The role of electron microscopy in the elucidation of bacterial structure and function.

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Ann. Rev. Microbial 1979. 33:459-79 Copyright @ 1979 by Annual Reviews Inc. All rights reserved

THE ROLE OF ELECTRON

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Annu. Rev. Microbiol. 1979.33:459-479. Downloaded from www.annualreviews.org by Boston University on 07/07/13. For personal use only.

MICROSCOPY IN THE ELUCIDATION OF BACTERIAL STRUCTURE AND FUNCTIONl J. W. Costerton Department of Biology, University of Calgary, Calgary, Alberta, Canada, T2N IN4

CONTENTS INTRODUCTION . . . . . . . . . . . .. . .. .. . . .. . . . . .. . . ... CHROMATIN . .. . ... . . . . . .. . . . . . .. . CYTOPLASM . . . .. . . . . . . . . .. . CYTOPLASMIC MEMBRANE.................................................................................... CELL WALL .. .. . . .. . . . . EXTRACELLULAR STRUCTURES . . . . . . . . . . .. BACTERIAL APPENDAGES ...................................................................................... CELL FRACTIONATION . . . . . . . . .. . . LOCALIZATION TECHNIQUES ................................................................................ EPILOGUE . ... .... . . . ..... . . . ..

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459 461 464 466 469 472 473 475 475 477

INTRODUCTION The improvement in resolution made available to the biologist by the devel opment of the electron microscope was dramatic indeed. The advance was most dramatic in microbiology, where the improvement from the effective 250-nm resolving power of the light microscope to the effective O.5-nm resolving power of the electron microscope changed our usual perception of the bacterial cell from a vaporous sausage to a highly structured cell with highly ordered appendages. To be fair, one must record that meticulous attention to detail by innovative microbiologists like Carl Robinow (67) IThis review is affectionately dedicated to Robert G. E. Murray who did many of these things. 459

0066-4227/79/1001-0459$01.00


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produced elegant images that revealed some detail in fixed cells, but gener ally, light microscopy served best by revealing the shape and behavior of living cells and its value persists in these studies. Balancing the improved resolution of the electron microscope was a series of problems associated with preparing living cells for introduction into its high vacuum in a state able to resist extreme heating and yet sufficiently thin to produce a shadowgram by using electrons with relatively low penetrating power. This was accomplished by immobilizing and tanning the cell con tents by fixation, initially in oxidative fixatives such as permanganate and osmium tetroxide, and more recently in aldehyde fixatives that depend largely on molecular cross-linking. The contents of aldehyde-fixed cells contained too few heavy metal atoms to deflect electrons and produce a well-defined image, and therefore staining with osmium, uranium, and lead salts was required. Problems of dehydration and embedding were gradually solved, and the fixed and stained cells were cut into sections as thin as 30 nm while firmly embedded in heat-resistant plastics. The obvious result of this complex series of chemical indignities is that many of the more delicate cellular structures are profoundly altered, and practitioners must anticipate and interpret these artifacts of preparation. Thus, the high-resolution forms of electron microscopy that examine thin sections of chemically fixed cells or dry depositions of structures thin enough to produce an image must often look for confirmation to material prepared by alternate methods, or to indirect biochemical or biophysical evidence. An excellent example is the preparative method developed by Moor & Miihlethaler (55), in which cells are frozen and cleaved, and after some sublimation of the enveloping ice, a replica of the cleaved surface is made and examined. This freeze-etching technique is enormously valuable, because although it is subject to its own artifacts of freezing and eutectic deposition (23), the cells can be prepared without chemical fixation and thus fixation and dehydration artifacts are avoided. Conclusions based on elec tron microscopy should be supported by biochemical and biophysical exam inations of live cells, wherever possible, and enzyme penetration evidence for membrane intactness (18) and X-ray analysis of hydrated cell compo nents (56) provide illustrative examples. We must also remember that elec tron microscopy can be invoked at different points in a study, using different methods, as when the chromosome-membrane association seen in sections of whole cells (69) was confirmed when Worcel & Burgi (86) isolated the chromosome and showed that it bore a membrane fragment clearly identifi able in negatively stained preparations. Generally, conclusions based on electron microscopy have agreed well with each other and with those based on other data, and this elegant technique has contributed the ability to examine individual cells, structures, and processes that would otherwise be lost in the heedless averaging charac-

BACTERIAL ULTRASTRUCTURE AND FUNCTION

461

teristic of more analytical biochemical methods. I propose to survey some of the important elements that comprise the bacterial cell and to aSsess the role of electron microscopy in building our present understanding of their structure and function.


CHROMATIN Both the existence and the circular nature of the bacterial chromosome had been anticipated by geneticists (43) before Kleinschmidt & Zahn (44) devel oped techniques that allowed its demonstration by using autoradiography and low-power light microscopy (11). Similarly, the sequential production of mRNA molecules along a length of bacterial DNA was anticipated long before the elegant visualization (32) of the process by electron microscopy. On the other hand, the intricate supercoiling of the bacterial chromosome had not been proposed before this structure was preserved by cell breakage under specific conditions and by careful electron microscopy (20). Jacob et al (42) suggested that the circular bacterial chromosome was replicated at an initiation point that corresponded to a membrane attachment point before Worcel & Burgi's demonstration (86) that the replicating bacterial chromosome is associated with the cytoplasmic membrane. Genetic evi dence for this association followed (87), as did evidence that the supercoiled structure of the chromosome was stabilized by association with the mem brane (25). Thus electron microscopy has confirmed the conclusions of genetics and cell biology with respect to the structure and function of the bacterial chromosome, and geneticists now detect the presence of plasmids by their circular appearance in cell lysates (15) and even assess their molecular size by direct measurement of their length in electron micro graphs (62). In more sophisticated studies annealed DNAs from different sources can be examined by electron microscopy to detect and quantitate non-complementary sequences (8) and thus shed some light on mechanisms of integration of extraneous DNA into the genome (51). A more vexing problem concerns the distribution of the very extensive DNA strand within the bacterial cell, and here the gel-like nature of this chromatin has created a will-o'-the-wisp. By light microscopy of living cells, Whitfield & Murray (82) showed that bacterial chromatin, which is dis persed throughout well-defined areas of the cytoplasm (52), could be sharply condensed by ionic manipulation. Since that time, electron micro scopists have accepted different degrees of condensation, from highly con densed electron-dense masses (Figure 1), to the skein-like fibrillar mass (Figure 2) produced by fixation by Ryter & Kellenberger's method (70), to the fine dispersed fibers seen in aldehyde-fixed cells (Figure 3), as being representative of the distribution of chromatin in the live cell. But freeze etching of unfixed cells (Figure 4) rarely shows any evidence of chromatin


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Figure 1

Section of cells of a Caryophanon species fixed in osmium in phosphate bulfer. Note the condensation of the chromatin to form electron-dense masses. The bar in this and subse

quent micrographs indicates 100

Figure 2

nm.

Section of cells of Myxococcus xanthus fixed in osmium by the method of Ryter

&; Kellenberger (70). Note the condensation of the chromatin to form a skein-like mass in the center of the cells and the production of mesosomes (arrows).

463



Figure 3

Section of a cell of Pseudomonas aeruginosa fixed in glutaraldehyde in cacodylate

buffer. Note the condensation of the chromatin to form a fine fibrillar mass, the resolution of aggregates of ribosomes in the cytoplasm, and the tripartite nature of both the cytoplasmic membrane and outer membrane.


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COSTERTON

at all (85), even when the cytoplasm is not disordered by sublimation (23), which indicates that chemical fixatives cause condensation and that chromatin is so dispersed in the living cell as to yield no aggregates thick enough to be resolved in freeze-etched cells. The conclusion thus forced upon us-that bacterial chromatin is con densed by chemical fixatives and by ionic manipulation (84)-has at least two disturbing sequellae. First, the organization of the cell must be pro foundly altered by the condensation of such a large linear structure that was previously distributed throughout large areas of its cytoplasm. Second, the condensation of the chromatin net must be expected to pull on the cytoplas mic membrane at the point where these structures are attached (69), and the membrane, having no tensile strength and no resistance to inward deformation, must be expected to invaginate. This deformation may be increased by direct effects of the fixative on the membrane (36). If these mechanisms are responsible for the formation (34) of mesosomes (Figure 2), one valuable conclusion can be salvaged from an otherwise depressing story of artifacts in that mesosomes would indicate actual chromosome membrane contact points and could be used to locate and quantitate these structures. It must be noted that this theory of the origin of mesosomes is by no means universally accepted (31). CYTOPLASM The cytoplasm is always defined somewhat negatively as the part of a bacterial cell that lies between the intensely productive chromatin and the very active enzymatic zone at the inner face of the cytoplasmic membrane. Because photosynthetic (46), chemosynthetic (80), and even specialized . heterotrophic (14) membranes are now known to be derived from and often connected to the cytoplasmic membrane (46), the cytoplasm seems only to consist of ribosomes, depots of stockpiled molecules, and enzymes not yet persuaded to hang on to membranes during cell breakage. All of this notwithstanding, the cytoplasm is fascinating once we understand the bases of the images seen in electron microscopy. In fixed and sectioned cells the cytoplasm appears to be tightly packed with aggregates of ribosomes, but this is largely a product of the superposition of these circa lO-nm bodies within the circa 50-nm sections, and very thin sections (Figure 3) or direct examination of the cytoplasmic cleavage plane in unsublimed freeze-cleaved cells shows very few ribosomes (Figure 4R) and much of the cytoplasm is nonparticulate. However, cytoplasmic detail is only very poorly preserved in freeze-etched cells, because the sublimation of ice from the frozen cyto plasm leaves the particulate elements in a collapsed and disorganized pile (Figure 5A). Very early examinations of lysed cells by van Iterson (75)


Figure" Replica of a freeze-cleaved preparation of cells of a marine pseudomonad. In this technique the frozen cells are cleaved and then replicated before any sublimation of ice can take place. Note the presence of a few ribosomes (R) in the cross-cleaved cytoplasm of one cell and the protein studs in the concave ( ) and convex ( ) cleavages of the cytoplasmic membrane. $ ,Angle of shadowing. �

Figure 5

�

Replica of a freeze-etched preparation of an Escherichia coli cell showing a major

cleavage plane in the cytoplasmic membrane (�, a weak cleavage plane in the outer mem brane (0), and a disordered pile of ribosomes (A) in the sublimed cytoplasm.


466

COSTERTON

suggested the presence of a fine fibrous net in the bacterial cytoplasm, and the recent discovery of an actin-like fibrous system in the bacterial cyto plasm (54) and a similar structure that controls chromatin distribution in the eukaryotic nucleus (37) should renew our interest in a central question: Are the organization of the cytoplasm and the distribution of chromatin really random in the bacterial cell? Randomness is in rapid decline in biological systems and the bacterial cytoplasm may reveal an elaborate secondary level of order when fixation methods are developed that prevent chromatin condensation and cytoplasmic collapse. Discrete cytoplasmic inclusions vary from very highly ordered gas va cuoles, through paracrystalline structures and membrane-enclosed vesicles, to the somewhat ephemeral cytoplasmic lipid concentrations often ex tracted by the organic solvents used in dehydration and not completely frozen by the -100°C temperatures used for freeze-etching. Before gas vacuoles were isolated and chemically characterized (78) their paracrystal line protein composition was anticipated, because of their regular non deformable shape (9) and their failure to produce a membrane-like tripartite (dark-light-dark) profile in sections or a cleavage plane in freeze-etched cells (77). Similarly, the lack of a tripartite profile in sections (Figure 6) and of a cleavage plane in freeze-etched material (Figure 7) showed the absence of an enclosing membrane in some carbohydrate (45) and protein (3) deposits and its presence in others (48). It is of considerable interest that the presence around carbohydrate deposits of the enzymes active in the deposition and mobilization of these structures may be indicated by a thin electron-dense "rim" (45) seen in sectioned material (Figure 6, arrow). Although valuable insights into the chemical nature of cytoplasmic inclusions may be deduced from such morphological data as fluid behavior at low temperatures (lipids), low contrast in electron micrographs (carbohydrate), high contrast and a tendency to volatilize on heating (phosphates), paracrystalline organization and globular subunits (protein), and the application of the few cytochemical techniques adapted for electron microscopy, perhaps electron microscopy serves best by monitoring the isolation and purification of these inclusions (45) so that their chemical nature can be determined by analytical methods. CYTOPLASMIC MEMBRANE The bacterial cytoplasmic membrane was first detected by electron micros copy of whole cells (68) and sections (12), and its tripartite structure subse quently was seen in electron micrographs of sectioned cells (58). Initially, this layered tripartite image supported erroneous membrane models in which protein was thought to be layered on either side of a bimolecular



Figure 6

467

Section of a partly disrupted cell of Clostridium pasteurianum showing the electron

dense rim (arrow) surrounding the large cytoplasmic polyglucose inclusions.

Figure 7

Replica of a freeze-etched preparation of Clostridium pasteurianum cell showing

the absence of cleavage planes in the polygiucose inclusions.


468

COSTERTON

leaflet composed of phospholipids (66). Later, Pinto da Silva & Branton (64) showed that membranes are medially cleaved in freeze-etched cells, and Fox (28) showed that the studs (Figures 4 and 5) seen in these medial cleavage planes are hydrophobic protein entities that traverse the membrane, so that electron microscopy contributed significantly to Singer's effective working model (73) of the molecular architecture of membranes. Now that we understand the significance of the studs in the cytoplasmic membrane cleavage plane (Figures 4 and 5) we have a useful means of locating proteins in face views of the membrane, and we can follow the exclusion of proteins from the liquid-crystal domains that form in bacterial membranes (76) at the transition temperature. One of the most useful products of this membrane work was the intense concentration of biochem istry, biophysics, physiology, and morphology on a single structure so that certain basic principles were established. One of these was that a well ordered and continuous bimolecular leaflet of phospholipids (or even of lipids) provides the linear weakness at low temperatures that produces a strong cleavage plane in freeze-etched cells. This principle can be neatly reversed to conclude that structures that provide no such cleavage plane (e.g. gas vacuoles) lack a planar phospholipid bilayer or that structures that cleave only poorly [e.g. the outer membrane (Figure 50) in many Gram negative cells] have lipid bilayers that are less planar or less continuous (41) than that of the cytoplasmic membrane. Thus, morphological data like the strength of the cleavage plane or the presence of studs in this median cleavage are important factors in the resolution of the structure and func tion of bacterial membranes. The bacterial cell resembles the plant cell in that its shape is maintained by an inelastic wall structure (81) against which the relatively maleable cytoplasmic membrane is adpressed by osmotic pressure when the cell is in a normal osmotic environment. In plants this osmotic pressure pushes the membrane into pockets in the cell wall, and the resultant protruding ectode smata may almost traverse this thick structure. The bacterial cytoplasmic membrane is pushed against the peptidoglycan of the cell wall (18) in hypotonic milieux and is withdrawn from this contact when the cell is plasmolyzed, in a hypertonic milieu, leaving the structures connected only at a variable number of adhesion points (1). Therefore it is possible mentally to visualize the state of the cytoplasmic membrane as it is pressed against the cell wall in the live cell, rather like a large balloon in a small wicker basket, and to postulate the effects of loss of osmotic pressure upon fixation and of the contraction of the chromatin connected to its inner surface at one or more points. Further, the cytoplasmic membrane is a fluid and dynamic structure in which most of the components are capable of rapid lateral movement (73), and interpretation of the distribution of the mem-



469

brane itself and its components must encompass the quicksilver nature of this structure and the effects of chemical fixatives and freezing. With these reservations in mind it is interesting to examine the valuable and unique contribution of electron microscopy to the study of the bacterial cytoplasmic membrane, because this structure, perhaps more than any other, coincides with the fovea centralis of biochemical methodology. In the eukaryotic cell, most important physiological processes are concentrated in organelles that can be isolated with minimal loss of enzymes and studied separately, but energy transduction, protein synthesis, photosynthesis, chemosynthesis, macromolecular transport, and many other processes are carried on in the bacterial cytoplasmic membranes and its connected deriva tives and the location of these processes in the mosaic of the membrane is beyond the unaided resolution of biochemical methods. Under normal os motic conditions the cytoplasmic membrane must enclose the cell at its perimeter, and it can then increase its area, to satisfy cellular demand for more space for such hydrophobic activities as photosynthesis, by simply invaginating to form membrane systems that ramify in the cytoplasm and may be detached to form virtually independent entities (38). The limitation of lateral component flow that produces such well-defined patches as the purple membrane of Halobacterium halobium (40) may involve the associ ation of protein components with each other and with neighboring rigid structures so that they form a raft that is moored in an essentially fluid system. Specialized domains are formed in photosynthetic and chemosyn thetic organisms, and these extensions of the cytoplasmic membrane often take the form of very extensive battery plates in the cytoplasm (80); thus it is important to remember that the noncytoplasmic spatial component of these structures constitutes the convoluted lumen of the basic membraneous invagination and communicates with the external milieu. Many other spe cialized domains are simply located in the cytoplasmic membrane at the cell perimeter (e.g. purple membrane), whereas other membrane processes (e.g. cell wall deposition) must be located at specific sites such as developing septa (35). It is clear that the localization of specific enzymes, and thus the localization of the processes they control, within the convoluted and fluid expanse of the prokaryotic cytoplasmic membrane will require the com bined techniques and imaginations of biochemists and morphologists armed, perhaps, with ferritin-coupled antibodies (see below). CELL WALL The cell wall of the bacterial cell is its life-support system in a wide and changeable variety of hostile environments. Its functions include osmotic protection by the provision of an inelastic girdle, and its layered structural

COSTERTON

470

arrangement has yielded well to electron microscopy so that much of what we know of its molecular architecture is derived from morphological data (22) and from the careful analysis of fractions whose derivation from the cell wall was monitored by electron microscopy

(71). Early in the develop

ment of ultrastructural techniques it became clear that bacterial cell walls are organized in two basic patterns, one of which contains a tripartite membrane in sectioned material, and a cleavage plane in freeze-etched


material, whereas the other does not. The type of cell that has this mem brane in its cell wall is usually negative in the Gram stain, whereas the type that lacks the membrane is usually Gram positive; however, this correspon dence breaks down in some organisms

(13),

notably those in which the

peptidoglycan is very thin or very thick (Figure

(0

in Figure

8a,b,

and

c) that

8).

The outer membrane

occurs in the generally Gram-negative type

of cell wall is very important in the regulation of molecular traffic through the wall, and cells that have this structure show important differences in antibiotic sensitivity, exoenzyme distribution, and toxicity for host animals

(18) so

that the determination of cell wall type is of more than academic

interest. Clearly then, electron microscopy has replaced the Gram the definitive method for the determination of cell wall type

(13),

stain as

and new

descriptive terms for these cell wall types are urgently required. Gibbons

& Murray (29) have proposed a new division (Gracilicutes) of the kingdom Procaryotae for those bacteria whose walls contain an outer membrane and are usually thin-these cells are usually Gram negative. They propose another new division (Firmacutes) for those bacteria whose walls lack the outer membrane and are usually thick-these cells are usually Gram posi tive.

A third new division (Mollicutes) is proposed to accommodate bacteria

lacking a cell wall and therefore uniformly Gram negative. The innermost component of both types of cell wall is the peptidoglycan, which is both robust and commendably avid for heavy metal stains so that it produces a well-defined image in sectioned material even when it is very thin

(P

in Figure

8b).

This layer has been shown to vary in thickness

depending on the metals bound in its interstices (5), and its virtual disap pearance following digestion with lysozyme

(60)

serves unequivocally to

identify it. In most cells with the firmacutes type of wall the peptidoglycan layer is thick (circa

40

)

nm

(Figure

8e) and

teichoic acids are also found

on biochemical analysis of the cell wall. In a recent paper Birdsell et al used concanavalin

A

(7)

to stabilize the teichoic acids and showed by electron

microscopy that they exist as a system of protruding fibers at the surface of the basically peptidoglycan cell wall. This important conclusion illus trates the effective use of the electron microscope in the spatial resolution of the molecular architecture of a complex layered structure.

471


BACfERIAL ULTRASTRUCfURE AND FUNCfION

Figure 8

(a) Gracilicutes wllll of a Pseudomonas aeruginosa cell that has an outer membrane

(0) and a peptidoglycan layer too thin to be resolved. The cell is Gram negative. (b) Gracili cutes wall of a Bacteroides ruminicola cell that has an outer membrane (0) and a thin peptidoglycan layer (P). The cell is Gram negative. (c) Gracilicutes wall of a

Megasphera elsdenii cell that has an outer membrane (0) and a very thick peptidoglycan layer (P). The cell is Gram variable. (d) Finnacutes wall of a ButyriJlibrio ./ibrisolvens cell that lacks an outer membrane and has a thin peptidoglycan wall. The cell is Gram negative. (e) Firmacutes waJl of a Clostridium pasteurianum cell that lacks an outer membrane and has a thick peptidogly can wall. The cell is Gram positive.


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COSTERTON

The outer membrane of the gracilicutes wall was discovered by electron microscopy (6, 58), and much of its molecular architecture can be deduced from morphological data. Its formation of a tripartite image in sectioned material (0 in Figures Sa,b, and c) indicates that it contains a highly ordered bimolecular arrangement of lipids, like that in the cytoplasmic membrane, and its formation of only a very weak cleavage plane (0 in Figure 5) in metabolically active wild-type strains (41) indicates that this ordered layer is either less planar or less continuous than that of the cyto plasmic membrane. The presence of a very large number of studs in the outer membrane cleavage plane (30) indicates that this hydrophobic struc ture is traversed by many proteins, and these proteins have now been purified and extensively characterized (72). Elements of the outer mem brane are exposed at the surface of the gracilicutes wall, and ferritin-coupled antibodies have been used to locate specific components, such as proteins (50) and lipopolysaccharide (57), at the surface of cells exposed by sublima tion of the ice in freeze-etched preparations (Figure 9). This method has been used to assess the pattern of insertion of lipopolysaccharide into the outer membrane (57), after its synthesis has been specifically switched on, and exquisite topographic localization is obtained by this technique. Mor phologists often noted in passing that a periplasmic space was always main tained between the peptidoglycan and the outer membrane in the gracilicutes wall, but we failed to deduce that there must be a spacer protein in the wall and thus were taken by surprise by Braun & Hantke's revelations (10) concerning the periplasmic lipoprotein. Thus electron microscopy has defined the basic layered organization of the bacterial cell wall, located certain components in a topographic mode, and monitored the recovery of specific layers (27) and derived structures (33) for biochemical analysis, so we have begun to evolve a working model (16) of the structure and function of this important cell component. EXTRACELLULAR STRUCTURES The study of bacterial structure by electron microscopy has reached both its zenith and its nadir in the examination of extracellular structures. The paracrystalline arrays of protein molecules at the surface of several species (39) have resisted the insult of chemical fixation and of freezing and, more notably, have resisted the disruptive forces involved in negative staining, to produce elegant micrographs of highly ordered and well-preserved struc tures (59, 79). On the other hand, the exopolysacchrides at the cell surface have not fared at all well in preparative procedures, because they are profoundly deranged by both dehydration and freezing and they lack both inherent electron density and an affinity for heavy metal stains. Bayer &



473

Thurow (2) led the way when they solved both of these problems and showed the true extent of the exopolysaccharide of Escherichia coli, by reacting the exopolysaccharide with specific antibodies that both stabilized the component fibers to prevent collapse during preparation and also im parted sufficient electron density to make them visible (Figure 10). The examination of bacteria in many natural environments indicates that these extracellular structures may be necessary for their survival in the presence of bacteriophage, and for their adhesion to certain surfaces (17). The ex opolysaccharide fibers of the bacterial glycocalyx sometimes pack tightly enough to exclude nigrosin and produce a positive capsule stain, but elec tron microscopy of the antibody-stabilized glycocalyx of several species have shown that many cells that fail to show capsules in the nigrosin stain are, in fact, totally surrounded by an extensive mass of exopolysaccharide fibers (Figure 10). BACTERIAL APPENDAGES Some bacterial appendages are large enough to be seen by light microscopy (stalks, spinae, prosthecae) whereas others are so small that electron mi croscopy is required (flagella and pili), but in all cases the relationship of the appendage to the cell can be established by electron microscopy. For example, stalks and prosthecae are extensions of the cell in which the cell wall forms a projection filled with cytoplasm and lined by the cytoplasmic membrane (63, 65). Flagella are open protein-lattice structures specialized at the proximal end and inserted by means of complex structural modifica tions into specialized sites in the cell wall and cytoplasmic membrane (53), whereas pili ( 61) and spinae (26) are tough protein-lattice structures that insert into the cell by association with the cell wall. The flagellum provides an illustrative example of the use of morphological and biochemical tech niques to define functional molecular architecture. Electron microscopy showed the flagellum to be an open protein-lattice with a strong repeating symmetry, and biochemistry showed the presence of a single protein that was seen to self-assemble (47). Electron microscopy showed the proximal differentiation of the flagellum into a hook, which was then separated from shaft material by differential thermal stability, checked for purity by elec tron microscopy, and analyzed by biochemical techniques to reveal a second self-assembling protein (24). The structure deduced from this data lacked the osmotic barrier and the structural sophistication necessary to function like the eukaryotic flagellum, by force generation along its length, so the presence of ATPase activity was examined by reaction product deposition and was found to be present in association with the basal structure (74). Subsequently, the ring-and-shaft structure of the base of the flagellum (21)


Figure 9 Replica of a freeze-etched preparation of a lipopolysaccharide-deficient rough strain of Escherichia coli that has been reacted with ferritin-conjugated antibody against alkaline phosphatase. The outer membrane (0) is exposed by cleavage and the cell surface (S) is exposed by sublimation to reveal the location of the enzyme by the position of the ferritin molecules

Figure 10

(P).

Section of streptococcal cells of Lancefield group B, type II, that were exposed

to specific antibodies before fixation and staining with ruthenium red. Note the radial arrange ment of exopolysaccharide fibers that comprise a highly ordered glycocalyx around these cells.

BACfERIAL ULTRASTRUCTURE AND FUNCTION

475

and the apparent association of one plate with the rigid peptidoglycan layer inspired the detailed and ingenious experiments that produced the rotary motor hypothesis of flagellar activity (4). From this example we can see how an appendage that is present only intermittently at the cell surface, whose component proteins would be a vanishingly minor component of a cell lysate, can be defined in both structural and functional terms by the cooper


ative use of appropriate electron microscopy and biochemical techniques.

CELL FRACTIONATION One is haunted, as are many colleagues, by the recollection of preparations made "just to check" some fraction of bacterial cell that revealed "cell wall" preparations that contained large numbers of whole cells, or "outer mem brane" preparations that contained large numbers of ribosomes! The most chilling facet was often that exhaustive biochemical studies of these prepa rations had been made and the results had been rationalized to a disquieting degree. The upshot of all of this is simply that the study of the structure and function of the prokaryotic cell involves many fractionation procedures and the complete microbiologist must include ultrastructural controls of his fractionation just as he would include controls in his biochemical protocol. Many important observations were made in the early days of electron microscopy by specialists in descriptive morphology, but modern microbi ology belongs to the person who is as comfortable with an electron micro scope as he is with a spectrophotometer and knows how to interpret the

results of both.

LOCALIZATION TECHNIQUES Two broad classes of techniques have been developed for the precise locali zation of cellular components. The first is the reaction product deposition technique in which the product of an enzyme is precipitated and rendered electron dense (19) so that the position of the enzyme can be deduced, and the second is the labeled antibody technique in which antibodies to a cell component are tagged with an electron-dense marker and reacted with whole cells or sections to locate the antigen (83). If the component to be localized is accessible to added substrate in the reaction product deposition preparation, or if it is located at the cell surface in the labeled antibody preparation, live cells can be used so that fixation effects are only operative after the specific localization reactions have taken place. In this way alkaline phosphatase has been located by reaction product deposition in the periplas mic space of Pseudomonas aeruginosa (Figure 11) and by the use of labeled antibodies (Figure 12) at the cell surface in rough mutants of Escherichia


476

Figure II

COSTERTON

Section of a Pseudomonas aeruginosa cell in which alkaline phosphatase has been

located by deposition of reaction product in the living cell with subsequent fixation and

embedding. Note the predominant location of the enzyme inside the outer membrane (0) within the periplasmic space.

F

/

Figure]2

Section of a cell of a lipopolysaccharide-deficient rough strain of Escherichia

coli

that had been reacted with ferritin-conjugated antibody against alkaline phosphatase before

fixation (as in Figure 9). The location of the enzyme at the outer surface ofthe outer membrane is revealed by the position of the e1ectron-dense ferritin molecules (F).



477

coli (49), and the specific enzymatic and antigenic reactions are uncom plicated by prior fixation of the molecule to be localized. Particularly useful topographical data are obtained when molecules at the surface of live cells are reacted with labeled antibodies, which are then visualized at the cell surface as it is exposed by ice sublimation in freeze-etching (Figure 9). When the enzyme to be localized is not freely accessible to added sub strate, the cells must be fixed to destroy the barrier to the substrate and the fixation with aldehyde fixatives must be sufficiently gentle to retain some enzyme activity (19). When internal molecules are to be localized using labeled antibodies, the cells are gently fixed in aldehyde fixatives so that the reactivity of the antigen is preserved, and the cells are frozen in sucrose and sectioned on an ultracryostat or embedded in a water-soluble plastic and sectioned on a conventional ultramicrotome. These sections contain the antigenically active target molecules at their cut surface and they can be reacted with labeled antibodies to reveal their location, but the resolution of most internal structures is so poor in these unstained preparations that localization is possible only in general terms, e.g. peripheral or cytoplasmic (49, 50). The further elucidation of bacterial structure and function will depend largely on these techniques, and on their subsequent modifications, to locate key enzymes and structural components with some precision and thus to provide the microbiologist with a clear perception of the location of functional units within the structural framework of the cell. One looks forward to the application of these location techniques to the question of specialized domains in the cytoplasmic membrane with some excitement. EPILOGUE When the microbiologist was first presented with the electron microscope he used it, quite reasonably, to describe cellular structures. Perceiving artifacts of preparation he sought to eliminate many, and to understand those that could not be avoided, and this sobering exercise led him to cross-reference his conclusions to biochemical and biophysical data. Slowly the electron microscope has ceased to be a specialists' descriptive tool and has become one of the many instruments that the microbiologist uses to understand both the structure and the function of the bacterial cell.

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