Physical and genetic analysis of the Bacillus subtilis (natto) fertility plasmid pLS20

The 64.2-kilobase Bacillus subtilis (natto) plasmid pLS20 encodes functions required for conjugal transmission of plasmid DNA among a variety of Bacillus species. Localization of the transfer region on this plasmid was accomplished via the generation of transposon insertions and subsequent deletion analysis. Utilization of the temperature-sensitive transposition selection vector pTV1 allowed the isolation of a collection of pLS20::Tn917 derivatives. Insertion of Tn917 outside of the 10.8-kb BglII fragment of pLS20 did not affect the transfer abilities of the host cells harboring these plasmid mutants. Insertion of the transposon within the 10.8-kb BglII fragment led to the identification of two distinct regions of pLS20 involved in plasmid-mediated DNA exchange. Insertions into one portion of the 10.8-kb BglII fragment abolished the ability of pLS20 to transfer itself but did not affect the plasmid\u27s ability to mobilize the tetracycline resistance plasmid pBC16. Mutants with insertions in another region of this fragment were almost completely transfer-deficient. Certain of the transposon-tagged derivatives, namely pLS20.3 and pLS20.4, incurred specific deletion following growth of host organisms for several generations in the presence of inhibitory levels of erythromycin. Deletion analyses revealed that loss of ca 15 kb of DNA encompassing of 10.8-kb BglII fragment results in total loss of conjugal transfer ability. Confirmation that this 10.8-kb BglII fragment contained all the DNA sequences necessary for conjugal DNA transfer came from cloning the fragment in the B. subtilis cloning vector pBD64. B. subtilis transformants carrying the pBD64 containing the cloned fragment were able to transfer the recombinant plasmid by conjugation at frequencies comparable to those obtained with B. subtilis donors carrying nondefective pLS20 or pLS20::Tn917 plasmids. The same region of pLS20 that is involved in transfer functions was also found to be involved in the suppression of motility of host organisms. Cells harboring pLS20 or the non-defective transposon-tagged derivatives appeared non-motile on 0.4% agar plates, and electron photomicrographs revealed the absence of flagella. However, cells cured of the plasmid or cells harboring the transfer-defective deletion derivatives pLS20.3$\Delta$1 and pLS20.4$\Delta$1 were flagellated and motile