Figure 1.

(A) BP348, a B. pertussis strain lacking ACT, makes more biofilm than WT BP338 B. pertussis. Strains were grown in 96 well microtiter plates and biofilm formation was assessed using the crystal violet assay at 96 hours. Bvg(−) BP347 serves as a negative control. Data expressed as the mean ± standard deviations, compiled from 3 experiments run in triplicate. * = p <0.05 and **** = p <0.0001 compared to WT BP338. (B) ACT inhibits biofilm in a concentration-dependent manner. WT BP338 biofilm formation in the presence of increasing concentrations of recombinant purified ACT (ng mL⁻¹) was assessed at 96 hours. Biofilm formation was measured by crystal violet assay. Bvg(−) BP347 serves as negative control. Data expressed as the mean ± standard deviations, compiled from 5 experiments run in triplicate. *** = p <0.001, **** = p <0.0001 compared to WT BP338 without ACT. (C) Schema of ACT truncated and enzymatically inactive mutant proteins (Lee et al., 1999). (D) The AC domain is necessary and sufficient for biofilm inhibition, although the catalytic activity of ACT is not required. ACT, iACT or other ACT mutant proteins were added to WT BP338 and biofilm formation was measured by crystal violet assay at 96 hours. AC domain was added at 10 ng mL⁻¹ and additional ACT proteins including ΔH, ΔHR1, ΔR, and ΔAC were all added to a final concentration of 100 ng mL⁻¹. Data expressed as the mean ± standard deviations, compiled from 3 experiments run in triplicate. *** = p <0.0005 compared to WT BP338 without ACT. (E) BP338 lacking the AC domain (BP338 ΔAC) makes more biofilm than the parental WT strain. Strains were grown in 96 well microtiter plates and biofilm formation was assessed using the crystal violet assay at 96 hours. Mean values represented by bars, error bars represent standard deviations. Bvg(−) strain serves as a negative control. Data expressed as the mean ± standard deviations, compiled from 3 experiments run in triplicate. ** = p <0.01 and **** = p <0.0001 compared to WT.