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Selection of several classes of mimosine-degradation-defective Tn3Hogus-insertion mutants of Rhizobium sp. strain TAL1145 on the basis of mimosine-inducible GUS activity

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Rhizobium sp. strain TAL1145 that nodulates Leucaena leucocephala degrades mimosine, a toxin produced by this tree legume. A cosmid clone, pUHR263, containing ~25 kb cloned DNA was isolated by plating Escherichia coli cells containing the cosmid clone library of TAL1145 on a minimal medium in which 3-hydroxy-4-pyridone (HP), a degradation product of mimosine, was used as the source of nitrogen. Cosmid pUHR263 was mutagenized by random insertions of Tn3Hogus, a transposon that makes transcriptional gus fusions when it is inserted in a gene in the correct orientation. Various pUHR263::Tn3Hogus derivatives that showed mimosine-inducible or mimosine-repressible GUS activities when transferred to the Rhizobium sp. strain TAL1145 were selected. Mutants of TAL1145 were constructed by transferring these Tn3Hogus insertions into the TAL1145 chromosome through double-homologous recombination. These mutants were classified into five classes on the basis of defects in mimosine degradation. The growth of these mutants was inhibited to different extents by mimosine applied to the growth medium. Mimosine forms a red-colored Fe-mimosine complex when FeCl3 is added to the medium. The inhibitory effect of Fe-mimosine on growth of the mutants was much less than that of mimosine.Key words: mimosine, mid and pyd genes, Leucaena leucocephala, tree legume, Tn3Hogus.

La souche TAL1145 du groupe de Rhizobium qui est responsable de la nodulation de Leucaena leucocephala dégrade la mimosine, une toxine produite par cet arbre légumineuse. Un clone cosmidique, pUHR263, contenant ~25 kb d'ADN cloné a été isolé en étalant des cellules d'Escherichia coli contenant une banque de clones cosmidiques TAL1145 sur un milieu minimal dans lequel le 3-hydroxy-4-opyridone (HP), un produit de dégradation de la mimosine, a été utilisé comme source d'azote. Nous avons provoqué des mutations dans le cosmide pUHR263 à l'aide d'insertions aléatoires de Tn3Hogus, un transposon qui effectue des fusions transcriptionnelles gus lorsqu'il est inséré à l'intérieur d'un gène dans la bonne orientation. Plusieurs clones dérivés de pUHR263::Tn3Hogus qui ont montré une activité GUS mimosine inductible ou mimosine répressible lorsque transférés dans la souche Rhizobium TAL1145 ont été sélectionnés. Des mutants de TAL1145 ont été construits par transfert de ces insertions de Tn3Hogus dans le chromosome TAL1145 par double recombinaison homologue. Ces mutants ont été organisés en cinq classes en se basant sur les irrégularités dans la dégradation de la mimosine. La croissance de ces mutants a été inhibée à différents degrés par la minosine appliquée au milieu. La mimosine forme un complexe Fe-mimosine de couleur rouge lorsque le FeCl3 est ajouté au milieu. L'effet d'inhibition de Fe-mimosine sur la croissance des mutants était bien en deçà que celui de la mimosine.Mots clés : mimosine, gènes mid et pyd, Leucaena leucocephala, arbre légumineuse, Tn3Hogus.[Traduit par la Rédaction]

Keywords: Leucaena leucocephala; Tn3Hogus; arbre légumineuse; gènes mid et pyd; mid and pyd genes; mimosine; tree legume

Document Type: Research Article

Publication date: June 1, 2001

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  • Published since 1954, this monthly journal contains new research in the field of microbiology including applied microbiology and biotechnology; microbial structure and function; fungi and other eucaryotic protists; infection and immunity; microbial ecology; physiology, metabolism and enzymology; and virology, genetics, and molecular biology. It also publishes review articles and notes on an occasional basis, contributed by recognized scientists worldwide.
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