| Bacteriophage are the most abundant entity in the biosphere and were the foundations of molecular biology work in the mid-20th century. Recently, interest in phage has resurfaced in the quest to fight antibiotic resistance in medicine. Bacteriophage P1, commonly used for generalized transductions, encodes a recombiniation enhancement function (ref) that acts as a RecA-dependent endonuclease. Ref can perform targeted cleavage of displacement loops (D-loops) formed by RecA and a targeting oligonucleotide. Ref is nickase which cleaves the two target strands of a D-loop in a prescribed order. Cutting the paired strand is rapid, occurs at many different sites within each D-loop, and does not require ATP hydrolysis by RecA. We hypothesize that Ref targets the strained phosphodiester bonds in active RecA filaments with some degree of sequence specificity. The displaced strand is cleaved much more slowly at a single site within each D-loop, requires RecA-mediated ATP hydrolysis, does not occur with Ref H153A, and also retains some degree of sequence specificity. We postulate that cleavage of the displaced strand requires disassembly of the RecA filament.;We also explored the in vivo role of Ref for phage P1. Ref is a lytic cycle gene, amplifying lysis by targeting RecA-bound DNA to enhance genome degradation. The presence of the ref gene in a P1 lysogen leads to an elevated bacterial SOS response. When cells are treated with ciprofloxacin, bacteriophage P1 lysogens reduce survival synergistically, an activity traceable to Ref. On its own, Ref expression is toxic to E. coli cells, and expression in combination with DNA damaging antibiotics enhances their effectiveness. Ref is especially toxic during the SOS response and in stationary phase, targeting aspects of the bacterial life cycle that are closely associated with the development of pathogen persistence. |
