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Isolation and characterization of a strong promoter element from the Streptomyces ghanaensis phage 119 using the gentamicin resistance gene (aacC1) of Tn1696 as reporter

Gabriele Labes

Mervyn Bibb

Wolfgang Wohlleben

Lehrstuhl für Genetik, Universität Bielefeld, Universitätsstr. 25, 33501 Bielefeld, Germany

ABSTRACT

A promoter-probe shuttle plasmid (pGL7011) containing the promoterless aminoglycoside-O-acetyltransferase I gene (aacC1) of Tn1696 was used to isolate DNA fragments from Streptomyces ghanaensis phage 119 that possessed promoter activity in Streptomyces lividans TK23. Analysis of gentamicin (Gm) resistance levels in Escherichia coli and in S. lividans TK23, and of aacC1 mRNA levels in S. lividans, identified a fragment (F14) that exhibited a high level of promoter activity in both species. Subsequent analysis revealed that the promoter activity of SF14 (a subcloned fragment of F14) was about twice that of ermEp^*, one of the strongest characterized actinomycete promoters. SF14 contained two tandemly arranged promoters, 14-1p and p14-llp, with overlapping and adjacent -10 and -35 regions, respectively. Both promoters appear to be recognized with different efficiencies by the major RNA polymerase holoenzyme (Es^hrdb) of Streptomyces coelicolor A3(2).

Author for correspondence: Gabriele Labes. Tel: +49 33432 82270. Fax: +49 33432 S2343. e-mail: glabes@zalf.de

Present address: Lehrstuhl für Mikrobiologie/Biotechnologie, Fakultät für Biologie, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 28, 72076 Tübingen, Germany.

Present address: Department of Genetics, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK.

Present address: Abteilung Mikrobenmonitoring, Institut fuRr Mikrobielle ökologie und Bodenbiologie, Zentrum für Agrarlandschafts- und Landnutzungsforschung e.V., Eberswalder Str. 84, 15374 Müncheberg, Germany.

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