Fig. 7: Cpx-signalling mediates transcriptional regulation of rpoE.

Cpx-signalling mediated repression of rpoE transcription (a). Quantitative RT-PCR was performed on the cDNA template, synthesised from the total RNA isolated from the Yptb-YPIII isogenic ‘in cis’ mutant strains: parental WT (intact Cpx-signalling), ΔcpxA (locked-on Cpx-signalling with excessive accumulation of active phosphorylated CpxR~P) and ΔcpxR (locked-off Cpx-signalling unable to produce CpxR), and from ‘in trans’ strains: ΔcpxR/pMMB208 (ectopic expression of IPTG-inducible pMMB208 plasmid in ΔcpxR null mutant—negative control), ΔcpxR/pCpxR_WT (ectopic expression of wild-type CpxR from pMMB208 plasmid in ΔcpxR null mutant) and ΔcpxR/pCpxR_Pneg (ectopic expression of phosphorylation defective CpxR_Pneg mutant from pMMB208 plasmid in ΔcpxR null mutant). Both in cis and in trans strains were cultured in LB with shaking (150 rpm) at 26 °C. The in cis strains were grown for 24 h (no IPTG) while the in trans strains were grown up to 6 h with IPTG-induction (10 µM) following subculture (1/20 dilutions). Expression of rpoE within the in cis strains, ΔcpxA and ΔcpxR was calculated relative to WT (left panel) whereas expression within the in trans strains, ΔcpxR/pCpxR_WT and ΔcpxR/pCpxR_Pneg was calculated relative to ΔcpxR/pMMB208 (right panel). Relative expression of rpoE from both in cis and in trans was monitored from three independent biological and three technical replicates of each strain with two house-keeping internal standards, gyrB and rpoC. Statistical significance was determined using One-way ANOVA with Tukey’s multiple comparisons test, with a single pooled variance. The difference in variance with a p-value of <0.05 was considered significant. The p-values are indicated by, <0.001 (***), <0.01 (**) and <0.05 (*). The active phosphorylated form of CpxR (CpxR~P) binds at the promoter of rpoE (b). EMSA with complete 5' intergenic regulatory DNA of rpoE with active CpxR~P. A single asterisk (*) indicates the target promoter DNA-CpxR_wt complex. Unbound promoter DNA is indicated with an arrowhead. A 16S rDNA fragment (148 bp) used as ‘non-specific’ negative control and its running location is indicated by an arrow (←). Inactive non-phosphorylated CpxR (CpxR_Pneg) was unable to bind target DNA. Lane-1: target promoter DNA, Lane-2: target promoter plus non-specific 16S rDNA, Lane-3: target promoter, non-specific 16S rDNA and CpxR_His6 (100 µM), Lane-4: target promoter, non-specific 16S rDNA and CpxR_wt (50 µM) and Lane-5: target promoter, non-specific 16S rDNA and phosphorylation defective CpxR_Pneg (100 µM). Each reaction contained 200 mM Acetyl phosphate to phosphorylate CpxR_wt in the respective lane. Gel were stained with 1× GelRed DNA-staining dye solution. A representative image from three independent experiments is shown. The representative unprocessed (raw) image of EMSA-gel can be seen in the Supplementary file.