F O R M A T I O N A N D S T R U C T U R E OF T H E

S P O R E O F B A C I L L U S C O A G U L A N S

D. F . O H Y E and W. G. M U R R E L L , D. Phil.

From the Commonwealth Scientific and Industrial Research Organization, Division of Food Preservation, Ryde, New South Wales, Australia

A B S T R A C T

Spore formation in Bacillus coagulans has been studied by electron microscopy using an epoxy resin (Araldite) embedding technique. The developmental stages from the origin of the initial spore septum to the mature spore were investigated. The two forespore membranes developed from the double layer of cytoplasmic membrane. The cortex was progressively deposited between these two membranes. The inner membrane finally became the spore protoplasmic membrane, and the outer membrane part of the inner spore coat or the outer spore coat itself. In the mature spore the completed integuments around the spore proto- plasm consisted of the cortex, a laminated inner coat, and a dense outer coat. No exosporium was observed. The method of formation of the cortex and the spore coats is discussed.

I N T R O D U C T I O N

Following certain nuclear events (38, 39) the early stage in spore formation involves the en- closing of the spore nucleus by two membranes derived from the centripetal invagination of the sporangial cytoplasmic membrane (5). This in- vaginatlon to form the initial spore septum is associated with considerable mesosome develop- ment (5). The association of certain peripheral bodies with cell division and the early stages of spore formation was observed by several workers (Figs. 2 and 3, reference 2; 3; 7; Fig. 14, reference 19; 33) before the cytological structure of these bodies (mesosomes) was clearly revealed by Fitz-James (5) and Giesbrecht (7). Although glimpses of certain stages in the development of the initial spore septum and "wal l" of the fore- spore had been obtained (17, 32-34), it was Young and Fitz-James (38, 39) who clearly demonstrated the origin of the septum and Fitz- James (5) who showed the association of meso- somes with septum development in several Bacilluf cereus strains, Bacillus megaterium, and Bacillus medusa. Hashimoto (16) has shown a similar

development of the septum in Clostridium sporogenes. Thus, the origin of the spore septum is now clear, and considerable detail is available on the struc- ture of the mature spore in several species (2, 4, 14, 16, 20, 26, 32, 34). However, until quite recently very little was understood of the stages between the early development and the complex structure of the mature spore, and of the mode of development of the structural components. In 1962 Young and Fitz-James (40) described in B. cereus var. alesti the formation of the cortex between the two membranes of the forespore. The spore coat apparently was formed external to the outer membrane but within the exosporium.

This study of Bacillus coagulans confirms the origin of the forespore membranes and the develop- ment of the cortex between the two membranes. It also reveals several differences in structure and perhaps in method of formation.

M A T E R I A L S A N D M E T H O D S

The observations reported in this study were made with a very heat resistant strain of B. coagulans

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isolated from c a n n e d cabbage. T h e o rgan i sm was grown in a m e d i u m of 0.5 per cent soya peptone (Oxoid), 0.5 per cent casamino acids (Oxold), 0.1 per cent yeast extract (Oxoid) a nd inorganic salts (final concentra t ions of Na2HPO4, 5 raM; NaC1, 5 raM; MgSO4, 0.125 raM; CaC12, 0.05 raM; MnSO4, 0.005 raM; FeSO4, 0.005 raM), p H 7.4. Synchronously sporing cultures of B. coagulans were obta ined as follows: 14 liters of m e d i u m were inoc- u la ted with about l0 s spores, s t eamed 1 hour to heat-act ivate the spores, and aera ted vigorously with a s t r eam of air for 15 to 16 hours at 50°C; 0.5 liter of this cul ture was added to fresh 14-liter ba tches of the same m e d i u m a n d similarly aerated. Spore

s e p t u m format ion occurred abou t 4 hours after inoculat ion in the final cul ture and spore format ion was complete wi thin 24 hours. Samples (100 ml) were r emoved at 15-min intervals and chilled in ice. Abou t 20 to 30 ml were fixed immedia te ly by the me thod of Kel lenberger , Ryter , and S6chaud (18). T h e fixed mater ia l in agar blocks was dehydra t ed by passage t h rough graded e thanol to 100 per cent and finally e m b e d d e d in Araldi te (I0).

Sections were cu t wi th a Por te r -Blum micro tome fitted with a d i a m o n d knife, f loated onto 20 per cent acetone in water , and picked up on collodion-coated grids a n d examined wi th a Siemens Elmiskop I electron microscope.

Explanation of Figures

SCM, sporangial cytoplasmic or p lasma m e m b r a n e

SPM, spore protoplasmic m e m b r a n e CM, cytoplasmic m e m b r a n e SPC, spore cy toplasm SPS, spore s e p t u m IM, inner m e m b r a n e OM, outer m e m b r a n e

CHR, chromat in , or nuc lea r mate r ia l TRS, t ransverse cell s e p t u m CW, cell wall IC, inner coat OC, outer coat M, mesosome CX, cortex

FIGURES 1 TO 4

Electron micrographs of early stages in the format ion of forespores in cells of B. coagulans fixed with OsO4. Only spore-forming ends of the rods are shown.

FIGURE l

An early stage in the development of the spore s e p t u m showing the involvement of the cytoplasmic m e m b r a n e and mesosome. Note also the dense outer marg in of the cell wall of this organism. X 140,000.

FIGURE 2

A later stage shows complet ion of the spore septum, and the double uni t m e m b r a n e na tu re of the septum. X 110,000.

FIGURE

T h e forcspore is a lmost free in the cy toplasm of the sporangium. T h e two m e m b r a n e s su r round ing the forespore cy toplasm are showing evidence of separat ion at several points (arrows). X 90,000.

~IGURE 4

T h e forespore is free in the cytoplasm of the sporangium. T h e ch roma t in mater ia l of the forespore is evident. T h e m e m b r a n e s are showing separat ion at more points in a vesicle-like fashion. Mesosomes occur at o ther points of the spo rang ium where di- vision m i g h t be expected to occur if sporulat ion had not commenced . The par t iculate debris on the outer surface of the cell wall in Figs. 1 to 4 suggests an outer layer in the vegetat ive cell wall of this species. T h e walls in Figs. 1, 3, and 4 also have a zoned appearance . X 110,000.

112 THE ~OURNAL OF CELL BIOLOGY • VOLUME 14, 1965

D. F. OHY~. AND W, G. MURRELL Spore Formation in B. coagulans 113

To overcome possible permeability difficulties during fixing and staining some mature spores were disrupted by shaking with glass beads before fixa- tion.

R E S U L T S

Development of the Forespore

The progressive development of the forcspore is shown in Figs. l to 4. The spore septum formed

by the invagination of the sporangial cytoplasmic

membrane is composed of two typical Bacillus unit membranes. The typical Bacillus unit membrane described by Fitz-James (5) consists of a dense backbone line bordered by two lighter zones with an over-all thickness of 140 to 150 A. Fitz-James suggests that the inner dense line, typical of a variety of animal cells, is obscured by the dense cytoplasm. The general appearance of the cyto-

FIGURE 5

An enlargement of the left hand portion of the spore septum of Fig. 2 which shows greater detail of the double membrane structure of the spore septum. The connection of the spore septum with the mesosome, in the upper right corner, is only partly resolved. The dense backbone lines of the two membranes show evidence of cross-banding at this stage. The inner line (adjacent to the cytoplasm) of the unit membrane is partly resolved, but is not nearly so dense as the dense backbone lines of the spore septum. X 320,000.

FIGURES 6 To 8

Electron micrographs of thin sections of cells of B. voagulans fixed with OsO 4.

FIGURE 6

A typical dividing vegetative cell shows development of the transverse cell wall. The large mesosome forms part of the invaginating plasma membrane. Cell wall material has been deposited between the invaginating membranes. X 90,000.

FIGURE 7

An oblique transverse section of a sporulating cell which shows well the double mem- brane structure of the spore septum, and the structure of a mesosome which appears to be enclosed within the forespore. The mesosome is probably joined to the spore septum at its centre, but the connecting membranes are poorly resolved. )< 90,000.

FmUnE 8

A well resolved spore septum at a later stage shows complete separation of the two membranes and a small mesosome clearly contiguous with the inner membrane of the forespore. X 120,000.

114 THE JOURNAL OF CELL BIOLOGY • VOLUME 14,, 196~

D. F. OHY• AN]) W. G. MVnR~LL Spore Formation in B. coagulan8 I I~

lq~OtrRE 9

A later stage in spore development than Fig. 8 shows a definite zonation of the cortical material between the two well defined membranes. The middle more dense zone is similar in density to the

plasmic or plasma membrane of B. coagulans is similar, but the over-all width of the plasma membrane is 85 to 105 A comprised of a centre dense line and two outer light zones of 25 to 30 A each, with a thin line next to the cytoplasm of 10 to 15 A thickness (Figs. 2, 4, 5 and 6). The thickness of the double membrane was 170 to 210

(Figs. 2 and 5) compared to 280 to 300 A ((5), his Fig. 16).

The description of the cytoplasmic membrane as used by Fitz-James (5) has been followed closely in this paper. It is appreciated, however, that the "inner dense line" of this membrane and of the inner and outer membrane of the forespore of B. coagulans is resolved in many instances (Figs. 2, 4, 5, 9). In such cases the plasma membrane resem- bles that described by Robertson (25) for animal cells, and by Glauert et al. (9) for B. subtilis. This resemblance is more marked if the lighter zone between the "dense back-bone line" and the cell wall is disregarded. The correct interpretation probably depends upon whether the osmiophilic layers are the "lipid lines" as discussed by Fitz- James (5).

The initial spore septum appeared particularly flexible in the early stages, but began to "round up" at the stage when the forespore moved towards the centre of the sporangium (Figs. 3 and 8). The two unit membranes are showing evidence of separation at several points in Figs. 3 and 4 with the suggestion of deposition between them of material of lower electron opacity (Figs. 8 and 9).

Mesosomes occur associated with the initial spore septum in many instances (Figs. I, 2, and 7), and with the inner membrane of the forespore at later stages in its development (Fig. 8). They are also observed at several other points on the sporangial cytoplasmic membrane, presumably where normal cell division would occur if sporo- genesis had not commenced (Fig. 4). The striking difference between membrane development in spore septum formation (Fig. 2) and ordinary cell division (Fig. 6) is again evident in this or- ganism (cf. Fitz-James, 5). The sporangium shows a fairly homogeneous granular appearance unlike that in some other organisms such as B. cereus (2, 38, 39) and B. rnegateriurn (2) which are usually filled with large granules believed to be of

sporangium wall. a X 80,000. b part of the same micrograph enlarged, X 160,000.

116 TH~ JOURNAL OF CELL BIOLOOY • VOLUME 14, 196~

/3-hydroxybutyrate. The cell wall is quite thick (ca. 250 A), and shows on the outer surface a considerable amount of dense granular material (Figs. 1 to 6). This may consist of adherent par- ticles of the growth medium as suggested by Glauert et al. (9) but the outer zone of the cell wall is sufficiently well defined to suggest an outer layer in the wall of this species (Figs. 1 to 4).

Development of the Forespore into a Mature Spore

As the terminology of the cytological compo- nents of the mature spore is still being resolved (cf. 16), it is necessary to define the terms used

FIGURE ]0

The cortical region between the two membranes is much more developed (the membranes are 750 A or more apart) and shows a gradual increase in density outward until a zone of reduced density is reached. The two membranes of the spore are not so well resolved as that of the sporangium. The outer membrane is vaguely resolved just beneath a broken ring of very dense material which is possibly the initial development of the dense outer coat of the spore envelope. X 90,000.

in this paper. The membrane enclosing the cyto- plasm and nucleus is called the "spore proto- plasmic membrane" rather than "spore wal l" (17, 27, 37). The spore protoplasm (nucleus + cytoplasm + spore protoplasmic membrane) is surrounded first by the cortex, then the inner coat and outer coat (or coat 1 and coat 2 respec- tively, 27). The terms "envelopes" or "integu- ments" are used collectively to include the layers external to the spore protoplasmic membrane.

The mature spore develops from the spore cytoplasm and nucleus which are initially en- closed by the two unit membranes. The stages in this development are shown in Figs. 8 to 1 I. The two membranes gradually move apart as cortical material and eventually the coat layers are formed. The spore cytoplasm remains im- mediately enclosed within the inner membrane clearly visible almost to complete maturity of the spore. Note in the membrane the typical dense backbone line cf 25 to 30 A bordered by the 2 less dense zones of ca. 25 to 30 A thickness (Figs. 9 and 10). Sections of completely mature spores (Fig. 12) usually show only poorly the inner membrane (now described as the spore proto- plasmic membrane), but this is probably due to difficulties in fixing and staining of the mature spore rather than to the disappearance of the plasma membrane. That the spore protoplasmic membrane is the same inner membrane of the spore septum would seem certain from the pro- gressive stages seen in Figs. 8 to 11.

The outer membrane of the septum of the early forespore gradually moves away from the inner membrane as the cortical material is de- posited between them, until finally its identity is lost in the outer regions of the complex spore envelopes (Figs. 10 and 11). In the intermediate stages there are 9 differentiated zones about the spore cytoplasm (Fig. 9). First can be seen the inner unit membrane of 3 zones with the outer less dense zone apparently merging into the corti- cal substance or 4th zone. Next is the cortical region about 300 A thick. This consists of less dense inner and outer zones with a centre zone very similar in density and granulation to the sporangial cell wall (Fig. 9). Eventually the thick- ness of the cortex increased to ca. 1200 A (0.12 #) (Fig. 11-13). Outside the cortical zone are the three layers of the outer membrane, that nearest the cortex again appearing thicker than usual due possibly to its blending with the cortical material (Fig. 9). As the gap between the two

D. F. OHx'~ ~ND W. G. ]V[IYRRELL Spore Formation in B. eoagulans 117

membranes increases the cortex assumes an ap-

pearance of several zones of varying electron

opacity (Figs. 10 and 11). This may result from

uneven pene t ra t ion of the fixative into the nearly

mature spore. Even at the stage of deve lopment

shown in Fig. 10 it is possible to make out density

differences hav ing the zonal appearance of the

Structure of the Integuments of the Mature Spore

With the fully mature spore of this organism it has not been possible to obta in sections in which the details of all the components of the spore protoplasm and its in teguments were clearly identifiable (Fig. 12). This may be due to poor

Fmt~m 11

A slightly more mature spore than in a previous figure which shows clearly the inner membrane, the very thick cortical region, the first two or three repeating layers of the laminated inner coat, and the dense outer coat. a X 60,000. b part of the same micrograph enlarged, X 290,000.

outer membrane . In Fig. 10 a broken ring of e lectron-opaque mater ia l appears jus t external to the outer membrane . This is probably the init ial deve lopment of the outer coat, which is possibly laid down on the surface of the outer m e m b r a n e of the spore septum. In Fig. 11 the lamellae of the inner coat are becoming evident, the cortex now being surrounded by a laminated inner coat and a very dense outer coat. At no stage in the deve lopment of the spore was there observed a m e m b r a n e having the loose flexible characteristics of the exosporium of B. cereus (2, 16, 26, 27) or Clostridium sporogenes (17).

pene t ra t ion of OsO4. M u c h more informat ion

on the na ture of the morphological components

of the ma tu re spore was, however, obta ined from

sections of spores which had been disrupted by

shaking wi th glass beads (35), which had been

hea t act ivated, or in which germina t ion had been

ini t iated (23). These sections show clearly tha t

the envelopes of the spore protoplasm consist of a

cortical region and two spore coats (Figs. 13 to

15). T h e cortex in these preparat ions is probably

largely residual insoluble mater ia l and appears

to have at its inner surface a m e m b r a n o u s zone

].18 THE JOURNAL'OF CELL BIOLOGY • VOLUME 14, 1962

or at least an orientated insoluble boundary, which fixes more OsO4 (Fig. 15), and which is distinct from the protoplasmic membrane. In the section of the heat activated spore the proto- plasmic membrane was again visible (Fig. 13). The cortex shows a less dense zone about a more dense inner one. The inner coat, with a thickness of about 375 A, can be seen as alternate light and dark zones having a repeating distance of about 75 A (Fig. 14). Altogether, up to 7 zones have been observed. The outer coat is a strongly electron-absorbing region of ca. 200 A thickness (Figs. 12 to 15). The outer membrane was at this stage masked by the very dense outer coat or the laminations of the inner coat.

D I S C U S S I O N

These studies have confirmed in another species the peculiar type of cell division involved in sporogenesis, and have revealed further detail of spore development and of the structure of the spore envelopes. The nature of the process of sporogenesis poses two very important questions. First, what are the chemical substances initiating this type of cell division and operating in the nuclear control mechanism responsible for this new phenotypic expression of the vegetative cell nucleus? Secondly, what are the mechanisms of the synthesis and deposition of the chemical substances composing the cortex and coat layers of the envelopes of the spore protoplasm? There is very little information available about the first point (22). With regard to the second, two hypotheses have been suggested for vegetative cell wall formation. First, mesosomes have been suggested as the site of synthesis and extrusion of polymerized wall material (5, 28). Secondly, cell wall synthesis may involve the synthesis of wall components in the cytoplasmic membrane itself with polymerization of the wall material over its surface or synthesis in the cytoplasm and poly- merization at any point on its surface where the chemical components are extruded through the plasma membrane.

Fitz-James (5) has discussed the function and structure of mesosomes. Although the present work tends to support the importance of these bodies in the formation of transverse septa and development of membranes, Murray (21) has suggested that they are not essential for cell division as they have not been seen in several bacterial species. The fact that mesosomes are associated with the formation and early develop-

ment of the spore septum and apparently not with later stages in the deposition of the inner and outer coats suggests that mesosomes are involved only in the active synthesis and unfolding of cyto- plasmic membrane material itself, and not in spore coat synthesis. We have looked for mesosomes, both on the inner and outer membranes, during the various stages in spore formation, and have ob- served them only on the inner membrane (Figs. 7 and 8), yet certainly in the intermediate and later stages of spore formation cortical and possibly coat material is being deposited between the membranes. The presence of mesosomes on the inner membrane does not exclude the possibility that this membrane only is involved in the syn- thesis and extrusion of cortical material by the first mechanism postulated for cell wall synthesis. If this membrane is involved in the synthesis of the cortex, one would expect the cortical material to be somewhat similar in composition to vegeta- tive cell walls. I t has been shown recently that the site of a , e-diaminopimelic acid (DAP)-hexosamine peptide containing material of the spore integu- ments, i.e. the material equivalent to the vegetative cell wall in Bacillus and other species, is the cortex (35). The evidence for this is the release by lyso- zyme and spore lytic enzymes of material con- taining DAP, hexosamine, and glutamate coinci- dent with the disappearance of cortical material seen in electron micrographs of thin sections of crude spore coat preparations. This had been suggested by Mayall and Robinow (20). Thus the cortex, in part at least, contains material similar in composition to that of the cell wall, and the presence of this material just external to the inner membrane suggests that the cortex is synthesised and deposited by a mechanism similar to that for vegetative cell wall formation. The accumulation of cell wall substrates in the cortex places these in a favourable position for assembly into the cell wall of the germ cell (20, 27) as the cortex is broken down by lyric enzymes during germination (24, 31).

The cytological evidence of the origin of the forespore membranes suggests that they are lipoprotein in composition like the vegetative cytoplasmic membrane (8, 36) and hence are likely to control permeability of the forespore.

There is a distinct possibility that, in the early stages of the forespore, before any marked changes in permeability of the integuments of the spore protoplasm have occurred, both the spore pro- toplasm and the sporangium are synthesising

D. F. OHYI~ AND W. G. MUURELL Spore ~brmation in B. coagulans 119

120 THE JOURNAL OF CELL BIOLOGY • VOLUME 14, 1962

mater ia l which diffuses into the region between the two membranes of the forespore. However, a t la ter stages changes in permeabi l i ty may cause the outer spore m e m b r a n e and the sporang ium to become involved more in the format ion of the two outer coats. The mechanism of format ion of these outer coats which are largely protein (29, 35) is still obscure.

There is evidence of mater ia l occurr ing as lamina ted entities bo th wi th in and outside an outer m e m b r a n e or exosporium, similar in com- position, appearance , and s taining properties, and sometimes contiguous wi th the lamina ted coat of the spore. This is of sufficient impor tance in regard to the mechan ism of format ion of the spore coats to wa r r an t e laborat ion. H a n n a y (14) has shown tha t the protein crystals of Fowler 's bacillus, a s t ra in of B. cereus, are formed wi th in a m e m b r a n e which also encloses the spore and tha t this m e m b r a n e bears some resemblance to a un i t plasma m e m b r a n e (cf. his Fig. 10). The crystals

of Bacillus thuringien~is and other parasporal bodies may remain a t tached to the spore after their lytic release f rom the sporangium (1, 13, 30) and are probably enclosed by an unresolved m e m b r a n e (1). H a n n a y (13) has also shown clearly tha t the parasporal body of Bacillus laterosporus L a u b a c h is contiguous with the lamina ted coat of the spore, consisting of lamellae adjacent and parallel to similar lamellae of the spore coat, suggesting a crystalline type of outgrowth of the coat itself (cf. Hannay , 13, his Figs. 12 and 13). Immedia te ly sur rounding the parasporal body of Fowler 's bacillus, a crystalline body with the lattices of the crystals or ientated at different angles, is a lami- na ted layer similar in appearance to the spore coat. This l amina ted layer resembles, also, cer tain m e m b r a n e systems and " la t t i ce" structures oc- curr ing in the sporangial cytoplasm external to the m e m b r a n e tha t surrounds the spore and the parasporal body (cf. Figs. 8 and l l of Hannay , 14). Tor tuous m e m b r a n e structures in the cytoplasm

FmuR~ 1£ TO 15

Sections of a mature spore in the sporangium, a heat-activated spore, a disrupted spore, and a germinated spore of B. coagulans.

FIGURE 1~

Transverse section of a sporangium containing a mature spore. The inner components of the spore are poorly preserved and of low contrast. The dense outer coat of the spore is resolved but the adjacent laminations of the inner coat are hardly resolved. There is no membrane present equivalent to a typical exosporium. X 90,000.

FIGURE 13

Section of a mature spore which was heat activated for 8 rain at 110°C. The inner membrane or spore protoplasmic membrane is evident again, surrounded by 2 cortical zones, the 1st slightly more dense than the outer zone. The 5 or 6 laminations of the inner coat can he seen surrounded by the very dense outer coat. No membrane equiva- lent to a loose exosporium is present. X 100,000.

FIGURE 14

An enlargement of the coat region of a section of a spore germinated in glucose (0.05 M) for 60 min at 50°C. The dense outer coat and the laminations of the inner coat can be seen. Four repeating light and dark units of 75 A thickness are present. )< 290,000.

FIGURE 15

Section of a disrupted spore after washing, showing the dense outer coat, the inner laminated coat, and residual fibrous cortical region. This region shows a number of partly resolved cross-banded layers as has also been observed by Robinow (private communication). The inner margin of the cortex is well defined, and has a membrane- like appearance which does not resemble the spore protoplasmic membrane. X 210,000.

D. F. OHYE AND W. G. MURRELL 8pore Formation in B. coagulans 121

around e lect ron-opaque particles and in cont inui ty wi th the spore coat have also been observed in B. subtilis (34). These lamina ted entities are ap- parent ly composed largely of prote in together with some basophilic lipid or phospholipid soluble material . T he crystals of B. thuringiensis are largely protein (15), the parasporal bodies of B. latero- ~porus phosphoprote in (6), and the staining and solubility properties suggest tha t in other cases these inclusions may be protein also (11-13). This evidence suggests tha t the lamina ted outer coat of the spore is a product of the metabol ism of the sporangium in the later stages of spore formation, and tha t this occurs as a type of ori- enta ted layering or crystal deve lopment wi th in or associated with a membrane , most probably the outer m e m b r a n e of the forespore. The mech- anisms possibly involved in the format ion of the spore coats could be some method of deposit ion of substrates (synthesised in situ or in the sporan- glum) onto a templa te m e m b r a n e or by the split- t ing of such a membrane . O n the other h a n d the outer m e m b r a n e may invaginate (evaginate?) and form concentr ic lamellae about the cortex by a method such as occurs dur ing the develop- men t of the myel in layers about the axon in the nerve cell (25). No evidence of the latter type of deve lopment has yet been observed. T he appa ren t occurrence of the spore coat external to the outer m e m b r a n e in B. cereus var. alesti (40) may per- haps suggest tha t a process of evaginat ion is involved in this species. Certainly, much more

R E F E R E N C E S

1. BARTHOLOMEW, J. W., and CLARE, P., Parasporal bodies of Bacillus spores, J. Bact., 1956, 71, 158.

2. CHAPMAN, G. B., Electron microscopy of ultra- thin sections of baeteria. II. Sporulation of Bacillus megaterium and Bacillus cereus, J. Bact., 1956, 71, 348.

3. CHAPMAN, G. B., and HILLIER, J., Electron microscopy of ultra-thin sections of bacteria. I. Cellular division in Bacillus cereus, J. Bact., 1953, 66, 362.

4. CHAPMAN, G. B., AND ZWORYXlN, K. A., Study of germinating Bacillus cereus spores employing television microscopy of living cells and electron microscopy of ultra-thin sections, or. Baet., 1957, 74, 126.

5. FITZ-JAMES, P. C., Participation of the cyto- plasmic membrane in the growth and spore formation of bacilli, J. Biophysic. and Biochem. Cytol., 1960, 8, 507.

detai led information is needed at this stage of deve lopment to elucidate the exact method of format ion of the coat layers.

Young and Fi tz-James (40) describe the com- pleted coat in B. cereus var. alesti as apparent ly a single s tructure of two zones of differing density. The dense outer layer is composed of two fine lines 70 A apart , and the inner zone merges into the cortex. This is in marked contrast to the two distinct coats of B. coagulans, unless the coat of B. cereus vat . alesti is a poorly developed laminated coat equivalent to the inner coat of B. coagulans. This would equate the exosporium of B. cereus var. alesti with the dense outer coat of B. coagulans as no loose outer m e m b r a n e (the typical exosporium of B. cereus strains) is observed in this species. Sections of spores of several o ther species tha t we have examined have the same sequence of in teguments as B. coagulans--cortex (simple or differentiated into zones of different density), l amina ted inner coat and outer coat. Also, a l though in m a n y cases the published electron rnicrographs are not ade- quate to confirm this accurately, the micrographs of B. megateriurn (7, 20, 27), C. butyricum (32), Fowler's bacillus (14) and possibly B. subtilis (34) suggest agreement. T h a t is, species with an exo- spor ium may have only one coat which is lami- nated. This may explain why the outer coat of some spores has been described as laminated (14), when the exosporium is disregarded.

Received for publication, March 5, 1962.

6. FITZ-JAMES, P. C., and YOUNG, I. E., Morpho- logical and chemical studies of the spores and parasporal bodies of Bacillus laterosporus, or. Biophysic. and Biochem. Cytol., 1958, 4, 639.

7. GmSBRECHT, P., Ueber 'organisierte' Mito- chondrien und andere Feinstrukturen von Bacillus megaterium, Zentr. Bakt., 1 Abt., Orig., 1960, 179, 538.

8. GILBY, A. R., FEw, A. V., and MCQUXLLEN, K., The chemical composition of the protoplast membrane of Micrococcus lysodeikticus, Biochim. et Biophysiea Acta, 1958, 29, 21.

9. GLAUERT, A. M., BRIEGER, E. M., and ALLEN, J. M., The fine structure of vegetative cells of Bacillus subtilis, Exp. Cell Research, 1961, 22, 73.

10. GLAUERT, A. M., ROGERS, G. E., and GLAUERT, R. H., A new embedding medium for electron microscopy, Nature, 1956, 178, 803.

11. HANNAY, C. L., Crystalline inclusions in aerobic

122 ThE JOURNAL OF CELL BIOLOGY • VOLUME 14, 196~

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D. F. OnYx. AND W. G. MURRELL Spore Formation in B. coagulans 123