**2.3 Why the** *B. subtilis* **host - An unprecedented genome vector**

Despite conceptual differences between *E. coli* and *B. subtilis* with respect to the cloning of DNA, *B. subtilis* is highly advantageous because it allows the use of small DNAs prepared by routine molecular cloning in *E. coli*. A high workload can be handled by *B. subtilis* via the integration of DNA fragments cloned once in an *E. coli* pBR322 plasmid or BAC. The plasmid transfer from *E. coli* to the *B. subtilis* genome shown in Fig. 1b renders the *B. subtilis*  genome a big cloning vector. The transfer of *E. coli*-borne plasmids to the *B. subtilis* genome is advantageous because after the DNA is stably integrated into the *B. subtilis* genome, it becomes part of its genome. A wide range of genetic manipulations is now possible in *B. subtilis* regardless of the origin of the integrated DNA. The number and effectiveness of tools available for genetic manipulations in *B. subtilis* far exceed the tools available in the *E. coli* K-12 system. They include the faithful and stable insertion and deletion of any DNA at designated loci. Another advantage of *B. subtilis* is its extraordinary stability, this eliminates the selection pressure to maintain DNA. This is in sharp contrast with the stringent requirements for the maintenance of plasmids in *E. coli*. During growth, plasmids that replicate independent of the host genome segregate out without selection pressure (Fig. 1b and Fig. 2). Indeed, we have demonstrated that *B. subtilis* stably accommodated DNAs far larger than those covered by *E. coli* BACs. The largest size was up to 3,500 kb (Itaya et al., 2005). We think that the stability of DNA integrated into the *B. subtilis* genome is attributable to the presence of a single-copy genome per bacterial cell. In subsequent sections we focus on the handling of BACs transferred to the *B. subtilis* genome and their recovery. This vector system can handle DNA manifesting sequence variations such as short- (mouse genome) or long repeats (IR), and different GC contents.
