The Site-Specific Recombination System of theEscherichia coliBacteriophage φ24B

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 shigatoxigenicEscherichia 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 enterohemorrhagicE. 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 phi 24(B)and bacterial DNA recombination was tested using bothin vitroandin siturecombination assays. It is well established that phage lambda, 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 minimalattPsequence required by phi 24(B)phage is between 350 and 427 bp. Unlike phage lambda, the minimal necessary flanking sequences for theattBsite do not appear to be equal in size, with a total length between 62 and 93 bp. Furthermore, we identified that the phi 24(B)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.