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  • 1
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: In response to phenolic compounds exuded by the host plant, symbiotic Rhizobium bacteria produce signal molecules (Nod factors), consisting of lipochitooligosaccharides with strain-specific substitutions. In Azorhizobium caulinodans strain ORS571 these modifications are an O-arabinosyl group, an O-carbamoyl group, and an N-methyl group. Several lines of evidence indicate that the nodS gene located in the nodABCSUIJ operon is implicated in the methylation of Nod factors. Previously we have shown that NodS is an S-adenosyl-l-methionine (SAM)-binding protein, essential for the l-[3H-methyl]-methionine labelling of ORS571 Nod factors in vivo. Here, we present an in vitro assay showing that NodS from either A. caulinodans or Rhizobium species NGR234 methylates end-deacetylated chitooligosaccharides, using [3H-methyl]-SAM as a methyl donor. The enzymatic and SAM-binding activity were correlated with the nodS gene and localized within the soluble protein fraction. The A. caulinodans nodS gene was expressed in Escherichia coli and a glutathione-S-transferase—NodS fusion protein purified. This protein bound SAM and could methylate end-deacetylated chitooligosaccharides, but could not fully methylate acetylated chitooligosaccharides or unmethylated lipo-chitooligosaccharides. These data implicate that the methylation step in the biosynthesis pathway of ORS571 Nod factors occurs after deacetylation and prior to acylation of the chitooligosaccharides.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The Azorhizobium caulinodans strain ORS571 nodu-lation genes nodSUIJ were located downstream from nodABC. Complementation data and transcriptional analysis suggest that nodABCSUIJ form a single operon. Mutants with Tn5 insertions in the genes nodS, nodU, and nodJ were delayed in nodulation of Sesbania rostrata roots and stems. The NodS amino acid sequences of ORS571, Bradyrhizobium japonicum, and Rhizobium sp. strain NGR234, contain a consensus with similarity to 5-adenosylmethionine (SAM)-utilizing methyltransferases. A naringenin-inducible nodS-dependent protein of approximately 25kDa could be cross-linked to radiolabelled SAM. By applying L-[methyl-3H]-methionine in vivo. Nod factors of ORS571, known to be N-methylated, could be labelled in wild type and nodU mutants but not in nodS mutants. Therefore, we propose that NodS is a SAM-utilizing methyltransferase involved in Nod factor synthesis.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Lipo-chitooligosaccharide (LCO) Nod factors are produced and secreted by rhizobia and trigger nodule development in leguminous host plants. The products of the bacterial nodlJ genes are related to transporters of capsular polysaccharides and were proposed to be involved in LCO transport. We have studied nodlJ of Azorhizobium caulinodans ORS571 by analysis of cell-associated and secreted radioactively labelled Nod factors in wild-type ORS571, a nodJ mutant and a complemented strain. Secretion was strongly reduced in the nodJ mutant, and restored to wild-type levels after complementation. Constructs were made for expression of combinations of different nod genes in Escherichia coli DH5a. The strain DH5α(pUCNABCSU) synthesized LCOs, but they were only secreted when a plasmid containing both nodl and nodJ was supplied in trans, nodi or nodJ alone was not sufficient. In E. coli as well as in Azorhizobium, the nod/J-encoded transporter showed a specificity for more hydrophilic LCOs.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The DNA region downstream of the nodABCSUIJ operon of Azorhizobium caulinodans was further characterized and two new genes, nodZ and noeC were identified in the same operon. The A. caulinodans wild-type strain produces a population of Nod factors that, at the reducing end, are either unmodified or carry a D-arabinosyl and/or an L-fucosyl branch. Nod factors produced by Tn5-insertion mutants in nodZnoeC, and the separate nolK locus, were analysed by thin-layer chromatography and mass spectrometry. Fucosylation of Nod factors depended on both nodZ and nolK. Arabinosylation depended on noeC and/or downstream genes. Protein extracts of A. caulinodans contained an enzymatic activity for fucose transfer from GDP-fucose to chitooligosaccharides and to Nod factors. By mutant analysis and expression of nodZ in Escherichia coli, the fucosyltransferase activity was ascribed to the protein encoded by nodZ. In addition, a Nod factor fucosyltransferase activity, independent of nodZ or other known nod genes, was detected in A. caulinodans. Finally, on the basis of sequence similarity of the nolK gene product, and mass spectrometric analysis of Nod factors produced by a nolK mutant, we propose that this gene is involved in the synthesis of GDP-fucose.
    Type of Medium: Electronic Resource
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  • 5
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 25 (1997), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The rhizobia–legume symbiosis is highly specific. Major host specificity determinants are the bacterial Nod factor signals that trigger the nodulation programme in a compatible host. Nod factors are lipo-chitooligosaccharides (LCOs) varying in the oligosaccharide chain length, the nature of the fatty acids and substitutions on the oligosaccharide. The nod genotype of rhizobia, which forms the genetic basis for this structural variety, includes a set of nodulation genes encoding the enzymes that synthesize LCOs. Allelic and non-allelic variation in these genes ensures the synthesis of different LCO structures by the different rhizobia. The nod genotypes co-evolved with host plant divergence in contrast to the rhizobia, which followed a different evolution. Horizontal gene transfer probably played an important role during evolution of symbiosis. The nod genotypes are particularly well equipped for horizontal gene transfer because of their location on transmissible plasmids and/or on ‘symbiosis islands’, which are symbiotic regions associated with movable elements.
    Type of Medium: Electronic Resource
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