Abstract
In order to investigate the mechanism by which exogenous plasmids are stabilized and maintained in Bacillus subtilis, a complex plasmid, pTUEB24 (14kb), composed of pBR322, pUB110 and the aroI+ tmrB genes from B. subtilis chromosomal DNA, was constructed. This plasmid was introduced into B. subtilis cells by the protoplast and competent cell transformation methods. Plasmids were re-extracted from Kmr transformants. Their molecular size was reduced to 6.3kb on average by protoplast transformation and to 5.7kb by competent cell transformation. These reductions in molecular size were caused by deletions in the pBR322 and aroI+ tmrB DNA regions of pTUEB24.
To determine the fate of pTUEB24 during the transformation procedure, DNAs were extracted from the medium, cell membrane and cytoplasm fractions. pTUEB24 was degraded unexpectedly on contact of the DNA with protoplasts and then degraded in the cytoplasm. Nuclease-deficient mutants of B. subtilis are desirable for the effective use of this bacteria in gene engineering.
