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 76

(a) (b) (c) (d)

Figure 4.4. AFM images of B. subtilis spores of different strains. The spores analyzed
were wild type (a), cotE (b), gerE (c) and cotEgerE (d). Images reproduced, with
permission from Refs. 11 and 12. © (2008) American Society for Microbiology.

Proper assembly of a multilayer spore coat of Bacillus spores is

dependent on a number of coat proteins. Loss of any of those proteins could

alter signiicantly the mechanisms of the spore coat assembly and the inal
spore coat structure. Indeed, as demonstrated in Fig. 4.4 , deletion of a single
spore coat protein could result in pronounced changes in the spore coat
architecture.

1

11,12

Thus, the AFM analysis demonstrated that intact wild-type B.

subtilis spores are completely or partially covered by a thin amorphous layer

lacking deined structure ( Fig. 4.4a ) . Directly below the amorphous layer is
a rodlet crystalline layer ( Fig. 4.4a ) , which has parameters similar to the B.

atrophaeus is a major spore coat morphogenetic

protein, and in its absence, the outer coat fails to assemble properly.

rodlet spore coat layer.

6

CotE

18

Indeed,

we have demonstrated that for most spores the outermost structure is

formed by 3-5 crystalline layers, each of which is ~6 nm thick ( Fig. 4.4b ),
which likely correspond to the inner coat layers.
Furthermore, surfaces of some

cotE

mutant spores exhibit patches or large
regions covering the spore of a hexagonal crystalline layer (located between
the rodlet layer and the inner coat multilayer structure) ( Fig. 4.4b ) . Surfaces
of

cotE

spores were found to lack completely both amorphous and rodlet
structures, being encased in several inner spore coat layers.

gerE

7

Finally, spores

lacking both spores) were found to lack all

outer and inner coat structures, with the spore cortex being the outermost
structure.

CotE and gerE proteins ( cotEgerE

12

Our recent comprehensive analysis of a wide range of B. subtilis

mutants, which lack various spore coat proteins, has provided improved

understanding of the spore coat architecture, assembly and function of coat
proteins.
To observe the structure of the

19

spore coat beneath the
amorphous shell, we developed procedures to remove the shells by chemical
treatment with various reducing agents and detergents or by physical
treatment using a French Press.

C. novyi-NT

When either French Press or chemical
treatments were used, the majority of the exposed spore coat surface is

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