A Note on the Fine Structure of a Spirochaete by A. V. GRIMSTONE

145

A note on the fine structure of a spirochaete By A. V. GRIMSTONE

(From the Department of Zoology, Downing St., Cambridge)

With 3 plates (figs, i to 3) Summary A description is given, based on high-resolution electron micrographs of thin sections, of the structure of a spirochaete from the gut of a cockroach, Cryptocercus punctulatus. There is a central cell-body, the structure of which resembles that of a bacterium. Two kinds of granule are abundant in the cell-body: large ones, 150 to 250 A in diameter, of unknown composition, and small ones, identified as ribosomes, which may be arranged in whorls on the inner surface of the bounding membrane. Numerous small chromatin bodies are present. The bounding membrane of the cell-body is complex in structure. The cell-body is loosely enveloped by a sheath, made up of a thin membrane supporting a coarsely filamentous outer layer. In the space between sheath and cell-body is a loose bundle of 60 to 100 fibres. These are 140 A in diameter and are coiled helically around the cell-body. They resemble bacterial flagella and are thought to be responsible for the movements of the organism. Introduction ALMOST all previous electron-microscope observations on spirochaetes have been made at comparatively low resolution on whole or fragmented organisms. In consequence, although a certain amount of information has been obtained about external morphology, almost nothing is known about the internal fine structure of these organisms. This paper describes the results of a study of sectioned material at relatively high resolution, which has revealed a number of interesting features of the organization of one member of this group.

Material and methods The spirochaete described in this paper comes from the hind gut of the cockroach, Cryptocercus punctulatus. This is a wood-feeding insect found in the Appalachians and elsewhere in North America. It harbours a large and remarkable fauna of symbiotic flagellates (Cleveland, Hall, Sanders, and Collier, 1934), as well as numerous spirochaetes and bacteria. The spirochaetes, although abundant, do not appear to have been described or studied previously; their taxonomic position will be considered later in this paper (see Discussion). Cryptocercus was collected from rotting timber in the woods around Moun- tain Lake Biological Station, Virginia, U.S.A. The spirochaetes were pre- pared for electron microscopy, along with the far more numerous flagellates, by techniques which have previously been successfully used for the flagellates of termites (Gibbons and Grimstone, i960). Gut contents, obtained by [Quart. J. micr. Sci., Vol. 104 pt. 1, pp. 145-53, 1963.] 2421 1 L 146 Grimstone—Fine structure of a spirochaete gently squeezing the abdomen of the cockroach or by removing and opening the gut, were allowed to fall directly into a fixative of the following composition:— sodium acetate (hydrated), 1 -95% 1 , sodium veronal, 2-95% J hydrochloric acid, o-i N ...... o-6 ml sodium chloride, 10% . . . . . 1 ml calcium chloride (anhydrous), 1% . . . 1 ml distilled water ...... 22 ml osmium tetroxide, 2% . . . . . 25 ml This fluid has a pH of about 7-9 and its tonicity is approximately equal to that of the gut fluid. Organisms were fixed for 30 to 60 min at room tempera- ture, dehydrated in a graded acetone series and embedded in araldite epoxy- resin (Glauert and Glauert, 1958). Changes of reagents were made by centrifugation. Sections were stained either in saturated uranyl acetate in 50% ethanol (Gibbons and Grimstone, i960) or by the lead staining method of Millonig (1961). They were examined in a Philips EM 200 electron microscope operated at 60 kV with a 25-/U. objective aperture. For accurate measurements the magnification of the microscope was calibrated with a diffraction grating replica. For light-microscope observations smears fixed in Schaudinn and stained either with iron haematoxylin or by Bodian's protein-silver method (a tech- nique adopted initially for the flagellates, see Kirby, 1950) were found particularly useful. Smears fixed in osmic vapour, hydrolysed in N HC1 at 6o° and stained in Giemsa (Robinow, 1942), were studied in an attempt to detect chromatin bodies. Observations Light-microscope observations Cryptocercus contains more than one species of spirochaete. The organism which will be described here was by far the commonest in all hosts examined. It is about 1 /j, in diameter and ranges in length from about 12 to 25 /x, the average of 20 measurements of fixed organisms being 19 JU.. The body tapers gradually to a point at each end (fig. 1, A). The other spirochaetes in Crypto-

FIG. 1 (plate). A, photomicrograph of spirochaetes fixed in Schaudinn and stained with iron haematoxylin. B, photomicrograph of spirochaetes fixed in Schaudinn and stained by Bodian's silver method. Note the indications of a helical band running round the body. c, electron micrograph of a transverse section, showing cell-body (c) bounded by a membrane (m), the sheath (J), and the fibres (/), here mostly cut transversely. A group of connecting fibres (c/) links the sheath and cell-body. (Stained with uranyl acetate, X 6o,ooo.) D, electron micrograph of an oblique longitudinal section. At the bottom of the picture the section passes near the centre of the cell-body, while at the top it runs almost tangentially to the membrane. Note the densely granular texture of the cell-body and also the lighter areas which are identified as chromatin bodies. Some of the fibres (/) run from one side of the cell-body to the other in a manner suggesting that they follow a helical path. (Lead- stained, X 62,000.) FIG. i A. V. GRIMSTONE Grimstone—Fine structure of a spirochaete 147 cercus are smaller and less common, though they appear to be essentially similar in fine structure. Living organisms occur either free in the gut fluid or attached to flagellates (compare Kirby, 1941; Grasse, 1938). In both cases they undulate vigor- ously, moving fairly rapidly if free. After fixation the body is usually gently flexed (fig. 1, A, B) but does not retain the one or more complete waves which living organisms usually display in movement. It has not been possible to determine whether the bending waves are two- or three-dimensional (see Sequeira, 1956). Silver-stained specimens sometimes have the appearance of being helically twisted or of having a helical ridge or band running round them (fig. 1, B). The origin of this appearance will be apparent from the electron-microscope observations to be described. Staining by a variety of techniques failed to reveal any internal heterogeneity at the light-microscope level. No evidence was found of either transverse septa or chromatin bodies.

Electron-microscope observations The main structural features of the organism can best be seen in transverse section (fig. 1, c). The central cell-body, round in cross-section and about 0-7 fj. in diameter, is loosely enveloped by a structure which will be called the sheath. Between the two is a voluminous space in which there is a number of longitudinal fibres. These structures will be described in turn. The surface of the cell-body is bounded by a membrane, or system of membranes, with a total thickness of 100 or 110 A. This is made up of an inner component 30 to 35 A thick, which stains particularly densely, and an outer, less dense layer with a thickness of 40 to 45 A. Between the two is a light space 20 to 30 A wide (figs. 1, c; 2, c). The difference in thickness and density of the inner and outer components of the membrane is constant and can be seen in sections stained with either lead or uranyl acetate. Some micrographs suggest that the outer layer may itself be triple-layered. At the ends of the organism, where the cell-body decreases in diameter, there are substantial additional membranous layers on the inner surface of the membrane (fig. 3, A). These have a total thickness of about 300 A and extend longitudinally for about 1 fi (fig. 3, B). There are at least 3 of these layers and in transverse sections (fig. 3, A) they appear to have a poorly defined beaded structure. Internally the bulk of the cell-body consists of granules, of which there are two kinds, differing in size and location. Small granules, averaging no A in diameter and somewhat angular in outline, are especially abundant peri- pherally. Where the membrane of the cell-body lies in the plane of the section these small granules can be seen densely packed on its inner surface (fig. 2, F). Frequently they occur in rows or whorls of a dozen or more, resembling in their arrangement the ribonucleoprotein particles, or ribosomes, of the rough-surfaced cytoplasmic membranes of higher cells (fig. 2, A, E). The small granules of the spirochaete are somewhat smaller than the ribosomes of most other cells, including bacteria, but their arrangement in 148 Grimstone—Fine structure of a spirochaete whorls, their density, and their shape, all suggest that they should indeed be regarded as ribonucleoprotein particles. Not all of them are associated with the cell membrane: some lie freely in the deeper parts of the cell-body. The second type of granule is larger, though less constant in diameter (150 to 250 A) and is distributed throughout the cell-body (figs. 1, D; 2, A, G; 3, D, E). These granules are homogeneous in texture, sometimes slightly irregular or angular in profile; they have no characteristic arrangement. After staining with lead most of them are of about the same density as the ribosomes; a few stain particularly densely. They are not stained by uranyl acetate (fig. 1, c). Nothing is known about the composition of these granules, but in their affinity for lead they resemble the glycogen granules of some mam- malian cells (Revel, Napolitano, and Fawcett, i960). Possibly they constitute polysaccharide reserves. The two kinds of granules, set in a fairly dense, more or less homogeneous ground substance, occupy much of the cell-body. They are absent from, and to some extent delimit, numerous rather irregularly shaped lighter areas which occur in the more central parts of the cell-body (figs, i, D; 2, A). These lighter areas measure about o-i to 0-2 /x across and contain small numbers of delicate fibres 20 to 40 A in diameter. The general appearance is similar to that of the chromatin bodies of bacteria (see Glauert, 1962). No internal membranes or other structures have so far been revealed in the cell-body. The sheath typically consists of two components: a delicate inner membrane and a coarse, irregular outer layer (figs. 1, c; 2, B, D, G; 3, c). The membrane, like that of the cell-body, is a triple structure, consisting of 2 more or less dense elements separated by a light space. However, its dimensions and appearance differ from those of the cell-body membrane, the outer dense component being 35 to 40 A thick and always much better defined and easier to demonstrate than the more tenuous inner layer, which is only about 20 A

FIG. 2 (plate). Electron micrographs of sections stained with lead, except D which is of a section stained with uranyl acetate. A, section through the cell-body. The most prominent structures are the large dense granules, set in a somewhat less dense matrix. The lighter areas containing delicate fibres (cb) are identified as chromatin bodies. At the bottom of the picture, where the section passes obliquely through the cell membrane, a row of four ribosomes (r) is seen. ( X 83,000.) B, section showing the texture of the sheath in surface view. Note also the apparent absence of any periodic or other substructure in the fibres (/). ( X 93,000.) c, section showing the membrane of the cell-body clearly resolved into a dense inner layer and a less dense outer layer, separated by a clear space. (x 120,000.) D, enlargement from fig. 1, c, to show more clearly the transversely cut fibres (/), and the delicate connecting fibres (c/) running between cell-body and sheath. (x 100,000.) E, tangential section through the membrane of the cell-body, showing whorls of ribosomes. (X 100,000.) F, tangential section through the membrane of the cell-body, showing densely packed ribosomes. (x 93,000.) c, longitudinal section showing groups of connecting fibres linking the cell-body and the sheath. Note also in the cell-body the abundant large dense granules, with the smaller ribosomes interspersed among them. (X 93,000.) FIG. 2 A. V. GRIMSTONE FIG. 3 A. V. GRIMSTONE Grimstone—Fine structure of a spirochaete 149 thick (fig. 3, c). The gap between the 2 layers is 30 to 40 A wide, giving a total thickness to the sheath membrane of about 90 A. The coarse outer layer of the sheath is about 300 A thick and typically appears to consist of an irregular network of branching filaments (fig. 2, B). In a substantial proportion of organisms this coarse outer layer is lacking: there may be either no outer layer at all (fig. 3, E), or else only a vague indication of a homogeneous layer of low density. The outer layer, where present, is probably sticky, since not infre- quently organisms are found apparently adhering at the points of contact of their sheaths (fig. 3, D). The sheath as a whole is irregular in form and, for the most part, not closely applied to the cell-body. This, of course, might well be an artifact arising during preparation, but it suggests that the adhesion between cell- body and its sheath, if it exists at all, can hardly be very strong. There are, however, occasional regions of close contact between the two (fig. 1, c) and there also exist well-defined structural connexions between them in the form of groups of short fibres, about 50 A thick and 500 A long, running between the membranes (figs. 1, c; 2, D, G). These fibrous connexions are uncommon and have been found in only about 1 % of all micrographs. The fibres which lie in the space between the cell-body and the sheath vary in number from about 60 to 100. They are not uniformly distributed but are grouped in a loose bundle lying to one side of the cell-body (figs. 1, c; 3, D, E). Longitudinal sections (fig. 1, D) suggest that they follow a helical path round the organism, and collectively, together with the overlying sheath, they probably form the basis of the helical ridge seen in silver-stained specimens examined in the light microscope (fig. i, B). The fibres are densely stained by uranyl acetate, but much less densely by lead. They are round and solid in cross-section (fig. 2, D) and have not so far been shown to have any periodic or other substructure. They measure 140 A in diameter. No definite connexions have been found between the fibres and either the sheath or the cell- body, and it is not known where they originate. The other spirochaetes found in Cryptocercus are apparently similar in the essential features of their structure, though they may be considerably smaller. Fig. 3, F shows a very small organism, 0-25 fi in diameter and with only 5 longitudinal fibres.

FIG. 3 (plate). Electron micrographs of sections stained with lead. A, transverse section near the tip of an organism. The cell membrane is thickened internally by the addition of layers of material with a poorly defined beaded structure. ( x 93,000.) B, longitudinal section through the tip of an organism, showing the thickening of the cell membrane. (X 83,000.) c, section showing the typical structure of the sheath. Beneath the coarsely textured external coat there is a membrane (m), made up of a dense outer layer separated by a clear space from a less dense inner component. (X 120,000.) D, two organisms apparently adhering at the points of contact of the outer layers of their sheaths. (X 62,000.) E, section through an organism in which the outer layer of the sheath is lacking. ( X 83,000.) F, a small organism in which there are only 5 fibres (/). As in the organism shown in E, the outer layer of the sheath is lacking. ( x 100,000.) 150 Grimstone—Fine structure of a spirochaete

Discussion The three main components of the spirochaete described here—cell-body, fibres, and sheath—have been demonstrated in whole or fragmented spirochaetes of various genera by a number of previous workers, most of whom have correctly deduced that the fibres lie outside the cell-body but under the sheath (see, for example, Bradfield and Cater, 1952; Swain, 1955, 1957; Czekalowski and Eaves, 1955; Simpson and White, 1961). This basic plan is probably common to all spirochaetes. Structurally the various genera appear to differ chiefly in the size and proportions of the body, the number of fibres, and the regularity and steepness of pitch of the coiling of the fibres around the body. The leptospires, for example, are always tightly coiled and appear to have only one fibre (Bradfield and Cater, 1952; Czekalowski and Eaves, 1955), compared with the rather lax coiling and 60 to 100 fibres of the organism described here. The present study confirms the view that the spirochaetes are to be placed among the bacteria, for while the loose sheath and enclosed fibres of the spirochaete may have no exact parallel elsewhere, the fine structure of the cell-body is in all respects similar to that of bacteria. The densely granular cytoplasmic texture and the ill-defined chromatin bodies are both highly characteristic of bacteria, as is the absence of complex cytoplasmic organelles and membrane-bounded nuclei. The bounding membranes of the spirochaete are not easy to interpret. In Gram-negative bacteria such as Escherichia coli the cell-body is bounded by both plasma membrane and cell wall, the former a unit membrane about 70 A thick, the latter about the same thickness, or thicker, and sometimes also triple-layered (Kellenberger and Ryter, 1958; Glauert, 1962). The membranes at the surface of the spirochaete cell-body cannot readily be inter- preted in these terms. If both cell wall and plasma membrane are, indeed, present it seems necessary to conclude that at least one of them is wholly or partly invisible in the present micrographs, since the total thickness (100 or 110 A) is too small to include both. This is not unreasonable, however, since the plasma membrane of E. coli was originally demonstrated only in 'phage- infected or otherwise abnormal bacteria (Kellenberger and Ryter, 1958) and cannot always readily be seen in normal cells. The membranes bounding the cell-body in the spirochaete are always closely applied to it and smooth in outline, and this suggests that the circular cross-sectional shape of the cell- body may be determined by turgor pressure operating against a relatively inextensible surface layer. This, of course, is the situation in bacteria, where osmotic swelling is prevented by a constraining cell wall (Weibull, 1956; Mitchell and Moyle, 1956). It is therefore not implausible to postulate a similar cell wall bounding the spirochaete body, and it is tentatively sug- gested that it is this which forms at least the outermost part of the triple- layered structure seen in the electron micrographs. However, this is clearly a topic which requires further work, preferably on more readily obtainable spirochaetes, in which it might be possible to correlate electron-microscope Grimstone—Fine structure of a spirochaete 151 observations with experiments on plasmolysis and osmotic swelling, and also with cytochemical studies. It is, of course, possible that the surface membranes of bacteria and spirochaetes are not in any sense homologous. The sheath is not dissimilar from the capsules of some bacteria (e.g. Corynebacterium (Glauert, 1962)), though an underlying membrane is probably not usually present in the latter. Most previous accounts of the fine structure of spirochaetes include a description of the sheath, sometimes referred to as the cell wall or slime layer. The former designation is now seen to be unsuitable, but the latter is probably not inappropriate in view of the known presence of polysaccharides in bacterial capsules. There is also a certain similarity in electron-microscope appearance between the coarse outer layer of the spirochaete sheath and the polysaccharide-containing layer at the surface of some amoebae (Pappas, 1959). The organisms which lack this outer layer can perhaps be regarded as analogous to the non-capsulated strains of, for example, pneumococci. Collectively the surface layers of the spirochaete (that is, the sheath and the membrane around the cell-body) to some extent resemble those of the blue- green algae (Hopwood and Glauert, i960; Ris and Singh, 1961). These also possess a sheath and inner and outer membranes. However, it does not seem particularly useful at present to try to trace detailed 'homologies' between these structures (see Grimstone, 1959). Probably the most interesting feature of the internal organization of the spirochaete described here is the arrangement of ribosomes on the inner surface of the bounding membrane. There is apparently no electron-microscope evidence that this state of affairs obtains in bacteria, except, perhaps, in the large sulphur bacterium, Thiovulum, in which there are deep invagina- tions of the cell membrane bearing granules which have been rather doubt- fully identified as ribosomes (Faure-Fremiet and Rouiller, 1958). However, there is now much biochemical evidence to suggest that the cell membrane of bacteria is a site of protein synthesis (McQuillen, i960), and the 'membrane complex' isolated from protoplasts of Bacillus megaterium, which incorporates amino-acids actively, has been shown by fractionation methods to consist of cell membranes with attached ribosomes (Godson, Hunter, and Butler, 1961). It seems probable, therefore, that the cell membrane both of bacteria and the spirochaete described here plays a role analogous to that of the ergastoplasmic membranes of plant and animal cells. It is not known whether the association of ribosomes and membranes is functionally necessary or advantageous: in the spirochaete, just as in some higher cells, there are many ribosomes lying free in the cytoplasm in addition to those attached to membranes. It should be noted that this association with ribosomes is probably not the only way in which, at the bacterial level of organization, the cell membrane may assume functions performed by intra-cytoplasmic membranes in higher cells: in certain bacteria the cell membrane has been shown to contain respiratory enzymes, and to some extent, therefore, resembles the mitochondrial membranes of plant and animal cells (Marr, i960). 152 Grimstone—Fine structure of a spirochaete Although in the present study chromatin bodies have not been demonstrated by light microscopy, it seems reasonable to identify as such the light, fibre-containing areas in the electron micrographs, on the grounds of their strong similarity in appearance to the chromatin bodies of bacteria (Kellen- berger, Ryter, and Sechaud, 1958; Murray, i960; Glauert, 1962). They differ from those of bacteria in being smaller and more numerous, an observa- tion which agrees with the fact that in another spirochaete Dyar (1947) found that Feulgen staining produced a stippled effect throughout the organism. The bodies described here are on the limit of resolution of the light microscope. The fibres wrapped around the cell-body are the only structures which seem likely to be responsible for the production of movements. It is not difficult to suppose that a bundle of helically wound contractile fibres might be able to produce sinusoidal bending waves, though until it is discovered how and where they are attached to the cell-body or sheath it seems unprofitable to speculate on their precise mode of action. Of more immediate interest is the fact that the diameter of the fibres closely resembles that of bacterial flagella. It is true that they do not show either the tubular structure or apparent 5-fold symmetry demonstrated in the flagella of Salmonella by Kerridge, Home, and Glauert (1962); nevertheless, taking into account the other similarities in cytoplasmic structure, it seems reasonable to suggest that the spirochaetes may have evolved from motile bacteria in which the flagella became coiled round the body and enclosed within the capsule. It remains to consider briefly the systematic position of the organism described here. The symbiotic flagellates of Cryptocercus are closely related to those of termites, so that in seeking relatives of the spirochaete it is natural to turn to those of termites. Spirochaetes are abundant in termites (Damon, 1926; Beckwith and Light, 1927; Kirby, 1941), and those of Calotermes militaris were described in detail by Dobell (1912), who placed them in the genus Treponema. Hollande (1922) removed these to the genus Cristispira but failed to provide adequate evidence of the crista or undulating membrane which characterizes this genus. Other species of Treponema have been described in Calotermes irridipennis (Duboscq and Grasse, 1927). In the broad sense the genus Treponema includes small to medium-sized spirochaetes which are not tightly coiled and lack obvious cytoplasmic differentiations. These characters are possessed both by the organisms which Dobell described and by the organism considered here. Furthermore, the electron micrographs published by Bradfield and Cater (1952) show that the general structure of T. duttoni and T. recurrentis is similar to that of the organism described here. The latter may therefore be placed with reasonable confidence among the treponemes. It is not proposed to assign it to a particular genus within this group or to give it a specific name, since it has not been compared directly with other spirochaetes and too little is known about its staining properties and biochemistry. It is adequately identified, for the time being, as the largest and commonest spirochaete in the hind gut of Cryptocercus punctulatus. Grimstone—Fine structure of a spirochaete 153 I am greatly indebted to Professor L. R. Cleveland, who enabled me to obtain Cryptocercus, and to Miss Audrey M. Glauert for her most helpful comments on the typescript. The work was made possible by grants from the Department of Scientific and Industrial Research.

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