Isolation and characterization of a strong promoter element from the Streptomyces ghanaensis phage I19 using the gentamicin resistance gene (aacC1) of Tn 1696 as reporter.
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 I19 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-Ip and p14-IIp, with overlapping and adjacent -10 and -35 regions, respectively. Both promoters appear to be recognized with different efficiencies by the major RNA polymerase holoenzyme (E sigma hrdB) of Streptomyces coelicolor A3(2).
Labes
Gabriele
G
Lehrstuhl für Genetik, Universität Bielefeld, Universitätsstr. 25, 33501 Bielefeld, Germany.
Bibb
Mervyn
M
Lehrstuhl für Genetik, Universität Bielefeld, Universitätsstr. 25, 33501 Bielefeld, Germany.
Wohlleben
Wolfgang
W
Lehrstuhl für Genetik, Universität Bielefeld, Universitätsstr. 25, 33501 Bielefeld, Germany.
D001426:Bacterial Proteins / Q000378:metabolism; D001435:Bacteriophages / Q000235:genetics*; D001483:Base Sequence; D004251:DNA Transposable Elements / Q000235:genetics*; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004352:Drug Resistance, Microbial / Q000235:genetics; D017930:Genes, Reporter; D005814:Genes, Viral; D005839:Gentamicins / Q000494:pharmacology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012329:RNA, Bacterial / Q000096:biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis; D012808:Sigma Factor / Q000378:metabolism; D013302:Streptomyces / Q000821:virology; D013379:Substrate Specificity
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; D005839:Gentamicins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D012808:Sigma Factor; C071941:HrdB protein, Streptomyces; D012321:DNA-Directed RNA Polymerases