Article Figures & Data
Figures
- FIG 1
Known components of the c-di-GMP signaling network in B. subtilis. The color code for protein domains is as follows: gray for the TM and putative soluble signaling domains, purple for the GGDEF domains, orange for the EAL domains, and blue for the PilZ domain. Inactive protein domains are illustrated with dashed lines. Note that the EAL domain activity of DgcW has not been tested so far in vitro.
- FIG 2
Single-molecule tracking of exponentially growing DgcK-mVenus in B. subtilis 3610 wild-type cells. (A) Representative static molecule of DgcK-mVenus in the wild type localized at the cell pole (D), and dynamic track of DgcK-mVenus moving along the cell pole. Scale bars correspond to 2 μm. (B, E) Corresponding projections of all tracks in these cells showing the static (B) or mobile (E) track in a color-coded manner. The origin is highlighted in red and the end in yellow. All other tracks in the same cell are displayed in gray. (C, F) Normalized intensity profile of the track shaded in gray. (G to I) Average dwell times of DgcK-mVenus and DgcP-mVenus in different B. subtilis NCIB 3610 strains. (G) The bar plots depict the change in the average dwell times (calculated with “SMM Track”) of DgcK-mVenus in the NCIB 3610 wild type (3610 WT), in a ydaK deletion strain (3610 ΔydaK), and in two strains overexpressing ydaK (3610 4% EtOH, cells stressed with 4% ethanol for 30 min; 3610 Pxyl-ydaKLMN, NCIB 3610 with Pxyl-ydaKLMN). The absence of YdaK leads to a decrease of the dwell time, whereas overexpression leads to an increase. (H, I) Dwell times of DgcK-mVenus (H) or DgcP-mVenus (I) in a stain lacking the c-di-GMP receptor dgrA (3610 ΔdgrA) and in the NCIB 3610 wild type. All data are from three biological replicates.
- FIG 3
Analyses of the mobility of DGC DgcK and DgcP by the Gaussian mixture model (GMM) in different B. subtilis NCIB 3610 strains. For the determination of fraction sizes and of diffusion coefficients, the GMM was used. (A to C) GMM of DgcK-mVenus in NCIB 3610 (wild-type) (A), NCIB 3610 ΔydaK (B), and NCIB 3610 ΔdgrA (C) cells. The histogram of the step-size distribution was fitted with a single fit and a double fit, which consists of a static/slow population and a mobile population, as indicated in the key. Because the double fit corresponds better to the data than the single fit, the double fit was taken for all determinations. (D, E) The bar plots depict the changes in the distributions of the two (static and mobile) subpopulations of DgcK-mVenus. (D) In the absence of the c-di-GMP receptor YdaK (NCIB 3610 ΔydaK), the size of the static DgcK-mVenus population decreases, whereas this population increased upon overexpression of YdaK (NCBI 3610 cells stressed with 4% ethanol for 30 min, or NCIB3610 Pxyl-ydaKLMN cells overexpressing YdaK via addition of xylose). (E) Deletion of the gene encoding the PilZ domain protein DgrA (NCIB 3610 ΔdgrA) leads to an increase of the mobile population of DgcK-mVenus compared to that of the wild type. Data are analyzed from three biological replicates. (F, G) GMM of DgcP-mVenus in NCIB 3610 (F) and NCIB 3610 ΔdgrA (G). (H) The bar plots depict the changes in the two subpopulations of DgcP-mVenus. Deletion of the dgrA gene encoding the c-di-GMP receptor DgrA (strain 3610 ΔdgrA) leads to an increase in the size of the mobile population of DgcK-mVenus compared to that of the wild type.
- FIG 4
Interaction of the DGC DgcK with its cognate receptor YdaK depends on an intact I-site within the GGDEF domain of YdaK. (A) In vitro interaction analysis of His6-DgcK-∆N175 and GST-YdaK-∆N111. Coomassie-stained SDS-PAGE of a pull-down assay employing His6-DgcK-∆N175 (bait), GST-YdaK-∆N111 (prey) and GST (control 2). 2 nmol His6-DgcK-∆N175 was immobilized on nickel-sepharose beads, followed by incubation with 20 nmol GST-YdaK-∆N111 with or without addition of c-di-GMP (cdG, second and fourth lane, respectively). First lane: His6-DgcK-∆N175 binding to nickel-sepharose beads. Third lane, Control 1: GST-YdaK-∆N111 does not bind to nickel-sepharose beads. Fifth lane, Control 2: His6-DgcK-∆N175 does not bind the affinity tag GST. (B) In vitro interaction analysis of GST-His6-YdaK-∆N111 and His6-DgcK-∆N175. Coomassie-stained SDS-PAGE of a pull-down assay employing GST-His6-YdaK-∆N111 (bait), His6-DgcK-∆N175 (prey). 2 nmol GST-His6-YdaK-∆N111 was immobilized on GST-sepharose beads, followed by incubation with 20 nmol His6-DgcK-∆N175 with or without addition of c-di-GMP (cdG, second and third lanes, respectively). First lane: GST-His6-YdaK-∆N111 binding to GST beads. Fourth lane: Control 1, His6-DgcK-∆N175 does not bind to GST-sepharose beads. Fifth lane: Control 2, His6-DgcK-∆N175 does not bind the affinity tag GST. (C) Coomassie-stained SDS-PAGE of a pull-down assay employing GST-His6-DgcK-∆N175 (bait), His6-YdaK-∆N111 (prey) and the I-site mutant His6-YdaK-∆N111_R202A. 2 nmol GST-His6-DgcK-∆N175 was immobilized on GST-sepharose beads, followed by incubation with 20 nmol His6-YdaK-∆N111 (first lane). Second and third lanes: GST-His6-DgcK-∆N175 does not bind His6-YdaK-∆N111_R202A with or without additional addition of c-di-GMP (cdG). Fourth lane: Control 1, His6-YdaK-∆N111_R202A does not bind to GST-sepharose beads. Fifth lane: Control 2, GST-His6-DgcK-∆N175 binding to GST beads.
- FIG 5
c-di-GMP alters the conformation of YdaK. (A) Amino acid residues of YdaK-ΔN111 are colored according to their differences in HDX profiles between c-di-GMP-bound YdaK-ΔN111 and apo-YdaK-ΔN111. The secondary structure of YdaK-ΔN111 based on a generated model is indicated. (B, left) Locations of regions with less HDX in the presence of c-di-GMP in a structural model of YdaK-ΔN111. The I-site and degenerated GGDEF motifs are in red and green, respectively. The I-site arginine 202 is shown as sticks. The position of c-di-GMP bound to the I-site is inferred from a superimposition of the YdaK-ΔN111 structural model upon the crystal structure of the GGDEF domain of Dcsbis from Pseudomonas aeruginosa (PDB accession number 4ZMM). (Right) Location of representative peptides in the structural model of YdaK-ΔN111. (C) Hydrogen/deuterium exchange profiles of representative YdaK peptides in the c-di-GMP-bound (red) and unliganded (blue) states. Data represent the means ± standard deviations (SD) of results from three technical replicates. t, time.
- FIG 6
Swarm expansion assays of pdeH mutant strains harboring additional dgc gene deletions in B. subtilis NCIB 3610. The defect in swarming motility of the pdeH mutant can be rescued by deleting all three dgc genes. Semiquantitative colony expansion assay of wild-type B. subtilis NCIB 3610 and of strains with pdeH deleted combined with additional deletions of the indicated dgc genes (DK391, pdeH; DS9305-DK391, ΔdgcK ΔpdeH; DS9537-DK391, ΔdgcP ΔpdeH; DS9883-DK391, ΔdgcW ΔpdeH; DS1809-DK391, ΔdgcK ΔdgcP ΔdgcW ΔpdeH) on 0.7% (wt/vol) LB agar for 1 h (T1), 2 h (T2), and 3 h (T3) after incubation at 37°C. All values are the averages of results from at least four experiments, which included three biological replicates. The colony diameters of all strains ranged between 0.8 and 0.9 cm at time zero. Asterisks indicate significant differences (*, P < 0.05; **, P < 0.01; ***, P <0.001, two-sided independent t test; ns, differences were not significant).
- FIG 7
Interaction of the PilZ protein DgrA with DgcK and DgcP. (A, left) In vitro interaction analysis of GST-His6-DgrA and His6-DgcK-ΔN175. Coomassie blue-stained SDS-PAGE gel from a pulldown assay employing GST-His6-DgrA (bait), His6-DgcK-ΔN175 (prey), and GST (control [Ctrl]). Two nmol of GST-His6-DgrA was immobilized on glutathione-Sepharose beads (first lane), followed by incubation with 20 nmol His6-DgcK-ΔN175 (second lane) and 2 mM c-di-GMP (cdG) (third lane). Control 1 (fourth lane) is His6-DgcK-ΔN175, which does not bind to glutathione-Sepharose beads. Control 2 (fifth lane) is His6-DgcK-ΔN175, which does not bind the affinity tag GST. (A, right) Input controls for: GST-His-DgrA (first lane), His-DgcK-ΔN175 (second lane), GST-His6 (third lane). (B, left) In vitro interaction analysis of DgcP-Strep and GST-His6-DgrA. Coomassie blue-stained SDS-PAGE gel from a pulldown assay employing DgcP-StrepII (bait) and GST-His6-DgrA (prey). Two nmol of DgcP-StrepII was immobilized on Strep-Sepharose beads (first lane), followed by incubation with 20 nmol GST-His6-DgrA (second lane), which shows that DgcP-StrepII can immobilize GST-His6-DgrA on Strep-Sepharose. In the third lane, 2 nmol DgcP-Strep was immobilized on Strep-Sepharose beads, followed by incubation with 20 nmol GST-His6-DgrA and of 2 mM c-di-GMP. Control 1 (fourth lane) was GST-His6-DgrA, which does not bind to Strep-Sepharose beads. (B, right) Input controls for DgcP-StrepII (first lane), GST-His6-DgrA (second lane).
- FIG 8
Molecule numbers. (A, B) Projection of tracks in exponentially growing B. subtilis NCIB 3610 wild-type cells onto bright-field images. All tracks of DgcK-mVenus (A) and DgcP-mVenus (B) are shown in red in their respective cells. Scale bars, 2 μm. (C, D) Distribution of the number of fluorophores per cell in exponentially growing cells. Cells expressing DgcK-mVenus (C) harbor on average 5.58 ± 0.16 fluorophores per cell, whereas cells expressing dgcP-mVenus (D) show 24.84 ± 0.13 fluorophores per cell. (E, F) Correlation between the number of fluorophores and cell length. For DgcK-mVenus cells (E) there is no correlation between the number of fluorophores and cell size, but a slight correlation can be seen for DgcP-mVenus (F). (G, H) The distributions of bleaching steps for DgcK-mVenus (G) and DgcP-mVenus (H) display two fractions of bleaching steps (monomers and dimers).
Tables
- TABLE 1
Determined diffusion coefficients and dwell times of DgcK-mVenus in different B. subtilis NCIB 3610 strainsa
- TABLE 4
Strains used in this study
- TABLE 5
B. subtilis plasmids and vectors used in this study
MOVIE S1
Exponentially growing B. subtilis cells expressing DgcK-mVenus from the original gene locus. Stream acquisition at 15 ms (66 frames per second [fps]) is shown in slow motion at 10 fps. Download Movie S1, AVI file, 0.8 MB.
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FIG S1
Heat maps of DGCs in B. subtilis wild-type cells and mutant strains. All tracks of each strain are projected into a 3- by 1-μm-large cell to visualize the subcellular distribution of the molecules using the SMTracker. The probability that a protein is located at a certain position in the cell is shown in a color-coded manner; the more likely a molecule is present at a specific point, the darker that point. (A to D) Heat maps of DgcK-mVenus in wild-type B. subtilis NCIB 3610 (A), NCIB 3610 ΔydaK (B), NCIB 3610 Pxyl-ydaKLMN (C), and wild-type NCIB 3610 stressed with 4% ethanol (induced ydaK expression) (D). When overexpressing the c-di-GMP receptor YdaK, the tracks are predominantly located at several positions at the cell membrane compared to where they occur in the ydaK deletion strain. (E to H) Same as panels A to D, but all tracks have an apparent diffusion lower than 0.5 μm2/s and an R2 of >0.7. Intensities are equalized between all four data sets. (I, J) Heat maps of DgcP-mVenus with wild-type NCIB 3610 (E) and NCIB 3610 ΔdgrA (F). (K, L) Same as panels I and J, but all tracks have an apparent diffusion lower than 0.5 μm2/s and an R2 of >0.7, as well as equalized data sets. Download FIG S1, TIF file, 0.4 MB.
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FIG S2
Single-molecule dynamics of CdaA-mVenus in different B. subtilis 3610 strains. (A to C) GMM of CdaA-mVenus in NCIB 3610 (wild-type) (A), NCIB 3610 ΔydaK (B), and NCIB 3610 ΔdgrA (C) cells. The histogram of the step size distribution was fitted with a single fit, shown in green, and a double fit, shown in red, which consists of a static/slow population (dotted red line) and a mobile population (dashed red line). (D, E) Bar plots depict the change in the distributions of the two subpopulations of CdaA-mVenus (static population in blue and mobile population in red). There was no change in population size in the absence of the c-di-GMP receptor YdaK (NCIB 3610 ΔydaK) (D) or DgrA (NCIB 3610 ΔdgrA) (E). (F, G) Equalized heat maps of CdaA-mVenus in wild-type B. subtilis NCIB 3610 (A) and NCIB 3610 ΔydaK (B). (H, I) Equalized heat maps of CdaA-mVenus in wild-type B. subtilis NCIB 3610 (A) and NCIB 3610 ΔdgrA (B). Download FIG S2, TIF file, 1.1 MB.
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FIG S3
Interaction of DGC DgcK with its cognate receptor YdaK. Coomassie blue-stained SDS-PAGE gel of His6-DgcK-ΔN175 and YdaK-ΔN111 coexpressed in E. coli BL21, after Ni-NTA chromatography (diluted elution fraction). Download FIG S3, JPG file, 0.1 MB.
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FIG S4
c-di-GMP content of purified proteins. (A and B) DgcK-ΔN175 (A) and YdaK-ΔN111 (B) proteins were denatured with chloroform, and the c-di-GMP was quantified by high-performance liquid chromatography at a wavelength of 260 nm. A solution containing 200 μM c-di-GMP served as the standard. The amount of c-di-GMP quantified equals approximately a loading of 25% of the DgcK molecules. Download FIG S4, TIF file, 0.5 MB.
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DATASET S1
HDX-MS data from HDX experiments. Download Data Set S1, XLSX file, 0.2 MB.
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FIG S5
YdaK_R202A is not responsive to c-di-GMP. (A and B) Difference in the hydrogen/deuterium exchange profiles of wild-type YdaK-ΔN111 (A) and YdaK-ΔN111_R202A (B) in the presence and absence of c-di-GMP. (C and D) Native YdaK and its R202A variant harbor similar conformations. (C) Relative hydrogen/deuterium exchange profiles of YdaK-ΔN111_R202A (top) and wild-type YdaK-ΔN111 (bottom) in the absence of c-di-GMP; (D) difference in the hydrogen/deuterium exchange profiles of YdaK-ΔN111_R202A and wild-type YdaK-ΔN111 in the absence of c-di-GMP. Download FIG S5, TIF file, 1.5 MB.
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FIG S6
DgcP-mV-YFP visualized by total internal reflection fluorescence microscopy (TIRFM) reveals distinct focal formation. TIRFM of mid-exponential-phase B. subtilis NCIB 3610 cells expressing dgcP-mV-yfp from the amylase locus (strain NCIB 3610-PB86, amyE::Pxyl-dgcP-mV-yfp) 45 min after induction with 0.01% (wt/vol) xylose. BF, bright field. Bar = 2 μm. Download FIG S6, TIF file, 0.2 MB.
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MOVIE S2
Exponentially growing B. subtilis cells expressing DgcP-mVenus from the original gene locus. Stream acquisition at 15 ms (66 fps) is shown in slow motion at 10 fps. Download Movie S2, AVI file, 2.1 MB.
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FIG S7
Determination of molecule numbers for YdaK-mVenus. (A and B) Distribution of the numbers of fluorophores per cell in exponentially growing cells. Cells expressing YdaK-mVenus during exponential growth (A) harbor on average 11.8 ± 0.1 fluorophores per cell, whereas cells stressed with 4% ethanol (B) show 14.4 ± 0.9 fluorophores per cell. (C, E) Correlation between the number of fluorophores and cell length during exponential growth (C) or after ethanol stress (D). There is no clear correlation between the number of fluorophores and cell size. Download FIG S7, TIF file, 0.9 MB.
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