Bacteriophage ϕ1 as a gene-cloning vector in Bacillus subtilis

Abstract

We attempted to use Bacillus subtilis phage ϕ1 as a gene-cloning vector since the ϕ1 genome was found to have few cleavage sites upon digestion with several kinds of restriction endonucleases. A ϕ1 stock supplied by J. Ito (University of Arizona, Tucson, USA) consisted of two phages, ϕ1E1 and ϕ1E2, having one and two EcoRI-cleavage sites in their genomes respectively. From the latter isolate a deletion mutant ϕ1E2Δ1 was induced to increase the size range of DNA segments to be cloned. It was demonstrated, by in vitro recombination experiments with phage ρ11 DNA, that ϕ1E2Δ1 can be used for cloning EcoRI fragments of various sizes. We analyzed the DNAs of ten ϕ1 clones isolated from independent transfectants and found that six of them carried ρ11 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 at the ρ11 DNA portion, whereas the parental ϕ1E2Δ1 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.