**2.1 What is** *B. subtilis?* **- A more detailed explanation**

We will place special emphasis on the use of natural transformation in the engineering of BACs (Fig. 1b). The general homologous recombination associated with *B. subtilis*  transformation is distinct from the induced recombination adopted by most *E. coli* DNA engineering systems. The *B. subtilis* 168 strain domesticated in the laboratory was isolated as a recipient that facilitates DNA-mediated transformation (Spizizen, 1958). The subsequent elucidation of its detailed molecular mechanisms revealed that competent *B. subtilis* cells can incorporate extracellular DNA into their cytoplasm via the process of natural transformation (Kidane & Graumann, 2005). It is possible to conduct genetic crosses in *B. subtilis* and like *E. coli* K-12, it has been subjected to extensive biochemical and genetic analyses. Although *E. coli* was thought to accept extracellular DNA via a chemical transformation process, this type of transformation was limited to the plasmid delivery method (Mandel & Higa, 1970; Hanahan, 1983). The cloning in and the propagation of BACs in *E. coli* are subsumed under the term DNA delivery. The process of DNA delivery through the cell membrane of *E. coli* involves elaborate physical and chemical treatments of the host (Fig. 1b). In contrast, the transformation system of *B. subtilis* applies to plasmid delivery as well as genomic gene engineering. Competent *B. subtilis* cells positively grab extracellular DNA and pull the fragment(s) into their cytoplasm in single-strand form. The molecular mechanisms, the processing of doublestranded DNA from the cell surface and its conversion into single-strand DNA for entry through the cellular membrane, are carried out in a concerted manner by several proteins encoded by *B. subtilis* genes (Kovács et al., 2009). This process is too complex to be explained here; in short, *B. subtilis* does, while *E. coli* does not possess a set of genes to conduct natural transformation.

As incorporated BACs cannot replicate in *B. subtilis* in plasmid form because there is no replication origin sequence for this host, BACs must enter cellular recombination pathways initiated by the existing *recA* protein (Kidane & Graumann, 2005). If homologous sequences are present in the *B. subtilis* chromosome (BAC\* in Fig. 1b), the incoming BAC is integrated into the homologous region. Such homologous recombination-mediated transformation yields the integrated form at high frequency in the *B. subtilis* genome (Fig. 1b). This well-known natural transformation system for the *B. subtilis* 168 strain has been used in genetic research targeting the original genome. Two striking results were the derivation of a 33-fold mutant of *B. subtilis* by repeating the transformation 33 times to introduce mutations at 33 chromosomal loci (Itaya & Tanaka, 1991) and the reduction of the genome to 75% of its original size by the consecutive deletion of genes unaffected by growth (Ara et al., 2007). Fortuitously *B. subtilis* 168 possesses no original/cognate plasmid.

*B. subtilis* has emerged as an appropriate host to supplement the use of BACs from *E. coli* (Itaya, 2009). Our favorite cloning host, *B. subtilis* 168, yet unfamiliar to many researchers, is a Gram-positive firmicute bacterium. It grows as rapidly and in the same media as *E. coli*. Consequently, many protocols used with *E. coli* can be used with *B. subtilis.* However, features inherent in *B. subtilis* facilitate natural transformation, in addition, the bacterium forms endospores that survive for extraordinarily long periods. *E. coli* lacks these features.

We will place special emphasis on the use of natural transformation in the engineering of BACs (Fig. 1b). The general homologous recombination associated with *B. subtilis*  transformation is distinct from the induced recombination adopted by most *E. coli* DNA engineering systems. The *B. subtilis* 168 strain domesticated in the laboratory was isolated as a recipient that facilitates DNA-mediated transformation (Spizizen, 1958). The subsequent elucidation of its detailed molecular mechanisms revealed that competent *B. subtilis* cells can incorporate extracellular DNA into their cytoplasm via the process of natural transformation (Kidane & Graumann, 2005). It is possible to conduct genetic crosses in *B. subtilis* and like *E. coli* K-12, it has been subjected to extensive biochemical and genetic analyses. Although *E. coli* was thought to accept extracellular DNA via a chemical transformation process, this type of transformation was limited to the plasmid delivery method (Mandel & Higa, 1970; Hanahan, 1983). The cloning in and the propagation of BACs in *E. coli* are subsumed under the term DNA delivery. The process of DNA delivery through the cell membrane of *E. coli* involves elaborate physical and chemical treatments of the host (Fig. 1b). In contrast, the transformation system of *B. subtilis* applies to plasmid delivery as well as genomic gene engineering. Competent *B. subtilis* cells positively grab extracellular DNA and pull the fragment(s) into their cytoplasm in single-strand form. The molecular mechanisms, the processing of doublestranded DNA from the cell surface and its conversion into single-strand DNA for entry through the cellular membrane, are carried out in a concerted manner by several proteins encoded by *B. subtilis* genes (Kovács et al., 2009). This process is too complex to be explained here; in short, *B. subtilis* does, while *E. coli* does not possess a set of genes to

As incorporated BACs cannot replicate in *B. subtilis* in plasmid form because there is no replication origin sequence for this host, BACs must enter cellular recombination pathways initiated by the existing *recA* protein (Kidane & Graumann, 2005). If homologous sequences are present in the *B. subtilis* chromosome (BAC\* in Fig. 1b), the incoming BAC is integrated into the homologous region. Such homologous recombination-mediated transformation yields the integrated form at high frequency in the *B. subtilis* genome (Fig. 1b). This well-known natural transformation system for the *B. subtilis* 168 strain has been used in genetic research targeting the original genome. Two striking results were the derivation of a 33-fold mutant of *B. subtilis* by repeating the transformation 33 times to introduce mutations at 33 chromosomal loci (Itaya & Tanaka, 1991) and the reduction of the genome to 75% of its original size by the consecutive deletion of genes unaffected by growth (Ara et al., 2007). Fortuitously *B. subtilis* 168

**2. The emerging** *B. subtilis* **host** 

conduct natural transformation.

possesses no original/cognate plasmid.

**2.1 What is** *B. subtilis?* **- A more detailed explanation** 

A homologous sequence is the sole requirement for extra DNA-engineering in *B. subtilis.* The transformation process shown in Fig. 2 can be repeated and the number of repetitions is practically unlimited. Consequently, due to its ability to repeat transformation, the assembly of large DNA fragments in the *B. subtilis* genome is possible. Typically, if DNA fragments with partial overlaps are prepared, repetitive integration by using the overlapping regions allows the reconstruction of the original DNA in the *B. subtilis* genome (Itaya & Tsuge, 2011). Even before its whole genome sequence determination in 1997, *B. subtilis* became a workhorse for the cloning and manipulations particularly of giant DNAs that cannot be handled by *E. coli.* As *B. subtilis* forms endospores that manifest significant resistance to vacuuming, dryness, and radiation, it has become a reservoir for giant DNA maintained at room temperature. Additional details will be presented in section 3-4.
