Fig. 4: Relative expression of the hms loci is influenced by accumulation of active phosphorylated CpxR.
From: Cpx-signalling facilitates Hms-dependent biofilm formation by Yersinia pseudotuberculosis
(a) Operon structure of the Yptb-YPIII hms loci. Genetic organisation of hmsHFRS and hmsCDE operons and unlinked hmsT and hmsP genes. The locus-tag (YPK_XXXX) of each loci mentioned underneath. PCR-amplified gene-specific (for qRT-PCR) and 5' UTR (for EMSA) fragments of corresponding loci are represented by respective vertical blue and horizontal black rectangle box. Numbers in parentheses indicate amino acid sequence identity with isofunctional homologues from Ype strain KIM10+. (b–e) Cpx-signalling mediated differential expression of hms loci. Quantitative RT-PCR was performed on the cDNA template, synthesised from the total RNA of Yptb-YPIII Cpx-signalling in cis strains, parental WT (intact Cpx-signalling), ΔcpxA (locked-on Cpx-signalling producing excessive 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/pCpxRPneg (ectopic expression of phosphorylation defective CpxRPneg 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 the hms loci within the in cis strains, ΔcpxA and ΔcpxR was calculated relative to WT (b–e; left panel) whereas expression of corresponding hms gene within the in trans strains, ΔcpxR/pCpxR-WT and ΔcpxR/pCpxRPneg was calculated relative to ΔcpxR/pMMB208 (b–e; right panel). Relative expression of hms loci from both in cis and in trans was monitored from three 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 <0.0001 (****), <0.001 (***), <0.01 (**), <0.05 (*) and >0.05 (ns).
