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. 2018 Jun 6;12(9):2114–2128. doi: 10.1038/s41396-018-0156-3

Fig. 6.

Fig. 6

Characterization of the EcCICFT073 alpA gene. a–c Schematic representation of the different blaZ transcriptional fusions generated. The relevant genes are shown. b–d Miller assay performed using the plasmids represented in (a–c). Results (average ± s.d.) of three independent assays are shown. A 2-way ANOVA with Tukey’s multiple comparisons test was performed to compare mean differences within rows. Adjusted p values were as follows: pJP1290 vs pJP1291 = 0. 0011**, pJP1288 vs pJP1289 = 0.0001***, PalpA  < 0.0001****, Pint  > 0.9999 ns. ns, not significant. e EcCICFT073 excision and replication after expression of the cloned alpA gene. A non-lysogenic derivative of strain C600 carrying EcCICFT073 was complemented with plasmid pBAD18 (empty plasmid) or plasmid pJP2037, which carries alpA under the control of the PBAD promoter. As controls, non-lysogenic derivatives of strain C600 carrying EcCICFT073 ∆int or EcCICFT073 ∆pri were complemented with plasmid pJP2037. One milliliter of each culture (optical density (OD)600nm = 0.3) was collected 2 h after treatment with 0.02% arabinose and used to prepare standard minilysates, which were resolved on a 0.7% agarose gel, Southern blotted and probed for EcCICFT073 DNA. In these experiments, because no helper phage is present, the excised EcCICFT073 DNA appears as covalently closed circular molecules (CCC). f Helper phages activate alpA transcription. Different lysogenic and non-lysogenic strains, containing plasmid pJP1290, were MC-induced and assayed for β-galactosidase activity. Results (average ± s.d.) of three independent assays are shown. Strains: C600 (non-lysogenic), JP10400 (C600 lysogenic for phage λ), 594 (non-lysogenic), JP12507 (594 lysogenic for phage 80) and JP15151 (594 lysogenic for phage 80 ∆terS). A 2-way ANOVA with Tukey’s multiple comparisons test was performed to compare mean differences within rows. Adjusted p values were as follows; C600 > 0.9999 ns, Phage Lambda < 0.0001****, 594 > 0.9999 ns, Phage 80 < 0.0001****, Phage 80 ∆terS < 0.0001****. ns, not significant