Cloning in Escherichia coli of the gene specifying the DNA-entry nuclease of Bacillus subtilis

Abstract

With the aim of cloning genes involved in transformation of Bacillus subtilis, a set of transformation-deficient mutants was isolated by means of insertional mutagenesis with plasmid pHV60 (Vosman et al., 1986). Analysis of these mutants showed that those mapping in the aroI region lacked the DNA-entry nuclease activity. Plasmid pHV60 derivatives, containing flanking chromosomal DNA fragments, were isolated from these mutants and were used to screen a library of B. subtilis chromosomal DNA in phage lambda EMBL4. In Escherichia coli lysates, prepared with the phages that hybridized to the pHV60-based probe, a prominent nuclease activity could be detected. The nuclease encoded by the phage DNA had the same Mr as the B. subtilis DNA-entry nuclease and its activity was strongly stimulated by Mn2+, which is also characteristic for the B. subtilis DNA-entry nuclease. From these results it was concluded that the gene specifying the B. subtilis DNA-entry nuclease had been cloned. It was shown that the nuclease activity was specified by a 700-bp EcoRI-PstI fragment.