Fig 1.
Schematic representation of the supramolecular organization of Che proteins.
(A) MCP form trimers of dimers (each dimer is shown as a green circle), which, in turn, form hexagons connected with rings composed of the CheAP5 domain (dark blue bars) and CheW (white bars). The light blue circles represent the CheAP4 domain and, the red circles the interface between the β-strands 3 and 4 of subdomain 1 of CheAP5 and the β-strands 4 and 5 of subdomain 2 of CheW. Rings containing six CheW proteins (shown at the center of the array) might serve to modulate the stability and activation of the system. A signaling unit is represented in the red box (Adapted from [23]). (B) FrzCD, FrzECheA, FrzA and FrzB proteins organization depicted by homology with Che proteins. (C) Schematic representation of the frz operon.
Fig 2.
The effect of FrzB on the IAA response and the location of the frzB gene in the Frz pathway.
(A) Motility and fruiting body formation phenotypes were photographed at 48h and 72h, respectively. (B) Box plots of reversal frequencies of single cells moving on agar pads supplemented or not with 0.15% IAA. The lower and upper boundaries of the boxes correspond to 25th and 75th percentiles, respectively. The median is shown as a line at the center of each box, and whiskers represents the 10th and 90th percentiles. For the reversal frequency measurements, a minimum of 30 cells from three to six biological replicates, were used. frzCDc corresponds to the allelic variant frzCDΔ6–182.
Fig 3.
Interaction of FrzB with FrzCD and FrzE.
(A) GST affinity chromatography co-purification of the indicated pairs. A, B, CD and E stand for GST-FrzA, GST-FrzB, 6His-FrzCD and 6His-FrzECheA, respectively. In the first gel, lanes are issued from the same gel and were grouped for simplicity. (B) In the left panel, BLi sensograms show the binding of different amounts of GST-FrzB with immobilized 6His-FrzCD. BlitZ ProTM was used to calculate the affinity constant (Kd = 0.7 μM), using a global 1:1 fit. The central panel shows the responses at 145 s (nm, x axis) (average of two biological replicates) of the binding of immobilized 6His-FrzCD with different concentrations of GST-FrzB (μM, y axis). Data were analyzed by GraphPad Prism 5.0, based on steady-state levels of the responses. In the right panel, BLi sensograms show the binding of different amounts of GST-FrzB with immobilized 6His-FrzCD or 6His-FrzECheA for comparison. A reference subtraction (sensor reference and GST-FrzB at 10 μM) was applied to account for non-specific binding, background, and signal drift to minimize sensor variability. (C) The effect of the FrzBβ4-β5 chimera on the FrzE phosphorylation. The FrzE kinase domain (FrzECheA) auto-phosphorylation was tested in vitro by incubation of FrzECheA in the presence of FrzA, FrzB or FrzBβ4-β5, the indicated different forms of FrzCD and ATPγP33 as a phosphate donor. (D) 3D models of the FrzEP4-P5:FrzA and FrzEP4-P5:FrzB complexes based on the T. maritima CheAP4-P5:CheW crystal structure (PDB 2ch4). The 20 amino acid region forming the β-strands 4 and 5 of subdomain 2 and corresponding to the CheW region important for the CheW:CheAP5 interaction are highlighted in red in the FrzA structure. These residues are missing in FrzB. (E) Protein structural alignments between the T. maritima CheW x-ray structure and M. xanthus FrzA and FrzB theoretical 3D models. CheW residues in atomic contact with CheA and MCP are shown as red stars and blue squares, respectively. Secondary structures determined from CheW x-ray structure are shown on top of the alignment.
Fig 4.
Localization of FrzB-mCherry in M. xanthus cells.
(A) (Left) Micrographs of mCherry-frzB cells stained with the DNA DAPI stain. (Middle) mCherry (red) and DAPI fluorescence (blue) profiles are shown with the fluorescence intensity (arbitrary units) represented on the y-axis and the cell length positions with -1 and +1 indicating the poles, on the x-axis. The cell boundaries were drawn manually from the phase-contrast images. (Right) Plot of correlation coefficients of DAPI and mCherry localization. R2 values > 0.5 indicate significant correlations. The table compares the R2 values between the indicated fluorescent fusion and DAPI staining. (B) (Left) Fluorescence micrographs of the indicated M. xanthus strains carrying FrzCD-gfp fusions. The cell boundaries were drawn manually from the phase-contrast images. (Right) For each indicated strain, more than 120 cells (x axis) from at least two biological replicates are represented as lines and ordered according to their length (pixels) in demographs. The GFP fluorescence intensity along the cell body is represented as colored pixels at the corresponding cell position (from -1 to +1 on the y axis). “0” is the cell center. On the right, a scale indicates the fluorescence intensity and the corresponding colors.
Fig 5.
Proposed models on how FrzB might confer plasticity to Frz clusters.
(A) FrzB participates to the formation of 6-CheW rings to stabilize small Frz clusters, which would otherwise collapse in one single cluster (C). (B) Alternatively FrzB allows the formation of small Frz clusters by breaking the networking of MCP trimers of dimers with CheP5-CheW rings.