From Rings to Layers: Surprising Patterns of Protein Deposition during Bacterial Spore Assembly

Elucidation of the mechanisms of assembly of subcellular structuresis often an elusive goal. The large number of component partsand the intricate ways in which they interact make experimentalanalysis a significant challenge and, in particular, frustrateattempts at traditional reductionist analysis. Frequently, keyinsights come from thoughtful cell biological observations thatidentify the most important steps in assembly. In the best cases,not only do these observations provide critical clues to oldproblems, but they also generate novel areas of investigation.An example of a finding that does both of these things comesfrom the work of van Ooij et al. (36a), published in this issue,who used careful cell biological analysis to uncover a noveldimension of the process by which bacterial spores assembletheir external armor plating. These authors found that the depositionof external proteins on the spore surface is unexpectedly dynamic,pointing towards as-yet-unknown regulatory mechanisms controllingspore formation.

SPORE FUNDAMENTALS

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Spore fundamentals

Bacterial spores are marvels of nature. They are formed by bacilliand clostridia in response to starvation during an approximately8-h developmental process, called sporulation, that is controlledby a complex cascade of cellular events (29). The result isa dormant, highly resistant cell that can endure almost anystress that nature has to offer. Spores can persist in the dormantstate 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 reintroductionof even minute amounts of nutrients to the milieu. The resultis the almost immediate conversion of the spore back to an activelygrowing cell, a process known as germination.

These remarkable properties have motivated intensive study ofspore formation and resistance since the first published descriptionof spores in 1874 (21). With the advent of molecular approachesand, subsequently, advanced cell biological approaches, sporeformation has become a well-developed model for elucidatingfundamental mechanisms of development and cellular assembly(34). It is in the context of the assembly of the outermostspore structures that van Ooij et al. have made their contribution.

To appreciate these findings, a brief review of Bacillus subtilisspore ultrastructure is needed. All Bacillus spores have a commonarchitecture (1, 18) (Fig. 1). At the center is the core, arelatively dry compartment that houses the spore DNA. Surroundingthis is a membrane (called the inner membrane) and then a thicklayer of peptidoglycan called the cortex. The cortex, in turn,is encased in a complex protein shell called the coat, whichis the focus of this discussion. (An additional membrane set,the outer membrane, may also be present between the cortex andcoat [9], and it is not illustrated in Fig. 1.) The coat hascritical roles in protecting the spore from a variety of toxicmolecules (6, 25, 32, 33, 39, 40) and in facilitating germination(1, 3, 4, 9). The coat is very likely to have additional functionsas 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.

A COMPLEX ASSEMBLY PROCESS

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A complex assembly process

We also need to consider how the spore is built. Early in sporulation,the starving cell's cytoplasm is divided by a specialized septuminto large and small compartments, known as the mother celland 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 offa protoplast. Coat proteins, which are synthesized in the mothercell cytoplasm, are deposited first on the mother cell sideof 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).

Initial interest in the coat as a model system for macromolecularassembly was motivated in large part by its morphological complexity(1, 38). In B. subtilis, thin-section electron microscopy hasrevealed multiple layers in the coat, which are organized intotwo major sets: the lightly staining inner coat, which has distinctivefine lamellae, and a darkly staining outer coat, whose sublayersare relatively coarse. Analysis of the coat surface by scanningelectron microscopy (5, 8) and atomic force microscopy (7) hasshown that the predominant features are ridges, most of whichrun along the long axis of the spore. Not surprisingly, thecoat 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, apreliminary model of coat assembly has emerged (9, 11, 17, 36).This model reveals the importance of a subset of coat proteinsthat guide the deposition of coat proteins from the mother cellcytoplasm into the appropriate coat layers, as well as directthe appearance of the ridges on the coat surface (7). One ofthese morphogenetic proteins (SpoIVA [12, 31, 35]) attachesthe 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 notmost cases, each species of coat protein ultimately forms ashell around the forespore or, in some cases, an incompleteshell, resulting in what appear to be caps at the foresporepoles (12, 13). The interconnections between coat proteins arealso becoming better understood. We now know the identitiesof several directly interacting coat proteins (16, 42) and twoof the coat proteins on the surface (20, 27). Overall, the modeldefines the major morphological steps in coat formation, identifiesa significant part of the complex network of coat protein interactions,and pinpoints coat proteins with pivotal roles in assembly.

UNANSWERED QUESTIONS

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Unanswered questions

As satisfying as the model is, it tells only part of the story.For example, it gives little insight into the precise sequenceof events that occur during the deposition of any given coatprotein. Detailed descriptions of these steps are essential,as it is at this level that we will ultimately understand themolecular interactions that drive coat assembly. van Ooij etal. used meticulous light microscopic examination and geneticmanipulation to address this deficit. They built strains bearingfusions of the recently identified coat proteins YabP, YheD,and YutH (13) to the green fluorescent protein (GFP) and monitoredtheir assembly into the coat over time by using fluorescencemicroscopy. The striking result is that these proteins do notcoalesce 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 intomother cell and forespore compartments, localizes to the septum(Fig. 2A). As septum migration proceeds, however, relativelyintense foci of fluorescence, in addition to the layer of fluorescenceat 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 axisof the forespore. This suggests the presence of a ring, whichappears as two dots when it is viewed in projection. The hypothesisthat the two foci are part of a ring of fluorescence in threedimensions was confirmed by deconvolution methods. In additionto this ring, a shell of fluorescence encircling the spore appearsat this stage. Formation of the shell, but not formation ofthe ring, depends on a coat protein encoded by a gene immediatelyupstream of yabP, called YabQ (2). Intriguingly, YabP-GFP islost from the forespore prior to release from the mother cell.

Even more striking than the localization of YabQ is the localizationof YheD. After forespore engulfment, YheD-GFP localizes to fourfoci that appear to correspond to two rings that are parallelto, but on either side of, the short axis of the forespore (Fig.2B). The mother cell pole-proximal foci are brighter than thefoci towards the forespore pole. Later, a contiguous shell surroundingthe forespore appears, and the rings are lost. The final fusionthat was examined, YutH-GFP, shows yet a third distinct patternof localization. Early in sporulation, a sole focus appearsat the septum (Fig. 2C). After engulfment, two foci at the extrememother cell pole of the forespore appear, suggesting that thereis a ring at this polar location. Soon after this, the ringbecomes a cap. Finally, a shell covering most of the foresporeforms.

These remarkable patterns of localization raise a number ofintriguing questions. First, what mechanisms guide the dynamicdeposition of these proteins? Although as-yet-unidentified proteinsare likely to be involved in the initial localization of atleast some coat proteins (30) (and, in the case of YabP, werespecifically shown to be involved by van Ooij et al.), thereis still the question of how any protein can distinguish onelocation on the forespore surface from another location. Perhapsthe process of forespore engulfment is in some manner intrinsicallyasymmetric, leaving behind telltale markers of position thatare recognized by coat proteins. Regardless of how coat proteinsinitially target to specific locations on the forespore surface,it seems that different mechanisms guide the formation of ringsthat appear after initial deposition. The latter events maybe the result of the intrinsic properties of the proteins inquestion, as well as the overall dynamics of coat assembly.

A second question is the purpose, if any, of the dynamic aspectof coat assembly characterized by van Ooij et al. One possibilityis that the properties of the coat would be fundamentally differentif the coat proteins were not assembled in these patterns. Alternatively,these patterns may not so much be critical to the final functioningof the coat but rather may form part of a process that coordinatesthe simultaneous assembly of a large number coat proteins andprevents deposition of one coat protein species from interferingwith deposition of another coat protein species. In any event,it is clear that an additional set of rules needs to be elucidatedif we are to fully understand how the spore builds its outershells. Like the early electron microscopy studies that identifiedthe coat layers and analyses of the coat surface that showedthe presence of ridges, the observations of van Ooij et al.uncover yet another layer of regulation of assembly that demandsmolecular characterization. As a model for macromolecular assembly,the coat is still full of surprises.

ACKNOWLEDGMENTS

I thank Jean Greenberg, David Keating, and Maike Müllerfor helpful suggestions.

Work in my laboratory is supported by grants GM53989 and AI53365from the NIH.

FOOTNOTES

* 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{at}lumc.edu.

The views expressed in this Commentary do not necessarily reflectthe views of the journal or of ASM.

REFERENCES

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