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. 2022 Jan 25;88(2):e01655-21. doi: 10.1128/AEM.01655-21

FIG 2.

FIG 2

FleR differentially regulates the expression of three T6SS clusters in PAO1 and repressed H1-T6SS is the major reason causing reduced anti-bacterial capacity of PAO1. (A) A diagram showing the three T6SS gene clusters in P. aeruginosa PAO1. (B) Relative expression of H1-T6SS genes clpV1, hcp1, vgrG1, and tse1 measured by RT-qPCR in the strains of PAO1, ΔfleR and ΔfleR with ectopic expression of fleR (ΔfleR(fleR)). (C) Relative expression of H2-T6SS genes hsiA2, hsiB2, clpV2, and vgrG2b measured by RT-qPCR in PAO1, ΔfleR, and ΔfleR(fleR) strains. (D) Relative expression of H3-T6SS genes hsiB3, hsiC3, hcp3, and pldB measured by RT-qPCR in PAO1, ΔfleR, and ΔfleR(fleR) strains. (E) Western blot analysis of Hcp1-His and Tse1-His in the cell-associated and concentrated supernatant protein fractions from PAO1, ΔfleR, and ΔfleR(fleR) strains. The RNA polymerase β subunit (β-RNAP) was selected as an internal control for the assay. (F) Bacterial killing assay between the PAO1 strains and the E. coli prey. Data are represented as the mean ± SD (n = 3). *, P <  0.05; **, P < 0.01; ***, P < 0.001 versus the indicated group based on paired Student's t test. n.s., not significant; EV, empty vector for the control.