Structure of ϕDI dimeric Dut.

<p>(A) Cartoon representation of the ϕDI Dut dimer with protomers coloured in pink and orange, respectively (left). The secondary structural elements are numbered and labeled in order from the N to C terminus (the ‘ indicates elements from the second protomer). A molecule of dUPNPP (sticks coloured by atom type) and two Mg ions (green spheres) occupies the active center of each protomer. In an orthogonal view (right) the conserved and variable (motif VI) regions of the dimeric Duts from <i>S</i>. <i>aureus</i> phages are colored in pink and yellow tones, respectively. The structural elements exploited to dimerize (helices α2 and α6, and the latch) are highlighted in dark tones. Notice that the nucleotide binding sites and the motifs VI map in opposite faces of the dimer. (B) Sequence of the ϕDI Dut, with residues that interact with the substrate highlighted with red stars and residues that interact across the dimer interface in blue text. The Ala residue mutated in the ϕDI^A73L is indicated in red. The locations of the five conserved motifs in the dimeric Duts are indicated. Structural elements are shown above the sequence coloured as (A) right panel. (C) Detailed view of the ϕDI Dut active center. The substrate dUPNPP is represented in stick with carbon atoms in cyan. The residues interacting with the nucleotide are shown in stick representation, with carbon atoms coloured according to the protomer to which they correspond and are labeled with a similar color text with the exception of A73 that is highlighted with red text. Nitrogen, oxygen, phosphorus atoms are coloured in dark blue, red and orange, respectively. The Mg ions are represented as green spheres.</p

<i>S</i>. <i>aureus</i> phage coded dimeric Duts show allelic variation.

<p>(A) Alignment of dimeric Duts from <i>S</i>. <i>aureus</i> phages ϕNM1, ϕDI, ϕDII, ϕO11, ϕO46, ϕMR25, ϕStauST398-3 (StauST), ϕHKU10-03 (HKU10) and ϕ55 are shown representing the nine different dimeric Dut families identified in the phylogenetic tree described below (Fig 1B). Colours indicate relative sequence conservation at each position, with red being most conserved and blue being least (alignment generated by PRALINE). The five conserved catalytic motifs in dimeric Duts are highlighted in magenta boxes and labeled. The bracket indicates the localization of the extra motif VI. (B) Radial phylogenetic tree of identified staphylococcal and <i>Staphylococcus</i> phage dimeric Duts. Radial tree showing the 9 different groups, constructed using the alignment of protein sequences of the dimeric Duts from staphylococcal phages (including the full sequence, not just the variable region) listed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006581#ppat.1006581.s008" target="_blank">S1 Table</a>. An NCBI BLAST of the known SaPI inducing ϕNM1 phage encoded dimeric Dut (full sequence) was used to select 59 dimeric Dut sequences that were annotated as staphylococcal phage Duts or as staphylococca<i>l</i> Duts. <i>S</i>.<i>aureus</i> does not encode a genomic Dut so any such annotations would also be phagic. Four trimeric staphylococca<i>l</i> phage Duts were also included in the alignment and tree to provide outliers and show the distance of these proteins from the dimeric Duts. The accession numbers for all sequences are listed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006581#ppat.1006581.s008" target="_blank">S1 Table</a>. The small box below shows the full tree, including the trimeric Dut outliers (red box), which were hidden for clarity in the more detailed tree above. This detailed tree consists of the area highlighted with the green box in the full depiction. Groups are shown by clustered leaf node names and the name of the representative used in the sequence alignment of panel (A) is highlighted with the same colour of the group. The tree was created using the following parameters; algorithm = Neighbour Joining, distance measure = Jukes-Cantor, bootstrap = 100 Replicates. Proteins are named by NCBI accession number with the exception of the group representative where the name of the corresponding phage is included used (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006581#ppat.1006581.s008" target="_blank">S1 Table</a>).</p

Interaction between Stl and the allelic variants of dimeric Duts from <i>S</i>. <i>aureus</i> phages and the effect on dUTPase activity.

<p>(A) The capacity of the ϕDI, ϕO11, ϕPhi55 and ϕDII dimeric Duts to form a complex with Stl was checked by Native-PAGE maintaining a constant amount of Dut proteins (17 μM) and identical or double concentrations of Stl. (B) Binding of Stl inhibits dUTPase activity. The enzymatic activity of the inducer dimeric Duts ϕDI and ϕO11 were inhibited by Stl whereas it was not affected for the non-inducer allelic variants ϕ55 and ϕDII even though a 10-fold excess of Stl was used (left panels). The dUTPase activity is inhibited by Stl in a dosage dependent manner for ϕDI and ϕO11 Duts (right panels). Average and standard deviation of six replicates are shown.</p

Transfer of SaPIbov1 and SaPIbov5 by dimeric Duts ФD1 and ФNM1.

<p>Transfer of SaPIbov1 and SaPIbov5 by dimeric Duts ФD1 and ФNM1.</p

The dUTP substrate but not the dUMP product reduces the Dut-Stl interaction.

<p>Native-Page gels were performed to analyze the effect of the substrate and product on the interaction of two inducing dimeric Duts, (A) ϕDI and (B) ϕO11, with Stl. Increasing concentrations (from 0 to 1000 μM) of dUPNPP (top gels) or dUMP (down gels) were added to equimolecular concentrations of each Dut with Stl, and the Dut-Stl complex formation was evaluated by Native-PAGE. Notice the reduction of the Stl-Dut complex band when the dUTP analogue but not the dUMP product was present. For ϕDI a new band (labeled with a red asterisk) corresponding to the complex between dUPNPP and the Dut can be observed when the Stl-Dut complex disappears. For each experiment a representative gel of 3 independents assays is shown.</p

Crystallographic statistics of Dut ϕDI structures.

<p>Crystallographic statistics of Dut ϕDI structures.</p

Structures of ϕDI <i>apo</i> and ϕDI^A73L dimeric Duts showed minimal conformational changes in the mutant, but a high impact on SaPI induction and Stl interaction.

<p>(A) Superimposition of three-dimensional structures of the ϕDI dimer in <i>apo</i> form (yellow tones), dUPNPP bound (magenta tones) and as A73L mutant (green tones) in cartoon representation. The nucleotide in the ϕDI-dUPNPP structure is represented in stick with carbon atoms in cyan and Mg as green spheres. (B) SaPIbov1 excision and replication following induction of the cloned ϕDI^A73L <i>dut</i> gene. Strain JP6774 containing SaPIbov1 was complemented with plasmids expressing the 3xFLAG-tagged ϕDI and ϕDI^A73L dimeric Dut or the empty pCN51 plasmid as a control. Samples were isolated at 3 hours after induction with 3μM CdCl₂ and Southern blots were performed using a probe for the SaPIbov1 integrase (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006581#ppat.1006581.s011" target="_blank">S4 Table</a>). The upper band is ‘bulk’ DNA, including chromosomal, phage, and replicating SaPI. CCC indicates covalently closed circular SaPI DNA. In these experiments, as no helper phage was present, the excised and replicating SaPI DNA appears as part of the bulk DNA or as CCC molecules, rather than the linear monomers that are seen following helper phage-mediated induction and packaging. (C) Native-PAGE experiment was performed to analyze the binding capacity of the ϕDI^A73L Dut vs the ϕDI wild-type form. Proteins were mixed with Stl in equimolecular relationship (in monomers). A clear reduction in the band corresponding to the Stl-ϕDI Dut^A73L complex with respect to the Stl-ϕDI Dut complex is observed. (D) Close-up view of the superimposed ϕDI-dUPNPP and ϕDI^A73L active centers. Residues responsible for substrate coordination and binding are represented in sticks with carbon atoms colored according to the structure to which they correspond. The Leu73 residue at the bottom of the active center of the ϕDI^A73L mutant that precludes the binding of the nucleotide is colored in yellow. The substrate dUPNPP in the ϕDI-dUPNPP structure is represented in stick with carbon atoms in cyan and Mg ions are represented as grey spheres.</p