Convergent evolution involving dimeric and trimeric dUTPases in pathogenicity island mobilization
(A) SaPIbov1 and SaPIbov5 excision and replication following induction of cloned ϕNM1, ϕO11, ϕDI, ϕDII, ϕO46, and ϕ55 dut genes. Strain JP6774 containing SaPIbov1 and strain JP16140 containing SaPIbov5 were complemented with the different plasmids expressing the 3xFLAG-tagged dimeric Duts 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 and SaPIbov5 integrase (S4 Table). 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. The lower panels below each Southern are western blots probed with antibody to the FLAG-tag carried by the Dut proteins. (B) The diagram represents a schematic of a blaZ transcriptional fusion generated in pJP674. β-lactamase assays were performed on strains containing pJP674 together with pCN51-derived plasmids expressing the ϕNM1, ϕO11, ϕDI, ϕDII, ϕO46, and ϕ55 dut genes or the empty pCN51 control (JP15105). Samples were taken after 5 hours in the absence or following induction with 5μM Cadmium. All data is the result of three 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; ϕNM1 = 0.0003***, ϕO11 = 0.0021**, ϕDI = 0.0075**, ϕDII = > 0.9999ns, ϕO46 = < 0.0001****, ϕ55 = > 0.9999 ns, pCN51 = > 0.9999 ns.