The mechanism of insertion of a segment of heterologous DNA into the chromosome of Bacillus subtilis

Abstract

An Escherichia coli plasmid, p1949, that is derived from pMB9 and pC194, and unable to replicate in Bacillus subtilis, can give rise to stable CmR transformants of the latter species if it is inserted into the bacterial chromosome. A purified segment of the B. subtilis chromosome, with transforming activity against pheA1 and nic-38 recipients, was used to direct the insertion of p1949 into the B. subtilis chromosome. Insertion of the ligated DNA segments occurred in the region of the chromosome from which the purified phe-nic segment was derived. Many of the properties of the resulting CmR transformants of B. subtilis are consistent with the occurrence of a Campbell recombination mechanism leading to integration. However, certain of these properties are more easily explained if it is proposed that integration occurs by a reciprocal recombination event involving a linear ligation product. Evidence is presented suggesting that the inserted sequences may be tandemly duplicated. This may effectively vitiate the use of p1949 as a convenient means for complementation analysis of recessive mutations in B. subtilis.