rhizobium trifolii, UR251, was obtained by transposon Tn5 mutagenesis of R. trifolii 0403 rif and recognized by its partially ineffective (Fix +/-) phenotype on white clover plants. UR251 had a single Tn5 insertion in plasmid DNA, a wild-type plasmid pattern, and no detectable Mu DNA sequences originally present in the vector used for Tn5 mutagenesis. Agglutination by the clover lectin trifoliin A and attachment to clover root hairs was higher with UR251 than with the wild-type strain. The capsular polysaccharide (CPS) of UR251 was altered, as shown by a slower rate of CPS depolymerization with a CPS beta-lyase, PD-I; more pyruvate and less acetate and 3-hydroxybutanoate noncarbohydrate substitutions as quantitated by 1H nuclear magnetic resonance; and a higher pyruvyl transferase activity (enzymatic pyruvylation of lipid-bound saccharides). The site of increased pyruvylation in the CPS of UR251 was on the terminal galactose of the branch of the repeating oligosaccharide unit. These results show that the level of noncarbohydrate substitutions of the CPS as well as pyruvyl transferase activity are altered in R. trifolii UR251 and that trifoliin A-binding ability and clover root hair attachment are improved in this mutant strain of R. trifolii 0403 rif.     

Cross-reactive antigens and lectin as determinants of symbiotic specificity in the Rhizobium-clover association.

Cross-reactive antigens of clover roots and Rhizobium trifolii were detected on their cell surfaces by tube agglutination, immunofluorescent, and radioimmunoassay techniques. Anti-clover root antiserum had a higher agglutinating titer with infective strains of R. trifolii than with noninfective strains. The root antiserum previously adsorbed with noninfective R. trifolii cells remained reactive only with infective cells, including infective revertants. When adsorbed with infective cells, the root antiserum was reactive with neither infective nor noninfective cells. Other Rhizobium species incapable of infecting clover did not demonstrate surface antigens cross-reactive with clover. Radioimmunoassay indicated twice as much antigenic cross-reactivity of clover roots and R. trifolii 403 (infective) than R. trifolii Bart A (noninfective). Immunofluorescence with anti-R. trifolii (infective) antiserum was detected on the exposed surface of the root epidermal cells and diminished at the root meristem. The immunofluorescent crossreaction on clover roots was totally removed by adsorption of anti-R. trifolii (infective) antiserum with encapsulated infective cells but not with noninfective cells. The cross-reactive capsular antigens from R. trifolii strains were extracted and purified. The ability of these antigens to induce clover root hair deformation was much greater when they were obtained from the infective than noninfective strains. The cross-reactive capsular antigen of R. trifolii 403 was characterized as a high-molecular-weight (greater than 4.6 times 10(6) daltons), beta-linked, acidic heteropolysaccharide containing 2-deoxyglucose, galactose, glucose, and glucuronic acid. A soluble, nondialyzable, substance (clover lectin) capable of binding to the cross-reactive antigen and agglutinating only infective cells of R. trifolii was extracted from white clover seeds. This lectin was sensitive to heat, Pronase, and trypsin. inhibition studies indicated that 2-deoxyglucose was the most probable haptenic determinant of the cross-reactive capsular antigen capable of binding to the root antiserum and the clover lectin. A model is proposed suggesting the preferential adsorption of infective versus noninfective cells of R. trifolii on the surface of clover roots by a cross-bridging of their common surface antigens with a multivalent clover lectin.     

Effect of nitrate supply on the in-vivo synthesis and distribution of trifollin A,a Rhizobium trifolii-binding lectin,in Trifolium repens seedlings

In-vivo synthesis of the white-clover lectin, trifoliin A, was examined by the incorporation of labeled amino acids into protein during heterotrophic growth of intact Trifolium repens L. seedlings. Lectin synthesis was quantified by measuring the level of labeled protein immunoprecipitated from root exudate, from the hapten (2-deoxyglucose) eluate of the roots, and from root and shoot homogenates. The presence of labeled trifoliin A was confirmed by non-denaturing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by fluorography and comparison with trifoliin A standards. In-vivo-labeled trifoliin A was detected in seedling root homogenate 2 h after the addition of labeled amino acids and on the root surface by 8 h. Incorporation of labeled amino acids into protein and trifoliin A was greatest with 2-d-old seedlings and was greater when the plants were grown continuously in the dark than when they were exposed to 14 h light daily. Significantly more labeled lectin accumulated on the root surface of seedlings grown with 1.5 mM KNO₃ than of seedlings grown either without N or with 15.0 mM KNO₃. The labeled lectin from the root surface in all nitrate treatments and from the rootexudate samples of seedlings grown N-free and with 1.5 mM KNO₃ was fully able to bind to Rhizobium trifolii. In contrast, only 2% of the immunoprecipitable protein found in the root exudate of seedlings grown with 15.0 mM KNO₃ was able to bind to the bacteria. Thus, excess nitrate does not repress the synthesis of trifoliin A in the root, but does affect the distribution and activity of this newly synthesized lectin in a way which reduces its ability to interact with R. trifolii. By using Western blot analysis, much more total trifoliin A is detected in the homogenates of shoots than roots. However, greater than 80% of the total labeled protein and 85–90% of the total labeled lectin were found in the root homogenates of 2-d-old dark-grown seedlings incubated for 5 h with labeled amino acids. In addition, Western blot analysis indicated that the shoot homogenate contained smaller-molecular-weight peptides which reacted with the specific anti-trifoliin A antibody. These studies indicate that stored trifoliin A in the seed is degraded in the shoots during seedling development, while newly synthesized trifoliin A in the roots is excreted to the root surface and external environment.Abbreviations IgG immunoglobulin G - LPS lipopolysaccharide - PBS 10 mM potassium-phosphate buffer, pH 7.0, containing 0.8% NaCl - PBS-T 20 mM phosphate-buffered saline, pH 7.4, containing 0.05% Tween 20 - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Adsorption of bacteria to roots as related to host specificity in the Rhizobium-clover symbiosis.

Quantitative microscope techniques were utilized to examine the adsorption of rhizobial cells to clover root hairs. Adsorption of cells of noninfective strains of Rhizobium trifolii or infective R. meliloti strains to clover root hairs was four to five times less than that of the infective R. trifolii strains. Attachment of the rod-shaped bacteria to clover root cells occurred in a polar, end-on fashion. Viable or heat-killed R. trifolii cells precoated with a clover lectin having 2-deoxyglucose specificity had increased adsorption to clover roots. Adsorption of bacteria to roots was not increased if the clover lectin was inactivated by heat or 2-deoxyglucose treatment prior to incubation with R. trifolii. Adsorption of R. trifolii to clover root hairs was inhibited by 2-deoxyglucose (30 mM) but not by 2-deoxygalactose or alpha-D-glucose. Adsorption of R. meliloti cells to alfalfa root hairs was not affected by 2-deoxyglucose at that concentration. These results suggest that expression of host specificity in the Rhizobium-clover symbiosis involves a preferential adsorption of infective cells to clover root hairs through a 2-deoxyglucose-sensitive receptor site.     

Molecular composition of Rhizobium meliloti when parathion was added at the start of incubation

Rhizobium meliloti 300h13 reached the stationary phase of growth very quickly and died rapidly when parathion was added to broth medium at the start of incubation. After 120 h cells changed their molecular composition. A striking diminution in poly-β-hydroxybutyrate (PHB) accumulation was noticed, it was accompanied neither by increase of its glucan precursors, nor by increase of polysaccharides excretion. Parathion-treated cells had decreased proteins, and augmented phospholipids (PL) and lipopolysaccharides (LPS) contents. The rise in PL was mainly due to increases in phosphatidyl glycerol (PG) and phosphatidyl choline (PC). LPS electrophoresis showed stacks of bands typical of LPS with increasing O-antigen chain lengths. Bacterial fatty acids profiles showed an increase of cis-vaccenic and palmitoleic acids, and a diminution of lactobacillic and palmitic acids.     

Properties of free and bound Citrobacter freundii lipopolysaccharides

Culture medium content of free lipopolysaccharide (LPS) components spontaneously released from a Citrobacter freundii culture grown in minimum synthetic medium was determined during early (8-hr culture) and late (24-hr culture) phases of growth. As judged by Limulus-lysate test, free LPS occurred in the medium as early as after 8 hrs of incubation, i.e. at the beginning of log growth phase. As the culture continued to grow the LPS amount released into culture medium kept rising, reaching 30% of endotoxin present in 24-hr Citrobacter culture. The released LPS complex was isolated by separation and its physicochemical, immunochemical and biological properties were determined and compared with those of cell-bound endotoxin recovered from cells by phenol extraction. Comparisons revealed distinct differences in the chemical composition and the degree of heterogeneity; free LPS was less heterogeneous. Immunologically, free LPS differed from bound LPS in the structure of macromolecules, but was identical with it in some antigenic determinants. The biological activity of free LPS preparation was greater than that of cell-bound LPS.