title: 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. DutNM1activity mutants were created and tested for dUTPase activity using a novel NMR-based assay. We found that all DutNM1null activity mutants interacted with the SaPIbov1 Stl C-terminal domain, formed DutNM1-Stl heterodimers, and caused the release of the Pstrpromoter. 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 DutNM1is active in Stl derepression and that dUTPase activity is not necessary for the mobilization of SaPIbov1 by DutNM1.
authors.affiliation: Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
authors.forename: Rosanne L L
authors.initials: RLL
authors.lastname: Hill
authors.affiliation: Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
authors.forename: Jiri
authors.initials: J
authors.lastname: Vlach
authors.affiliation: Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond, VA 23298, USA.
authors.forename: Laura K
authors.initials: LK
authors.lastname: Parker
authors.affiliation: Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond, VA 23298, USA.
authors.forename: Gail E
authors.initials: GE
authors.lastname: Christie
authors.affiliation: Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
authors.forename: Jamil S
authors.initials: JS
authors.lastname: Saad
authors.affiliation: Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA. Electronic address: dokland@uab.edu.
authors.forename: Terje
authors.initials: T
authors.lastname: Dokland
mesh_terms: D001435:Bacteriophages / Q000201:enzymology; D003856:Deoxyuracil Nucleotides / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism; D055786:Gene Knockout Techniques; D044404:Genomic Islands; D006378:Helper Viruses / Q000201:enzymology*; D011485:Protein Binding; D011755:Pyrophosphatases / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000235:genetics / Q000378:metabolism* / Q000821:virology
chemical_list: D003856:Deoxyuracil Nucleotides; D004791:Enzyme Inhibitors; D012097:Repressor Proteins; C027078:deoxyuridine triphosphate; C007267:2'-deoxyuridylic acid; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase
keywords: Staphylococcus aureus pathogenicity island; bacteriophage; molecular piracy; nuclear magnetic resonance (NMR); staphylococcal repressor