Development of genetic engineering in Bacillus megaterium.

The opportunities for industrial genetic engineering in several species of Bacillus other than B. subtilis and B. thuringiensis are now becoming a reality. Many species have advantages for certain industrial applications, such as the lack of alkaline proteases, stable plasmid maintenance, and production of thermostable enzymes. It is now possible to increase production levels in many Bacillus strains that are already high producers of amylases, proteases, penicillinases, and penicillin amidases, by the introduction of such genes on high-copy-number plasmids. Possible problems in gene regulation, expression, limits on protein production, and secretion will be encountered, but recent reports on comparisons of expression of bacterial and eukaryotic genes in B. subtilis and B. megaterium (Shivakumar et al. 1989; Donovan et al. 1989a, 1989c; Ginsburgh et al. 1989) suggest that some species may prove to be better expression hosts for specific genes than others. What is needed is extensive comparative studies in promising species to better understand the parameters affecting cloning, gene expression, and protein secretion in the bacilli. In this chapter we have emphasized the development of genetic analysis and rDNA methods in B. megaterium. While the chromosomal map is still not a complete circle, many gene loci have been mapped, some with three-factor crosses, and have been characterized by enzyme assays (leu, trp, hem, cob, cbl) as well as complementation with B. subtilis genes (trp, dnaK, pur, met, ssp) E. coli genes (ATPase), hybridization (spoVG, abrB, sigK, ssp), and by growth on intermediates (trp, leu, thy, gua, ade, pur, pyr, his, arg). A mapping kit of 12 strains is available, and can facilitate the mapping of new mutations rapidly. This is of great advantage in strain construction since genes can be transferred at will by cotransduction. A partial physical map generated by pulse gel electrophoresis is also available (Muse 1990). It remains to be determined whether gene conversion occurs in B. megaterium as it does in B. subtilis. This is a powerful tool for rescuing genes and mutations of interest from the chromosome. There are now over 600 mutants available in our laboratory, over 100 in the laboratory of J.C. Vary, and several more that have been characterized in various laboratories throughout the world. Many of these are available in stock culture collections, although this source needs to be expanded. Mutants that should be useful for genetic engineering include recombination negative, protease negative, and plasmidless wild type, as well as Lac negative and several auxotrophs in the plasmidless background, and many sporulation negative mutants.