Abstract
BackgroundThe Red recombinase system of bacteriophage lambda has been used to inactivate chromosomal genes in E. coli K-12 through homologous recombination using linear PCR products. The aim of this study was to induce mutations in the genome of some temperate Shiga toxin encoding bacteriophages. When phage genes are in the prophage state, they behave like chromosomal genes. This enables marker genes, such as antibiotic resistance genes, to be incorporated into the stx gene. Once the phages' lytic cycle is activated, recombinant Shiga toxin converting phages are produced. These phages can transfer the marker genes to the bacteria that they infect and convert. As the Red system's effectiveness decreased when used for our purposes, we had to introduce significant variations to the original method. These modifications included: confirming the stability of the target stx gene increasing the number of cells to be transformed and using a three-step PCR method to produce the amplimer containing the antibiotic resistance gene.ResultsSeven phages carrying two different antibiotic resistance genes were derived from phages that are directly involved in the pathogenesis of Shiga toxin-producing strains, using this modified protocol.ConclusionThis approach facilitates exploration of the transduction processes and is a valuable tool for studying phage-mediated horizontal gene transfer.
Highlights
The Red recombinase system of bacteriophage lambda has been used to inactivate chromosomal genes in E. coli K-12 through homologous recombination using linear PCR products
Excision of the prophage DNA from bacterial DNA occurred without phage particle formation
The spontaneous release and loss of stx genes occurring in some strains carrying stx-prophages, as already described [38] decreases this method's efficacy
Summary
The Red recombinase system of bacteriophage lambda has been used to inactivate chromosomal genes in E. coli K-12 through homologous recombination using linear PCR products. Once the phages' lytic cycle is activated, recombinant Shiga toxin converting phages are produced These phages can transfer the marker genes to the bacteria that they infect and convert. As the Red system's effectiveness decreased when used for our purposes, we had to introduce significant variations to the original method These modifications included: confirming the stability of the target stx gene increasing the number of cells to be transformed and using a three-step PCR method to produce the amplimer containing the antibiotic resistance gene. The analysis of microbial sequences has revealed that a substantial fraction of the genome of some bacteria corresponds to prophage DNA [1,2] Such prophage DNA is inserted into the bacterial chromosome after infection by free phage particles.
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