Biochemical analysis of bacteriophage Orf family recombinases

Abstract

Bacterial pathogen evolution is an important field of study in light of the emergence of new diseases and drug resistant strains. Genomic rearrangements facilitated by temperate bacteriophages provide a major route for bacteria to acquire new pathogenic traits and disseminate them across species barriers. In phage λ, the Red pathway comprising the exo, bet and gam genes, promotes recombination in Escherichia coli at elevated frequency and with only limited sequence homology leading to numerous illegitimate exchanges. λ encodes another recombinase, Orf, which appears to supply similar functions to that of the host RecFOR proteins but has been considerably less well studied. This thesis describes the characterisation of mutations in Orf located within a proposed central DNA binding channel and a RecA interaction module. The mutants were found to impact upon single-stranded DNA binding by Orf and, in some cases, affect homodimerisation. Similar characterisation of Orf homologues from phage φETA from Staphylococcus aureus (ETA20) and E. coli K-12 cryptic prophage DLP12 (Orf151) was undertaken. In addition, another  DNA binding protein (NinH) has been subjected to bioinformatic analysis, uncovering a relationship with helix-turn-helix proteins involved in site-specific recombination and gene regulation.