Figure 7.

H-NS-mediated silencing is either alleviated or enhanced by an active T7-dependent promoter when positioned upstream of PicsP in two orientations, independently of VirB. (A) Schematic of pDivergent and pParallel T7 RNAP-dependent Pφ10 cloned in two orientations upstream of PicsP-lacZ. The localized changes in supercoiling generated in the icsP promoter region predicted by the twin-domain model of transcription-coupled supercoiling (90) are depicted for each construct. (B) Normalised GFP fluorescence is depicted as a proxy for Pφ10 activity for each reporter construct (PicsP-lacZ, pDivergent, and pParallel) with either pEmpty or pT7-RNAP in E. coli wild-type or hns mutant. (C) β-Galactosidase activity as a proxy for icsP promoter activity for each of the reporter constructs (PicsP-lacZ, pDivergent, or pParallel) either with pEmpty or pT7-RNAP in E. coli wild-type or hns mutant. Assays were done with three biological replicates and repeated three times. Representative data are shown. Significance was calculated using a three-way ANOVA with post hoc Bonferroni, *p < 0.05 (solid lines compare wild-type and dashed lines compare hns mutant). Complete statistical analysis provided in Supplementary Table S4.