Vol. 32, No. 3 Printed in U.S.A .
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1976, p. 438-440 Copyright ©D 1976 American Society for Microbiology
Fine Structure of the Bacillus thuringiensis Spore' PHILIPP GERHARDT,* H. STUART PANKRATZ, AND REN12 SCHERRER Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan 48824
Received for publication 13 February 1976
The thin-sectioned spore ofBacillus thuringiensis resembles that of Bacillus cereus in fine structure. Planar inclusions occur between the exosporium and spore coat and are structured differently from the parasporal crystal outside the exosporium. Because of its importance as an insect pathogen and insecticide, Bacillus thuringiensis has been investigated intensively (7, 11, 12). The organism resembles B. cereus in general morphology and other characteristics, differing mainly in the formation of a toxic parasporal protein crystal (2, 5, 8). Indeed, some authorities designate the organism as B. cereus var. thuringiensis (5). The present study was undertaken to compare the sporal fine structure of B. thuringiensis with that of B. cereus (4, 6) and similar species (1, 3, 10). B. thuringiensis (ATCC no. 10792) was grown, the spores and crystals were separated, and electron microscopy was managed essentially as described previously (1, 4), with the exception that 0.4% uranyl acetate was incorporated into the osmium fixative. An illustrative micrograph depicting all of the main features of a thin-sectioned B. thuringiensis spore is shown in Fig. 1A, and an enlarged portion is shown in Fig. 1B. A thinsectioned parasporal crystal is shown in Fig. 2. The exosporium of B. thuringiensis was structured with a basal layer and a peripheral nap like that of B. cereus (4) and similar types (1, 3). The spore coat of B. thuringiensis contained a granular, loosely fitting outer layer of irregular thickness. A similar if less pronounced outer layer was observed in spores of B. cereus strain T prepared the same way. The inner layer of the spore coat was laminated in both species. When sectioned, the lamellae of the inner coat layer appear either stacked or comblike because this layer consists of a patchwork of striated packets (see Fig. 9 in reference 1 and Fig. 3A in reference 6). The spore cortex and core structures of B. thuringiensis were similar to those of B. cereus. Planar inclusions were found between the
exosporium and coat of the B. thuringiensis spore, similar to those in B. cereus (4), B. anthracis (10), and exosporium-forming strains of B. megaterium (1). The structure of the planar inclusions of the B. thuringiensis spore appeared quite different from that of the parasporal crystal. Other investigators have dealt with the formation and structure of the crystal (9, 14) and its antigenic relationship to spore structure (13). In all, thin-sectioned spores ofB. thuringiensis and B. cereus appeared alike in fine structure.
Journal article no. 7565 from the Michigan Agricultural Experiment Station.
438
LITERATURE CITED 1. Beaman, T. C., H. S. Pankratz, and P. Gerhardt. 1972. Ultrastructure of the exosporium and underlying inclusions in spores of Bacillus megaterium strains. J.
Bacteriol. 109:1198-1209. 2. Buchanan, R. E., and N. E. Gibbons (ed.). 1974. Bergey's manual of determinative bacteriology, 8th ed. Williams and Wilkins Co., Baltimore. 3. Gerhardt, P. 1967. Cytology of Bacillus anthracis. Fed. Proc. 26:1504-1517. 4. Gerhardt, P., and E. Ribi. 1964. Ultrastructure of the exosporium enveloping spores of Bacillus cereus. J. Bacteriol. 88:1774-1789. 5. Gordon, R. E., W. C. Haynes, and C. H.-N. Pang. 1973. The genus Bacillus. U.S. Department of Agriculture handbook no. 427. U.S. Government Printing Office, Washington, D.C. 6. Hashimoto, T., and S. F. Conti. 1971. Ultrastructural changes associated with activation and germination of Bacillus cereus T spores. J. Bacteriol. 105:361-368. 7. Heimpel, A. M. 1967. A critical review of Bacillus thuringiensis var. thuringiensis Berliner and other crystalliferous bacteria. Annu. Rev. Entomol. 12:287-322. 8. Krieg, A. 1968. A taxonomic study of Bacillus thuringiensis Berliner. J. Invertebr. Pathol. 12:366-378. 9. Labaw, L. W. 1964. The structure of Bacillus thuringiensis Berliner crystals. J. Ultrastruct. Res. 10:6675. 10. Moberly, B. J., F. Shafa, and P. Gerhardt. 1966. Structural details of anthrax spores during transformation into vegetative cells. J. Bacteriol. 92:220-228. 11. Norris, J. R. 1969. Sporeformers as insecticides, p. 485516. In G. W. Gould and A. Hurst (ed.), The bacterial spore. Academic Press Inc., London. 12. Norris, J. R. 1970. Sporeformers as insecticides. J. Appl. Bacteriol. 33:192-206.
IA
.i t,
is FIG. 1. (A) Thin-sectioned spore of B. thuringiensis. Bar = 100 nm. (B) Enlarged portion of (A), with identifying labels: XN, exosporium nap; XBL, exosporium basal layer; PI, planar inclusion; OCL, outer coat layer; ICL, inner coat layer; C, cortex; CW, core wall; CM, core membrane; N, nucleoplasm. Bar = 100 nm. 439
440
NOTES
APPL. ENVIRON. MICROBIOL.
FIG. 2. Thin-sectioned parasporal crystal of B. thuringiensis. The edge corrugations were spaced 24 nm apart. The arrow indicates the orientation of laminations within the crystal, which were spaced 5.0 nm apart when measured at greater magnification. Bar = 100 nm. 13. Short, J. A., P. D. Walker, R. 0. Thomson, and H. J. Somerville. 1974. The fine structure ofBacillus finitimus and B. thuringiensis spores with special reference to the location of crystal antigen. J. Gen. Microbiol. 84:261-276.
14. Young, I. E., and P. C. Fitz-James. 1959. Chemical and morphological studies of bacterial spore formation. II. Spore and parasporal protein formation in Bacillus cereus var. alesti. J. Biochem. Biophys. Cytol. 6:483498.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1976, p. 438-440 Copyright ©D 1976 American Society for Microbiology
Fine Structure of the Bacillus thuringiensis Spore' PHILIPP GERHARDT,* H. STUART PANKRATZ, AND REN12 SCHERRER Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan 48824
Received for publication 13 February 1976
The thin-sectioned spore ofBacillus thuringiensis resembles that of Bacillus cereus in fine structure. Planar inclusions occur between the exosporium and spore coat and are structured differently from the parasporal crystal outside the exosporium. Because of its importance as an insect pathogen and insecticide, Bacillus thuringiensis has been investigated intensively (7, 11, 12). The organism resembles B. cereus in general morphology and other characteristics, differing mainly in the formation of a toxic parasporal protein crystal (2, 5, 8). Indeed, some authorities designate the organism as B. cereus var. thuringiensis (5). The present study was undertaken to compare the sporal fine structure of B. thuringiensis with that of B. cereus (4, 6) and similar species (1, 3, 10). B. thuringiensis (ATCC no. 10792) was grown, the spores and crystals were separated, and electron microscopy was managed essentially as described previously (1, 4), with the exception that 0.4% uranyl acetate was incorporated into the osmium fixative. An illustrative micrograph depicting all of the main features of a thin-sectioned B. thuringiensis spore is shown in Fig. 1A, and an enlarged portion is shown in Fig. 1B. A thinsectioned parasporal crystal is shown in Fig. 2. The exosporium of B. thuringiensis was structured with a basal layer and a peripheral nap like that of B. cereus (4) and similar types (1, 3). The spore coat of B. thuringiensis contained a granular, loosely fitting outer layer of irregular thickness. A similar if less pronounced outer layer was observed in spores of B. cereus strain T prepared the same way. The inner layer of the spore coat was laminated in both species. When sectioned, the lamellae of the inner coat layer appear either stacked or comblike because this layer consists of a patchwork of striated packets (see Fig. 9 in reference 1 and Fig. 3A in reference 6). The spore cortex and core structures of B. thuringiensis were similar to those of B. cereus. Planar inclusions were found between the
exosporium and coat of the B. thuringiensis spore, similar to those in B. cereus (4), B. anthracis (10), and exosporium-forming strains of B. megaterium (1). The structure of the planar inclusions of the B. thuringiensis spore appeared quite different from that of the parasporal crystal. Other investigators have dealt with the formation and structure of the crystal (9, 14) and its antigenic relationship to spore structure (13). In all, thin-sectioned spores ofB. thuringiensis and B. cereus appeared alike in fine structure.
Journal article no. 7565 from the Michigan Agricultural Experiment Station.
438
LITERATURE CITED 1. Beaman, T. C., H. S. Pankratz, and P. Gerhardt. 1972. Ultrastructure of the exosporium and underlying inclusions in spores of Bacillus megaterium strains. J.
Bacteriol. 109:1198-1209. 2. Buchanan, R. E., and N. E. Gibbons (ed.). 1974. Bergey's manual of determinative bacteriology, 8th ed. Williams and Wilkins Co., Baltimore. 3. Gerhardt, P. 1967. Cytology of Bacillus anthracis. Fed. Proc. 26:1504-1517. 4. Gerhardt, P., and E. Ribi. 1964. Ultrastructure of the exosporium enveloping spores of Bacillus cereus. J. Bacteriol. 88:1774-1789. 5. Gordon, R. E., W. C. Haynes, and C. H.-N. Pang. 1973. The genus Bacillus. U.S. Department of Agriculture handbook no. 427. U.S. Government Printing Office, Washington, D.C. 6. Hashimoto, T., and S. F. Conti. 1971. Ultrastructural changes associated with activation and germination of Bacillus cereus T spores. J. Bacteriol. 105:361-368. 7. Heimpel, A. M. 1967. A critical review of Bacillus thuringiensis var. thuringiensis Berliner and other crystalliferous bacteria. Annu. Rev. Entomol. 12:287-322. 8. Krieg, A. 1968. A taxonomic study of Bacillus thuringiensis Berliner. J. Invertebr. Pathol. 12:366-378. 9. Labaw, L. W. 1964. The structure of Bacillus thuringiensis Berliner crystals. J. Ultrastruct. Res. 10:6675. 10. Moberly, B. J., F. Shafa, and P. Gerhardt. 1966. Structural details of anthrax spores during transformation into vegetative cells. J. Bacteriol. 92:220-228. 11. Norris, J. R. 1969. Sporeformers as insecticides, p. 485516. In G. W. Gould and A. Hurst (ed.), The bacterial spore. Academic Press Inc., London. 12. Norris, J. R. 1970. Sporeformers as insecticides. J. Appl. Bacteriol. 33:192-206.
IA
.i t,
is FIG. 1. (A) Thin-sectioned spore of B. thuringiensis. Bar = 100 nm. (B) Enlarged portion of (A), with identifying labels: XN, exosporium nap; XBL, exosporium basal layer; PI, planar inclusion; OCL, outer coat layer; ICL, inner coat layer; C, cortex; CW, core wall; CM, core membrane; N, nucleoplasm. Bar = 100 nm. 439
440
NOTES
APPL. ENVIRON. MICROBIOL.
FIG. 2. Thin-sectioned parasporal crystal of B. thuringiensis. The edge corrugations were spaced 24 nm apart. The arrow indicates the orientation of laminations within the crystal, which were spaced 5.0 nm apart when measured at greater magnification. Bar = 100 nm. 13. Short, J. A., P. D. Walker, R. 0. Thomson, and H. J. Somerville. 1974. The fine structure ofBacillus finitimus and B. thuringiensis spores with special reference to the location of crystal antigen. J. Gen. Microbiol. 84:261-276.
14. Young, I. E., and P. C. Fitz-James. 1959. Chemical and morphological studies of bacterial spore formation. II. Spore and parasporal protein formation in Bacillus cereus var. alesti. J. Biochem. Biophys. Cytol. 6:483498.
