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 75

treatment 8 revealed crystalline rodlet ( Fig. 4.3c ) and hexagonal honeycomb
( Fig. 4.3d ) outer spore coat structures, respectively.
As seen in Fig. 4.3c , the ~10 nm thick rodlet layer of B. cereus spores is
formed by multiple randomly oriented domains, comprising parallel subunits
with a periodicity of ~8 nm. The size of the domains is typically 100-200 nm.
In contrast to the multi-domain rodlet structure of the spore coat,

typically only a single continuous domain or several domains were found
to be present on the outer coat of

B. cereus

B. atrophaeus ( Fig. 4.3b ) and B. subtilis

( Fig. 4.4a ) spores. Complete removal of the exterior rodlet layer by

sonication revealed an underlying honeycomb structure ( Fig. 4.3e ) similar to
the exterior spore coat layer of

B. cereus

( Fig. 4.3d ) . For both species,
the lattice parameter for the honeycomb structure is ~9 nm, with ~5-6 nm
holes/pits ( Fig. 4.3d,e ) . In case of

B. thuringiensis

spores, rodlet structures
were not observed as an integral component of the spore coat 4,6 ; however, as
illustrated in Fig. 4.3f , patches of extrasporal rodlet structures were observed
adsorbed to the substrate. 4,6 Rodlet width and thickness ( Fig. 4.3.f ) were
similar to those observed for

B. thuringiensis

structures, which indicates that the similar rodlet proteins could be present
during the sporulation in these three species of

B. atrophaeus , B. subtilis and B. cereus spore coat

spores.
Similar rodlet and honeycomb crystalline structures to those seen in
Fig. 4.3 were observed in freeze-etching electron microscopy (EM) studies
of several species of

Bacillus

Bacillus spores 13 and AFM studies of fungal spores. 14

Note that in the case of B. thuringiensis spore coat rodlet structures were not

observed in freeze-etching EM. 13
The assembly, physical properties and proteomic nature of these
bacterial spore rodlets are poorly understood. The closest structural and
functional orthologs to the

Bacillus species rodlet structure ( not its protein

sequence) are found outside the genus. Several classes of proteins,

with divergent primary sequences, were found to form similar rodlet
structures on the surfaces of cells of Gram-negative

Bacillus

Escherichia coli and

Salmonella enterica , as well as on spores of Gram-positive streptomycetes

and various fungi (for a review, see Ref. 15). Hydrophobins, a new class of
structural proteins, 16 were shown to be an integral component of rodlet
fungal spore surface structures. However, it has not been possible to identify
orthologs of hydrophobin-like proteins in bacterial spores. 17 Similarities in
crystalline outer coat layer motifs found in prokaryotic and eukaryotic spore
types are a striking and unexpected example of the convergent evolution of
critical biological structures. Further investigation is required to determine
the molecular composition of prokaryotic endospore rodlets and their
evolutionary relationship to eukaryotic rodlet structures.