RESOLVING THE HIGH-RESOLUTION ARCHITECTURE, ASSEMBLY AND FUNCTIONAL REPERTOIRE OF BACTERIAL SYSTEMS BY IN VITRO ATOMIC FORCE MICROSCOPY - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 77

formed by a ~8-10 nm thick honeycomb layer with a periodicity of 8.7 ± 1
nm ( Fig. 4.5 ) .

(a) (b) (c) (e)

(d)

Figure 4.5. C. novyi-NT spore coats—high-resolution AFM height images. (a) Removal
of the amorphous shell by physico-chemical treatments reveals the underlying
honeycomb layer. (b) Most of the honeycomb layers disappeared from the spores
within ~1 hour during the germination process. Remaining honeycomb patches
(left, lower sides) could be easily removed by scanning with increased force. Below
the honeycomb layer, several underlying coat layers (upper right) are revealed.
(c-e). Typical growth patterns seen on C. novyi-NT spore surface after removing the
honeycomb layers. (c) Whole spore with several ~6 nm thick layers exposed on the
surface. (d) Zoom-in of the centre of (c) showing that spore coat layers originate at
screw dislocations. (e) Zoom-in of the area indicated in (c). The circle in (e) denotes a
fourfold screw axis. Many dislocation centres show depressions reminiscent of hollow
cores (arrow). Images reproduced, with permission from Ref. 8. © (2007) American
Society for Microbiology.

As seen in Fig. 4.5b-e , the removal of the honeycomb layer revealed a
multilayer structure formed by ~6 nm thick smooth layers. Typically, there
were 3-6 layers exposed on the spore surface, similar to ones observed for

B.

subtilis (data are not shown here) spores. The spore

coat surface patterns ( Fig. 4.5b-e ) were very similar to ones observed on the
surfaces of inorganic, organic and macromolecular crystals. 20-23
These patterns include steps and growth spirals originating from screw
dislocations, such as those previously described in studies of the crystallization
of semiconductors, 24 salts 25 and biological macromolecules. 22,23 In the middle
of the growth centres, the dislocations cause depressions, typically <15 nm,
which are known as

( Fig. 4.4 ) and B. anthracis

in crystal growth theory and are formed by
the stress associated with the dislocations. 26
Thus, the presence of the aforementioned growth patterns conirms
the crystalline nature of the coat layers. However, while AFM resolution is
typically suficient for visualization of crystal lattices on a molecular scale
for a wide range of protein crystals, 23,27 we were not able to resolve a regular,
crystalline lattice on the spore coat layers. In this case, the lattice periodicity
is assumed to be smaller than ~1 nm, which is the resolution associated with
the sharpest AFM tips used. Such a periodicity would be small compared

hollow cores