Saturation mutagenesis in Escherichia coli of a cloned Xanthomonas campestris DNA fragment with the lux transposon Tn4431 using the delivery plasmid pDS1, thermosensitive in replication |
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Authors: | Dorothee Steinmann Heinrich -G Wiggerich Bärbel Klauke Uwe Schramm Alfred Pühler Ursula B Priefer |
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Institution: | (1) Lehrstuhl für Genetik, Universität Bielefeld, Postfach 100131, D-33501 Bielefeld, Germany;(2) Ökologie des Bodens, RWTH Aachen, Botanisches Institut, Worringer Weg 1, D-52056 Aachen, Germany |
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Abstract: | A system allowing transposon mutagenesis of cloned DNA fragments in Escherichia coli with Tn4431, which carries the promotorless luciferase (lux) operon of Vibrio fischeri, has been developed. The transposon delivery plasmid, pDS1, based on an IncF replicon, is thermosensitive in replication and mobilizable to many Gram-negative bacteria. We used pDS1 for Tn4431-saturation mutagenesis of a 10-kb DNA fragment of Xanthomonas campestris pv. campestris (X.c.c.) in E. coli and showed that the expression of the lux operon was dependent on orientation and location of the transposon. Transfer of a specific Tn4431 insertion to X.c.c. allowed the determination of the bioluminescence phenotype in planta.
Correspondence to: U. B. Priefer |
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