Electron Microscope Study of Sporulation and Parasporal

JOURNAL OF BACTERIOLOGY, Sept. 1976, p. 1472-1481Copyright 0 1976 American Society for Microbiology

Vol. 127, No. 3Printed in U.S.A.

Electron Microscope Study of Sporulation and ParasporalCrystal Formation in Bacillus thuringiensis

DONALD B. BECHTEL AND LEE A. BULLA, JR.*

U.S. Grain Marketing Research Center, Agricultural Research Service, U.S. Department ofAgriculture,Manhattan, Kansas 66502

Received for publication 11 June 1976

A comprehensive ultrastructural analysis of sporulation and parasporal crys-tal development is described for Bacillus thuringiensis. The insecticidal crystalofB. thuringiensis is initiated at the start of engulfment and is nearly completeby the time the exosporium forms. The crystal and a heretofore unobservedovoid inclusion develop without any clear association with the forespore septum,exosporium, or mesosomes. These observations contradict previous hypothesesthat the crystal is synthesized on the forespore membrane, exosporium, ormesosomes. Formation of forespore septa involves densely staining, double-membrane-bound, vesicular mesosomes that have a bridged appearance. Fore-spore engulfment is subpolar and also involves mesosomes. Upon completion ofengulfment the following cytoplasmic changes occur: decrease in electron den-sity of the incipient forespore membrane; loss of bridged appearance of incipientforespore membrane; change in stainability of incipient forespore, forespore, andmother cell cytoplasms; and alteration in staining quality of plasma membrane.These changes are involved in the conversion of the incipient forespore into aforespore and reflect "commitment" to sporulation.

Bacillus thuringiensis is a rod-shaped, aero-bic, sporeforming bacterium uniquely charac-terized by the production, during the sporula-tion cycle, of one or more proteinaceous para-sporal crystals. The organism is distinguishedfrom other bacilli such asB. cereus by its patho-genicity for larvae of Lepidoptera, which is at-tributed to the proteinaceous crystal (2). In ad-dition to insecticidal properties, the protein ap-parently is capable of regressing tumors (26)and enhancing the overall immune response inmammals (27).Found to be glycoprotein (L. A. Bulla, Jr.,

K. J. Kramer, D. B. Bechtel, and L. I. David-son, in D. Schlessinger [ed.], Microbiology-1976, in press), the crystal is formed outsidethe exosporium during stages III to VI of sporu-lation. Electron microscope studies of the pro-teinaceous crystal primarily have been con-cerned with its physical arrangement. The sur-face was examined using metal-shadowed prep-arations (11) and carbon replicas (18). X-raydiffractograms provided the crystal unit cell di-mensions (15), and thin sections revealed theinternal structure (24) from which Norris (23)constructed a hypothetical crystal model. Nega-tively stained and freeze-etched samples pre-pared by Norris (23) corroborated the crystalmodel originally proposed by Holmes andMonro (15).

Although there have been several develop-mental studies of B. thuringiensis reported (1,29, 32), a comprehensive ultrastructural analy-sis of the sporulation cycle involving parasporalcrystal formation in this organism has not beenpublished. Our study provides an overview ofB. thuringiensis spore and crystal morphogene-sis and describes certain aspects of the sporula-tion process that are unique to this organism.Particularly, evidence is presented for involve-ment of vesicular mesosomes in the formationof forespore septa and of forespore membrane.Also, our data contradict the hypotheses thatthe crystal is synthesized and assembled onforespore membrane (8) or on the exosporium(33, 34).

MATERIALS AND METHODSOrganism and cultural conditions. B. thuringien-

sis strain HD-1 was isolated from Dipel, a commer-cial insecticide manufactured by Abbott Laborato-ries, North Chicago, Ill. The strain (HD-1) used inthis study has been identified as B. thuringiensissubsp. kurstaki by H. de Barjac, Institut Pasteur,Paris, France, and is the most toxic for lepidopteraninsects. Spore stocks of HD-1 were maintained onmodified GYS (21) agar slants at 4°C. Spores, heat-shocked for 30 min at 80°C as previously described(22), were used to inoculate 50 ml of prewarmed(28°C) modified GYS medium contained in a 250-mlErlenmeyer flask aerated by rotary agitation at 200rpm. Midexponential cells (10% inoculum) were

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SPORULATION AND PARASPORAL CRYSTAL FORMATION 1473

transferred three times in the GYS medium main-tained at 28°C to obtain synchronously dividingcells. The cells were harvested at midexponentialphase by centrifugation at 40C, and the pellet wassuspended in 50 ml of cold GYS medium. The sus-pension was then equally distributed among five250-ml Erlenmeyer flasks containing 50 ml of pre-warmed (280C) GYS medium. The cells were incu-bated in this medium at 280C with rotary agitationat 200 rpm. Samples (10 ml) of the 50-ml cultureswere removed at 30-min intervals, quickly chilled,and centrifuged at 40C. Sporulation was monitoredby phase-contrast and electron microscopy. Greaterthan 95% of the cells reached stages III and IVsynchronously.

Electron microscopy. The pellets were suspendedin 4% glutaraldehyde in 0.01 M phosphate-bufferedsaline at pH 7.2 for 5 min at 40C. The bacteria werepelleted at 2,000 rpm and suspended in warm (5500)2% water agar. The agar was immediately cooled to40C, cut into 1-mm cubes, and placed into fresh coldfixative for 1 h. Samples were washed four times incold buffer for a total of 80 min. After washing, thebacteria were postfixed in buffered 1% OsO4 at 40Cfor 1 h. Samples were washed in double-distilledwater for 30 min and stained overnight in 0.5%aqueous uranyl acetate. The bacteria were dehy-drated by passing them through a graded acetoneseries and embedded in either Epon 812 (20) or Spurrresin (35). Samples were cut with a diamond knife

on a Porter-Blum MT-2b ultramicrotome, stainedwith lead citrate (28), and examined in a PhilipsEM 201 electron microscope operated at 60 kV. Se-rial sections, 150 nm thick, were placed on slottedgrids previously coated with Formvar and carbonand examined in the microscope at 100 kV.

RESULTS

Overview of sporulation. There are certainsporogenic events, excluding crystal formation,that are unique to B. thuringiensis subsp. kur-staki although the overall pattern of sporula-tion in this organism is similar to that of otherbacilli (4, 6, 16, 25). The sequence of spore de-velopment and parasporal crystal formation inB. thuringiensis is diagramed in Fig. 1 and issummarized here according to the conventionalsporulation stages: stage I (7 h), axial filamentformation in which there is no apparent in-volvement of mesosomes with the nucleoid;stage II (7 to 8 h), forespore septum formationinvolving mesosomes; stage III (8 to 9 h), en-gulfment with mesosome involvement, first ap-pearance of ovoid inclusion and parasporalcrystal, change in stainability of membranesand cytoplasm, and formation of forespore;stages IV to VI (9 to 12 h), formation of exospo-

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FIG. 1. Diagramatic scheme of sporulation in B. thuringiensis. Abbreviations: M, mesosome; CW, cellwall; PM, plasma membrane; AF, axial filament; FS, forespore septum; IF, incipient forespore; OI, ovoidinclusion; PC, parasporal crystal; F, forespore; IM, inner membrane; OM, outer membrane; PW, primordialcell wall; E, exosporium; LC, lamellar spore coat; OC, outer spore coat; C, cortex; IMC, incorporated mothercell cytoplasm; S, mature spore in an unlysed sporangium.

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rium, primordial cell wall, cortex, and sporecoats accompanied by transformation of thespore nucleoid; stage VII (post-12 h), spore mat-uration.Forespore septum formation - stage II (7 to

8 h). The forespore septa were initiated imme-diately after stage I and were recognizable asinvaginations of the plasma membrane at sub-polar regions of the cell (stage II, 7 to 8 h; seearrowhead, Fig. 2a). Mesosomes were associ-ated with the invaginations and were preva-lent throughout forespore septum development(Fig. 2a and b). Upon completion ofthe septum,the area of cytoplasm destined to be incorpo-rated into the forespore was termed the incipi-ent forespore (Fig. 2b and c). The foresporeseptum was easily distinguished from the vege-tative division septum because the formerlacked visible cell wall material and was simi-lar in appearance to mesosomal membranes.Engulfment-stage III (8 to 9 h). Once the

forespore septum was complete, engulfment ofthe incipient forespore commenced. The septumcharacteristically formed an apex pointed to-ward the mother cell cytoplasm (see arrow, Fig.2c). This phenomenon was followed by themovement of the junction of the forespore sep-tum and plasma membrane toward one pole ofthe cell. The movement was generally greateron one side and resulted in subpolar ratherthan polar engulfment. Mesosomes were pres-ent at the junction of the septum and plasmamembrane during engulfment (Fig. 2d). Subpo-lar engulfment by B. thuringiensis is typifiedin both cross- and longitudinally sectioned cells(Fig. 2e and f).Completion of engulfment occurred when the

septum became detached from the plasmamembrane, isolating the incipient foresporefrom the mother cell cytoplasm. Immediatelyafter septum detachment, several changes oc-curred which lead to development of the fore-spore. One of the most noticeable changes wasthe decrease in electron density of the incipientforespore membrane (Fig. 3a). The incipient

forespore membrane also lost its mesosome-likeappearance and was transformed into the innerand outer forespore membranes (compare Fig.3b and c). A second significant change was inthe staining qualities of the mother cell (spor-angial) and forespore cytoplasms. The sporan-gial cytoplasm of cells that had completed en-gulfment stained more heavily and appearedmore granular than either the cytoplasm offorespores or cells not having completed theprocess (compare cells in Fig. 3a). A third, lessconspicuous change occurred in the appearanceof the plasma membrane. Prior to engulfmentthe plasma membrane was irregular and notdistinctly trilaminar (see cell A, Fig. 3d). Afterengulfment, however, the membrane was dis-tinctly trilaminar, with the outer electron-dense portion being thicker than the inner one(see cell B, Fig. 3d).Spore wall development -stages IV to VI (8

to 13 h). Spore wall development is summa-rized diagrammatically in Fig. 4 and is detailedin Fig. 3e to i and 5d. The primordial cell wallwas the first part of the spore wall to form andwas secreted between the inner and outer fore-spore membranes (stage IV, 8 to 9 h; Fig. 3e andf). Concomitant to primordial cell wall appear-ance, electron-dense, fibrous masses appearedwith the spore nucleoid (Fig. 3e and g); also themother cell nucleoid began to fragment (Fig. 3gand 5b and c). As the primordial cell wall wassecreted, ribosome-like spherical particles be-came aligned along the lateral sides ofthe sporewhere the lamellar spore coat was initiated(arrowheads, Fig. 3h) and remained attachedduring development of the lamellar coat (stageV, 11 to 13 h; arrowheads, Fig. 3i and 5d). Whilethe lamellar coat was being formed, three othermodifications also occurred: (i) the electron-dense, fibrous spore nucleoid (Fig. 3e and g)became an electron-transparent homogeneousstructure (Fig. 5d); (ii) the primordial cell wallthickened (Fig. 4); and (iii) the exosporium en-gulfed the spore (Fig. 5d).

After the primordial cell wall had attained

FIG. 2. Forespore septum development and engulfment in B. thuringiensis. (a) Forespore septum develop-ment during stage II. Lower septum (FS) has vesicular mesosomes (M) closely associated, whereas upperseptum (arrowhead) is still represented as an invagination. x67,500. Bar = 0.25 pm. (b) Two newlycompleted forespore septa (FS) that have isolated the incipient forespore cytoplasm (IF) from the mother cellcytoplasm (MC). Note mesosomes (M) still continuous with septa. x54,000. Bar = 0.25 pm. (c) Initiation ofengulfment (stage III). Forespore septum extends (arrow) toward mother cell center (MC) forming ovalincipient forespore (IF). x61,500. Bar = 0.25 pm. (d) Stage III cell showing predominance ofmesosomes (M)at one junction of engulfment membrane. Note that engulfment membranes are continuous with plasmamembrane at several locations (arrows). IF, Incipient forespore. x61,500. Bar = 0.25 pLm. (e) Cross sectionthrough cell containing incipient forespore (IF) showing connection ofengulfment membranes on lateral sideof cell (arrowheads). x67,500. Bar = 0.25 pAm. (f) Longitudinal section through subpolar engulfment (stageIII) of incipient forespore (IF). Note subpolar attachment of engulfment membrane to plasma membrane(arrowheads). x38,200. Bar = 0.5 pmn.

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maximum thickness, the cortex developed(stages IV to VI, 11 to 12 h) between the primor-dial cell wall and the outer forespore membrane(Fig. 4). After cortex and lamellar spore coatdevelopment, the outer fibrous spore coatformed. The mature lysed spore (stage VII) hasseveral distinct layers. Beginning with the in-ner membrane (see Fig. 4) they are as follows:primordial cell wall, cortex, outer membrane,incorporated mother cell cytoplasm, lamellarspore coat, outer fibrous spore coat, and theexosporium.Parasporal crystal development -stages III

to VI (8 to 12 h). The parasporal crystal of B.thuringiensis was observed first during engulf-ment (stage III, 8 h; Fig. 5a) and possessedcrystal lattice fringes at this early stage of de-velopment. The crystal was almost full-sized bythe time the exosporium appears (stage IV, 9 h;Fig. 1 and 5b and c). An ovoid inclusion devel-oped simultaneously to crystal appearance(Fig. 3e and 5b and c). Some cells lacked thisinclusion, however, as evidenced in Fig. 5a.The ovoid inclusion was easily distinguishedfrom the diamond-shaped parasporal crystal inthat it lacked crystal lattice fringes and alwaysappeared ovoid. More than one crystal can de-velop within a cell, but only one ovoid inclusionper sporangium was ever observed. Several dif-ferent types of crystal lattice shapes were seen;many parasporal crystals lacked lattice fringes.The distance between the parallel latticefringes is 8.9 nm. Both the parasporal crystaland ovoid inclusion were not consistently asso-ciated with a specific structure. Serial sections(not shown) reveal that the crystal was not

connected to mesosomes or to membranes of theincipient forespore, forespore, or exosporium.Although in some micrographs there appears tobe an association of the crystal with the fore-spore membrane, serial sections revealed thatthe parasporal crystal and forespore were al-ways separated by cytoplasm.

DISCUSSIONOne of the most striking aspects ofB. thurin-

giensis sporogenesis is the synthesis of the par-asporal crystal. Our study reveals that the crys-tal is initiated during engulfment and is vir-tually full sized by the completion of this proc-ess. The lattice fringes observed during its for-mation support the view that this body is pro-gressively assembled from subunits to ulti-mately produce a crystal that refracts light(30). The absence of crystal lattice images insome crystals and the variation of images inothers are probably the result of the angle ofthe crystal axis to the electron beam (17). Ka-neko and Matsushima (17) demonstrated thatby changing the axis of B. subtilis crystals inthe electron beam, they were able to show var-ious lattice shapes within the same crystal. Weobserved a similar phenomenon with B. thurin-giensis crystals (micrographs not shown). Theovoid inclusion observed in this study has notbeen reported before for B. thuringiensis. Itsgeneral appearance suggests that it is not of thesame composition as the crystal because it doesnot display crystal lattice fringes and usuallystains differently from the crystal. Whether theovoid inclusion possesses insecticidal propertiesis not known.

FIG. 3. Forespore and spore wall formation in B. thuringiensis. (a) Comparison of cells undergoingengulfment (A) and those that have completed engulfment (B). Note that incipient forespore cytoplasm (IF)and mother cell cytoplasm (MC) of cell A stain differently than the cytoplasms of either the forespore (F) ormother cell of cell B. Also observe the change in stainability of forespore membranes and engulfmentmembranes. x34,000. Bar = 0.5 ,um. (b) High magnifiation of engulfment membranes (G). Note densestaining and bridged appearance. IF, Incipient forespore. x235,000. Bar = 0.05 ,um. (c) High magnificationof forespore membranes (IM, inner membrane; OM, outer membrane) after engulfment (stage III). Notetrilaminar appearance ofeach membrane. Also, there is a decrease in electron density and a loss of bridgedappearance. F, Forespore. x 225,000. Bar = 0.05 ,um. (d) High-magnification comparison ofcell undergoingengulfment (A) and one that has completed engulfment (B). Note differences between plasma membranes(PM) and staining qualities ofcytoplasm. x93,000. Bar = 0.10 pm. (e) Early stage IV cell showing inner (IM)and outer (OM) membranes with innerspaced primordial cell wall (PW). Observe fibrous regions (Fr) withthe spore nucleoid (SN). OI, Ovoid inclusion; E, exosporium. x72,500. Bar = 0.1 pm. (f) High magnificationof spore (S) showing developing primordial cell wall (PW) between inner (IM) and outer (OM) membranes.PM, Plasma membrane; CW, cell wall. x120,000. Bar = 0.10 pm. (g) Low magnifwation ofsporulating cell(stage IV). Observe nucleoid completely transformed into fibrous region (Fr) and fragmentation ofmother cellnucleoid (N). Note well-developed parasporal crystal (PC) near newly initiated exosporium (E). x22,500. Bar= 0.5 pm. (h) High magnification of developing spore (S) showing thickened primordial cell wall (PW)between inner (IM) and outer (OM) membranes. Note ribosome-like particles lined up in region wherelamellar spore coat will develop (arrowheads). x215,000. Bar = 0.05 um. (i) High magnification ofspore (S)as lamellar spore coat (LC) develops. Note ribosome-like particles (arrowheads) on outer surface of lamellarspore coat (LC). Outer membrane (OM) is observed in oblique section. IM, Inner membrane; PW, primordialcell wall; PM, plasma membrane; CW, cell wall. xl 70,000. Bar = 0.10 pm.

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The unique subpolar type of engulfment ex-hibited by B. thuringiensis is in contrast to thepolar kind described for other sporeforming ba-cilli (4, 7, 13, 16, 25, 37). Moreover, the largenumbers of vesicular mesosomes accompanyingthe engulfing membrane is unlike the singlelarge mesosome (usually morphological type IIor IX according to the categorization of Ghosh[10]) associated with engulfment of some otherbacilli (4, 7, 12-14, 16, 25). We also observed thevesicular mesosomes (type VIII after Ghosh) ofB. thuringiensis in close association with celldivision septa (unpublished data) as well aswith sporulation septa. They stain densely, aredouble layered, and are not distinctly trilami-nar. Ellar and Lundgren (6) described vesicularareas inB. cereus that apparently functioned inforespore septum formation.Some variation in morphological types of

mesosomes has been shown to be greatly af-fected by fixation procedures (10). Aldehyde fix-ation (used in this study) tends to reveal meso-somal membranes as being "bridged"; such astructural arrangement usually is not retainedwith osmium tetroxide fixation (10). For B. thu-ringiensis fixed in aldehyde, the mesosomes,the terminal ends of cell division septa, theforespore septa, and the engulfment mem-branes all appeared as bridged. Holt et al. (16)fixed B. sphaericus in aldehydes and observedlamellar mesosomes without the bridged ap-

pearance. Presumably, the difference in alde-hyde fixation qualities between the mesosomesofB. thuringiensis and B. sphaericus indicatesthat the mesosomal membranes of these twoorganisms are different. Whether the meso-somes of B. thuringiensis are morphologicallydistinct from those of the other bacilli reportedwill probably be resolved by freeze-etching. Be-cause the mesosomes and the septa and engulf-ment membranes of B. thuringiensis allstained similarly and because of the close asso-ciation of mesosomes and septa, we believe thatthere is a direct involvement of mesosomes inthe synthesis of membrane during cell divisionand sporulation. However, our studies showedno physical relationship between mesosomesand developing crystals as did Kaneko andMatsushima (17) for B. subtilis.The ultrastructural changes that occurred

within the sporulating cell after completion ofengulfment could not be related to crystal for-mation. The dramatic loss of electron density inthe incipient forespore membrane probably re-sulted from its transformation into two distincttrilaminar layers (see Fig. 3a to c). Highton (14)observed a similar reduction in electron densityof the forespore membrane of B. cereus 569H/24. We have observed that B. thuringiensiscells that have reached this particular stage ofsporulation are incapable of returning to vege-tative growth (unpublished data) and, there-

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(PC) at opposite end of incipient forespore (IF). x34,800. Bar = 0.5 ,um. (b) Sporulating cell (stage IV) withparasporal crystal (PC) and forespore (F) at one end and the ovoid inclusion (OI) at the other end. Noteexosporium (E) and fragmenting mother cell nucleoid (N). x22,000. Bar = 0.5 ,um. (c) Sporulating cell (stageIV) with parasporal crystal (PC) at the opposite end offorespore (F) and ovoid inclusion (OI). E, Exosporium;N, fragmenting mother cell nucleoid. x29,000. Bar = 0.5 pm. (d) Sporulating cell (stage IV to V) of B.thuringiensis showing lamellar spore coat (arrowheads) forming on lateral sides of spore (S). Note thatexosporium (E) has nearly enveloped the spore and that the spore nucleoid (SN) has been transformed into anelectron-translucent region. Both parasporal crystal (PC) and ovoid inclusion (OI) are near their maximumsize. x29,500. Bar = 0.5 pm.

fore, we conclude that the decrease in electrondensity of the forespore membrane reflects"commitment" to sporulation. Whether the in-creased electron density and granulation of themother cell (sporangial) cytoplasm is character-istic of senescence or reflects a change inplasma membrane permeability or both is notknown. The increase in density of the outerportion of the plasma membrane upon comple-tion of engulfment has been interpreted as a

change in the molecular structure of the mem-brane, which consequently alters its permeabil-ity (14). Freeze et al. (9) demonstrated diminu-tion of the membrane-associated glucose-phos-phoenolpyruvate transferase system after vege-tative growth of B. subtilis. Lang and Lund-gren (19) showed quantitative changes in mem-brane lipid composition during sporulation ofB. cereus. Also, Bulla et al. (3) observed de-repression of membrane-bound enzyme activi-

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ties of B. thuringiensis involved with energyproduction by the electron transport systemand oxidation of acetyl coenzyme A via termi-nal respiratory pathways, i.e., tricarboxylicacid and glyoxylic acid cycles. Another implica-tion of the change in electron density of theplasma membrane is the active transport ofcations (5, 31). Scribner et al. (31) describedactive transport of potassium, magnesium, cal-cium, and manganese and demonstrated thateach cation transport system is independentlyregulated during sporulation, resulting in var-ious rates of cation uptake. Coincidently, cal-cium and potassium uptake was high duringforespore formation (31, 36).The results of our observations indicate that

there is no involvement of the exosporium withcrystal formation and is in contrast to the pos-tulation that the parasporal crystal of B. thu-ringiensis is synthesized on this structure (33,34). Our present study and that of Young andFitz-James (38) reveal that the crystal isformed during engulfment and is almost com-plete by the end of this process. Furthermore,the exosporium forms at least 1 h after comple-tion of engulfment (see Fig. 1). These observa-tions indicate no involvement of the exospo-rium with crystal formation. The fact that thecrystal can develop with no apparent associa-tion of the forespore membrane dispels the no-tion that it is synthesized on this membrane (8).That the crystal is not synthesized on the fore-spore membrane was substantiated by serialsections and numerous high-magnification mi-crographs (not shown). We surmise that theparasporal crystal ofB. thuringiensis is formedwithin the cytoplasm without any apparent in-volvement of mesosomes, forespore septa, fore-spore membrane, or exosporium.

LITERATURE CITED1. Aronson, J. N., P. A. Bowe, and J. Swafford. 1967.

Mesosomes in m-tyrosine-inhibited Bacillus thurin-giensis. J. Bacteriol. 93:1174-1176.

2. Buchanan, R. E., and N. E. Gibbons. 1974. Bergey'smanual of determinative bacteriology, p. 536. TheWilliams & Wilkins Co., Baltimore, Md.

3. Bulla, L. A., Jr., G. St. Julian, and R. A. Rhodes. 1971.Physiology of sporeforming bacteria associated withinsects. III. Radiorespirometry of pyruvate, acetate,succinate, and glutamate oxidation. Can. J. Micro-biol. 17:1073-1079.

4. Decker, S., and S. Maier. 1975. Fine structure of meso-somal involvement during Bacillus macerans sporu-lation. J. Bacteriol. 121:363-372.

5. Eisenstadt, E., and S. Silver. 1972. Calcium transportduring sporulation in Bacillus subtilis, p. 180-186. InH. 0. Halvorson, R. Hanson, and L. L. Campbell(ed.), Spores V. American Society for Microbiology,Washington, D.C.

6. Ellar, D. J., and D. G. Lundgren. 1966. Fine structureof sporulation in Bacillus cereus grown in a chemi-cally defined medium. J. Bacteriol. 92:1748-1764.

7. Fitz-James, P. C. 1960. Participation of the cytoplasmicmembrane in growth and spore formation of bacilli.J. Biophys. Biochem. Cytol. 8:507-528.

8. Fitz-James, P. C. 1962. The initial formation of para-sporal inclusions in bacilli, p. R-10. In Sidney S.Breese, Jr. (ed.), Fifth International Congress forElectron Microscopy, Philadelphia, August 29-Sep-tember 5, 1962. Academic Press Inc., New York.

9. Freeze, E., W. Klofat, and E. Gallius. 1970. Commit-ment to sporulation and induction of glucose-phos-phoenolpyruvate transferase. Biochim. Biophys. Acta222:265-289.

10. Ghosh, B. K. 1974. The mesosome-a clue to the evolu-tion of the plasma membrane. Sub-Cell. Biochem.3:311-367.

11. Hannay, C. L., and P. C. Fitz-James. 1955. The proteincrystals of Bacillus thuringiensis Berliner. Can. J.Microbiol. 1:694-710.

12. Highton, P. J. 1969. An electron microscopic study ofcell growth and mesosome structure of Bacillus li-cheniformis. J. Ultrastruct. Res. 26:130-147.

13. Highton, P. J. 1970. An electron microscopic study ofthe structure of mesosomal membranes in Bacilluslicheniformis. J. Ultrastruct. Res. 31:247-259.

14. Highton, P. J. 1972. Changes in the structure and meso-somes and cell membrane of Bacillus cereus duringsporulation, p. 13-18. In H. 0. Halvorson, R. Hanson,and L. L. Campbell (ed.), Spores V. American Societyfor Microbiology, Washington, D.C.

15. Holmes, K. C., and R. E. Monro. 1965. Studies on thestructure of parasporal inclusions ofBacillus thurin-giensis. J. Mol. Biol. 14:572-581.

16. Holt, S. C., J. J. Gauthier, and D. J. Tipper. 1975.Ultrastructural studies of sporulation in Bacillussphaericus. J. Bacteriol. 122:1322-1338.

17. Kaneko, I., and H. Matsushima. 1975. Crystalline in-clusions in sporulating Bacillus subtilis cells, p. 580-585. In P. Gerhardt, R. N. Costilow, and H. L. Sadoff,(ed.), Spores VI. American Society for Microbiology,Washington, D.C.

18. Labaw, L. W. 1964. The structure of Bacillus thurin-giensis Berliner crystals. J. Ultrastruct. Res. 10:66-75.

19. Lang, D. R., and D. G. Lundgren. 1970. Lipid composi-tion ofBacillus cereus during growth and sporulation.J. Bacteriol. 101:483-489.

20. Luft, J. 1961. Improvements in epoxy resin embeddingmethods. J. Biophys. Biochem. Cytol. 9:409-414.

21. Nickerson, K. W., and L. A. Bulla, Jr. 1974. Physiologyof sporeforming bacteria associated with insects: min-imal nutritional requirements for growth, sporula-tion, and parasporal crystal formation in Bacillusthuringiensis. Appl. Microbiol. 28:124-128.

22. Nickerson, K. W., and L. A. Bulla, Jr. 1975. Lipidmetabolism during bacterial growth, sporulation,and germination: an obligate nutritional require-ment in Bacillus thuringiensis for compounds thatstimulate fatty acid synthesis. J. Bacteriol. 123:598-603.

23. Norris, J. R. 1971. The protein crystal toxin ofBacillusthuringiensis biosynthesis and physical structure, p.229-246. In H. D. Burges and N. W. Hussey (ed.),Microbial control of insects and mites. AcademicPress Inc., London.

24. Norris, J. R., and D. H. Watson. 1960. An electronmicroscope study of sporulation and protein crystalformation in Bacillus cereus var. alesti. J. Gen. Mi-crobiol. 22:744-749.

25. Ohye, D. F., and W. G. Murrell. 1962. Formation andstructure of the spore of Bacillus coagulans. J. CellBiol. 14:111-123.

26. Prasad, S. S. S. V., and Y. I. Shethna. 1974. Purifica-tion, crystallization and partial characterization of

J. BACTERIOL.


the antitumour and insecticidal protein subunit fromthe S-endotoxin of Bacillus thuringiensis var. thurin-giensis. Biochim. Biophys. Acta 363:558-566.

27. Prasad, S. S. S. V., and Y. I. Shethna. 1975. Enhance-ment of immune response by the proteinaceous crys-tal of Bacillus thuringiensis var. thuringiensis. Bio-chem. Biophys. Res. Commun. 62:517-523.

28. Reynolds, E. S. 1963. The use of lead citrate at high pHas an electron-opaque stain in electron microscopy. J.Cell Biol. 17:208-212.

29. Ribier, J., and M. M. Lecadet. 1974. Bacillus thurin-giensis var. Berliner 1715: sporulation d'un mutantcristal-participation des me'sosomes a la fafmation es

enveloppes sporales (exosporium et tunique). C.R.Acad. Sci. Ser. D 278:1131-1134.

30. Rogoff, M. H., and A. A. Yousten. 1969. Bacillus thu-ringiensis: microbial considerations. Annu. Rev. Mi-crobiol. 23:357-386.

31. Scribner, H. E., J. Mogelson, E. Eisenstadt, and S.Silver. 1975. Regulation of cation transport duringbacterial sporulation, p. 346-355. In P. Gerhardt, R.N. Costilow, and H. L. Sadoff (ed.), Spores VI. Ameri-can Society for Microbiology, Washington, D.C.

32. Short, J. A., P. D. Walker, R. 0. Thomson, and H. J.

Somerville. 1974. The fine structure ofBacillus finiti-mus and Bacillus thuringiensis spores with specialreference to the location of crystal antigen. J. Gen.Microbiol. 84:261-276.

33. Somerville, H. J. 1971. Formation of the parasporalinclusion of Bacillus thuringiensis. Eur. J. Biochem.18:226-237.

34. Somerville, H. J., and C. R. James. 1970. Association ofthe crystalline inclusion of Bacillus thuringiensiswith the exosporium. J. Bacteriol. 102:580-583.

35. Spurr, A. R. 1969. A low-viscosity epoxy resin embed-ding medium for electron microscopy. J. Ultrastruct.Res. 26:31-43.

36. Vinter, V. 1969. Physiology and biochemistry of sporu-lation, p. 73-123. In G. W. Gould and A. Hurst (ed.),The bacterial spore. Academic Press Inc., London.

37. Walker, P. D. 1970. Cytology of spore formation andgermination. J. Appl. Bacteriol. 33:1-12.

38. Young, I. E., and P. C. Fitz-James. 1959. Chemical andmorphological studies of bacterial spore formation. II.Spore and parasporal protein formation in Bacilluscereus var. alesti. J. Biophys. Biochem. Cytol. 6:483-498.

VOL. 127, 1976

Documents

Electron Microscope Study of Sporulation and Parasporal