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(one major domain for each spore),
B. cereus
(a patchy multi-domain motif )
and
(extrasporal rodlets) appear to be a consequence of
species-speciic nucleation and crystallization mechanisms that regulate the
assembly of the outer spore coat. In the case of
B. thuringiensis
outer coat assembly,
the surface free energy
38
for crystalline phase nucleation appears to be low
enough to allow the formation of multiple rodlet domains resulting in cross-
patched and layered assemblies. During the assembly of the outer coat of
B. atrophaeus
B. cereus
spores, the surface free energy may be considerably higher,
reducing nucleation to the point that only one major domain is formed covering
the entire spore surface. In addition to the possible differences in the surface
free energies of the underlying inner coat, the pronounced difference in the
nucleation rate of the outer coat rodlet layers for different
species
could be caused by different supersaturation levels of the sporulation media
during rodlet self-assembly. Since the molecular mechanisms of self-assembly
of spore coat structural layers appear to be very similar to those described
for nucleation and crystallization of inorganic and macromolecular single
crystals,
22,23,38
fundamental and applied concepts developed for the nucleation
and growth of inorganic and protein crystals can be applied successfully to
understanding the assembly of the spore coat. Thus, based on experimentally
observed rodlet structural properties, we have developed a model for rodlet
spore surface assembly, which was derived from well-developed molecular-
scale crystallization/self-assembly mechanisms.
6
The consequence of spore coat crystalline assembly process is that
similar to inorganic and macromolecular crystallization, and conditions
during sporulation such as salt concentration, pH, the presence of impurities,
nucleation rates of crystalline self-assembly of spore coat layers and random
variations in the number of screw dislocations on spores could change the
growth rate and hence the thickness of the spore coat.
Furthermore, these observations suggest that spore coat architecture and
assembly are not purely genetically determined but could also be strongly
inluenced by the modiications of sporulation media, which in turn could
affect spore germination competence and physicochemical properties.
However, the effects of environmental and chemical perturbations on spore
coat structure have not been investigated before.
By observing spore coat high-resolution structures, AFM analysis could
be utilized to reconstruct the environmental conditions that were present
during spore formation. Thus, we have demonstrated for the irst time the
pronounced differences in the spore coat architecture of
Bacillus
B. thuringiensis
spores grown under different sporulation conditions. Thus, for spores grown
in NB medium, only honeycomb crystalline layers were seen on the spore
coat (
Fig. 4.6a,b
)
accompanied by the extrasporal rodlets. However, for
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