Fig 2. The X-ray crystal structures for RpfR_Ct(PAS)–C12:0 and RpfR_Ct(PAS)–BDSF.

(A) Cartoon representation of RpfR_Ct(PAS) (orange) in complex with C12:0 (pink/red balls and gray sticks). Disordered residues 207–212 are shown as a dashed line. C12:0 alone is depicted in the same orientation as the bound ligand below the model. (B) C12:0 (pink/red balls and gray sticks) interacting with the hydrophilic RpfR_ct(PAS)-binding site residues (orange sticks). Hydrogen bonds (black dashed lines) between hydrophilic residues and the C12:0 carboxylic acid group are shown with their measured distances. RpfR–C12:0 hydrophobic interactions are depicted as a smooth orange contour line. (C) Stereo diagram of C12:0 (pink/red balls and gray sticks) and corresponding 2F_o-F_c electron density contoured at 1.0 σ. (D) Cartoon representation of RpfR_ct(PAS) (cyan) in complex with BDSF (purple/red balls and gray sticks). Disordered residues 206–212 are shown as a dashed line. Below the structure, BDSF alone is depicted in the same orientation of the modeled ligand. (E) BDSF (purple/red balls and gray sticks) forming hydrogen bonds (black dashed lines with their measured distances) with the same RpfR_ct(PAS)-binding site residues as C12:0. N202 forms an additional interaction with C3 of BDSF. R187 forms an additional hydrogen bond with the BDSF carboxylic acid. RpfR–BDSF hydrophobic interactions are depicted as a smooth cyan contour line. (F) Stereo diagram of BDSF and corresponding 2F_o-F_c electron density contoured at 1.0 σ (purple/red balls and gray sticks). (G) Biofilm fitness advantages of B. cenocepacia strains containing chromosomally encoded RpfR_Bc BDSF-binding site point mutations in direct competition with WT strain HI2424. Fitness is the difference in selective rate constants (s) over 24 h of attachment and biofilm assembly on a polystyrene bead. Each mutant is significantly more fit than WT (ANOVA with Tukey posthoc testing, P < 0.001), and N171A is more fit than N201A or S168A (P < 0.05) [34,35]. (H) Measurement of the average c-di-GMP pool per cell at 24 h in planktonic cultures of WT B. cenocepacia or strains containing RpfR_Bc BDSF-binding site point mutants. C-di-GMP levels are significantly greater in the mutant strains than in the WT (***Welch’s unpaired t test, P < 0.0006). The numerical values underlying panels G and H can be found in S1 Data. BDSF, Burkholderia DSF; C3, carbon 3; C12:0, dodecanoic acid; c-di-GMP, bis-(3′-5′)-cyclic dimeric guanosine monophosphate; DSF, diffusible signal factor; GMP; PAS, Per-Arnt-Sim; WT, wild-type.