An improved method of prophage transformation in Bacillus subtilis.

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Abstract
The gene cloning system in Bacillus subtilis, using temperature phages .vphi.105 (prophage transformation), was improved by introducing a chloramphenicol resistance (Cmr) marker into the vector phage DNA to facilitate the primary selection of transformants. Two vector phages, .vphi.CM and .vphi.CL, 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 .vphi.105 lysogen about tenfold by using a non-inducible mutant of .vphi.105, .vphi.105ind-l, as prophage. When the .alpha.-amylase gene previously cloned from Bacillus amyloliquefaciens was transferred with the new vector-prophage system, .alpha.-amylase-producing clones were isolated at a frequency of about 10% of the Cmr 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 .vphi.105 prophage.