Transduction-Mediated Transfer of Unmarked Deletion and Point Mutations through Use of Counterselectable Suicide Vectors

Abstract

ABSTRACT A challenge in strain construction is that unmarked deletion and nucleotide substitution alleles generally do not confer selectable phenotypes. We describe here a rapid and efficient strategy for transferring such alleles via generalized transduction. The desired allele is first constructed and introduced into the chromosome by conventional allelic-exchange methods. The suicide vector containing the same allele is then integrated into the mutant chromosome, generating a tandem duplication homozygous for that allele. The resulting strain is used as a donor for transductional crosses, and selection is made for a marker carried by the integrated suicide vector. Segregation of the tandem duplication results in haploid individuals, each of which carries the desired allele. To demonstrate this mutagenesis strategy, we used bacteriophage P22H int for generalized transduction-mediated introduction of unmarked mutations to Salmonella enterica serovar Typhimurium. This method is applicable to any species for which generalized transduction is established.