Mechanism of integrating foreign DNA during transformation of Bacillus subtilis.

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Abstract
Genes encoding thymidylate synthetase from B. subtilis bacteriophages were cloned in Escherichia coli. Chimeric plasmids pCD1 and pCD3 were constructed from site-specific endonuclease digests of bacteriophage .vphi.3T DNA cloned in pMB9 in E. coli. Similar cloning techniques with bacteriophage .beta.22 DNA yielded chimeric plasmids pCD4, pCD5 and pCD6. Endonuclease digests of DNA from pCD1 and pCD3 propagated in E. coli or from DNA isolated from bacteriophage .vphi.3T propagated in B. subtilis transformed B. subtilis from Thy- to Thy+. Intact DNA from bacteriophage .beta.22, endonuclease digests of .beta.22 DNA, and a chimeric plasmid (pCD5) composed only of the thy.beta.22 gene and pMB9 did not transform B. subtilis from Thy- to Thy+ even though pCD5 could transform Thy- E. coli to Thy+. If the thy.beta.22 fragment from pCD5 was introduced into another chimeric plasmid, pCD2, that contains a region of homology to the chromosome of B. subtilis besides pMB9, transformation of Thy- clones of B. subtilis was possible. Southern hybridization analyses of the digests of chromosomal DNA from the Thy+ transformants established that the entire chimeric plasmid was incorporated into the chromosome of B. subtilis. Treatment of these plasmids with site-specific endonucleases abolished transformation. These results indicated that the entire chimeric plasmid can be incorporated into the chromosome of B. subtilis by a Campbell-like model. Therefore, an additional mechanism for transformation exists whereby plasmids can be integrated if sufficient chromosomal homology is maintained.