DgrA is a member of a new family of cyclic diguanosine monophosphate receptors and controls flagellar motor function in Caulobacter crescentus

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   Fig. 1.

   Isolation of c-di-GMP-binding proteins from C. crescentus. (A) Coomassie blue-stained SDS/polyacrylamide gel with protein fractions used for UV cross-linking with c-[³³P]di-GMP. Lane 1, 100.000 × g supernatant; lane 2, 60% ammonium sulfate precipitation; lane 3, 0.4–0.7 M NaCl eluate from Blue Sepharose; lane 4, 0.7–0.9 M NaCl eluate from Blue Sepharose; and lane 5, 125 mM NaCl eluate from GTP-Sepharose column. (B) Autoradiograph of SDS/polyacrylamide gel shown in A. C-di-GMP-binding proteins a, b, and d were identified by MS/MS. Protein c was identified by MS/MS as hypothetical protein CC1599 and was renamed DgrA.

   
   
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   Fig. 2.

   DgrA is a member of a new family of c-di-GMP-binding proteins. (A) UV cross-linking of purified His₆-tagged diguanylate receptor proteins with c-[³³P]di-GMP. The following proteins were used: DgrA (CC1599; C. crescentus), DgrB (CC3165; C. crescentus), PA4608 (P. aeruginosa), YcgR (S. typhimurium), and BSA (control). The Coomassie blue-stained gel (Left) and the autoradiograph (Right) are shown. (B) UV cross-linking of 10 μM DgrA in the presence of 60 nM ³³P-labeled c-di-GMP. Autoradiograph (Upper) and Coomassie-stained gel (Lower). (Left) Samples were supplemented with increasing concentrations of nonlabeled nucleotides as indicated. (Right) Controls were carried out in the absence of UV irradiation or with BSA.

   
   
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   Fig. 3.

   DgrA and DgrB are involved in motility control by c-di-GMP. Motility behavior of C. crescentus wild-type strain CB15 and mutants is shown on semisolid agar plates. Three different colonies from independent conjugation experiments are shown. (A) The following strains containing plasmid pUJ142::dgcA or control plasmid pUJ142 were analyzed: CB15/pUJ142::dgcA (a), CB15ΔdgrA/pUJ142::dgcA (b), CB15dgrAW75A/pUJ142::dgcA (c), CB15ΔdgrB/pUJ142::dgcA (d), CB15ΔdgrAΔdgrB/pUJ142::dgcA (e), and CB15/pUJ142 (f). (B) Overexpression of dgrA or dgrB from the lactose promoter (Plac) repressed C. crescentus motility. (a) CB15/pBBR (vector control). (b) CB15/pBBR::dgrA. (c) CB15/pBBR::dgrB. (C) Levels of class II, class III, and class IV structural components of the C. crescentus flagellum were determined by immunoblot analysis for the following strains: CB15/pBBR (wild-type), CB15/pBBR::dgrA (DgrA), CB15/pBBR::dgrB (DgrB), and the extragenic diguanylate receptor motility suppressors CB15dms0541 pBBR::dgrA (dms0541). The motility behavior of each strain is shown on top of the graph.

   
   
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   Fig. 4.

   Combined amide ¹H and ¹⁵N shift differences (Δδ) between PA4608 in its free and ligand-bound form. Shift differences are color-coded on the structure of free PA4608 (PDB 1YWU, model 12). Combined chemical shift differences were calculated as . These data are also shown in SI Fig. 8. Residue Trp⁹⁹ is shown as sticks, and N^ε1 and H^ε1 are shown in red to highlight the large Δδ value (1.67 ppm) for these atoms.

   
   
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   Fig. 5.

   C-di-GMP binding and motility control of DgrA mutants. (A) UV cross-linking of different DgrA mutant proteins with c-[³³P]di-GMP. Coomassie blue-stained SDS/PAGE (Upper) and autoradiograph (Lower) with purified wild-type and mutant DgrA proteins (10 μM) are shown. (B) Motility behavior of C. crescentus wild-type CB15 overexpressing different dgrA alleles: CB15/pBBR::dgrA (a), CB15/pBBR::dgrAR11AR12A (b), CB15/pBBR::dgrAD38A (c), CB15/pBBR::dgrAV74A (d), CB15/pBBR::dgrAR11AR12AV74A (e), CB15/pBBR::dgrAW75A (f), and CB15/pBBR (vector control) (g). Three different colonies from independent conjugation experiments are shown.

   
   
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   Fig. 6.

   Sequence alignment of the c-di-GMP-binding proteins DgrA, DgrB, YcgR, and PA4608 according to the PilZ Pfam entry PF07238. The PilZ domain is highlighted in green. DgrA residues shown to be important for c-di-GMP binding and in vivo function (red) and the positions of intragenic dms suppressor mutations (black) are highlighted above the alignment. Residues of PA4608 with large chemical shift differences upon c-di-GMP binding (blue) are indicated below the alignment.