Fig. 2: Identification of the conserved MexL-binding motif within promoters.
a Determination of binding ability of MexL with truncated mexJ, phz1, phz2 and phzM promoter region by EMSA, respectively. Representative images from three independent experiments. b The predicted MexL-binding consensus motif (5’-TGTAATTT-3’) obtained by MEME online tools. c Sequence of the mexJ, phz1, phz2 and phzM promoter regions carrying potential MexL binding site. The predicted MexL binding site were shown in red letter, and the location of palindromes in each promoter region was marked by inverted arrows. d Four binding site (BS-mexJ, BS-phz1, BS-phzM and BS-phz2) were tested by EMSAs. Microscale thermophoresis (MST) analysis the binding affinities of MexL to BS-mexJ, BS-phz1, BS-phz2 and BS-phzM promoter, respectively, and the corresponding KD (dissociation constant) value was shown under the image. Representative images were from three independent experiments. e Confirming the MexL-binding sites in phz1 and phzM promoters. The left panel is the schematic diagrams of the phz1 and phzM promoter regions and corresponding promoter::gfp fusion reporter plasmids. ΔBS-phz1::gfp indicates the phz1::gfp reporter without the BS-phz1 site. ΔBS-phzM::gfp is phzM::gfp reporter without the BS-phzM site. The column at the right is the fluorescence intensity of corresponding promoter::gfp fusion reporter plasmid in PAO1 and ΔmexL strains post 24 h of growth. (n = 3 independent experiments). P values were determined using two-tailed Student’s t test. Significance was indicated by a P value. ns, non-significant, ***P < 0.001, **P < 0.01. e P = 0.001528 (PAO1/phz1::gfp vs PAO1/ΔBS-phz1::gfp), P = 0.000198 (PAO1/phzM::gfp vs PAO1/ΔBS-phzM::gfp).
