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. 2019 Feb 28;14(2):e0212909. doi: 10.1371/journal.pone.0212909

Fig 7. RcsB regulation on the expression of kvhAS, yfdX, and hde genes.

Fig 7

(A) The promoter regions of yfdX and hde genes, and predicted RcsB binding sites (KMRGAWTMWYCTGS, W = A or T, K = G or T, M = A or C, R = A or G, Y = C or T and S = C or G) are marked. (B) The promoter activity was assessed by monitoring the expression of β-galactosidase on the plasmid pLacZ15 cloned with the promoter regions of target genes on ΔlacZ and ΔlacZΔrcsB strains, respectively. Bacteria grown to the exponential phase were resuspended in the acidic LB broth (pH 4.4) for 1 h for acid adaptation and then measured the promoter activity. Error bars indicate standard deviations of three independent experiments done in triplicate. (C) EMSA for the interaction between the recombinant RcsB and the putative promoter of yfdX or hdeB1. The different reaction mixtures of the recombinant His6-RcsB and the biotin-labeled probe PyfdX* or PhdeB1* were resolved on the polyacrylamide gel, and the binding specificity was investigated by adding nonlabeled probe PyfdX or PhdeB1 at a 300-fold concentration. To determine the phosphorylation effect in the interaction, different concentrations of acetyl phosphate was added in the reaction buffer.