**3. Type of engineered BACs in the** *B. subtilis* **genome (BGM) vector**

Hereafter, DNA introduced into the *B. subtilis* host will be called guest DNA. Guest DNA cloned in the host becomes integrated into the host genome. The single-copy genome of each *B. subtilis* cell can accommodate chloroplast and mitochondrial guest genomes (Itaya et al., 2008) and the genome of the bacterium *Synechocystis* PCC6803 (Itaya et al., 2005). *B. subtilis* strains developed as hosts for these DNAs are called BGM vectors, an acronym for *Bacillus* GenoMe vector. The wild-type strain *B. subtilis* 168 was the first BGM vector to host one pBR322 sequence (Itaya, 1993). This sequence was successfully introduced at different genomic loci (Itaya et al., 2005). The integrated pBR322, a 4.3 kbp *E. coli* plasmid, served as a common cloning locus in a manner reminiscent of the integration by homologous recombination illustrated in Fig 1b. Before the creation of the BAC vector, pBR322 and its derivatives were widely used for various gene-cloning experiments as they offered several advantages, e.g. a small size, a medium-sized copy number, and an ability to carry DNA up to 30 kbp. DNA cloned in pBR322 via the *E. coli* molecular cloning system immediately became guest DNA in the pBR322-based BGM. Integration required only two homologous sequences and appropriate selection markers for the bacterium. The DNA flow from *E. coli* BACs to the BGM vector is shown in Fig. 1b; it is similar to the pBR322-based system but requires major modifications.
