PubMed Health⌕ Search

Biomedical subjects

W J Broughton

Publications and source records attributed to W J Broughton.

54 records · Page 3Linked to original sources
PubMed1987

Cloning of Rhizobium leguminosarum genes for competitive nodulation blocking on peas.

One type of competitive interaction among rhizobia is that between nonnodulating and nodulating strains of Rhizobium leguminosarum on primitive pea genotypes. Pisum sativum cv. Afghanistan nodulates effectively with R. leguminosarum TOM, and this can be blocked in mixed inoculations by R. leguminosarum PF2, which does not nodulate this cultivar. We termed this PF2 phenotype Cnb+, for competitive nodulation blocking. Strain PF2 contains three large plasmids including a 250-kilobase-pair symbiotic (Sym) plasmid. Transfer of this plasmid, pSymPF2, to nonblocking rhizobia conferred the Cnb+ phenotype on recipients in mixed inoculations on cultivar Afghanistan with TOM. A library of the PF2 genome constructed in the vector pMMB33 was used to isolate two cosmid clones which hybridize to pSymPF2. These cosmids, pDD50 and pDD58, overlapped to the extent of 23 kilobase pairs and conferred a Cnb+ phenotype on recipient Cnb- rhizobia, as did pSD1, a subclone from the common region.

Cloning, Molecular↗
PubMed1986

Identification of Rhizobium plasmid sequences involved in recognition of Psophocarpus, Vigna, and other legumes.

Symbiotic DNA sequences involved in nodulation by Rhizobium must include genes responsible for recognizing homologous hosts. We sought these genes by mobilizing the symbiotic plasmid of a broad host-range Rhizobium MPIK3030 (= NGR234) that can nodulate Glycine max, Psophocarpus tetragonolobus, Vigna unguiculata, etc., into two Nod- Rhizobium mutants as well as into Agrobacterium tumefaciens. Subsequently, cosmid clones of pMPIK3030a were mobilized into Nod+ Rhizobium that cannot nodulate the chosen hosts. Nodule development was monitored by examining the ultrastructure of nodules formed by the transconjugants. pMPIK3030a could complement Nod- and Nif- deletions in R. leguminosarum and R. meliloti as well as enable A. tumefaciens to nodulate. Three non-overlapping sets of cosmids were found that conferred upon a slow-growing Rhizobium species, as well as on R. loti and R. meliloti, the ability to nodulate Psophocarpus and Vigna, thus pointing to the existence of three sets of host-specificity genes. Recipients harboring these hsn regions had truly broadened host-range since they could nodulate both their original hosts as well as MPIK3030 hosts.

Base Sequence↗
PubMed1984

Plasmid-linked nif and "nod" genes in fast-growing rhizobia that nodulate Glycine max, Psophocarpus tetragonolobus, and Vigna unguiculata.

Forty-nine fast-growing Rhizobium strains from the nodules of 26 different tropical legume genera were screened to find isolates that would (i) nodulate, e.g., winged beans, so producing large nodules for RNA and protein isolation; (ii) also nodulate various small-seeded legumes, thus allowing screening of large numbers of mutants; and (iii) harbor plasmids containing nif structural genes as well as other functions involved in nodulation. On the basis of six different criteria, this rhizobial group appeared intermediate between classical fast- and slow-growing organisms, yet all contained plasmids. Plasmid numbers varied from one to five. Hybridizations between DNA prepared from nifDH and the putatative "nod" region of R. meliloti and these plasmids bound to nitrocellulose filters suggested that nif-nod genes are linked on a single sym plasmid. A broad-host-range strain containing a single sym plasmid was chosen for further study. Its plasmid, pMPIK3030a, was isolated on cesium chloride gradients and cloned in the cosmid pJB8, and the overlapping fragments were mapped by homology with the nif and nod regions of R. meliloti. As the wild-type plasmid pMPIK3030a was not self-transmissible, confirmation that the nod genes detected by homology were responsible for nodulation was obtained by introducing the mobilization functions of RP(4) (together with Tn5) and selecting transconjugants resistant to kanamycin and neomycin. Transconjugants (obtained at a frequency of about 10(-6) per recipient) in Agrobacterium tumefaciens cured of the Ti plasmid produced ineffective nodules on Vigna unguiculata, those in nonnodulating (Nod(-)) R. meliloti were partially effective, while those in Nod(-)R. leguminosarum were often fully effective.

Journal Article↗
PubMed1983

Morphology of root nodules and nodule-like structures formed by Rhizobium and Agrobacterium strains containing a Rhizobium meliloti megaplasmid.

We examined expression of the megaplasmid pRme41b of Rhizobium meliloti in two different Rhizobium sp. Strains and in Agrobacterium tumefaciens. Transfer of pRme41b into these bacteria was facilitated by insertion of a recombinant plasmid coding for mobilization functions of RP4 into the nif region (Kondorosi, A., E. Kondorosi, C.E. Pankhurst, W. J. Broughton, and Z. Banfalvi, 1982, Mol. Gen. Genet., 188:433-439). In all cases, transconjugants formed nodule-like structures on the roots of Medicago sativa. These structures were largely composed of meristematic cells but they were not invaded by bacteria. Bacteria were found only within infection threads in root hairs, and within intercellular spaces of the outermost cells of the structures. The donor strain of R. meliloti containing pAK11 or pAK12 in pRme41b initially produced nodules on M. sativa that did not fix nitrogen (Fix-). In these nodules, bacteria were released from infection threads into the host cells but they did not multiply appreciably. Any bacteroids formed degenerated prematurely. In some cases, however, reversion to a Fix+ phenotype occurred after 4 to 6 wk. Bacteria released into newly infected cells in these nodules showed normal development into bacteriods.

Fabaceae↗
PubMed1983

Transfer of an indigenous plasmid of Rhizobium loti to other rhizobia and Agrobacterium tumefaciens.

Rhizobium loti strains NZP2037 and NZP2213 were each found to contain a single large plasmid: pRlo2037a (240 MDal) and pRlo2213a (120 MDal), respectively. Plasmid DNA present in crude cell lysates of each strain and purified pRlo2037a DNA did not hybridize with pID1, a recombinant plasmid containing part of the nitrogen fixation (nif) region of R. meliloti, indicating that nif genes were not present on these plasmids. The transposon Tn5 was inserted into pRlo2037a and this plasmid was then transferred into R. leguminosarum, R. meliloti and Agrobacterium tumefaciens. All transconjugants failed to nodulate Lotus pedunculatus, suggesting that the ability to nodulate this legume was also not carried on pRlo2037a. Transfer of pRlo2037a to R. loti strain NZP2213 did not alter the Nod+ Fix- phenotype of this strain for L. pedunculatus. Determinants for flavolan resistance, believed to be necessary for effective nodulation of L. pedunculatus, were not carried on pRlo2037a. These data suggest that nodulation, nitrogen fixation and flavolan resistance genes are not present on the large plasmid in R. loti strain NZP2037.

Conjugation, Genetic↗
PubMed1980

Dynamics of Rhizobium competition for nodulation of Pisum sativum cv. Afghanistan.

Nodulation of Pisum sativum cv. Afghanistan by Rhizobium leguminosarum strain Tom can be blocked by the nonnodulating R. leguminosarum strain PF2. This system was used to study the dynamics of rhizobial binding to seedling root surfaces. Appreciable numbers of 35S-labeled rhizobia bound to the root surface within 2h after inoculation. PF2 colonized the root surfaces earlier and in greater numbers that did Tom. Radioactive extracellular polysaccharides and lipopolysaccharides from either organism failed to bind to the seedling roots in measurable quantities.

Fabaceae↗
PubMed1976

Comparative structural and immunochemical properties of leghaemoglobins.

Circular dichroism studies on leghaemoglobins from snake bean, lupin, serradella and other plants show that, in common with soybean (reported earlier) they have a similar overall polypeptide chain conformation and haem environment and orientation. Immunochemical studies, on the other hand, suggest that the antigenic determinants on the surface of the leghaemoglobins vary considerably. Thus, firstly the alpha-helix content of the leghaemoglobins as a class is very similar (60-65%) and approaches that of the myoglobins, secondly, the sign, magnitude and shape of their circular dichroism spectra in the near ultraviolet, Soret and visible regions suggest close similarities in the environment and orientation of a structurally important tryptophan residue and of the haem moiety, and thirdly, there is comparatively weak haem-protein interaction. The extent of immuno cross-reactivity was found to be best deomonstrated using the Farr radioimmunoassay procedure. The results were (a) 5 leghaemoglobins from one plant (soybean) crossreacted completely but with varying affinities. (b) The extent of cross reactivity between leghaemoglobins from different plants was compared to that within a single plant; the reaction of antiserum to a soybean leghaemoglobin with a serradella leghaemoglobin was weak, with a snake bean leghaemoglobin still weaker (and incomplete) while lupin leghaemoglobins showed no cross reactivity at all. (c) The "rapid" attenuation of cross reactivity among different plant leghaemoglobins is explicable in terms of the extensive amino acid substitutions which have been demonstrated in the literature and in the present studies. (d) In view of this rapid divergence it is not surprising that sperm whale and horse heart myoglobins showed no cross reactivity with soybean leghaemoglobins. In summary, amino acid substitutions in the leghaemoglobin family are conformationally but not immunochemically conservative.

Amino Acid Sequence↗
PubMed1972

Molecular properties of lupin and serradella leghaemoglobins.

1. Leghaemoglobins were extracted from the root nodules of lupin (Lupinus luteus L.) and serradella (Ornithopus sativus Brot.) plants and fractionated into different leghaemoglobin components on DEAE-cellulose-acetate columns. 2. The first two fractions eluted from columns loaded with either lupin or serradella leghaemoglobins were in the Fe(3+) oxidation state. 3. These components have protohaem IX as the prosthetic group and glycine as the N-terminal amino acid. 4. Other properties are: lupin component I, pI5.08, molecular weight 19000; lupin component II, pI5.13, molecular weight 20600; serradella component I, pI5.00, molecular weight 17500; serradella component II, pI5.05, molecular weight 19100. 5. Leghaemoglobins are thus heterogeneous with respect to size and charge.

Amino Acid Sequence↗
PubMed1971

Control of leghaemoglobin synthesis in snake beans.

1. The finding that the plant is the genetic determinant of leghaemoglobin production in legume nodules was further tested by inoculating snake beans with two strains of Rhizobium selected to give large genetic differences. Carbohydrate requirement patterns, immunological techniques and DNA base ratio determinations were used to demonstrate genetic differences between the two rhizobial strains. 2. Partially purified preparations of the haemoglobins from the nodules produced by the two strains showed no differences when examined by electrophoresis, isoelectric focusing or ion-exchange chromatography. 3. Two different leghaemoglobins from each type of nodule were separated by chromatography on DEAE-cellulose. One of these was isolated in the Fe(3+) form and accounted for two-thirds of the total leghaemoglobin. When it was examined in the analytical ultracentrifuge and by amino acid analysis, this major component did not vary with the inoculant rhizobial strain. The molecule had an s(20,w) of 1.88S, a diffusion coefficient of 10.7x10(-7)cm(2).s(-1) and a mol. wt. of 16700. 4. These results strongly support the hypothesis that the mRNA for leghaemoglobin is transcribed from plant DNA.

Amino Acids↗
PubMedDate not supplied

nodSU, two new nod genes of the broad host range Rhizobium strain NGR234 encode host-specific nodulation of the tropical tree Leucaena leucocephala.

Rhizobium species strain NGR234 nodulates at least 35 diverse genera of legumes as well as the nonlegume Parasponia andersonii. Most nodulation genes are located on the 500-kilobase pair symbiotic plasmid, pNGR234a. Previously, three plasmid-borne host range determinants (HsnI, HsnII, and HsnIII) were identified by their ability to extend the nodulation capacity of heterologous rhizobia to include Vigna unguiculata. In this study, we show that HsnII contains two new nod-box linked hsn genes, nodS and nodU.nodS controls nodulation of the tropical tree Leucaena leucocephala, while the nodSU genes regulate nodulation of the pasture legume Desmodium intortum and the grain legume V. unguiculata. Regulation of the nod-box upstream of nodSU by the flavonoid naringenin was shown using a fusion with a promoterless lacZ gene. Determination of the nucleotide sequence of the nodS gene did not reveal homology with any gene in the EMBL library, although Bradyrhizobium japonicum USDA110 contains both nodS and nodU (M. Göttfert, S. Hitz, and H. Hennecke, Molecular Plant-Microbe Interactions 3:308-316, 1990). We suggest that broad host range in NGR234 is controlled in part by a nodD gene which interacts with a wide range of flavonoids, and in part by host-specific nod genes such as nodS.

Amino Acid Sequence↗
PubMedDate not supplied

Biological activity of Rhizobium sp. NGR234 Nod-factors on Macroptilium atropurpureum.

The broad host range of Rhizobium sp. NGR234 is based mainly on its ability to secrete a family of lipooligosaccharide Nod factors. To monitor Nod-factor purification, we used the small seeded legume Macroptilium atropurpureum, which responds evenly and consistently to Nod factors. At concentrations between approximately equal to 10(-11) M and 10(-9) M, this response takes the form of deformation of the root hairs. Higher concentrations (approximately equal to 10(-9) to 10(-7) M), provoked profound "shepherd's crook" type curling of the root hairs. Similar concentrations of Nod factors of Bradyrhizobium japonicum, Rhizobium leguminosarum, and R. meliloti also provoked marked curling of the root hairs, but the latter two species are unable to nodulate Macroptilium. On the other hand, plant hormones, hormone-like substances, inhibitors of hormone action, as well as substituents of Nod factors were without effect in this bioassay. We thus conclude that only Nod factors are capable of inducing shepherd's crook type curling of Macroptilium root hairs. Perturbations in the auxin-cytokinin balance induced "pseudo" nodulation on M. atropurpureum, as did NodNGR factors at concentrations between 10(-7) and 10(-6) M. Concomitant inoculation of Macroptilium with a NodABC- mutant of NGR234 and sulfated NodNGR factors (NodNGR[S]) gave rise to plants that slowly greened, showing that the NodNGR factors permitted entry of the Nod- mutant into the roots.

Bacterial Proteins↗
PubMedDate not supplied

Accumulation of transcripts encoding a lipid transfer-like protein during deformation of nodulation-competent Vigna unguiculata root hairs.

A cDNA library was constructed from RNA of Vigna unguiculata root hairs harvested 1 day and 4 days after inoculation with Rhizobium sp. NGR234. A heterologous probe was used to identify a cDNA clone, the predicted 99-amino-acid sequence of which shares homology with a nonspecific lipid transfer protein (LTP) of Hordeum vulgare. Other characteristics, including an estimated molecular weight of 10.4 kD, an isoelectric point of 8.6, and a signal peptide with a hydrophobic region at the amino-terminal end, are shared by most LTPs. A transcript of 630 nt was found in all tissues tested, except nodules. Levels of mRNA increased in root hairs 24 hr after treatment with Rhizobium sp. NGR234, with different hormones, or with Nod factors. Amounts of transcripts were dependent on the concentration of Nod factors. Accumulation of transcripts during nodule development correlated with root hair deformation, the first visible step in the Rhizobium-legume symbiosis.

Amino Acid Sequence↗