Sak and Sak4 recombinases are required for bacteriophage replication in Staphylococcus aureus

Abstract

DNA-single strand annealing proteins (SSAPs) are recombinases frequently encoded in the genome of many bacteriophages. As SSAPs can promote homologous recombination among DNA substrates with an important degree of divergence, these enzymes are involved both in DNA repair and in the generation of phage mosaicisms. Here, analysing Sak and Sak4 as representatives of two different families of SSAPs present in phages infecting the clinically relevant bacterium Staphylococcus aureus, we demonstrate for the first time that these enzymes are absolutely required for phage reproduction. Deletion of the genes encoding these enzymes significantly reduced phage replication and the generation of infectious particles. Complementation studies revealed that these enzymes are required both in the donor (after prophage induction) and in the recipient strain (for infection). Moreover, our results indicated that to perform their function SSAPs require the activity of their cognate single strand binding (Ssb) proteins. Mutational studies demonstrated that the Ssb proteins are also required for phage replication, both in the donor and recipient strain. In summary, our results expand the functions attributed to the Sak and Sak4 proteins, and demonstrate that both SSAPs and Ssb proteins are essential for the life cycle of temperate staphylococcal phages.

Pirating conserved phage mechanisms promotes promiscuous staphylococcal pathogenicity island transfer

“…In silico scrutiny looking at the genes located upstream of the ssb genes revealed that staphylococcal phages encode at least 4 distant SSAP families, including Erf, Redβ, and the aforementioned Sak and Sak4 ( Supplementary file 3 ). All the staphylococcal phages encode one SSAP, in accordance with the fact that these proteins are essential for the phage ( Neamah et al, 2017 ). To test the possibility that these other unrelated recombinases also induced SaPI2, we characterised in detail those present in phages ϕSLT (ORF 17) and ϕN315 (SA1794), which belong to the Erf and Redβ families of SSAPs, respectively, and have completely different sequences ( Figure 5—figure supplement 4 ).…”

“…The 80α ORF16 protein belongs to the Sak family of single strand annealing proteins (SSAP, also called recombinases) involved in homologous recombination ( Lopes et al, 2010 ; Scaltriti et al, 2011 ). Although for many of these proteins their role in the phage cycle has not been established yet, we have recently demonstrated that this protein is essential for 80α phage replication ( Neamah et al, 2017 ). Note, however, that the chimeric Sak-Ssb protein is still functional for the phage, as demonstrated by the fact that the mutant phage encoding this protein still replicates and forms plaques in a sensitive recipient strain.…”

“…This protein belongs to a distinct family of SSAPs, Sak4 ( Lopes et al, 2010 ). While Sak4 and Sak are not homologous in sequence ( Figure 5—figure supplement 3 ), we have recently demonstrated that they are both SSAPs (recombinases) performing a similar function in their cognate phages ( Neamah et al, 2017 ).…”

Molecular Mechanisms That Contribute to Horizontal Transfer of Plasmids by the Bacteriophage SPP1

“…These recombinases have gained increased attention in recent years because of their abundance in phage genomes ( Lopes et al, 2010 ; Delattre et al, 2016 ), and also due to their wide use in recombineering systems ( Datta et al, 2008 ; Sun et al, 2015 ). Many of these recombinases, including G 35 P, are essential for the phage life cycle ( Zecchi et al, 2012 ; Neamah et al, 2017 ).…”

“…Similarly, we found that the SPP1 SSB protein, G 36 P, only slightly contributes to the mechanisms of plasmid transduction. However, in some phages the recombinases require the activity of their cognate SSB proteins to perform their function ( Neamah et al, 2017 ).…”