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Biomedical subjects

S G Pueppke

Publications and source records attributed to S G Pueppke.

51 records · Page 3Linked to original sources
PubMed1984

Mode of infection, nodulation specificity, and indigenous plasmids of 11 fast-growing Rhizobium japonicum strains.

Eleven fast-growing strains of Rhizobium japonicum were characterized with respect to indigenous plasmids and abilities to infect (Inf+) and nodulate (Nod+) cowpea, siratro, wild soybean, and three commercial cultivars of soybean. All strains caused infection via infection threads in root hairs and consistently nodulated cowpea, siratro, and wild soybean in growth pouches. Interactions with commercial cultivars of soybean were strikingly strain specific. Some combinations were Nod-, and infection was delayed in others. The ratios of infections to nodules and the distribution of nodules on primary and lateral roots also varied substantially. A modified in-gel lysis procedure was devised for electrophoretic separation of plasmids from the strains. Plasmids (ranging in size from 35 to greater than 300 megadaltons) were reproducibly detected in all strains.

Fabaceae↗
PubMed1984

Adsorption of tumorigenic Agrobacterium tumefaciens cells to susceptible potato tuber tissues.

Cells of tumorigenic Agrobacterium tumefaciens strain B6 were labeled with [35S]methionine and used to estimate bacterial adsorption to potato tuber discs. After 10 min at pH 7.2, about 5 X 10(5) bacteria adsorb to each 9.0-mm disc. Lengthening the incubation period to 90 min increases adsorption to about 11 X 10(5) bacteria per disc. Bacterial adsorption is reduced under both alkaline and acid pH conditions and increases 2.3-fold if the number of bacterial cells in the inoculum is doubled. Adsorption is increased if citrus pectin, polygalacturonic acid, or demethylated pectin is included in the inoculum; methylated polygalacturonic acid is inactive. The activity of citrus pectin is abolished, however, if the compound is applied to the discs as a pretreatment and then removed prior to inoculation. Lipopolysaccharide preparations from five of six A. tumefaciens strains have no effect on bacterial adsorption. The sixth preparation, from nontumorigenic strain NT-1, reduces adsorption by about 20%.

Adsorption↗
PubMed1981

Examination of Le and lele Genotypes of Glycine max (L.) Merr. for Membrane-Bound and Buffer-Soluble Soybean Lectin.

Membrane fractions from seedlings of four soybean [Glycine max (L.) Merr.] lines were examined by radioimmunoassay and hemagglutination assay for the 120,000 dalton soybean lectin. Two of the lines (Sooty and T-102) are genotypically lele and lack buffer-soluble soybean lectin; the remaining two lines (Beeson and Harosoy 63) are Le and produce seeds that contain the lectin (Su et al. 1980 Biochim. Biophys. Acta 629: 292-304). Both Triton X-100 (0.5% v/v) and nonidet P-40 (0.05% v/v) solubilized soybean lectin from membrane fractions of Le cotyledons. Triton X-100 interfered substantially with the assay of protein and hemagglutinating activity and was unacceptable for use in quantitative measurements. The nonidet P-40-solubilized soybean lectin from Le cotyledons was consistently present both in washed 13,000g and 82,500g membrane fractions, but it accounted for less than 1.5% of the total (buffer-soluble plus membrane-bound) soybean lectin. The membrane lectin was purified by the affinity chromatography procedure devised for soluble soybean lectin, and it was immunologically indistinguishable from authentic soybean lectin. Membrane fractions from Le cotyledons contained insignificant amounts of radioisotope-labeled soybean lectin that had been added during homogenization, and purified membrane fractions did not bind the lectin in the presence of the hapten, d-galactose. These controls make it unlikely that the membrane soybean lectin was of cytoplasmic origin. Soybean lectin and other hemagglutinins were not present in buffer-soluble or membrane fractions from lele cotyledons or from roots and hypocotyls of any of the lines.

Journal Article↗
PubMed1980

Lectins and the soybean-Rhizobium symbiosis. I. Immunological investigations of soybean lines, the seeds of which have been reported to lack the 120 000 dalton soybean lectin.

Seeds of six soybean lines (Glycine max (L.) Merr. cv. Columbia, D68-127, Norredo, Sooty, T-102, Wilson 5) have been reported to lack the 120 000 dalton soybean lectin. Immunodiffusion and radioimmunoassay using anti-soybean lectin immunoglobulin failed to detect the lectin in seeds of five lines, but D68-127 seeds contained as much soybean lectin as the control line, Harosoy 63. The D68-127 seed lectin could be purified by affinity chromatography on Sepharose-N-caproylgalactosamine, and was indistinguishable from the conventional soybean lectin by the following criteria: electrophoretic migration in acidic and alkaline buffers, subunit molecular weight and composition, analytical isoelectric focusing, gel filtration chromatography. Phosphate-buffered saline extracts of roots, hypocotyls, stems, and leaves of 3--66-day-old Norredo and Harosoy 63 plants lacked soybean lectin, as determined by hemagglutination and radioimmunoassay (detection limit: 1.4 micrograms soybean lectin/g dry weight tissue). Cotyledons of Harosoy 63 (but not Norredo) contained large quantities of the lectin, which diminished as the plants aged. 5-day-old roots and hypocotyls of 20 soybean lines did not contain soybean lectin. Roots of Columbia, Norredo, Sooty, T-102, Wilson 5, and Harosoy 63 (control) were nodulated by a variety of strains of Rhizobium japonicum and Rhizobium sp.

Lectins↗
PubMed1980

Differentiation of Rhizobium japonicum strain derivatives by antibiotic sensitivity patterns, lectin binding, and utilization of biochemicals.

Several strains of Rhizobium japonicum have been reported to consist of mixtures of stable derivativess having distinct colony morphologies and physiological characteristics. We isolated derivatives from strains of R. japonicum and systematically compared them with previously isolated derivatives with respect to the utilization of biochemicals, antibiotic sensitivity, and soyben lectin binding. With the exception of a pair of derivatives from 3Ilb110, one of which utilized pyruvate and one of which did not, sibling derivatives had essentially identical biochemical utilization patterns. The sibling derivatives of parental strains 3Ilb110 and 3Ilb140 exhibited marked variation in their sensitivities to several antibiotics, including gentamicin, sul famethoxazole, and tetracycline. Compared with the derivative with small colony morphology, derivatives with large colony morphology were in general more sensitive to these antibiotics. With one exception, the binding of soybean lectin to the derivatives was quantitatively the same as that to the parental strain. The anomaly was 110-Y which, in contrast to its parental strain and sibling derivatives, failed to bind detectable amounts of the lectin. 110-Y, as well as all the other derivatives and parental strains, nodulated Disoy soybean.

Anti-Bacterial Agents↗
PubMed1980

Interaction of lectins from soybean and peanut with rhizobia that nodulate soybean, peanut, or both plants.

Four of 14 strains of Rhizobium japonicum from soybean nodulated peanut (Arachis hypogaea L. cultivar Jumbo Virginia), and 3 of 8 Rhizobium sp. strains from peanut nodulate soybean (Glycine max (L.) Merr. cultivar Harosoy 63). Cells of three peanut rhizobia bound fluorescent- and radioisotope-labeled soybean lectin. Two of these strains failed to nodulate soybean, and conversely, two peanut strains that nodulated soybean did not bind to soybean lectin. Both culture medium and age had pronounced effects of the number of peanut rhizobia cells that bound fluorescent-labeled soybean lectin. Harosoy 63 soybean root exudates stimulated the growth of peanut rhizobia, but had no consistent influence on the number of cells that bound soybean lectin. Although extracellular soybean lectin receptors were present in culture fluids from each of the peanut rhizobia whose cells bound the lectin, the titer of receptors was greatest for strains 3G4b5. The affinity constants for the adherence of soybean lectin to Rhizobium sp. 3G4b5 cells from cultures of various ages ranged from 4.2 X 10(6) to 4.9 X 10(6) M-1, and the number of lectin binding sites per cell decreased as cells aged. Cells of the soybean and peanut rhizobia did not bind fluorescent- or radioisotope-labeled peanut lectin. The results indicate that there is no relationship between the ability of peanut and soybean rhizobia to nodulate the reciprocal host plant and their ability to bind to the lectin of that plant.

Arachis↗
PubMed1979

Purification and characterization of a lectin from seeds of the winged bean, Psophocarpus tetragonolobus (L.)DC.

A lectin from seeds of the winged bean, Psophocarpus tetragonolobus (L.)DC, has been purified to homogeneity by affinity chromatography on a Bio-Gel lactobionate adsorbent. The lectin is rich in acidic amino acids, and after isoelectric focusing in polyacrylamide gel, appeared as a single band of pI5.5 The molecular weight of the lectin is 46 000 +/- 2000 as determined by analytical ultracentrifugation. Dodecyl sulfate gel electrophoresis detected a single type of subunit with a molecular weight of 29 000 +/- 3000. The lectin has specificity for receptors containing D-galactose and agglutinates trypsinized human red blood cells of all three ABO types. Of the tested inhibitors of hemagglutination caused by winged bean lectin, p-nitrophenyl-beta-D-galactoside was the most potent. Lactose was the most active oligosaccharide tested, and other oligosaccharides containing alpha-linked D-galactose were much less inhibitory.

Amino Acids↗
PubMed1979

Distribution of Lectins in the Jumbo Virginia and Spanish Varieties of the Peanut, Arachis hypogaea L.

Peanut lectin was purified from seed meal of the Spanish and Jumbo Virginia varieties of peanut (Arachis hypogaea L.) by affinity chromatography on lactose coupled to Sepharose 4B. Polyacrylamide gel isoelectric focusing resolved the lectin preparation from Jumbo Virginia seeds into seven isolectins (pI 5.7, 5.9, 6.0, 6.2, 6.3, 6.5, and 6.7). Seed meal from the Spanish variety contained six isolectins which were indistinguishable from the pI 5.7, 5.9, 6.2, 6.3, 6.5, and 6.7 isolectins from Jumbo Virginia. Quantitative, lactose-specific hemagglutination was used to examine the lectins in tissues of both peanut varieties. In young (3- to 9-day-old) seedlings of each variety, more than 90% of the total amount of lectins detected in the plants was in the cotyledons. Most of the remainder was in hypocotyls, stems, and leaves; young roots contained no more than 4 micrograms of lectin per plant. Lectins were present in all nonroot tissues of 21- to 30-day-old seedlings, except 27-day-old Spanish hypocotyls. As cotyledons of each variety senesced, several of the more basic isolectins decreased to undetectable levels, but the acidic isolectins remained until at least 15 days after planting. Some of the seed isolectins and several apparently new lactose-binding lectins were also identified in affinity-purified extracts of 5-day-old roots and hypocotyls. Rabbit antibodies raised against the Jumbo Virginia seed isolectin preparation reacted with seed, cotyledon, and hypocotyl lectin preparations from both varieties. Analysis of seed lectin preparations from seven varieties of A. hypogaea and of a related species (A. villosulicarpa) indicated that isolectin composition in Arachis may be a characteristic of both the species and the subspecies (botanical type) to which the variety belongs.

Journal Article↗
PubMed1978

Role of Lectins in Plant-Microorganism Interactions: II. Distribution of Soybean Lectin in Tissues of Glycine max (L.) Merr.

Three different assay procedures have been used to quantitate the levels of soybean (Glycine max [L.] Merr.) lectin in various tissues of soybean plants. The assays used were a standard hemagglutination assay, a radioimmunoassay, and an isotope dilution assay. Most of the lectin in seeds was found in the cotyledons, but lectin was also detected in the embryo axis and the seed coat. Soybean lectin was present in all of the tissues of young seedlings, but decreased as the plants matured and was not detectable in plants older than 2 to 3 weeks. Soybean lectin isolated from seeds of several soybean varieties were identical when compared by several methods.

Journal Article↗
PubMed1977

Role of lectins in plant-microorganism interactions: I. Binding of soybean lectin to rhizobia.

Highly purified soybean lectin (SBL) was labeled with fluorescein isothiocyanate (FITC-SBL) or tritium ((3)H-SBL) and repurified by affinity chromatography. FITC-SBL was found to bind to living cells of 15 of the 22 Rhizobium japonicum strains tested. The lectin did not bind to cells of the other seven R. japonicum strains, or to cells of any of the nine Rhizobium strains tested which do not nodulate soybean. The binding of the lectin to the SBL-positive strains of R. japonicum was shown to be specific and reversible by hapten inhibition with d-galactose or N-acetyl-d-galactosamine.The lectin-binding properties of the SBL-positive R. japonicum strains were found to change substantially with culture age. The percentage of cells in a population exhibiting fluorescence after exposure to FITC-SBL varied between 0 and 70%. The average number of SBL molecules bound per cell varied between 0 and 2 x 10(6). While most strains had their highest percentage of SBL-positive cells and maximum number of SBL-binding sites per cell in the early and midlog phases of growth, one strain had a distinctly different pattern. The SBL-negative strains did not bind lectin at any stage of growth.Quantitative binding studies with (3)H-SBL indicated that the affinity constant for binding of SBL to its receptor sites on R. japonicum is approximately 4 x 10(7)m(-1). Many of the binding curves were biphasic. An inhibitor of SBL binding was found to be present in R. japonicum culture filtrates.

Journal Article↗
PubMedDate not supplied

Sequence and analysis of the nodABC region of Rhizobium fredii USDA257, a nitrogen-fixing symbiont of soybean and other legumes.

We cloned and analyzed nodABC from Rhizobium fredii USDA257. These genes are thought to have common functions in initiation of nitrogen-fixing nodules by all rhizobia. In USDA257, they were located in a 9.2-kb EcoRI fragment that was not closely linked to either of two copies of the regulatory gene, nodD. nodABC was present in a 3,094-base pair (bp) sequenced region, which also included a consensus nod-box promoter. The three open reading frames contained 654, 642, and 1,239 bp, respectively, and encoded deduced proteins of 21.9, 23.4, and 44.7 kD. The sequence of the nodABC region of USDA257 was generally homologous with corresponding regions from other rhizobia, but it diverged significantly in the 5' non-translated region and in the 3'terminus of nodC. nodC was not translationally coupled to nodSU, as in another soybean symbiont, Bradyrhizobium japonicum, and the deduced NodC protein was the shortest of any such proteins yet described. Site-directed mutagenesis of the 9.2-kb EcoRI fragment confirmed that nodA, nodB, and nodC are essential for nodulation of soybean, but failed to identify other linked nod genes. Daidzein, a major isoflavone from soybean roots, was the most potent of nine tested flavonoids in activating a plasmid-borne nodC::lacZ fusion. The 9.2-kb fragment complemented nodA-, nodB-, and nodC- mutants of R. meliloti to the Nod+ phenotype on Medicago sativa, M. truncatula, and Trigonella foenum-graecum. Nodule numbers, percentage of nodulated plants, and shoot dry weights, however, were considerably less than in plants inoculated with mutants complemented with nodABC from R. meliloti.

Acyltransferases↗
PubMedDate not supplied

Repetitive sequences with homology to Bradyrhizobium japonicum DNA and the T-DNA of Agrobacterium rhizogenes are closely linked to nodABC of Rhizobium fredii USDA257.

We have detected strong homology between a 9.2-kb EcoRI restriction fragment from Rhizobium fredii USDA257 that contains nodABC and eight additional EcoRI fragments in DNA digests from this organism. A series of repetitive sequences responsible for this hybridization lies within a 0.95-kb HindIII/SalI subfragment about 1-kb upstream of nodA. This subfragment also hybridizes to multiple restriction fragments from nine other strains of R. fredii, but only one is common to all strains. The 0.95-kb subfragment does not hybridize to genomic DNA from 17 other strains of fast-growing rhizobia, but there is weak homology to two fragments from Rhizobium sp. strain NGR234. We sequenced 2,432 base pairs (bp) of the region encompassing the repetitive sequences. It contains 65 separate 8- to 11-bp inverted and direct repeats, as well as two large open reading frames (ORFs) that overlap on opposite strands. ORF1 reads in the same direction as nodABC, contains 1,071 bp, and encodes a 40.6-kD protein. It has 74% sequence homology to an ORF within the T-DNA of Agrobacterium rhizogenes and similar homology to a series of repetitive sequences from Bradyrhizobium japonicum. ORF2 (981 bp) reads in the opposite direction, encodes a 34.7-kD protein, and has partial identity with a second ORF from A. rhizogenes. We could detect no poly(A)+ nodule transcripts with homology to ORF1 and ORF2. The eight sets of repetitive sequences found in other EcoRI fragments of the genome were cloned from USDA257 on separate cosmids. Some of these cosmids appear to overlap, and two have fragments with homology to nifKDH.

Amino Acid Sequence↗