Easy cloning of mini-Tn10 insertions from the Bacillus subtilis chromosome.
Delivery vectors for mini-Tn10 transposons function in Bacillus subtilis (M. A. Petit, C. Bruand, L. Janniére, and S. D. Ehrlich, J. Bacteriol. 172:6736-6740, 1990). Using this system, we identified a new gene (sytA) whose inactivation affected regulation of genes of sucrose metabolism. For cloning the sytA::Tn10 insertion in Escherichia coli, we developed a methodology similar to that commonly used for B. subtilis Tn917 insertions. We constructed a plasmid which can be used to insert (by in vivo recombination) a ColE1 origin linked to a spectinomycin resistance gene (ori-spc element) into mini-Tn10 transposons inserted into the B. subtilis chromosome. DNA extracted from a sytA::Tn10::ori-spc transformant was cut with restriction enzymes that do not cut into the Tn10::ori-spc sequence; plasmids containing the sytA::Tn10 insertion were cloned by self-ligation, followed by transformation of E. coli. To obtain the wild-type sytA region, one of these plasmids was ligated with an E. coli-B. subtilis shuttle vector conferring erythromycin resistance, and the hybrid was used to transform the wild-type B. subtilis strain. Erythromycin-resistant transformants, detected as spectinomycin sensitive, resulted from conversion of the insertion mutation by the resident wild-type locus. The shuttle plasmid containing the wild-type locus could then be recovered in E. coli.
Steinmetz
M
M
Laboratoire de Génétique des Microorganismes, URA537 du Centre National de La Recherche Scientifique, Institut National de la Recherche Agronomique, Thiverval-Grignon, France.
Richter
R
R
D001412:Bacillus subtilis / Q000235:genetics*; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular / Q000379:methods*; D004251:DNA Transposable Elements / Q000235:genetics*; D005798:Genes, Bacterial / Q000235:genetics*; D005822:Genetic Vectors; D013395:Sucrose / Q000378:metabolism
D004251:DNA Transposable Elements; D013395:Sucrose