Umeå University's logo

Bacteria often carry multiple genes encoding anti-phage defense systems, clustered in defense islands and phage satellites. Various unrelated anti-phage defense systems target phage-encoded homologous recombinases (HRs) through unclear mechanisms. Here, we show that the phage satellite SaPI2, which does not encode orthodox anti-phage defense systems, provides antiviral immunity mediated by Stl2, the SaPI2-encoded transcriptional repressor. Stl2 targets and inhibits phage-encoded HRs, including Sak and Sak4, two HRs from the Rad52-like and Rad51-like superfamilies. Remarkably, apo Stl2 forms a collar of dimers oligomerizing as closed rings and as filaments, mimicking the quaternary structure of its targets. Stl2 decorates both Sak rings and Sak4 filaments. The oligomerization of Stl2 as a collar of dimers is necessary for its inhibitory activity both in vitro and in vivo. Our results shed light on the mechanisms underlying antiviral immunity against phages carrying divergent HRs.

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.

Replication is a crucial cellular process. Replicative helicases unwind DNA providing the template strand to the polymerase and promoting replication fork progression. Helicases are multi-domain proteins which use an ATPase domain to couple ATP hydrolysis with translocation, however the role that the other domains might have during translocation remains elusive. Here, we studied the unexplored self-loading helicases called Reps, present in Staphylococcus aureus pathogenicity islands (SaPIs). Our cryoEM structures of the PriRep5 from SaPI5 (3.3 Å), the Rep1 from SaPI1 (3.9 Å) and Rep1-DNA complex (3.1Å) showed that in both Reps, the C-terminal domain (CTD) undergoes two distinct movements respect the ATPase domain. We experimentally demonstrate both in vitro and in vivo that SaPI-encoded Reps need key amino acids involved in the staircase mechanism of translocation. Additionally, we demonstrate that the CTD's presence is necessary for the maintenance of full ATPase and helicase activities. We speculate that this high interdomain flexibility couples Rep's activities as initiators and as helicases.

Single-strand annealing (SSA) is a key mechanism in non-homologous DNA repair, with recombinases playing a central role in both bacterial and phage genomes. Phage-encoded single-strand annealing proteins (SSAPs) facilitate genome concatemerization and recombination, contributing to phage replication, spread and diversity.  While phage recombinases (PRs) such as RecT, Redβ, Sak and Sak4 have been studied, the RAD52-like Erf family remains both biochemically and structurally uncharacterised. Here, we present the first cryo-EM structures of ErfΦSLT, an Erf-family PR from the Staphylococcus aureus phage ΦSLT, in apo (3.16 Å), ssDNA-bound (2.5 Å), and dsDNA-bound (2.5 Å) states. We reveal a dynamic beta hairpin motif that shifts to a loop conformation in apo state, deviating from conventional RAD52-like SSAPs. We also show that Erf_ΦSLT stabilizes and holds ssDNA into nucleotide triplets, diverging from the use of nucleotide quartets seen in all other RAD52- and Redβ-like SSAPs. Erf_ΦSLT utilizes bulky amino acids conserved in the Erf family. The structures presented here highlight conformational changes during ssDNA binding and annealing, revealing an unwound dsDNA intermediate, a first in the RAD52 superfamily. These findings provide a first look into Erf-based SSA and suggest a greater functional variety in the RAD52 superfamily than previously observed.

Homologous recombination is a vital mechanism for genome maintenance, driven by homologous recombinases (HRs) such as RAD51 in eukaryotes and RecA in prokaryotes. Many bacteriophages encode their own HR proteins to promote mosaicism and coordinate phage genomic replication. Among these, Sak4 recombinases form a distinct subgroup, yet their function remains poorly understood. Here, we structurally and biochemically characterize the RecA-like HR Sak4 from bacteriophage 52A (Sak4_52A).

We present a 2.1 Å cryo-EM structure of Sak4_52A as an ATPγS-bound nucleoprotein filament, revealing a tightly packed helical architecture distinct from RecA and RAD51. Unlike canonical HRs, Sak4_52A employs an alternative loop for ssDNA interaction and base stacking. Functional assays confirmed that Sak452A binds ssDNA, hydrolyzes ATP, and catalyzes both single-strand annealing and dsDNA strand exchange, the latter being the first reported for a phage-derived HR. Interestingly, strand exchange was observed in the presence of ATP but not ATPγS, suggesting further mechanistic differences from RecA.

A comparison between the Sak4_52A-ssDNA filament with Sak4_52A bound to the SaPI2-encoded Stl repressor revealed that Stl2 forces Sak4_52A into a severely underwound conformation. These findings highlight Sak4_52A as a minimal RecA-like HR with strand exchange activity and potential functional implications for phage biology that should be explored further.