AN IMPROVED METHOD OF PROPHAGE TRANSFORMATION IN BACILLUS SUBTILIS

Abstract

The gene cloning system in Bacillus subtilis, using temperate phage φ105 (prophage transformation), was improved by introducing a chloramphenicol resistance (Cm^r) marker into the vector phage DNA to facilitate the primary selection of transformants. Two vector phages, φCM and φCL, were constructed; the former has BamHI and BglII sites for cloning and the latter BglII and ClaI sites. Another improvement was increasing the transformation efficiency of the φ105 lysogen about tenfold by using a non-inducible mutant of φ105, φ105ind-1, as prophage. When the α- amylase gene previously cloned from Bacillus amyloliquefaciens was transferred with the new vector-prophage system, α-amylase-producing clones were isolated at a frequency of about 10% of the Cm^r transformants. When the transformants were non-inducible, the induction- negative prophages could be made induction-positive by repeating the transformation using the recipient lysogen harboring wild type φ105 prophage.