Table 1.
B. subtilis strains used in this study.
Figure 1.
AFM images of B. subtilis wild-type spores.
(a) Height and (b) phase images of spores with surface ridges (coincidental in both images) extending along the entire length of spores (several surface ridges noted by light blue arrows). (c) High-resolution height image of an area on a surface of a single spore showing surface ridges (light blue arrow), patches of an amorphous outermost layer (green arrows), and a rodlet layer (red arrows) seen beneath the amorphous layer. (d) A cross section line drawn perpendicular to rodlets (indicated with red arrows in (c)) showing a periodicity of ∼8.2 nm. (e) High-resolution height image of an area on the surface of a single spore showing patches of an amorphous outermost layer (green arrow and green rectangle), and a rodlet layer (red arrows) seen beneath the amorphous layer.
Figure 2.
AFM images of decoated B. subtilis wild-type spores.
Surface ridges extending along the entire length of spores are indicated with light blue arrows in height (a, b) and phase (c, d) images. Patches of rodlet structures are indicated with red arrows in (b–d). The green arrows in (a–c) indicate remnants of the amorphous outermost layer. High resolution height (e) and phase (f) images showing coincidental patches of rodlet structures denoted with red arrows.
Figure 3.
AFM images of cotA and cotB spores.
Height images of cotA (a) and cotB (b) spores exhibit surface ridges similar to those in wild-type spores (light blue arrows). High-resolution phase images of single cotA (c) and cotB (d) spores show an irregular outermost amorphous layer (green arrows) as well as underlying rodlets (red arrows). In addition to the amorphous layer and rodlets seen on these spores' outermost surface, a strong undulating topography from a sub-surface layer is also present (red circles). Surface ridges in (c,d) are indicated with light blue arrows.
Figure 4.
AFM height images of safA spores.
(a–c) Several surface ridges are indicated with light blue arrows, and in (a) and (b) spores with an oversized sacculus are marked with adjacent green stars. A spore with at most minimal ridges is indicated with a dark blue arrow in (a). In panel (c), two patches of rodlet structure are indicated with red arrows, and a patch of an amorphous layer is indicated with a green arrow.
Figure 5.
(a) Height image of spores with surface ridges extending along the entire length of spores (light blue arrows). (b,c) High-resolution height images of areas on surfaces of single spores showing a dense fiber structure forming a granular structure (b; brown arrow) and individual fibers (c; light yellow arrows). In panel (c), three layers (terraces) of inner coat structure are numbered 1, 2, and 3 in purple. Step edges representing boundaries of each layer (one marked with a purple arrow) are visible. In panels (b) and (c), nanodots are marked with black arrows and one area with a high density of nanodots is circled in panel (c).
Figure 6.
(a) Height image of spores with surface ridges extending along the entire length of spores (light blue arrows). (b) High-resolution height image of a spore surface area showing the upper surface area (green rectangle) covered with an amorphous layer (green arrow) and rodlets (red arrows). The lower part of the outermost layer-free area (black rectangle) is covered with nanodots (black arrow). One of the surface ridges in (b) is indicated with a light blue arrow. In panel (c), a two–layer inner coat structure (two purple arrows noting the two layers) decorated with nanodots can be seen.
Figure 7.
(a,b) Height images of spores that exhibit surface ridges (light blue arrows), and several spores with an oversize sacculus are labeled with green stars. In (a) a spore with no apparent ridges is indicated with a dark blue arrow. (c) Height image of a multilayer inner coat structure. Three layers are indicated with numbers, and a kink on a step edge is marked with a purple arrow. Several holes in the layered structure are also indicated with purple circles. The hole in the middle circle corresponds to a pinning point on the step. (d) Height image of a multilayer layer structure similar to ones seen in Figs. 5b,c, 6c, and 7c, as seen on the surface of a trypsin crystal. Similar to the spore coat layers in (c), three layers, kinks and several holes are indicated with purple numbers, arrows and circles, respectively. The insert in (d) is a larger area of the crystal surface seen in (d). The same holes and three layers seen in (d) are indicated in the insert. The red line in (d) denotes the step contour, which was utilized for the measurement of the sinuosity index. Panel (d) is reprinted with permission from Plomp M, McPherson A, Larson SB, Malkin AJ (2001). Growth mechanisms and kinetics of trypsin crystallization. J Phys Chem B 105: 542–551. [52]. © (2001) American Chemical Society.
Figure 8.
(a) High-resolution height (a) and phase (b) images of the spore surface showing (coincidental in both images) a rodlet structure (red arrows) covered with patches of an amorphous layer (green arrows). (c) High-resolution height image of the spore surface with an insert with a cross section line drawn perpendicular to rodlets showing the periodicity of ∼7.2 nm.
Figure 9.
(a,b) High-resolution height images of the spore surface showing a honeycomb structure (orange arrows in (a)) and patches of rodlets on top seen in (a) (red arrows). The insert in (b) is a cross section line along a honeycomb structure (indicated with a black line and red arrows in (b)) showing a periodicity of ∼8.5 nm. (c) A portion of a loose honeycomb layer (orange arrows) with remnants of rodlet structures (red arrows), which were seen in cotE spore preparations.
Figure 10.
AFM height images of gerE spores.
(a) gerE spores are either completely (black stars) or partially (grey stars) covered with coat material. (b) A spore that is completely encased in the coat material, and (c) a spore with patches of coat material (grey arrow).
Figure 11.
AFM height images of cotE gerE spores.
(a) Spores which appeared to be devoid of spore coat material. Closely packed spores are more deformed than ones that are not surrounded by other spores. Some spores exhibit 80–100 nm wide and 30–40 nm deep depressions (black circles). The insert in (a) is a cross section line (indicated with a white line) drawn across the ∼100 nm wide depression showing a depth of ∼40 nm. (b) Image showing small patches of coat material (grey arrow) on the spore surface. (c) High-resolution image of a spore devoid of any obvious coat material, and showing a textured outermost surface.
Figure 12.
AFM height images of sspoVID spores.
(a) Many of the spoVID spores are devoid of obvious spore coat material, although some spoVID spores are encased in loosely fitting coat sacculi (green stars); insert: an empty intact sacculus (blue star) present in a spore preparation. (b,d) Severely deformed spores without any visible coat material are indicated with white stars. (c, d) Spores with partially sloughed off coat sacculii are indicated with grey stars.
Figure 13.
High-resolution AFM height images of spoVID spores.
(a) External and internal surfaces of the empty coat sacculus in Fig. 12a, insert exhibit morphology similar to that of the outermost wild-type spore layer seen in Fig. 1b. The surface is comprised of rodlets (red arrows) and patches of amorphous material (green arrows). In (b) a pitted layer (pink arrow) is seen beneath a layer of coat material (grey arrow).
Figure 14.
Model of the spore coat architecture of a single B. subtilis spore.
The layers of the spore coat and the cortex are depicted as: (1) an outermost amorphous layer (the crust); (2) the rodlet layer; (3) the honeycomb layer; (4) the fibrous/granular layer, (5) the nanodot layer on top of a multilayer structure (6) ((with a 2D nucleus (indicated with *) seen on the upper layer)); and the basement layer (7), which is on the top of the cortex's outer pitted surface (8). Structural features of spore coat layers are not shown to scale.
Figure 15.
2D nucleation and growth of inner spore coat layers.
Panel (a) shows two putative 2D nuclei (purple arrows) on the inner coat surface of a cotO spore. Panel (b) shows 2D nuclei (purple arrows) on the surface of a satellite tobacco mosaic virus (STMV) crystal. This illustration is reproduced with permission from Malkin AJ, Kuznetsov YuG, Land TA, DeYoreo JJ, McPherson A (1995) Mechanisms of growth for protein and virus crystals. Nature Struct Biol. 2: 956–959 [48]. © (1995) Nature Publishing Group. (c) At a relatively small impurity (indicated as small balls) density, the average impurity distance dimp is larger than dcrit and steps are able to advance. (d) At higher impurity densities, dimp<dcrit, the curvature of step segments between impurities increases and steps are halted. Panels (c) and (d) are reproduced, with permission from Plomp M, McPherson A, Malkin AJ (2003). Repair of impurity-poisoned protein crystal surfaces. Proteins: Struct, Function, Bioinform 50: 486–495 [82]. © (2003) John Wiley and Sons.