Derepression of SaPIbov1 Is Independent of φNM1 Type 2 dUTPase Activity and Is Inhibited by dUTP and dUMP

Abstract

Staphylococcus aureus is an opportunistic human pathogen able to transfer virulence genes to other cells through the mobilization of S. aureus pathogenicity islands (SaPIs). SaPIs are derepressed and packaged into phage-like transducing particles by helper phages like 80α or φNM1. Phages 80α and φNM1 encode structurally distinct dUTPases, Dut80α (type 1) and DutNM1 (type 2). Both dUTPases can interact with the SaPIbov1 Stl master repressor, leading to derepression and mobilization. That two structurally distinct dUTPases bind the same repressor led us to speculate that dUTPase activity may be important to the derepression process. In type 1 dUTPases, Stl binding is inhibited by dUTP. The purpose of this study was to assess the involvement of dUTP binding and dUTPase activity in derepression by DutNM1. DutNM1 activity mutants were created and tested for dUTPase activity using a novel NMR-based assay. We found that all DutNM1 null activity mutants interacted with the SaPIbov1 Stl C-terminal domain, formed DutNM1–Stl heterodimers, and caused release of the Pstr promoter. However, promoter release was inhibited in the presence of dUTP or dUMP. We tested two φNM1 mutant phages that had null enzyme activity and found they could still mobilize SaPIbov1. These results show that only the apo form of DutNM1 is active in Stl derepression, and that dUTPase activity is not necessary for mobilization of SaPIbov1 by DutNM1.

Convergent evolution involving dimeric and trimeric dUTPases in pathogenicity island mobilization

“…However, the structural characterization of the ϕDI Dut A73L mutant has shown that this mutation promotes a compact conformation that could be stabilizing the dimer, supporting our proposition. During the review of this manuscript, Dokland and collaborators published that Stl interaction with the dimeric Dut from phage ϕNM1 was inhibited by dUTP in close agreement with our results and proposition [ 24 ]. However, the authors indicate that the product dUMP also has an inhibitory capacity for the Stl-ϕNM1 Dut interaction, in contrast to the results obtained here for two alternative inducing dimeric Duts.…”

Pirating conserved phage mechanisms promotes promiscuous staphylococcal pathogenicity island transfer

“…Importantly, dimeric and trimeric Duts are completely unrelated both in sequence and structure, representing a nice example of convergent evolution ( Penadés et al, 2013 ). While the 80α and ϕ11 phage-encoded trimeric Duts were initially described as the SaPIbov1 inducers ( Tormo-Más et al, 2010 ; 2013 ), the dimeric Dut from phage ϕNM1 also induces SaPIbov1 ( Hill and Dokland, 2016 ; Hill et al, 2017 ). The fact that both dimeric and trimeric Duts induce SaPIbov1 raised the interesting possibility that the Stl repressors could target different phage proteins, significantly increasing the capacity of the SaPIs to be induced and transferred.…”

HDX and Native Mass Spectrometry Reveals the Different Structural Basis for Interaction of the Staphylococcal Pathogenicity Island Repressor Stl with Dimeric and Trimeric Phage dUTPases

“…also Supplementary Figure S2). However, we also note here that specific mutation of K159 to alanine did not abolish DUTφNM1:Stl complex formation either in vitro or in vivo, thus this residue is not an essential factor in the protein–protein interaction [15].…”

“…In parallel to these studies it has also been revealed that not only the homotrimeric phage dUTPases but a homodimeric dUTPase from φNM1 phage is also capable to interact with the Stl of SaPIbov1 [14,15]. Hill et al provided clear evidence also for the direct interaction of the φNM1 phage dUTPase and Stl [14].…”

“…The two active sites of dimeric dUTPases are built up symmetrically on the interface of the dimer by 5 motifs as one protomer provides motif 1,2,4,5 and the other donates motif 3. Although it has turned out that in case of φNM1 phage dUTPase the enzymatic activity is not essential for SaPI mobilization [15], the two structurally highly different dUTPase families have only the dUTP binding ability in common, so it is suggestive to speculate on the role of this region in Stl binding.…”