How do temperate phages acquire moonlighting proteins as directionality factors for their large serine integrases?

Site-specific recombination catalysed by large serine recombinases (LSRs) is used by temperate phages and mobile elements to insert their genomes into that of their host bacteria as stably integrated prophages during lysogeny. During the lytic phase, a second protein called the recombination directionality factor (RDF) binds the LSR and modifies its specificity to promote the reverse ‘excision’ reaction. The diversity of proteins used by LSRs as RDFs raises the question of how phages evolve functional RDF – LSR pairs. One likely mechanism is that phages repurpose existing proteins to acquire a 'moonlighting' role as RDFs; this hypothesis is supported by demonstration of dual functions of some known RDFs. The aim of this proposal is to use computational and biochemical approaches to understand how LSRs recruit phage (or host) proteins to function as RDFs. I propose that phage LSRs acquire RDFs by mutating their RDF-binding interface to recognize existing phage proteins with suitable structural features and temporal expression profiles to allow them to moonlight as RDFs. To validate this new paradigm, I will show that mutating the RDF-interacting interface of one or more well-studied LSRs can facilitate acquisition of a novel RDF, or recognition of a non-cognate RDF protein.