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Journal of Bacteriology, July 2004, p . 4423-4426, Vol . 186,
No . 14
From
Rings to Layers: Surprising Patterns of Protein Deposition during Bacterial
Spore Assembly
Adam Driks*
Department of Microbiology and Immunology, Loyola University Medical Center,
Maywood, Illinois 60153
Elucidation of the mechanisms of assembly of subcellular structures
is often an elusive goal . The large number of component parts and the
intricate ways in which they interact make experimental analysis a
significant challenge and, in particular, frustrate attempts at
traditional reductionist analysis . Frequently, key insights come from
thoughtful cell biological observations that identify the most
important steps in assembly . In the best cases, not only do these
observations provide critical clues to old problems, but they also
generate novel areas of investigation . An example of a finding that
does both of these things comes from the work of van Ooij et al . (36a),
published in this issue, who used careful cell biological analysis to
uncover a novel dimension of the process by which bacterial spores
assemble their external armor plating . These authors found that the
deposition of external proteins on the spore surface is unexpectedly
dynamic, pointing towards as-yet-unknown regulatory mechanisms
controlling spore formation .
Bacterial spores are marvels of nature . They are formed by bacilli
and clostridia in response to starvation during an approximately 8-h
developmental process, called sporulation, that is controlled by a
complex cascade of cellular events (29) . The result is
a dormant, highly resistant cell that can endure almost any
stress that nature has to offer . Spores can persist in the dormant
state for very long and perhaps even geological time scales (28,
37) . Nonetheless, the spore is not insensitive to its
surroundings . Rather, it is continuously poised to react to the
reintroduction of even minute amounts of nutrients to the milieu . The
result is the almost immediate conversion of the spore back to an
actively growing cell, a process known as germination .
These remarkable properties have motivated intensive study of
spore formation and resistance since the first published description
of spores in 1874 (21) . With the advent of molecular
approaches and, subsequently, advanced cell biological approaches,
spore formation has become a well-developed model for elucidating
fundamental mechanisms of development and cellular assembly (34) .
It is in the context of the assembly of the outermost spore
structures that van Ooij et al . have made their contribution .
To appreciate these findings, a brief review of Bacillus subtilis
spore ultrastructure is needed . All Bacillus spores have a common
architecture (1, 18) (Fig.
1) . At the center is the core, a relatively dry
compartment that houses the spore DNA . Surrounding this is a membrane
(called the inner membrane) and then a thick layer of peptidoglycan
called the cortex . The cortex, in turn, is encased in a complex
protein shell called the coat, which is the focus of this discussion .
(An additional membrane set, the outer membrane, may also be present
between the cortex and coat [9], and it is not
illustrated in Fig . 1.) The coat has critical roles
in protecting the spore from a variety of toxic molecules (6,
25, 32, 33,
39, 40) and in facilitating germination
(1, 3, 4,
9) . The coat is very likely to have additional functions
as well, including the ability to act as an elastic material (7,
10) and the ability to perform enzymatic reactions (14,
15, 19, 24,
26) .
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FIG . 1 . Diagram of a B . subtilis spore in cross section . The
outer and inner coat layers are indicated by dark blue and light blue,
respectively . The thick yellow layer is the cortex, which surrounds the
inner membrane, indicated by a black line . At the center is the core,
which is white.
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A COMPLEX ASSEMBLY PROCESS
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We also need to consider how the spore is built . Early in sporulation,
the starving cell's cytoplasm is divided by a specialized septum
into large and small compartments, known as the mother cell and
the forespore, respectively (29) (Fig . 2A) .
Following this, the rim of the septum, where it meets the mother cell
envelope, migrates in the direction of the forespore so as to pinch
off a protoplast . Coat proteins, which are synthesized in the mother
cell cytoplasm, are deposited first on the mother cell side of
the septum and later on the engulfed forespore surface .
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FIG . 2 . Patterns of localization of coat protein-GFP fusions during
spore formation . Spore formation proceeds from top to bottom . The cells
in a row are at approximately equivalent stages . (A) YabP-GFP . Early in
spore formation, immediately after the septum appears, YabP-GFP (blue)
localizes to the mother cell side of the septum (top cell) . Mother cell
(MC) and forespore (FS) compartments are indicated . As forespore
engulfment continues (next cell down), foci (blue dots) appear at the
leading edge of the septum . In fully engulfed forespores, the foci are
located on either side of the short axis of the forespore (next cell
down) . YabP-GFP is delocalized prior to mother cell lysis (bottom cell) .
(B) YheD-GFP . After engulfment, four foci (pink) are present, on either
side of the short axis of the forespore, suggesting that there are two
rings (upper cell) . The sizes of the dots indicate relative levels of
fluorescence . These rings become a contiguous shell (lower cell) . (C)
YutH-GFP . A focus (orange) is present after septum formation (top cell) .
This focus becomes a ring after forespore engulfment (next cell down)
and then a cap at the mother cell pole (next cell down) . Finally, the
cap becomes a complete shell (bottom cell).
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Initial interest in the coat as a model system for macromolecular
assembly was motivated in large part by its morphological complexity
(1, 38) . In B . subtilis,
thin-section electron microscopy has revealed multiple layers in the
coat, which are organized into two major sets: the lightly staining
inner coat, which has distinctive fine lamellae, and a darkly
staining outer coat, whose sublayers are relatively coarse . Analysis
of the coat surface by scanning electron microscopy (5,
8) and atomic force microscopy (7) has
shown that the predominant features are ridges, most of which
run along the long axis of the spore . Not surprisingly, the coat is
biochemically complex . It is composed largely of protein (22)
and is comprised of as many as 60 polypeptide species (23,
24), most of which are unique to the bacilli and
clostridia . From studies of about 30 of the B . subtilis coat
proteins, a preliminary model of coat assembly has emerged (9,
11, 17, 36) .
This model reveals the importance of a subset of coat proteins that
guide the deposition of coat proteins from the mother cell cytoplasm
into the appropriate coat layers, as well as direct the appearance of
the ridges on the coat surface (7) . One of these
morphogenetic proteins (SpoIVA [12, 31,
35]) attaches the coat to the underlying spore
surface; another (CotE [41]) is responsible for
nucleating the formation of the outer layer . From cell biological
analyses, it is clear that in many if not most cases, each species of
coat protein ultimately forms a shell around the forespore or, in
some cases, an incomplete shell, resulting in what appear to be caps
at the forespore poles (12, 13) .
The interconnections between coat proteins are also becoming better
understood . We now know the identities of several directly
interacting coat proteins (16, 42) and two
of the coat proteins on the surface (20,
27) . Overall, the model defines the major morphological steps in
coat formation, identifies a significant part of the complex network
of coat protein interactions, and pinpoints coat proteins with
pivotal roles in assembly .
As satisfying as the model is, it tells only part of the story . For
example, it gives little insight into the precise sequence of events
that occur during the deposition of any given coat protein . Detailed
descriptions of these steps are essential, as it is at this level
that we will ultimately understand the molecular interactions that
drive coat assembly . van Ooij et al . used meticulous light
microscopic examination and genetic manipulation to address this
deficit . They built strains bearing fusions of the recently
identified coat proteins YabP, YheD, and YutH (13)
to the green fluorescent protein (GFP) and monitored their assembly
into the coat over time by using fluorescence microscopy . The
striking result is that these proteins do not coalesce into shells in
one step . Instead, and quite unexpectedly, the localization pattern
of each protein is distinct and dynamic . YabP-GFP, which is
synthesized just after the cell divides into mother cell and
forespore compartments, localizes to the septum (Fig .
2A) . As septum migration proceeds, however, relatively intense
foci of fluorescence, in addition to the layer of fluorescence at the
septum, appear at the leading edge of the engulfing septum, where it
meets the mother cell . After engulfment is complete, the two foci
reside opposite each other, along the short axis of the forespore .
This suggests the presence of a ring, which appears as two dots when
it is viewed in projection . The hypothesis that the two foci are part
of a ring of fluorescence in three dimensions was confirmed by
deconvolution methods . In addition to this ring, a shell of
fluorescence encircling the spore appears at this stage . Formation of
the shell, but not formation of the ring, depends on a coat protein
encoded by a gene immediately upstream of yabP, called YabQ (2) .
Intriguingly, YabP-GFP is lost from the forespore prior to release
from the mother cell .
Even more striking than the localization of YabQ is the localization
of YheD . After forespore engulfment, YheD-GFP localizes to four
foci that appear to correspond to two rings that are parallel to, but
on either side of, the short axis of the forespore (Fig .
2B) . The mother cell pole-proximal foci are brighter than the
foci towards the forespore pole . Later, a contiguous shell surrounding
the forespore appears, and the rings are lost . The final fusion
that was examined, YutH-GFP, shows yet a third distinct pattern of
localization . Early in sporulation, a sole focus appears at the
septum (Fig . 2C) . After engulfment, two foci at the extreme
mother cell pole of the forespore appear, suggesting that there
is a ring at this polar location . Soon after this, the ring becomes a
cap . Finally, a shell covering most of the forespore forms .
These remarkable patterns of localization raise a number of
intriguing questions . First, what mechanisms guide the dynamic
deposition of these proteins? Although as-yet-unidentified proteins
are likely to be involved in the initial localization of at least
some coat proteins (30) (and, in the case of YabP, were
specifically shown to be involved by van Ooij et al.), there is
still the question of how any protein can distinguish one location on
the forespore surface from another location . Perhaps the process of
forespore engulfment is in some manner intrinsically asymmetric,
leaving behind telltale markers of position that are recognized by
coat proteins . Regardless of how coat proteins initially target to
specific locations on the forespore surface, it seems that different
mechanisms guide the formation of rings that appear after initial
deposition . The latter events may be the result of the intrinsic
properties of the proteins in question, as well as the overall
dynamics of coat assembly .
A second question is the purpose, if any, of the dynamic aspect of
coat assembly characterized by van Ooij et al . One possibility is
that the properties of the coat would be fundamentally different if
the coat proteins were not assembled in these patterns . Alternatively,
these patterns may not so much be critical to the final functioning
of the coat but rather may form part of a process that coordinates
the simultaneous assembly of a large number coat proteins and
prevents deposition of one coat protein species from interfering with
deposition of another coat protein species . In any event, it is clear
that an additional set of rules needs to be elucidated if we are to
fully understand how the spore builds its outer shells . Like the
early electron microscopy studies that identified the coat layers and
analyses of the coat surface that showed the presence of ridges, the
observations of van Ooij et al . uncover yet another layer of
regulation of assembly that demands molecular characterization . As a
model for macromolecular assembly, the coat is still full of
surprises .
I thank Jean Greenberg, David Keating, and Maike Müller for helpful
suggestions .
Work in my laboratory is supported by grants GM53989 and AI53365
from the NIH .
* Mailing address: Department of Microbiology and Immunology,
Loyola University Medical Center, 2160 South First Avenue, Maywood, IL 60153 .
Phone: (708) 216-3706 . Fax: (708) 216-9574 . E-mail: adriks@lumc.edu .
The views expressed in this Commentary do not necessarily
reflect the views of the journal or of ASM.
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