Figure 1. Induction of SaPIbov1 and SaPIbov5 by the ϕO11 dimeric Dut.

(A) SaPIbov1 and SaPIbov5 excision and replication following induction of the cloned ϕO11 dut gene. Strains JP6774 and JP11634, containing SaPIbov1 and SaPIbov5 respectively, were complemented with a plasmid expressing the 3xFLAG-tagged ϕO11 dimeric Dut. Samples were isolated at 0’ or 3 hr after induction with 0.5 μM CdCl₂ and Southern blots were performed using a probe for the SaPIbov1/SaPIbov5 integrase. 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. (B) Derepression of str transcription by ϕO11 Dut expression. The diagram represents a schematic of a blaZ transcriptional fusion generated in pJP674. β-lactamase assays were performed on strains containing pJP674 together with a pCN51-derived plasmid expressing the ϕO11 Dut (JP14818) or the empty pCN51 control (JP15105). Samples were taken after 5 hr in the absence or following induction with 5 μM Cadmium. All data is the result of five independent experiments. Error bars represent SEM. A 2-way ANOVA with Sidak's multiple comparisons test was performed to compare mean differences within rows. Adjusted p values were as follows: ϕO11 = 0.0004^***, pCN51 = 0.9579^ns. ns, not significant. (C) Affinity chromatography of the ϕO11 Dut for the His-tagged SaPIbov1 Stl. Strains were induced with 10 mM isopropyl-β-d-thiogalactoside (IPTG) and samples taken at 3 hr. Cells were disrupted and expressing proteins were applied to a Ni²⁺ column and eluted. Lane 2, elution fraction for His₆-Stl_SaPIbov1 and Dut_ΦO11 (JP14832). Lane 1, corresponding elution fraction for Stl_SaPIbov1 and Dut_ΦO11 (JP14833, no His₆-tag). Proteins were confirmed by Mass Spectrometry analysis.

Figure 1—figure supplement 1. Sequence alignment for the ϕNM1 (accession number ABF73061) and ϕO11 (accession number EGA96175) dimeric dUTPases.

Colours indicate relative sequence conservation at each position, with red being most conserved and violet being least (adapted from alignment generated by PRALINE).