Fig. 6: Interaction of ClpX and ClpP is not required for phage induction.

a The clpX wt gene (clpX_wt) or a clpX gene encoding a ClpX mutant unable to interact with ClpP (ClpX_I265E) were cloned into the inducible expression plasmid pCN51, introduced into either the RN4220 wt or its ΔclpX mutant derivative lysogenic for Φ11 or 80α and induced by MitC. Expression from these plasmids was maintained throughout the experiment by the addition of 1 μM CdCl₂. Plaque formation was assessed on a lawn of RN4220. Bold horizontal lines in each boxplot represent the median and lower and upper hinges of the first and third quartiles, respectively (n = 3 biological replicates). Assessment of statistically significant differences between groups was performed using ANOVA followed by Tukey’s HSD post-test. b Samples of the same strains as in a were taken for DNA extraction at the time points indicated. Crude DNA lysates for Southern blotting analysis were then separated by agarose gel electrophoresis, transferred onto a nitrocellulose membrane, and replicating phage DNA visualised using a phage-specific DIG-labelled DNA probe. c Bacterial Two-Hybrid assay of either the Φ11 full-length CI protein (CI_wt) or the post-cleavage CI N-terminal domain only (CI_G131*). The gene encoding either the Φ11 full-length CI protein (CI_wt) or c the post-cleavage CI N-terminal domain only (CI_G131*) were cloned into pKT25 (pJP2636 or pJP2632, respectively), while genes encoding either WT clpX (clpX_wt) or ClpX unable to interact with ClpP (clpX_I265E) were cloned into pUT18c (pJP2642, pJP2638, respectively). The pUT18c- and pKT25-derivative plasmids were co-transformed into E. coli strain BTH101 and a single colony selected. Serial dilutions of an overnight culture were plated onto LB supplemented with kanamycin (30 µg ml⁻¹), ampicillin (100 µg ml⁻¹), 100 µM isopropyl β-d−1-thiogalactopyranoside (IPTG) and 20 µg ml⁻¹ X-gal. BTH101 transformed with pUT18c-zip and pKNT25-zip or pUT18c and pKT25 served as positive or negative controls for protein–protein interactions, respectively.