Figure 5. ImmA is required for full activation of SaPI3.
(a) 80α lysogens carrying SaPIbov1 and the indicated SaPI3 islands were MC induced and the transfer of SaPI3 assessed in RN4220. (b) The SaPI3 immR or stir’ genes were cloned into pUT18c (pJP2531 or pJP2532, respectively) and the immA gene was cloned into pKNT25 (pJP2530). The pUT18c- and pKNT25-derivative plasmids were co-transformed into E. coli strain BTH101. Serial dilutions of an overnight culture were plated onto LB supplemented with kanamycin, ampicillin and 100 μM IPTG and 20 μg ml-1 X-gal. BTH101 transformed with pUT18c-zip and pKNT25-zip or pUT18c and pKNT25 served as positive or negative controls for protein-protein interactions, respectively. (c) RN4220 80α lysogens carrying the SaPI3 seb::tetM WT or the ΔimmA mutant were transformed with either an empty control plasmid (pCN51), a plasmid expressing the wild-type ImmA (pJP2520) or a plasmid expressing an ImmA version mutated in the protein’s active site (pJP2521, ImmA E51A/E52A). Cultures were grown to early exponential phase, MC treated and the expression of ImmA was induced with 1 μM CdCl2. To analyze SaPI3 transfer, the lysates were transduced in RN4220. Error bars indicate ±s.d. (n=3 biological replicates). Statistical analysis was performed using log10 transformed data and an unpaired, two-sided Student’s t-test (a) or One-Way ANOVA followed by Sidak’s multiple comparisons test (c). p-values are as follows: * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, ns not significant. Additional information is provided in the supplementary data file.