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Bacteria have been locked in an evolutionary arms race against bacteriophages (viruses targeting bacteria) since time immemorial. To defend against phage infection, bacteria can carry mobile genetic elements (MGEs) that have evolved to partake in phage parasitism, where they inhibit and hijack the bacteriophage replication machinery. By meddling with key bacteriophage replication pathways, parasitic MGEs such as Staphylococcus aureus Pathogenicity Islands (SaPIs) drastically reduce phage spread. In the hijacking process, phage particles end up carrying SaPI DNA instead, leading to horizontal SaPI spread and an increase in resistance against the infecting phage in the bacterial population. SaPI-like elements are therefore widespread in nature.

SaPI activity is repressed by the master regulator Stl until phage-specific proteins trigger de-repression. In this thesis, we structurally and biochemically characterize the Stl from SaPI2 (Stl2), a dual-role transcriptional repressor. Stl2 releases its operator DNA upon binding to phage recombinases (PRs), which are essential for phage replication and homologous recombination. PRs, despite their functional similarity, exhibit diverse structures, with Stl2-interacting PRs falling into four subgroups: Sak, Erf, Redβ/RecT, and Sak4.

Here, we determine the cryo-EM structures of three Stl2-interacting PR subgroups: Sak, Erf, and Sak4. Sak and Erf form RAD52-like rings, whereas Sak4 is a RAD51/RecA-like helical ATPase. The cryo-EM structures of Erf and Sak4 reveal significant mechanistic differences from their homologs, with the Erf structures providing the first complete visualization of ssDNA annealing in a RAD52 homolog. Additionally, we show that Stl2 forms megadalton-sized complexes with Sak, Erf, and Sak4, adapting to each protein’s quaternary structure. When interacting with Sak, Stl2 binds and blocks the protein-interacting C-terminal domain (revealed for the first time), effectively inhibiting Sak’s function in vitro.

Finally, we explore the previously uncharacterized SaPI replicative helicase (Rep), responsible for unwinding the SaPI genome, inducing genomic replication. We determine the structures of Rep proteins from SaPIs 1 and 5, revealing unknown structure-function capabilities.

In summary, the work done here advances our understanding of SaPI-phage interactions, revealing novel regulatory mechanisms and structural adaptations that push the ongoing molecular arms race.