In vitro mutagenesis of Bacillus subtilis by using a modified Tn7 transposon with an outward-facing inducible promoter.
A Tn7 donor plasmid, pTn7SX, was constructed for use with the model gram-positive bacterium Bacillus subtilis. This new mini-Tn7, mTn7SX, contains a spectinomycin resistance cassette and an outward-facing, xylose-inducible promoter, thereby allowing for the regulated expression of genes downstream of the transposon. We demonstrate that mTn7SX inserts are obtained at a high frequency and occur randomly throughout the B. subtilis genome. The utility of this system was demonstrated by the selection of mutants with increased resistance to the antibiotic fosfomycin or duramycin.
Bordi
Christophe
C
Department of Microbiology, Wing Hall, Cornell University, Ithaca, NY 14853-8101, USA.
Butcher
Bronwyn G
BG
Shi
Qiaojuan
Q
Hachmann
Anna-Barbara
AB
Peters
Joseph E
JE
Helmann
John D
JD
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001412:Bacillus subtilis / Q000235:genetics*; D001430:Bacteriocins / Q000494:pharmacology; D004251:DNA Transposable Elements; D024881:Drug Resistance, Bacterial / Q000235:genetics; D005578:Fosfomycin / Q000494:pharmacology; D023061:Gene Order; D016254:Mutagenesis, Insertional / Q000379:methods*; D010455:Peptides / Q000494:pharmacology; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D000198:Spectinomycin / Q000494:pharmacology; D014994:Xylose / Q000378:metabolism
D000900:Anti-Bacterial Agents; D001430:Bacteriocins; D004251:DNA Transposable Elements; D010455:Peptides; C040522:duramycin; D005578:Fosfomycin; D000198:Spectinomycin; D014994:Xylose