Overexpression of dccA results in a dose-dependent increase in c-di-GMP-dependent gene expression. (A) β-Glucuronidase reporter activity in 630Δerm pPpilA1-UTR-gusA (RT1050), 630Δerm pPpilA1-UTRA70G-gusA (RT1051), 630Δerm::PcprA-dccA pPpilA1-UTR-gusA (RT1054), and 630Δerm::PcprA-dccA pPpilA1-UTRA70G-gusA (RT1055) grown on 70:30 sporulation agar supplemented with 2 μg/ml thiamphenicol in the absence or presence of 0.1 μg/ml or 0.5 μg/ml nisin at H12 and H15. (B) Ethanol-resistant sporulation assays of 630Δerm and 630Δerm::PcprA-dccA (RT993) grown on 70:30 sporulation agar in the presence or absence of 0.5 μg/ml nisin at H24. The means and standard errors of the means from three biological replicates are shown. *, P < 0.05; **, P < 0.01 (by one-way ANOVA and Dunnett’s posttest comparing values to that of the WT vector, with 0.5 μg/ml nisin).