Dataset Information

The Site-Specific Recombination System of the Escherichia coli Bacteriophage ?24B.

ABSTRACT: Stx bacteriophages are members of the lambdoid group of phages and are responsible for Shiga toxin (Stx) production and the dissemination of Shiga toxin genes (stx) across shigatoxigenic Escherichia coli (STEC). These toxigenic bacteriophage hosts can cause life-threatening illnesses, and Stx is the virulence determinant responsible for the severe nature of infection with enterohemorrhagic E. coli, a subset of pathogenic STEC. Stx phages are temperate, and in the present study, the identification of what is actually required for Stx phage ?24B and bacterial DNA recombination was tested using both in vitro and in situ recombination assays. It is well established that phage ?, which underpins most of what we understand about lambdoid phage biology, requires its own encoded phage attachment site (attP) of 250 bp, a host-encoded attachment site (attB) of 21 bp, and a host-encoded DNA binding protein known as integration host factor (IHF). The assays applied in this study enabled the manipulation of the phage attachment site (attP) and the bacterial attachment site (attB) sequences and the inclusion or exclusion of a host-encoded accessory element known as integration host factor. We were able to demonstrate that the minimal attP sequence required by ?24B phage is between 350 and 427 bp. Unlike phage ?, the minimal necessary flanking sequences for the attB site do not appear to be equal in size, with a total length between 62 and 93 bp. Furthermore, we identified that the ?24B integrase does not require IHF to drive the integration and the recombination process. Understanding how this unusual Stx phage integrase works may enable exploitation of its promiscuous nature in the context of genetic engineering.

SUBMITTER: Mohaisen MR

PROVIDER: S-EPMC7581858 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Publications

The Site-Specific Recombination System of the Escherichia coli Bacteriophage Φ24B.

Mohaisen Mohammed Radhi MR McCarthy Alan John AJ Adriaenssens Evelien M EM Allison Heather Elizabeth HE

Frontiers in microbiology 20201009

Stx bacteriophages are members of the lambdoid group of phages and are responsible for Shiga toxin (Stx) production and the dissemination of Shiga toxin genes (stx) across shigatoxigenic Escherichia coli (STEC). These toxigenic bacteriophage hosts can cause life-threatening illnesses, and Stx is the virulence determinant responsible for the severe nature of infection with enterohemorrhagic E. coli, a subset of pathogenic STEC. Stx phages are temperate, and in the present stu ...[more]

PMID: 33162958

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Project description:Recombineering, in vivo genetic engineering with bacteriophage homologous recombination systems, is a powerful technique for making genetic modifications in bacteria. Two systems widely used in Escherichia coli are the Red system from phage λ and RecET from the defective Rac prophage. We investigated the in vivo dependence of recombineering on DNA replication of the recombining substrate using plasmid targets. For λ Red recombination, when DNA replication of a circular target plasmid is prevented, recombination with single-stranded DNA oligonucleotides is greatly reduced compared to that under replicating conditions. For RecET recombination, when DNA replication of the targeted plasmid is prevented, the recombination frequency is also reduced, to a level identical to that seen for the Red system in the absence of replication. The very low level of oligonucleotide recombination observed in the absence of any phage recombination functions is the same in the presence or absence of DNA replication. In contrast, both the Red and RecET systems recombine a nonreplicating linear dimer plasmid with high efficiency to yield a circular monomer. Therefore, the DNA replication requirement is substrate dependent. Our data are consistent with recombination by both the Red and RecET systems occurring predominately by single-strand annealing rather than by strand invasion. Bacteriophage homologous recombination systems are widely used for in vivo genetic engineering in bacteria. Single- or double-stranded linear DNA substrates containing short flanking homologies to chromosome targets are used to generate precise and accurate genetic modifications when introduced into bacteria expressing phage recombinases. Understanding the molecular mechanism of these recombination systems will facilitate improvements in the technology. Here, two phage-specific systems are shown to require exposure of complementary single-strand homologous targets for efficient recombination; these single-strand regions may be created during DNA replication or by single-strand exonuclease digestion of linear duplex DNA. Previously, in vitro studies reported that these recombinases promote the single-strand annealing of two complementary DNAs and also strand invasion of a single DNA strand into duplex DNA to create a three-stranded region. Here, in vivo experiments show that recombinase-mediated annealing of complementary single-stranded DNA is the predominant recombination pathway in E. coli.

Dataset Information

The Site-Specific Recombination System of the Escherichia coli Bacteriophage ?24B.

Publications

The Site-Specific Recombination System of the <i>Escherichia coli</i> Bacteriophage Φ24<sub>B</sub>.

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