Derepression of SaPIbov1 Is Independent of φNM1 Type 2 dUTPase Activity and Is Inhibited by dUTP and dUMP. Issue 10 (19th May 2017)

• Record Type: Journal Article
• Authors: Hill, Rosanne L.L.; Vlach, Jiri; Parker, Laura K.; Christie, Gail E.; Saad, Jamil S.; Dokland, Terje
• Is Part Of: Journal of molecular biology. Volume 429:Issue 10(2017)
• Page Start: 1570
• Page End: 1580
• Publication Date: 2017-05-19
• Subjects: SaPI S. aureus pathogenicity island -- DutNM1 type 2 dUTPase from phage φNM1 -- CTD C-terminal domain -- WT wild-type -- EMSA electrophoretic mobility shift assay; bacteriophage -- Staphylococcus aureus pathogenicity island -- molecular piracy -- staphylococcal repressor -- nuclear magnetic resonance (NMR); Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2017.04.001
• Languages: English
• ISSNs: 0022-2836

Abstract

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 the release of the P str 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 that 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 the mobilization of SaPIbov1 by DutNM1 . Graphical Abstract: Highlights: SaPIs are mobilized by helper phages. SaPIbov1 Stl is derepressed by the type 2 dUTPase from phage φNM1 (DutNM1 ). Mobilization of SaPIbov1 by φNM1 does not require DutNM1 dUTPase activity. Derepression of Stl by DutNM1 is inhibited by dUTP and dUMP.