A specialized transducing phage constructed from Bacillus subtilis phage phi 105.

Abstract

Chromosomal DNA of Bacillus subtilis 168 (trpC2) prepared from defective phage P BSX was digested by restriction endonuclease Eco RI and ligated in vitro with DNA fragments of page phi 105C digested by the same endonuclease. The ligated DNA was used to transform a competent culture of B. subtilis (trpC2 lys3 metB10) which was lysogenic for phi 105, and transformants of the auxotroph markers were selected. The bacterial DNA ligated to the phage DNA fragments could be integrated into the prophage genome by transformation. The transformants in toto were treated with mitomycin C and the lysate was used to transduce B. subtilis (trpC2 lys3 metB10). Among metB+ transductants, one clone appeared to be a double lysogen carrying both plaque forming and metB+ transducing phage genomes. The latter defective phage was designated phi 105dmetB. Physical mapping of these phages was carried out by agarose gel electrophoresis of the restriction endonuclease digests and also by electron microscopic analysis of heteroduplex DNA. These results indicate that two adjacent fragments Eco RI-G and E of phi 105 DNA had been substituted with a foreign fragment Eco RI-M in phi 105dmetB DNA. Transformation experiments showed that the metB+ gene resided on the fragment Eco RI-M. This fragment was found to have a BamHI-sensitive site. The transforming activity for the metB marker, however, was not affected by the treatmment with BamHI.