Figure S2.

CdgB Overexpression Has No Phenotypic Effect on a bldD Mutant, c-di-GMP Binding Is Required for BldD Activity In Vivo, and Motifs 1 and 2 Are Both Required for c-di-GMP Binding In Vitro, Related to Figures 3 and 5

(A) Scanning electron micrographs showing that overexpression of CdgB has no effect on the phenotype of the bldD mutant and that an allele of bldD encoding a protein defective in c-di-GMP binding (with the DGR-X₈-DQDR mutation) cannot complement a bldD mutant. CdgB was overexpressed from the ermEp^∗ promoter and the mutagenized bldD gene was expressed from its native promoter. Strains were grown for 40 hr at 30°C.

(B) BldD CTD Motifs 1 and 2 are both required for c-di-GMP binding. The double mutants DGR-X₈-RQDD (Motif 1 mutated) and RGD-X₈-DQDR (Motif 2 mutated), in which R114 and D116 or R125 and D128 were changed to D and R, respectively, as well as the quadruple mutant, DGR-X₈-DQDR, (R114, D116, R125 and D128 of Motifs 1 and 2 were changed to D, R, D and R, respectively) of BldD Full-Length (FL) were generated using a four-primer/two-step PCR protocol (Germer et al., 2001) and tested in DRaCALAs (Roelofs et al., 2011) for c-di-GMP binding.