Specific pattern of instability of Escherichia coli HisG gene cloned in Bacillus subtilis via the Staphylococcus aureus plasmid pCS194

Abstract

The plasmid pCS194, generated in vivo by recombination of two Staphylococcus aureus plasmids, pC194 and pS194, coding, respectively, for chloramphenicol (Cm) and streptomycin (Sm) resistance, can be replicated also in Bacillus subtilis in the presence of either of the two antibiotics. In their absence, no segregation of the individual components is observed, but the whole plasmid is lost at a rate of about 10% per generation. The unique EcoRI site of pCS194 is located in the Sm R determinant. EcoRI-cleaved pCS194 has been joined to an EcoRI-linearized Escherichia coli replicon, the in vitro recombinant p HisG plasmid, composed of the vector pBR313 plus a BglII-segment of E. coli chromosomal DNA, containing a functional hisG gene. The ligation mixture has been used to transform either E. coli or B. subtilis. Following E. coli transformation and selection for Ap R and Cm R (the latter is expressed in E. coli by the pC194 determinant), two his + clones were picked at random and the plasmids extracted. These appear identical and contain the original segments. Conversely, after transformation of B. subtilis and selection for Cm R, only his − clones have been obtained. From them, deleted plasmids have been extracted. They have lost part or, more frequently, all of the E. coli DNA insert. In the latter case also most of the bracketing pS194 sequence has been lost, and the resulting plasmids are almost identical to pC194, the Cm R parent of pCS194. When the intact recombinant plasmids, isolated from his + Ap R Cm R E. coli clones, have been used to transform B. subtilis cells for Cm R, again deleted plasmids almost identical to pC194 have been obtained. The events causing these rearrangements occur after in vitro ligation, during either transformation or early propagation of the plasmids, and are probably caused by a translocatable element present in pCS194. A detailed physical map of pC194, carrying the restriction sites for HindIII, HaeIII, HpaII, MboII, AluI, HhaI, and BglI, is presented.