Mini-Mulac transposons with broad-host-range origins of conjugal transfer and replication designed for gene regulation studies in Rhizobiaceae

  • Ratet P
  • Schell J
  • de Bruijn F
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Abstract

Novel mini-Mu derivatives were constructed, carrying a truncated lacZYA operon fused to the terminal 117 bp of the Mu S-end, for the isolation of translational lac fusions by mini-Mu-mediated insertion mutagenesis. Different selectable markers (chloramphenicol resistance; gentamycin resistance) were introduced to allow selection for mini-Mu insertions in different replicons and bacterial strains. A mini-Mulac derivative carrying the site for conjugal transfer of plasmid RP4 (oriT) and the origin of replication of the Agrobacterium rhizogenes Ri plasmid (oriRiHRI) was constructed to enable one-step lac-fusion mutagenesis of cloned (plasmid-borne) regions in Escherichia coli and efficient conjugal transfer of gene fusions to a variety of Gram-negative bacteria. The conjugation frequency, stability and copy number of replicons carrying mini-Mulac derivatives with oriT and ori'RiHRI in members of the Rhizobiaceae such as Rhizobium meliloti, Azorhizobium caulinodans ORS571 and Agrobacterium tumefaciens C58 was examined. © 1988.

Author-supplied keywords

  • Agrobacterium rhizogenes Ri origin
  • Agrobacterium tumefaciens
  • Azorhizobium caulinodans ORS571
  • Rhizobium meliloti
  • Translational lac fusions
  • mini-Mu transposon
  • recombinant DNA
  • replicon stability
  • selectable markers

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Authors

  • P. Ratet

  • J. Schell

  • F. J. de Bruijn

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