FleR regulates H1-T6SS in a c-di-GMP-dependent manner. (A) Relative expression of tse1 measured by RT-qPCR in the PAO1, ΔfleR, ΔsadC, and ΔfleR ΔsadC strains. (B) Western blot analysis of Tse1-His in the cell-associated protein fractions from PAO1, ΔfleR, ΔsadC, and ΔfleR ΔsadC strains. (C) Relative expression of tse1 measured by RT-qPCR in the strains of PAO1, ΔfleR, and ΔfleR with ectopic expression of DGCs and PDEs. (D) Western blot analysis of Tse1-His in the cell-associated protein fractions from the PAO1, ΔfleR, and ΔfleR strains with ectopic expression of DGCs and PDEs. (E) Bacterial killing assay between the indicated PAO1 strains and the E. coli prey. Data are represented as the mean ± SD (n = 3). **, P < 0.01; ***, P < 0.001 versus the indicated group based on paired Student's t test (A) or versus PAO1(EV) based on one-way ANOVA (C, E). n.s., not significant; EV, empty vector for the control.