Fig. 4: Feedback regulation of pyocyanin on its own production through MexL.
a Docking of PYO to the structure of MexL15-212. Left, hydrophobic surface of subunit A of MexL15-212, blue represents hydrophilic surface, orange represents hydrophobic surface. Middle, ribbon diagram of subunit A of MexL15-212. The ligand substrate tunnel shown in gray. Right, the interaction between MexL15-212 and PYO. b Detection of the interaction between MexL and PYO in vitro. Left panel, thermal-shift analyzes the purified MexL bound to PYO and PCA. The MexL sample were incubated with DMSO (no phenazines), 250 μM PYO (red trace), 250 μM PCA (blue trace), respectively. Middle panel, the corresponding Tm of MexL in the presence of different concentrations of PYO and PCA, respectively. Right panel, Octet Red 96 determine the binding affinity (KD) between MexL and PYO. The KD value was 84.2 ± 1.8 µM. (n = 3 independent experiments). c EMSA analysis the interaction between MexL and promoter in the presence of PYO and PCA, respectively. Representative images were from three independent experiments. d Microscale thermophoresis (MST) analysis the binding affinities of MexL to mexJ, phz1, phz2 and phzM promoter in the presence or absence of 1 mM PYO, respectively. KD was the dissociation constant. (n = 3 independent experiments). e Analysis of promoter activity of phz1, phz2 and phzM in PAO1 and ΔmexL by corresponding promoter::gfp reporter plasmids in the presence of 500 μM PYO. (n = 3 independent experiments). f RT-qPCR determine the transcriptional level of mexJ in the presence of PYO. (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. b P = 0.00071 (0 μM vs 50 μM), 0.000152 (0 μM vs 250 μM), 0.000168 (0 μM vs 500 μM), 0.000732 (50 μM vs 250 μM) and 0.000659 (50 μM vs 500 μM). e P = 0.000464 (phz1), 0.000326 (phzM) and 0.000625 (phz2). f P = 0.009945 (0 μM vs 250 μM) and 0.0023 (0 μM vs 500 μM).
