A new bacteriophage vector for cloning in Bacillus subtilis and the use of phi 105 for protein synthesis in maxicells

Zabarovsky and Allikmets [Gene 42 (1986) 119-123] have described a cloning procedure based on partial filling-in of vector and target DNA cohesive ends, which strongly enriches for recombinant molecules with single insertions. Improved Bacillus subtilis bacteriophage phi 105 vectors containing unique cloning sites for SalI have been constructed to take advantage of the partial fill-in method. The new vectors have been used to construct B. subtilis genomic libraries from which several sporulation loci have been isolated, including five not previously cloned. On inserting a promoterless lacZ gene into the cloning site, beta-galactosidase (beta Gal) was detected at a late stage in lytic phage growth, indicating that phage transcription is directed through the cloning site. When UV-irradiated cells ('maxicells') were infected with the recombinant phage containing the lacZ gene, in the presence of labelled amino acids, a protein of the expected Mr for beta Gal was visualised, in addition to the phage proteins. This system should provide a useful general approach for the identification of the products of cloned genes from B. subtilis and other Gram-positive organisms.