Identification of pBM300 as the source plasmid for clone III by Southern hybridization and separation of pBM300 into PV361. (A) Agarose gel electrophoresis of plasmids. QM B1551, the wild-type strain, contains all seven plasmids; QM B1551/26::spc contains plasmids isolated after spc gene insertion; the PV203 strain does not contain plasmid pBM300 (used as a negative control); and PV634 contains only pBM300::spc. The DNA was transferred to a nitrocellulose membrane. Then the membrane was hybridized with different probes, as indicated above panels B to E, sequentially after the old probe was stripped. (B to E). Hybridization with plasmids and derivatives from B. megaterium QM B1551 with the following specific probes: pBM500 (clone I) (40) (B), pBM400 (clone II) (38) (C), a PCR fragment specific for the clone III probe (distal to the rep gene in pBM300) (D), and the spc gene probe (E). Note that the blots in panels B to E were derived from the gel shown in panel A.