Fig. 2.

Structure of the megacloned Synechocystis genome in the B. subtilis genome. Schematic drawings of the genomes of Synechocystis PCC6803 (green circle) (17), B. subtilis 168 (brown circle), and strain BEST7613 (mosaic with green and brown). Seven plasmids reported for Synechocystis are omitted. B. subtiis 168 has no indigenous plasmid. Regions A, B, and C are from the B. subtilis genome and regions [I]-[IV] are from the Synechocystis genome. The BEST7613 genome (7,700 kb) shows approximate size because the B. subtilis strain used in this study, BEST7003 (13), had certain deletions and was shorter than the reported strain (26). The locations of oriC and terC are known for B. subtilis only. Antibiotic resistance markers inserted in the BEST7613 genome are shown with relevant genes and locations: resistance to blasticidin S (bsr), tetracycline (tet), spectinomycin (spc), neomycin (neo), and phleomycin (phl). The GpBR (indicated by the striped boxes) is the megacloning locus prepared at the proB, leuB and ytqB loci of the B. subtilis genome. I-PpoI sites indicated by bars inside the BEST7003 and BEST7163 circle are created at the ends of GpBRs and of three megacloned Synechocystis genomes. The GpBR sequences that remained in BEST7613 after removal manipulations are shown. GpBR between regions A and [I] was removed after BEST7155 (Fig. 3). The GpBR sequence between regions [II] and B was replaced by tet before BEST7566 (Fig. 3) to reduce the overlap of regions [II] and [III]. GpBR between regions B and [IV] was replaced by spc before BEST7374 (Fig. 3). The I-PpoI site is left behind upon all of these removals. Overlaps of 5.984 kb for regions [II] and [III], 4.479 kb for regions [III] and [IV], and 0.334 kb for regions [IV] and [I] remained in BEST7613. Six fragments yielded from BEST7613 by I-PpoI digestion are shown in Fig. 4.