Site-Specific Mutagenesis of Bacillus subtilis Phage SPO1

Abstract

This chapter describes the procedure that we have used to introduce suppressible nonsense mutations into various genes of Bacillus subtilis bacteriophage SPO1. The targeted gene is cloned in a B. subtilis/Escherichia coli shuttle vector. Using an in vitro enzymatic procedure dependent on mutant oligonucleotide primers, a mutation is inserted into the cloned gene, replacing an early lysine codon (AAA or AAG) with a nonsense codon (TAG or TAA). The mutant plasmid is recovered by transformation into E. coli, and is then transformed into B. subtilis carrying a suppressor that inserts lysine at TAG or TAA codons. Recombination is allowed between the mutant plasmid and superinfecting wild-type SPO1, and mutant progeny phage are identified by plaque-lift hybridization to labeled oligonucleotides having the mutant sequence. This procedure is adaptable for other types of mutations, and for other phage–bacteria combinations for which appropriate strains and plasmids are available.