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/μg of DNA segments were obtained. Hybrids represented 11–18% of transformants. Mean m. wt. of cloned DNA segments was about 1 × 10 6 , substantially lower than 3 × 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/μg of donor DNA. Several B. subtilis insertional-inactivation cloning vectors were examined. One, pHV41, allows inactivation of the kanamycin-resistance (Km R ) gene by insertion into its unique Bgl II site. In two other vectors, pHV11 and pHV23, insertion in their unique Kpn site inactivates the tetracycline-resistance (Tc R ) gene. pHV23 replicates both in E. coli and B. subtilis , and carries unique sites for seven restriction endonucleases ( BamHI , Eco RI, Hpa I, Kpn I, Pst I, Sal I, Xba I). This makes it one of the most versatile B. subtilis cloning vectors yet described.