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 Φ24B and bacterial DNA recombination was tested using both in vitro and in situ recombination assays. It is well established that phage λ, 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 Φ24B phage is between 350 and 427 bp. Unlike phage λ, 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 Φ24B 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.
Highlights
Shigatoxigenic Escherichia coli (STEC) emerged in the early 1980s as the causal agents of a variety of clinical symptoms and sequelae ranging from mild diarrhea to life-threatening conditions such as hemolytic uremic syndrome and thrombotic thrombocytopenic purpura (Paton and Paton, 1998; Fogg et al, 2011)
Escherichia phage λ is generally used as a textbook illustration of temperate phage behavior
Our research shows that temperate phage integration, including the presence of host factors and recognition sites, is not generalizable from λ, in particular for the Shiga-toxin encoding phage 24B
Summary
Shigatoxigenic Escherichia coli (STEC) emerged in the early 1980s as the causal agents of a variety of clinical symptoms and sequelae ranging from mild diarrhea to life-threatening conditions such as hemolytic uremic syndrome and thrombotic thrombocytopenic purpura (Paton and Paton, 1998; Fogg et al, 2011). DNA from most temperate phages, including phage λ and Stx phages, can, alternatively, become integrated in the host genome, becoming a prophage (Madsen et al, 1999; Saunders et al, 2001) or, in a few other phages, surviving in the cell as a replicating plasmid (Łobocka et al, 2004). For those phage destined to become integrated, the incoming phage produces a site-specific recombinase enzyme that directs phage recombination within the bacterial genome. This recombinase enzyme is known as integrase (Groth and Calos, 2004)
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