DISCUSSION

The work reported in this chapter details attempts to shotgun-clone the B. subtilis trpC gene and the B. licheniformis 749/C penP gene into the unique EcoRI site of the plasmid vector pAB22<f. Attempts met with repeated failure for apparently unknown reasons.

Although at the outset o f this work there had been only one report of a successful shotgun-cloning of chromosomal genes in B. subtilis (Keggins et ah, 1978) several other successes have been recently reported (e.g . Palva, 1982). Why then have some workers been able to shotgun-clone chromosomal genes in B. subtilis whereas others have been unsuccessful? It is likely that several factors such as the plasmid vector used, the particular gene to be cloned, and the size of the DNA fragment required, are important.

The failure to shotgun-clone chromosomal genes is often attributed to the inherent properties of the competence transformation system employed, i.e. only plasmid oligomers are active in transformation of competent cells and attempts to increase the number of recombinant plasmids produced a fte r ligation decreases the transforming efficiency of the DNA. The rationale behind this is that, as reported by Canosi et al. (1978), monomeric plasmid D N A is inactive in the transformation of competent B. subtilis cells. Also, as the ra tio of insert DNA to vector DNA increases, the likely hood of forming oligom eric vector decreases. Therefore one would predict that attempts to shotgun-clone chromosomal genes directly in competent B. subtilis cells would result in (1) a low transformation

frequency and (¡1) a low Insertion frequency. These tw o factors probably

contributed to the failure to shotgun-clone chromosomal genes using competent B. subtilis cells in this work. However, this explanation cannot be applied to the transformation of B. subtilis protoplasts since, as stated previously, monomeric plasmid DNA and linear plasmid DNA are active in the transformation of B. subtilis protoplasts.

Very little is known of the actual plasmid species arising after ligation of chromosomal and plasmid DNAs and also how variations in the vector to insert DNA ratio and DNA concentration a ffe c t the final composition of plasmid species

produced a fter ligation. These factors are clearly important and probably

markedly a ffe c t the outcom e of a shotgun-cloning experiment. Also, nothing is known about the structure of plasmid species, containing chromosomal DNA inserts, as in a shotgun-cloning, which give rise to transformants when introduced into competent B. subtilis cells. Are contiguous vector molecules required or can two or more vector molecules be separated by a length of chromosomal DNA? Also, are linear hybrids containing contiguous vector molecules in addition to a

chromosomal DNA fragm ent active in transformation? The answer to these

questions are not yet known but are clearly important to an understanding of plasmid DNA transformation of B. subtilis.

It has been demonstrated that it is possible to shotgun-clone chromosomal

genes using competent B. subtilis cells, e.g. Palva (1982) obtained a

transformation frequency o f 10^ transformants per 2.5 pg of DNA with an insertion frequency of 13.%. Thus the B. amvloliquefaciens a-amylase gene was successfully cloned. A system atic study of random-segment cloning (Michel et al., 1980) also demonstrated that under the correct conditions, a high transformation and insertion frequency can be obtained. One important finding of Michel et al. (1980) and P a lva (1982), was that the average size of the cloned DNA fragments was sm aller than the average size o f the original chromosomal DNA fragments used for ligation to vector DNA. Michel et al. (1980) suggested that the selective cloning o f small DNA fragments may explain the failure of

several workers to shotgun-clone genes in B. subtilis. The reasons for the

selective cloning of small D N A fragments reported by Michel et al. (1980) is unknown. These workers suggested that it could be due to the instability of large

DNA fragments or, altern atively, small hybrid plasmids, produced from

predominate and displace any large plasmid molecules present. The latter explanation is generally more acceptable since it may not be so much the size of a cloned DNA fragment that is important to its stability as the nature of the particular cloned sequence itself. This would apply to both the competence and protoplast transformation systems.

If the shotgun-cloning of large DNA fragments cannot be overcome, it may still be possible to clone many genes by cloning relatively small fragments produced by digestion with enzymes such as Sau3A or Mbol. Palva (1982) used Mbol-generated fragments of 1.4 to 3.4 Md to clone the B. amyloliquefaciens a - amylase gene. However in this work from 90,000 to 150,000 clones had to be screened and only one desired clone was found. The use of insertional inactivation vectors, such as those described by Michel et al. (1980), may be useful to rapidly and simply determine the insertion frequency. This can be done by screening transformants for inactivation of an antibiotic-resistance marker carried on the vectors.

In summary, it has been demonstrated in several cases that direct shotgun-cloning in B. subtilis employing plasmid vectors is possible. However, for unknown reasons, the shotgun-cloning of various genes has proved to be extremely difficult. Therefore, unless there is a specific reason for wanting to shotgun- clone directly in B. subtilis. it is probably simpler to perform the initial shotgun­ cloning in E. coli and subsequently introduce the cloned gene into B. subtilis. The use of bifunctional plasmid vectors capable of autonomous replication in both E. coli and B. subtilis may prove to be particularly useful in this respect.

CHAPTER IV