Figure 4
BsmR degrades c-di-GMP to regulate bacterial swimming motility and biofilm formation. (a) BsmR protein degrades c-di-GMP in vitro. Purified BsmR protein was incubated at 28 °C in reaction buffer together with the synthesized 32P-labeled c-di-GMP. At the indicated time points, aliquots of equal volume were retrieved and separated by thin layer chromatography. (b) Overexpressing bsmR significantly decreased the cellular c-di-GMP level. The FRET-based c-di-GMP biosensor was used to measure the amounts of intracellular c-di-GMP. The table of FRET values and c-di-GMP concentrations was generated by incubating the purified c-di-GMP biosensor with increasing concentrations of c-di-GMP. (c) Quantification of cellular c-di-GMP level by LC-MS/MS. (d) Biofilm of the bacterial strains stained with crystal violet. (e) Quantification of the relative bacterial levels in the biofilm shown in (d). (f) Swimming motility of the indicated strains grown on rich NYG medium plates containing 0.15% agar. (g) Relative quantification of bacterial swimming zone diameters. (h) Quantification of the cellular c-di-GMP level of strains induced by different concentrations of IPTG. (i) Relative quantification of the biofilm formed by strains induced by different concentrations of IPTG. (j) Relative quantification of the diameters of the swimming zones formed by strains induced by different concentrations of IPTG. The data are representatives of three independent experiments. Each value is the average of three replicates. Error bars represent the standard deviation (n ≥ 3). *p < 0.05, as determined by AVONA. The data are representative of at least triplicate repeatable experiments.
