Transcription analysis of a Bacillus subtilis arg gene following cloning in Escherichia coli in an initially unstable hybrid plasmid

Abstract

Following shotgun cloning of EcoRI fragments of Bacillus subtilis DNA in pBR322, a hybrid plasmid pUL710 was isolated which complements argC but no other auxotrophs of E. coli K12. Restriction mapping, Southern blotting and other evidence suggest that pUL710 carries an insert of 1.6 kbp, and derives, by deletion of both vector and insert sequences, from a larger but unstable initial hybrid which carried a 12 kbp EcoRI fragment from the B. subtilis chromosome. RecE-dependent integration of pUL710 into the B. subtilis chromosome demonstrated homology between the insert DNA and the argO locus of B. subtilis. pUL710 was found to confer appreciable tetracycline resistance even though the deletion presumed to stabilise the hybrid had inactivated the tet promoter. The results of analysis by Tn5 mutagenesis, transcriptional fusions and run-off in vitro transcription suggest that both the cloned argC gene and the tetracycline gene in pUL710 are expressed from a B. subtilis promoter located very close to the EcoRI cloning site.