Bacteriophage phi 1 as a gene-cloning vector in Bacillus subtilis.

Abstract

We attempted to use Bacillus subtilis phage phi 1 as a gene-cloning vector since the phi 1 genome was found to have few cleavage sites upon digestion with several kinds of restriction endonucleases. A phi 1 stock supplied by J. Ito (University of Arizona, Tucson, USA) consisted of two phages, phi 1E1 and phi 1E2, having one and two EcoRI-cleavage sites in their genomes respectively. From the latter isolate a deletion mutant phi 1E2 delta 1 was induced to increase the size range of DNA segments to be cloned. It was demonstrated, by in vitro recombination experiments with phage rho 11 DNA, that phi 1E2 delta 1 can be used for cloning EcoRI fragments of various sizes. We analyzed the DNAs of ten phi 1 clones isolated from independent transfectants and found that six of them carried rho 11 DNA fragments inserted at either of the two EcoRI-cleavage sites. Some of the hybrid phage DNAs were found to be cleaved with BamHI and HaeIII endonucleases and the rho 11 DNA portion, whereas the parental phi 1E2 delta 1 DNA was insensitive to any of these enzymes. These hybrid phages would therefore be useful vectors for cloning foreign DNA fragments generated by cleavage with BamHI or HaeIII endonucleases.