Figure 1.
Electron micrographs of ΔywdL and wild-type spores.
(A) Negatively stained ΔywdL spores frequently have a fragile exosporium that is no longer intact or (B) is completely detached from the rest of the spore. Spore sections (C) show more electron dense deposits visible in the interspace region in ΔywdL spores compared to wild-type (D). Scale bars are 0.5 µm corresponding to the full size images in (A–D).
Table 1.
Spore properties of the ΔywdL mutant in comparison to wild-type.
Figure 2.
Immunogold labelling of the exosporium with polyclonal anti-YwdL IgG and a secondary antibody conjugated to 10 nm gold.
(A) B. cereus ΔywdL whole spores (B) B. cereus wild-type whole spores, (C) B. cereus wild-type exosporium and (D) B. thuringiensis kurstaki 4D11 exosporium. (E) B. cereus ΔywdL spores and fragmented exosporium. Samples were stained with uranyl formate and analysed by TEM. Scale bars are 0.5 µm.
Figure 3.
Detection of YwdL in unwashed and fully washed exosporium using Western blotting.
An anti-YwdL antibody was used to locate YwdL in unwashed and fully washed B. cereus ATCC 10876 and B. thuringiensis kurstaki 4d11 exosporium. Lanes 1 and 2 contain unwashed and washed ΔywdL exosporium, lane 3 and 4 unwashed and fully washed B. cereus exosporium and lanes 5 and 6 unwashed and washed B. thuringiensis exosporium respectively.
Figure 4.
Analysis of ΔywdL fragments by TEM.
Isolated exosporium fragments from (A) B. cereus wild-type and (B) ΔywdL were negatively stained and analysed by TEM. Both preparations appear similar. The hairy nap can be seen in both strains protruding out from the hexagonal basal layer in both crystal fragments shown. Fourier transforms (top right corner) were taken from the whole crystal image or from a smaller boxed crystalline area in A and B respectively. First order spots are at a spatial frequency of ∼1/75 Å-1. Scale bars are 50 nm.
Figure 5.
Contour and greyscale projection maps of negatively stained wild-type and ΔywdL exosporium crystals.
Each map is the average of 5 independent crystal images taken from the respective exosporium preparation showing (A, B) B. cereus wild-type and (C, D) ΔywdL exosporium crystals. p3 symmetry-averaging has been applied in both cases. One unit cell of edge of 80 Å is outlined. Solid contours (A, C) represent densities below average (protein) whilst dashed contours represent densities above average (stain). White regions (B, D) represent stain-excluding density (protein) whilst black areas represent stain-accumulating regions.
Figure 6.
Analysing BclA content of wild-type and ΔywdL spores using immunogold labelling.
Whole (A) B. cereus ATCC 10876 wild-type and (B) ΔywdL spores were immunogold labelled using anti-BclA antibodies and a secondary antibody conjugated to 10 nm gold particles. Scale bars are 0.5 µm. Note the fragile exosporium in Figure B. Figure (C) and (D) represent zoomed in areas of (A) and (B) respectively. Scale bars are 0.5 µm.
Figure 7.
Analysis of the glycoprotein content of wild-type and ΔywdL exosporium.
(A) Western blot analysis with anti-BclA antibodies. Lane 1 shows negative control (vegetative cells), lane 2 and 3 unwashed and fully washed B. cereus wild-type exosporium and lanes 4 and 5 show unwashed and fully washed ΔywdL exosporium respectively. (B) Total glycoprotein analysis of wild-type and ΔywdL exosporium samples. Lane 1 shows negative control (sodium metaperiodate replaced with 100 mM acetate buffer), lane 2 and 3 unwashed and fully washed wild-type exosporium and lanes 4 and 5 show unwashed and fully washed ΔywdL exosporium respectively. Samples in (B) were extracted under harsher conditions, using solubilisation buffer (see Materials and Methods).
Figure 8.
Silver stain PAGE of exosporium of ΔywdL compared to wild-type.
Lane 1 corresponds to molecular weight protein standards. Lane 2 shows fully washed B. cereus wild-type exosporium and lane 3 shows fully washed ΔywdL exosporium. The same amount of total protein (15 µg) was applied to each lane for comparison. Samples were treated with solubilisation buffer plus a 90°C incubation step to disrupt protein complexes.
Figure 9.
Germination of ΔywdL spores compared to wild-type.
Germination in response to (A) CaDPA; (B) L-alanine (1 mM, with 10 mM OCDS as an inhibitor of alanine racemase) and (C) inosine (0.3 mM). Filled symbols represent B. cereus wild-type and open symbols the ΔywdL mutant. Circles represent samples with germinant, and triangles samples without germinant. Data are representative of several experiments.