Antonie van Leeuwenhoek 61: 265-268, 1992. 9 1992 Kluwer Academic Publishers. Printed in the Netherlands.
The occurrence of disporous Bacillus thuringiensis ceils George B. Chapman, Anneke Slob-van Herk & Jos6 M. Eguia
Department of Biology, Georgetown University, Washington, DC20057, USA Received 2 June 1991; accepted I0 December 1991
Key words: B. thuringiensis, disporous, sporulation Abstract
Ultrathin sections of sporulating Bacillus thuringiensis were examined in a transmission electron microscope. Less than 1% of the about 2,000 approximately sagittal sections of the bacterial cells examined contained two endospores per cell. This finding clarifies the majority of textbook and research reports (which tend to be ambiguous), contradicts several of the most recent textbook reports, and confirms three unillustrated textbook reports, in relation to the occurrence of disporous bacilli. Electron microscopic evidence of the observation is presented, apparently for the first time.
Introduction
Bacillus thuringiensis and its subspecies, aerobic, sporeforming, soil bacilli, produce one or several parasporal bodies or paracrystalline inclusions during sporulation. These crystals are toxic to the larvae of a wide range of insects, including more than 100 lepidopteran species, and several dipteran and coleopteran species (Whiteley & Schnepf 1986). This toxicity and the fact that the crystalline proteins produced are biodegradable and do not affect other orders of insects, other animals, and plants make these bacilli an important biocontrol agent. During attempts over several years to determine whether varying culture conditions would have associated changes in crystal ultrastructure, we reviewed endospore occurrence. Considerable ambiguity and contradiction occur in the literature relative to the number of endospores produced by each sporulating bacillus. Of the 17 microbiology textbooks we reviewed, the majority were either ambiguous or non-committal in their statements relative to the number of spores formed by a single sporangium. One (Davis et al. 1980) of these in-
cluded a drawing (Fig. 5b of their Fig. 1-4) that strongly suggests the occurrence of two spores in each of two cells and three spores in one cell of Bacillus anthracis. The state of the art represented in this figure, taken from the work of Ferdinand Cohn, does not, however, permit visualization of cross-walls. Therefore, the true number of cells (and spores per cell) is unknown. Eight of the most recent texts (Alcamo 1990; Atlas 1984; Boyd 1988; Brock & Madigan 1991; Cregar et al. 1990; Jensen & Wright 1989; Prescott et al. 1990; Stanier et al. 1984) and several older classic texts (Freeman 1979; Joklik et al. 1980) specified, or strongly implied, that one spore is produced per cell. One textbook (Van Demark & Batzing 1986) specifically stated that Bacillus thuringiensis cells produce only one endospore. In the second volume of Bergey's Manual of Systematic Bacteriology (Claus & Berkeley 1986), a four-volume revision of the eight-edition classic Bergey's Manual of Determinative Bacteriology, it is stated that bacilli form not more than one endospore per cell. Three text references reported disporous cells. Of these, Fuerst (1983) reported disporogeny as a rare occurrence,
266 Lamanna & Mallette (1965) related the disporogeny to 'certain bacilli occurring in the gut of both warm and cold blooded animals and which are not well known to bacteriologists,' and Wilson & Miles (1964) stated that disporogeny occurs 'in a few larger forms of bacteria.' The fact that none of the above three references included an electron micrograph showing a disporous cell is especially disappointing to any modern bacterial cytologist. Five International Bacterial Spore Symposia (Barker et al. 1974; Barker et al. 1977; Chambliss & Vary 1978; Halvorson et ai. 1971; Levinson et al. 1981) were reviewed. The symposia contained various references to asporogenous and oligosporogenous variants (variants with a low-frequency of sporulation), but none of these symposia mentioned disporous cells. Electron microscopy of ultrathin sections provides a very accurate method of determining the presence of membrane septa and cross-walls, and hence the number of endospores per cell. The purpose of this study was to seek facts that would remove the ambiguity and resolve the contradictions associated with this fundamental feature of bacterial cytology.
Materials and methods
B. thuringiensis cultures (Ward's Natural Science Establishment Inc., catalogue number 85 W 1839) were grown on trypticase soy agar (BBL Microbiology Systems) at the species specific optimum of 30~C. After growth for 18 to 96 h, bacilli were fixed for 18h in 0.2M sodium phosphate (pH7.3) buffered 1% (w/v) osmium tetroxide. Subsequently, the preparations were washed with buffer four times, 10 min each. Cells were dehydrated by exposure to a graded ethanol series (25%, 50%, 70%, 85%, 95%, 100%, and 100%), with 10rain in each concentration, followed by two 10-min changes of propylene oxide. Epon prepared in a conventional manner (Luft 1961) was employed as the embedding medium. The bacilli were suspended in a 1:1 solution of propylene oxide and Epon accelerated mixture for 18 h. The 1:1 solution was removed and a 3:1 solution of Epon and propylene oxide was
added for 8 h. After the removal of the 3:1 solution, the bacilli were suspended in Epon and transferred to plastic capsules. The Epon was polymerized at 60~ for 48h. (The above procedure was sometimes varied in minor ways, e.g., different buffer or dehydrating agent, which had no bearing on the sporulation process). Ultrathin sections were cut with a Sorvall Porter-Blum MT2-B ultramicrotome and a Du Pont diamond knife. Sections were stained in saturated alcoholic uranyl acetate (Gibbons & Grimstone 1960) and secondarily stained in 0.3% lead citrate (Venable & Coggeshall 1965). An RCA EMU-2D transmission electron microscope was used to examine the sections. Approximately 2,000 different, sporulating, appropriately (i.e., nearly sagittally) sectioned bacilli were viewed.
Results and discussion
A sporulating cell with two endospores is shown in Fig. 1. The conclusion that there are two endospores in a single cell is substantiated by the fact that there is no cross-wall deposition between the two endospores. Additional support is the fact that the exosporium of the left endospore nearly extends to the surface of the right endospore's cortex. In each disporous cell in Figs. 2 and 3, the initial stage (arrows) in the centripetal deposition of cell wall material leading to the formation of a cross-wall is clear. (This event is not shown in Fig. 1). This observation indicates that sporulation can occur before cellular division is complete and raises the questions of 1) whether sporulation can arrest or abort cellular division and 2) whether cellular division would have been completed if the cells had been undisturbed. Of additional interest is the occurrence (Fig. 3) of two parasporal bodies within a single disporous cell. The low frequency of occurrence of disporous cells, viz., about ten per 2,000 sagittal sections, suggests that they do not result from a mutation. They could, however, be the result of non-viable mutations. We interpret this occurrence of disporous bacilli simply as an aberration in the mechanisms controlling cell division and sporulation. As
267
Fig. 1. Transmission electron micrograph of ultrathin section of Bacillus thuringiensis with two endospores(S) forming in the one cell. Bar, 1/am. Fig. 2, Transmission electron micrograph of ultrathin section of Bacillus thuringiensis with two endospores(S). Arrows designate sites of initial centripetal deposition of cross-wall material. Bar, 1/am. Fig. 3. Transmission electron micrograph of ultrathin section of Bacillus thuringiensis with two endospores(S) and two parasporal bodies (PB). The arrow designates a site of initial centripetal deposition of cross-wall material. Bar, 1tam.
s p o r u l a t i o n is a c o m p l e x p r o c e s s that is still n o t fully u n d e r s t o o d , with s o m e b a c t e r i o l o g i s t s r e g a r d ing it as a t y p e o f d i f f e r e n t i a t i o n ( B o y d 1988), a n d o t h e r s c o n s i d e r i n g it a v a r i a t i o n in the p r o c e s s o f
r e p l i c a t i o n ( A t l a s 1984), k n o w l e d g e o f the existence of d i s p o r o u s cells c o u l d b e o f s o m e r e s e a r c h value in clarifying the p r o c e s s .
268 References Alcamo IE (1990) Fundamentals of Microbiology. Third Edition. The Benjamin/Cummings Publishing Company, Inc., Redwood City, California, 960 pp Atlas RM (1984) Microbiology: Fundamentals and Applications. Macmillan Publishing Company, New York, New York, 880 pp Barker AN, Gould GW & Wolf 5 (Eds) Spore Research 1973. Academic Press, London, England, 277 pp Barker AN, Wolf J, Ellar DJ, Dring GJ & Gould GW (Eds) (1977) Spore Research 1976. Vol. 1. Academic Press, London, England, 449 pp Boyd RF (1988) General Microbiology. Second Edition. Times Mirror/Mosby, St. Louis, Missouri, 863 pp Brock TD & Madigan MT (1991) Biology of Microorganisms. Sixth Edition. Prentice Hall, Englewood Cliffs, New Jersey, 874 pp Chambliss G & Vary 5L (Eds) (1978) Spores VII, Seventh International Spore Conference, Madison, Wisconsin. American Society for Microbiology, Washington, D.C., 354 PP Claus D & Berkeley RCW (1986) In: Bergey's Manual of Systematic Bacteriology. Vol. 2 Sneath PHA (Ed) (pp 965-1599) Williams & Wilkins Company, Baltimore, Maryland Creager JG, Black JG & Davison VE (1990) Microbiology: Principles and Applications. Prentice Hall, Englewood Cliffs, New Jersey, 839 pp Davis BD, Dulbecco R, Eisen HN & Ginsberg HS (1980) Microbiology. Third Edition. Harper and Row, Hagerstown, Maryland, 1355 pp Freeman, BA (1979) Burrows Textbook of Microbiology. Twenty-first Edition. WB Saunders Company, Philadelphia, Pennsylvania, 1138 pp Fuerst R (1983) Microbiology in Health and Disease. Fifteenth Edition. WB Saunders Company, Philadelphia, Pennsylvania, 688 pp Gibbons JR & Grimstone AV (1960) On flagellar structure in certain flagellates. J. Biophys. Biochem. Cytol. 7:697-710
Halvorson HO, Hanson R & Campbell LL (Eds) (1971) Spores V, Fifth International Spore Conference, Fontana, Wisconsin. American Society for Microbiology, Washington, D.C., 471 pp Jensen MM & Wright DN (1989) Introduction to Microbiology for the Health Sciences. Second Edition. Prentice-Hall, Englewood Cliffs, New Jersey, 704 pp Joklik WK, Willett HP & Ames DB (1980) Zinsser Microbiology. Seventeenth Edition. Appleton-Century-Crofts, New York, New York, 1539 pp Lamanna C & Mallette MF (1965) Basic Bacteriology. Third Edition. Williams & Wilkins, Baltimore, Maryland, 1001 pp Levinson HS, Sonenshein AL & Tipper DJ (Eds) (1987) Spores VIII, Eighth International Spore Conference, Woods Hole, Massachusetts. American Society for Microbiology, Washington, D.C., 317 pp Luft 5H (1961) Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol 9:409-413 Prescott LM, Harley JP & Klein DA (1990) Microbiology. WC Brown Publishers, Dubuque, Iowa, 883 pp Stanier RY, Ingraham 5L, Wheelis ML & Painter PR (1984) The Microbial World. Fifth Edition. Prentice-Hall, Englewood Cliffs, New Jersey, 689 pp Van Demark PJ & Batzing BL (1986) The Microbes: An Introduction to Their Nature and Importance. The Benjamin/ Cummings Publishing Company, Inc., Menlo Park, California, 991 pp Venable JM & Coggeshall R (1965) A simplified lead citrate stain for use in electron microscopy. J. Cell Biol. 25:407 Whiteley HR & Schnepf HE (1986) The molecular biology of parasporal crystal body formation in Bacillus thuringiensis. In: Ornston LN, Balows A & Baumann P (Eds) Annual Review of Microbiology 40: 549-576. Annual Reviews, Inc., Palo Alto, California, 712 pp Wilson GS & Miles AA (1964) Topley and Wilson's Principles of Bacteriology and Immunity. Fifth Edition. Williams & Wilkins Company, Baltimore, Maryland, 1191 pp
The occurrence of disporous Bacillus thuringiensis ceils George B. Chapman, Anneke Slob-van Herk & Jos6 M. Eguia
Department of Biology, Georgetown University, Washington, DC20057, USA Received 2 June 1991; accepted I0 December 1991
Key words: B. thuringiensis, disporous, sporulation Abstract
Ultrathin sections of sporulating Bacillus thuringiensis were examined in a transmission electron microscope. Less than 1% of the about 2,000 approximately sagittal sections of the bacterial cells examined contained two endospores per cell. This finding clarifies the majority of textbook and research reports (which tend to be ambiguous), contradicts several of the most recent textbook reports, and confirms three unillustrated textbook reports, in relation to the occurrence of disporous bacilli. Electron microscopic evidence of the observation is presented, apparently for the first time.
Introduction
Bacillus thuringiensis and its subspecies, aerobic, sporeforming, soil bacilli, produce one or several parasporal bodies or paracrystalline inclusions during sporulation. These crystals are toxic to the larvae of a wide range of insects, including more than 100 lepidopteran species, and several dipteran and coleopteran species (Whiteley & Schnepf 1986). This toxicity and the fact that the crystalline proteins produced are biodegradable and do not affect other orders of insects, other animals, and plants make these bacilli an important biocontrol agent. During attempts over several years to determine whether varying culture conditions would have associated changes in crystal ultrastructure, we reviewed endospore occurrence. Considerable ambiguity and contradiction occur in the literature relative to the number of endospores produced by each sporulating bacillus. Of the 17 microbiology textbooks we reviewed, the majority were either ambiguous or non-committal in their statements relative to the number of spores formed by a single sporangium. One (Davis et al. 1980) of these in-
cluded a drawing (Fig. 5b of their Fig. 1-4) that strongly suggests the occurrence of two spores in each of two cells and three spores in one cell of Bacillus anthracis. The state of the art represented in this figure, taken from the work of Ferdinand Cohn, does not, however, permit visualization of cross-walls. Therefore, the true number of cells (and spores per cell) is unknown. Eight of the most recent texts (Alcamo 1990; Atlas 1984; Boyd 1988; Brock & Madigan 1991; Cregar et al. 1990; Jensen & Wright 1989; Prescott et al. 1990; Stanier et al. 1984) and several older classic texts (Freeman 1979; Joklik et al. 1980) specified, or strongly implied, that one spore is produced per cell. One textbook (Van Demark & Batzing 1986) specifically stated that Bacillus thuringiensis cells produce only one endospore. In the second volume of Bergey's Manual of Systematic Bacteriology (Claus & Berkeley 1986), a four-volume revision of the eight-edition classic Bergey's Manual of Determinative Bacteriology, it is stated that bacilli form not more than one endospore per cell. Three text references reported disporous cells. Of these, Fuerst (1983) reported disporogeny as a rare occurrence,
266 Lamanna & Mallette (1965) related the disporogeny to 'certain bacilli occurring in the gut of both warm and cold blooded animals and which are not well known to bacteriologists,' and Wilson & Miles (1964) stated that disporogeny occurs 'in a few larger forms of bacteria.' The fact that none of the above three references included an electron micrograph showing a disporous cell is especially disappointing to any modern bacterial cytologist. Five International Bacterial Spore Symposia (Barker et al. 1974; Barker et al. 1977; Chambliss & Vary 1978; Halvorson et ai. 1971; Levinson et al. 1981) were reviewed. The symposia contained various references to asporogenous and oligosporogenous variants (variants with a low-frequency of sporulation), but none of these symposia mentioned disporous cells. Electron microscopy of ultrathin sections provides a very accurate method of determining the presence of membrane septa and cross-walls, and hence the number of endospores per cell. The purpose of this study was to seek facts that would remove the ambiguity and resolve the contradictions associated with this fundamental feature of bacterial cytology.
Materials and methods
B. thuringiensis cultures (Ward's Natural Science Establishment Inc., catalogue number 85 W 1839) were grown on trypticase soy agar (BBL Microbiology Systems) at the species specific optimum of 30~C. After growth for 18 to 96 h, bacilli were fixed for 18h in 0.2M sodium phosphate (pH7.3) buffered 1% (w/v) osmium tetroxide. Subsequently, the preparations were washed with buffer four times, 10 min each. Cells were dehydrated by exposure to a graded ethanol series (25%, 50%, 70%, 85%, 95%, 100%, and 100%), with 10rain in each concentration, followed by two 10-min changes of propylene oxide. Epon prepared in a conventional manner (Luft 1961) was employed as the embedding medium. The bacilli were suspended in a 1:1 solution of propylene oxide and Epon accelerated mixture for 18 h. The 1:1 solution was removed and a 3:1 solution of Epon and propylene oxide was
added for 8 h. After the removal of the 3:1 solution, the bacilli were suspended in Epon and transferred to plastic capsules. The Epon was polymerized at 60~ for 48h. (The above procedure was sometimes varied in minor ways, e.g., different buffer or dehydrating agent, which had no bearing on the sporulation process). Ultrathin sections were cut with a Sorvall Porter-Blum MT2-B ultramicrotome and a Du Pont diamond knife. Sections were stained in saturated alcoholic uranyl acetate (Gibbons & Grimstone 1960) and secondarily stained in 0.3% lead citrate (Venable & Coggeshall 1965). An RCA EMU-2D transmission electron microscope was used to examine the sections. Approximately 2,000 different, sporulating, appropriately (i.e., nearly sagittally) sectioned bacilli were viewed.
Results and discussion
A sporulating cell with two endospores is shown in Fig. 1. The conclusion that there are two endospores in a single cell is substantiated by the fact that there is no cross-wall deposition between the two endospores. Additional support is the fact that the exosporium of the left endospore nearly extends to the surface of the right endospore's cortex. In each disporous cell in Figs. 2 and 3, the initial stage (arrows) in the centripetal deposition of cell wall material leading to the formation of a cross-wall is clear. (This event is not shown in Fig. 1). This observation indicates that sporulation can occur before cellular division is complete and raises the questions of 1) whether sporulation can arrest or abort cellular division and 2) whether cellular division would have been completed if the cells had been undisturbed. Of additional interest is the occurrence (Fig. 3) of two parasporal bodies within a single disporous cell. The low frequency of occurrence of disporous cells, viz., about ten per 2,000 sagittal sections, suggests that they do not result from a mutation. They could, however, be the result of non-viable mutations. We interpret this occurrence of disporous bacilli simply as an aberration in the mechanisms controlling cell division and sporulation. As
267
Fig. 1. Transmission electron micrograph of ultrathin section of Bacillus thuringiensis with two endospores(S) forming in the one cell. Bar, 1/am. Fig. 2, Transmission electron micrograph of ultrathin section of Bacillus thuringiensis with two endospores(S). Arrows designate sites of initial centripetal deposition of cross-wall material. Bar, 1/am. Fig. 3. Transmission electron micrograph of ultrathin section of Bacillus thuringiensis with two endospores(S) and two parasporal bodies (PB). The arrow designates a site of initial centripetal deposition of cross-wall material. Bar, 1tam.
s p o r u l a t i o n is a c o m p l e x p r o c e s s that is still n o t fully u n d e r s t o o d , with s o m e b a c t e r i o l o g i s t s r e g a r d ing it as a t y p e o f d i f f e r e n t i a t i o n ( B o y d 1988), a n d o t h e r s c o n s i d e r i n g it a v a r i a t i o n in the p r o c e s s o f
r e p l i c a t i o n ( A t l a s 1984), k n o w l e d g e o f the existence of d i s p o r o u s cells c o u l d b e o f s o m e r e s e a r c h value in clarifying the p r o c e s s .
268 References Alcamo IE (1990) Fundamentals of Microbiology. Third Edition. The Benjamin/Cummings Publishing Company, Inc., Redwood City, California, 960 pp Atlas RM (1984) Microbiology: Fundamentals and Applications. Macmillan Publishing Company, New York, New York, 880 pp Barker AN, Gould GW & Wolf 5 (Eds) Spore Research 1973. Academic Press, London, England, 277 pp Barker AN, Wolf J, Ellar DJ, Dring GJ & Gould GW (Eds) (1977) Spore Research 1976. Vol. 1. Academic Press, London, England, 449 pp Boyd RF (1988) General Microbiology. Second Edition. Times Mirror/Mosby, St. Louis, Missouri, 863 pp Brock TD & Madigan MT (1991) Biology of Microorganisms. Sixth Edition. Prentice Hall, Englewood Cliffs, New Jersey, 874 pp Chambliss G & Vary 5L (Eds) (1978) Spores VII, Seventh International Spore Conference, Madison, Wisconsin. American Society for Microbiology, Washington, D.C., 354 PP Claus D & Berkeley RCW (1986) In: Bergey's Manual of Systematic Bacteriology. Vol. 2 Sneath PHA (Ed) (pp 965-1599) Williams & Wilkins Company, Baltimore, Maryland Creager JG, Black JG & Davison VE (1990) Microbiology: Principles and Applications. Prentice Hall, Englewood Cliffs, New Jersey, 839 pp Davis BD, Dulbecco R, Eisen HN & Ginsberg HS (1980) Microbiology. Third Edition. Harper and Row, Hagerstown, Maryland, 1355 pp Freeman, BA (1979) Burrows Textbook of Microbiology. Twenty-first Edition. WB Saunders Company, Philadelphia, Pennsylvania, 1138 pp Fuerst R (1983) Microbiology in Health and Disease. Fifteenth Edition. WB Saunders Company, Philadelphia, Pennsylvania, 688 pp Gibbons JR & Grimstone AV (1960) On flagellar structure in certain flagellates. J. Biophys. Biochem. Cytol. 7:697-710
Halvorson HO, Hanson R & Campbell LL (Eds) (1971) Spores V, Fifth International Spore Conference, Fontana, Wisconsin. American Society for Microbiology, Washington, D.C., 471 pp Jensen MM & Wright DN (1989) Introduction to Microbiology for the Health Sciences. Second Edition. Prentice-Hall, Englewood Cliffs, New Jersey, 704 pp Joklik WK, Willett HP & Ames DB (1980) Zinsser Microbiology. Seventeenth Edition. Appleton-Century-Crofts, New York, New York, 1539 pp Lamanna C & Mallette MF (1965) Basic Bacteriology. Third Edition. Williams & Wilkins, Baltimore, Maryland, 1001 pp Levinson HS, Sonenshein AL & Tipper DJ (Eds) (1987) Spores VIII, Eighth International Spore Conference, Woods Hole, Massachusetts. American Society for Microbiology, Washington, D.C., 317 pp Luft 5H (1961) Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol 9:409-413 Prescott LM, Harley JP & Klein DA (1990) Microbiology. WC Brown Publishers, Dubuque, Iowa, 883 pp Stanier RY, Ingraham 5L, Wheelis ML & Painter PR (1984) The Microbial World. Fifth Edition. Prentice-Hall, Englewood Cliffs, New Jersey, 689 pp Van Demark PJ & Batzing BL (1986) The Microbes: An Introduction to Their Nature and Importance. The Benjamin/ Cummings Publishing Company, Inc., Menlo Park, California, 991 pp Venable JM & Coggeshall R (1965) A simplified lead citrate stain for use in electron microscopy. J. Cell Biol. 25:407 Whiteley HR & Schnepf HE (1986) The molecular biology of parasporal crystal body formation in Bacillus thuringiensis. In: Ornston LN, Balows A & Baumann P (Eds) Annual Review of Microbiology 40: 549-576. Annual Reviews, Inc., Palo Alto, California, 712 pp Wilson GS & Miles AA (1964) Topley and Wilson's Principles of Bacteriology and Immunity. Fifth Edition. Williams & Wilkins Company, Baltimore, Maryland, 1191 pp
