Bacteriophage P22 transduction of integrated plasmids: single-step cloning of Salmonella typhimurium gene fusions.
Transcriptional fusions to Salmonella typhimurium chromosomal genes were constructed by integration of a suicide fusion vector into the chromosome by homologous recombination with random cloned chromosomal fragments. We describe here a transductional method using the generalized transducing phage of S. typhimurium, P22, to clone these fusions directly from the bacterial chromosome, in a single step, without the use of restriction enzymes. In this transduction, the phage packages the chromosomal fragment containing the integrated plasmid. Once introduced into the recipient, the plasmid circularizes by homologous recombination between the duplicated region determined by the cloned fragment. Although RecA mediates the majority of these events, the plasmid can circularize in a recA recipient. However, in this case, the event occurs at a much lower frequency and only when the transduction is done at a high multiplicity of infection. In addition to integrated fusion constructs, we also show that autonomously replicating low-copy-number plasmids can be transduced. In this case, transduction is dependent on homologous recombination between the plasmid and the donor chromosome via cloned sequences, in which the transducing particle effectively traps the integrated plasmid.
Mahan
M J
MJ
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115.
Slauch
J M
JM
Mekalanos
J J
JJ
D017100:Bacteriophage P22 / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli; D005798:Genes, Bacterial; D005838:Genotype; D016254:Mutagenesis, Insertional; D010957:Plasmids; D011938:Rec A Recombinases / Q000378:metabolism; D011995:Recombination, Genetic; D012486:Salmonella typhimurium / Q000235:genetics*; D014158:Transcription, Genetic; D014161:Transduction, Genetic; D016662:Virus Integration
D011938:Rec A Recombinases