DNA cloning in Bacillus subtilis. III. Efficiency of random-segment cloning and insertional inactivation vectors.

ABSTRACT

Random segments of Bacillus amyloliquefaciens and yeast Saccharomyces cerevisiae DNA were used to determine two parameters pertinent to cloning in Bacillus subtilis, the yield of hybrids and the mean size of cloned segments. 10(3) to 10(4) hybrids/micrograms of DNA segments were obtained. Hybrids represented 11--18% of transformants. Mean m. wt. of cloned DNA segments was about 1 x 10(6), substantially lower than 3 x 10(6) found for donor DNAs after digestion with restriction endonucleases. We have cloned a B. amyloliquefaciens DNA segment which complemented a deficiency in B. subtilis hisH and E. coli hisC genes, which encode imidazolylacetolphosphate aminotransferase. The cloning efficiency for this gene was 10 transformed hosts/micrograms of donor DNA. Several B. subtilis insertional-inactivation cloning vectors were examined. One, pHV41, allows inactivation of the kanamycin-resistance (KmR) gene by insertion into its unique Bg/II site. In two other vectors, pHV11 and pHV23, insertion in their unique Kpn site inactivates the tetracycline-resistance (TcR) gene. pHV23 replicates both in E. coli and B. subtilis, and carries unique sites for seven restriction endonucleases (BamHI, EcoRI, HpaI, KpnI, PstI, SalI, XbaI). This makes it one of the most versatile B. subtilis cloning vectors yet described.