APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1982, p. 1449-1455 Vol. 44, No. 6 0099-2240/82/121449-07$02.00/0 Copyright C 1982, American Society for Microbiology
Ultrastructural Analysis of Spores and Parasporal Crystals Formed by Bacillus sphaericus 2297 ALLAN A. YOUSTENI* AND ELIZABETH W. DAVIDSON2 Microbiology Section, Biology Department, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 240611 and Department of Zoology, Arizona State University, Tempe, Arizona 852872 Received 19 March 1982/Accepted 27 July 1982 Bacillus sphaericus 2297, growing from a boiled, relatively nontoxic spore inoculum, increased about 30-fold in toxicity for mosquito larvae during early exponential growth but showed an approximately 1,000-fold toxicity increase during the late-exponential phase, as spores began to appear in the culture. The development of spores in the bacterial cells was accompanied by the formation of parasporal crystals. These parasporal crystals appeared during stage III as the forespore septum engulfed the incipient forespore. The paraspores were separated from the forespores by a branch of the exosporium across the cell. Measurements of the parasporal substructure revealed a 6.3-nm distance between the striations. When spores and paraspores were fed to mosquito larvae and the larvae were fixed 15 min after feeding, it was found that the spores remained relatively unchanged but that the matrix of the paraspores was dissolved. After dissolution of the paraspore matrix, a meshlike envelope remained which retained the paraspore shape and which was often in contact with the cross-cell portion of the exosporium. The parasporal crystals may be a source of the mosquito larval toxin in this strain of B. sphaericus, but proof will require their isolation from other cellular components.
Some strains of Bacillus sphaericus originally spore. It has not been reported that the para- isolated from dead mosquito larvae have been spore of strain 1593 dissolves in the larval gut. B. shown to produce a toxin(s) which is lethal when sphaericus 2297 produces a particularly large fed to healthy larvae (7, 13, 16). The toxic effect and easily detected inclusion, but it is unknown of the B. sphaericus toxin resembles that of the if the inclusion possesses the crystal-like lattice B. thuringiensis 8-endotoxin in that both rapidly or if it dissolves in the larval gut. We examined affect the midguts of the intoxicated larvae (10, the formation of this inclusion (a parasporal 12). In B. thuringiensis, the 8-endotoxin is con- crystal) during the course of sporulation and the tained within a parasporal body or crystal which fate of the inclusion after ingestion by mosquito is formed at the time of sporulation (1, 3, 8): larvae. Although the toxicity of B. sphaericus 1593 has also been shown to increase at the time of MATERIALS AND METHODS sporulation (15), initial studies did not detect any Bacteria. B. sphaericus 2297 was obtained from S. inclusion bodies within the cell. Subsequently, Singer, Western Illinois University, Macomb, Ill. This and is the strain isolated by Wickremesinghe and Mendis electron microscopy (5, 6) light microscopy and designated MR4 in their paper (17). (14, 17) were used to demonstrate the presence Growth conditions. Spores of B. sphaericus 2297 to of inclusions in some strains. Several strains, be used as inocula in growth curve experiments were including some that are nontoxic, produce dark- produced by smearing the bacteria onto the surface of staining elliptical or oval bodies that do not NYSM agar (nutrient agar [Difco Laboratories, De- dissolve during passage through the larval gut (5, troit, Mich.], 0.05% yeast extract, 5 x 10-5 M MnCl2, 6). Because of the failure to dissolve in the gut, 7 x 10-4 M CaCl2, 10-3 M MgC12) and incubating the they were judged to be unlikely sites of toxin. In plates at 30°C for 48 h. The sporulated cells were addition to the oval and four of washed off the plates with sterile distilled water and elliptical bodies, washed three times with sterile distilled water, and the the most highly toxic strains (1593, 2013-4, 1691, final pellet was frozen and lyophilized. The spore and 2297) also formed polyhedral inclusions. powder was held at -20°C; it had an LC50 of 150 ng/ml The polyhedral inclusion in strain 1593 was (LC50 is the dry weight of bacterial cells that killed shown to have a crystal-like lattice structure. In 50% of the test insect population in 3 days). Sporulated this paper, this type of inclusion will be referred cells to be fed to larvae were prepared by growing the to as a parasporal crystal or simply as a para- bacteria with shaking at 150 rpm in a model G25 1449 1450 YOUSTEN AND DAVIDSON APPL. ENVIRON. MICROBIOL. incubator shaker (New Brunswick Scientific Co., New in Fig. 1. After inoculation of the flask with Brunswick, N.J.) at 28°C in NYSM broth. The bacte- boiled spores, approximately 90% of the spores ria were then washed free of medium and suspended in which had survived boiling germinated (lost heat tap water. resistance) during the first 4 h of incubation. A Growth curve experiments were begun by suspend- small increase in total cell number was observed ing dried spores in sterile distilled water (12 mg/ml) and boiling the suspensions for 15 min. This treatment as early as 2 h after inoculation, and exponential either completely destroyed the toxicity of the spores growth occurred from 4 to 12.5 h. The toxicity of or allowed a very small percentage of the original the inoculum was very low, and it changed little toxicity to remain. Also, approximately 99.99% of the for the first 3 h of growth. However, after 3 h the spores lost viability after this treatment. These nonvia- toxicity of the cell mass began to increase at a ble spores retained refractility and remained visible as rather steady rate. From 3 to 8 h, the vegetative free, nongerminated spores throughout the course of population increased about 1,000-fold, the heat- the experiment. A 3-ml portion of the spore suspen- stable spore count failed to increase, and the sion was added to 200 ml of NYSM broth in a 2-liter toxicity increased about 30-fold. From 8 to 14 Erlenmeyer flask which was shaken (175 rpm) at 30°C h, on a model G25 incubator shaker. Growth was moni- the vegetative population increased about 100- tored by following absorbance with a Klett-Summer- fold, the spore count increased about 1,000-fold, son photoelectric colorimeter and red filter. All total and the toxicity of the cell mass increased about viable counts, spore counts, and the 11-, 12.5-, and 14- 2,800-fold. h samples for bioassay were taken from a single flask. Thin sections were prepared to determine the The cells used for bioassay at 0, 3, 5, 7, and 9 h were sporulation stage at which the inclusion first recovered from 200 ml of broth taken from other appeared. Examination of cells at stage II (fore- flasks. The larger volumes were required in the early spore septum formation) did not reveal the pres- hours because of the low toxicity of the cells. The absorbance of the entire content of each flask did not ence of inclusions (Fig. 2A). At stage III, as the differ by more than 0.03 from that of the flask from forespore septum engulfed the forespore, the which total viable and spore counts were performed. inclusion became visible (Fig. 2B). The assem- Total viable cell counts were carried out by plating bly of the inclusion appeared to be very rapid on NYSM agar. Spore counts were carried out by after the completion of the forespore septum and plating on NYSM agar after a 1.5-ml sample had been the beginning of forespore engulfment. In Fig. heated at 80°C for 12 min. 2C, the inclusion is visible in a cell during stage Electron microscopy. Samples from flasks used for III and in a cell in which spore cortex is being growth curve experiments were fixed by the method of Kellenberger et al. (11) and embedded in Epon 812. Sections were stained for 3 min with 2% uranyl acetate and for 2 min with 0.4% alkaline lead citrate before examination with a Jeolco 100-B electron microscope operating at 80 kV. Feeding of larvae and preparation for electron mi- d croscopy. Second-instar Culex quinquefasciatus larvae .~~~~~~7 were placed in a suspension of sporulated B. sphaeri- cus 2297 cells (ca. 107 ml).The larvae were recovered 15 min after being placed into the bacterial suspension. and the heads and siphons were removed. The bodies / ~~~zs were fixed in 5% glutaraldehyde (0.15 M cacodylate / buffer, pH 7.3) for 2.5 h, washed in distilled water, and postfixed in 1% osmium tetroxide for 2 h. The larvae 106 /03J were rinsed, held overnight in 2% uranyl acetate, and 0 dehydrated, embedded in Spurr resin. Thin sec- U10', \ / l40 tions were stained with uranyl acetate and alkaline lead citrate and observed in a Philips EM300 electron ot /\. =o microscope. Bioassays. Bioassays were conducted with second- instar C. quinquefasciatus larvae as previously de- scribed (15), except that final observations were made after 3 days rather than 4 days. Toxic activities are reported by LC50 values. Dry weights were deter- mined in triplicate by drying 1-ml samples of the cell suspensions at 110°C for 24 h.
RESULTS 0 2 4 6 8 10 12 14 HOURS The data showing growth, sporulation, and FIG. 1. Growth, sporulation, and toxin production toxicity development of B. sphaericus 2297 in a by B. sphaericus 2297. Symbols: 0, total colony- typical growth curve experiment are presented forming units; A, spores; *, LC50. VOL. 44, 1982 SPORE AND PARASPORE ULTRASTRUCTURE 1451 A B
FIG. 2. B. sphaericus 2297 at different stages of sporulation. (A) Stage II. The forespore septum (ar- rows) is complete but there is no paraspore present. Bar = 0.2 ,um. (B) Stage III. The incipient forespore (IF) is being engulfed by the membrane (arrows). The parasporal inclusion (PI) is well developed adjacent to the membrane. Bar = 0.2 ,um. (C) Stages III and IV. At stage III, the IF is being engulfed, and the PI is adjacent. The cell at stage IV has a well-developed PI adjacent to the forespore (F), which is developing cortex (C). Bar = 0.4 ,um.
graph revealed a distance of 6.3 nm between the striations present in the paraspore. Very rarely, a cell was seen which had terminal swelling and a paraspore present within this area but which lacked a forespore septum (Fig. 4). Cells having a paraspore without a forespore septum or other indications of spore development always had terminal swelling typical of stage III sporulation. Because B. sphaericus exerts its toxic effect in the midguts of mosquito larvae, we were inter- ested in determining the fate of spores and particularly paraspores after ingestion by the formed at stage IV. The inclusion was separated filter-feeding larvae. After 15 min of feeding on from the outer spore coat by a halo of low- spores and paraspores, the only evidence of density-staining material (Fig. 3). The ultrastruc- parasporal bodies in larval midguts was the ture of the parasporal inclusion was visible in presence of residual envelopes (Fig. 5A). The this section (Fig. 3), and the inclusion was parasporal envelopes retained the shape of the confirmed as a parasporal crystal. Measure- paraspores and appeared to have a meshlike ments made from enlargements of this photo- substructure (Fig. SB). In the partially digested 1452 YOUSTEN AND DAVIDSON APPL. ENVIRON. MICROBIOL.
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FIG. 3. B. sphaericus 2297 at stage V. The spore coats (SC) are developing, and the parasporal inclusion (PI) is adjacent to a lightly staining material surrounding the spore. Note the striations on the paraspore. Bar = 0.1 ,um. bacterial cells, the exosporium branched and peared (5). However, signs of larval intoxication separated the paraspore from the spore (Fig. appeared shortly after the larvae were placed 5B). This branch of the exosporium crossed the into the suspension of B. sphaericus 2297. cell between the paraspore and the halo of low- density-staining material. This halo of unidenti- DISCUSSION fied material was also observed in nonpathogen- During B. sphaericus 2297 exponential ic strain 9602 by Holt et al. (9). Only a few growth, the toxicity per dry weight unit of the paraspores found in the most anterior end of the cells increased; i.e., the LC50 decreased. Al- larval midgut, near the stomodeal valve, re- though the synthesis of some toxin by vegetative tained their electron-dense appearance and pre- cells cannot be excluded by these experiments, sumably had not yet been dissolved by digestive the decrease in the LC50 cannot be accounted fluids (Fig. 5C). In Fig. SC, the separation of the for by the increase in numbers of vegetative spore from the paraspore by a branch of the cells. The reproduction of vegetative cells hav- exosporium is visible. The spores did not appear ing the same amount of toxin would have result- to change significantly in structure for up to 1 h ed in an unchanged LC50. Rather, the increase in after ingestion, the longest this strain was ob- the number of spores in the culture seems to be served. An earlier study with B. sphaericus 1593 directly correlated with the increasing toxicity. showed that about 6 h were required before For B. sphaericus 9602, Holt et al. (9) found 6 to signs of spore germination and outgrowth ap- 7 h to be required from the time of the onset of VOL. 44, 1982 SPORE AND PARASPORE ULTRASTRUCTURE 1453 dissolution of the paraspore was often seen in contact with the cross-cell portion of the exo- sporium. The meshlike structure of the envelope is similar to that which has been shown to be present on the paraspores of B. thuringiensis serovar israelensis, another mosquito pathogen (1, 10). It also has some similarity to the hexago- nally arranged subunit structure shown by Holt et al. (9) on the exosporium of B. sphaericus 9602. The parasporal matrix was probably dis- solved by the alkaline pH (9 to 10) of the larval gut (4) as well as by the digestive enzymes present in the gut. The resistance of the exospor- ium and the parasporal envelope to the condi- tions in the larval gut which dissolved the para- sporal matrix, the similarity of the strain 2297 parasporal envelope and the B. sphaericus 9602 exosporium, and the proximity of the parasporal envelop to the cross-cell portion of the exospor- ium in strain 2297 suggest a possible relationship between these structures. The striations seen within the paraspore were determined to be 6.3 nm apart. This contrasts with 7.9 nm, reported to be the distance between striations in a thin section of the B. thuringiensis serovar israelensis parasporal crystal (2). More- FIG. 4. A parasporal inclusion (PI) present within over, the distance observed was similar to the the swollen end of the bacterial cell. Note the lack of a forespore septum. Bar = 0.4 ,um. approximately 7-nm separation between the out- er coat lamellar layers and the exosporium lay- ers found in measurements made on the B. sphaericus 9602 photographs of Holt et al. (9). sporulation to the development of mature spores The dissolution of the parasporal matrix under in a medium similar to the one used in this study. conditions which could be expected to liberate a This indicates that a small number of B. sphaeri- toxin from the ingested cells indicates that these cus 2297 cells probably initiated sporulation as inclusions could be a source of the toxin. How- early as 2 or 3 h after inoculation, and the early ever, even if these inclusions contain toxin, they increase in toxicity could be attributed to the are certainly not the only site of the B. sphaeri- appearance of these forms in the primarily vege- cus mosquito larval toxin. For example, some tative cell population. It is possible that the strains, such as SSII-1, do not form inclusions at earliest appearing spores were the result of all, and yet they possess some toxicity (6, 13). microcycle sporulation. Between 12.5 and 14 h, Also, purified cell walls of the highly toxic strain the vegetative cell population had stopped in- 1593 have been shown to be toxic, as have creasing, whereas the spores and sporulating spores cleaned by sonication (14). There does cells constituted a higher percentage of the total appear to be a correlation between presence of population. The largest increase in toxicity took the parasporal crystals and high toxicity. David- place in this time interval. The number of heat- son and Myers (6) found that strains 1593, 2297, resistant spores continued to increase after 14 h 2013-4, and 1691, which produced polyhedral (to 5.2 x 108/ml at 24 h), but the LC50 did not bodies (which we now call parasporal crystals), decline further. have a lower LC50 than strains which produce The large parasporal crystal was first ob- no inclusions or which produce only the dark- served during the engulfment of the forespore staining elliptical or oval inclusions lacking a (stage III). This is the same as the time of lattice substructure. The strains which produced parasporal appearance in B. thuringiensis (1). the paraspores were found to be approximately Whereas in most serovars of B. thuringiensis the equivalent in toxicity to one another. The toxici- paraspore is located outside the exosporium, the ty of the strain 2297 paraspores can only be paraspore of B. sphaericus 2297 is partially proven after their isolation from other cellular enclosed by the exosporium and yet is separated components. from the spore by what appears to be an exten- The rare appearance of the parasporal crystal sion or branch of the exosporium across the cell. in cells having terminal swelling but lacking a The parasporal envelope which remained after forespore septum seems to indicate an uncou- 1454 YOUSTEN AND DAVIDSON APPL. ENVIRON. MICROBIOL.
FIG. 5A. Spores and parasporal envelopes within the midguts of C. quinquefasciatus larvae. Bar = 0.5 ,m. (B) Parasporal envelope (double arrows) showing its meshlike substructure. The exosporium (EX) has branched (arrow), separating the spore from the para- spore. Bar = 0.2 ,um. (C) Spore and undissolved paraspore in the anterior portion of a larval midgut. Bar = 0.2 ,um.
pling of paraspore formation from the normal sporulation process. It also indicates that termi- nal swelling is not invariably linked to engulf- ment of the forespore. However, terminal swell- ing is not a direct consequence of the formation of the paraspore, because many strains of B. sphaericus which undergo terminal swelling at sporulation do not produce the parasporal crys- tal (5). ACKNOWLEDGMENTS This research was supported by grant PCM 8004262 from the National Science Foundation and by financial assistance from the United Nations Development Program/World Bank/ World Health Organization Special Programme for Research and Training in Tropical Diseases. Sharon Zielinsky provided technical assistance. LITERATURE CITED 1. Bulla, L., D. Bechtel, K. Kramer, Y. Shethna, A. Aronson, and P. C. Fitz-James. 1980. Ultrastructure, physiology, VOL. 44, 1982 SPORE AND PARASPORE ULTRASTRUCTURE 1455 and biochemistry of Bacillus thuringiensis. Crit. Rev. 10. Huber, H., and P. Luthy. 1981. Bacillus thuringiensis Microbiol. 8:147-204. delta endotoxin: composition and activation, p. 207-234. 2. Charles, J.-F., and H. de Barjac. 1982. Sporulation and In E. W. Davidson (ed.), Pathogenesis of invertebrate cristallogenese de Bacillus thuringiensis var. israelensis microbial diseases. Allanheld, Osmun and Co., Totowa, en microscopie electronique. Ann. Microbiol. (Paris) N.J. 133A:425-442. 11. Kellenberger, E., A. Ryter, and K. Sechaud. 1958. Elec- 3. Cooksey, K. 1971. The protein crystal toxin of Bacillus tron microscope study of DNA-containing plasms. II. thuringiensis: biochemistry and mode of action, 247-274. Vegetative and mature phage DNA as compared with In H. D. Burges and N. W. Hussey (ed.), Microbial normal bacterial nucleoids in different physiological control of insects and mites. Academic Press, Inc., Lon- states. J. Cell Biol. 4:671-678. don. 12. Luthy, P., and H. Ebersold. 1981. Bacillus thuringiensis 4. Dadd, R. 1975. Alkalinity within the midgut of mosquito delta endotoxin: histopathology and molecular mode of larvae with alkaline-active digestive enzymes. J. Insect action, p. 235-267. In E. W. Davidson (ed.), Pathogenesis Physiol. 21:1847-1853. of invertebrate microbial diseases. Allanheld, Osmun and 5. Davidson, E. 1981. A review of the pathology of bacilli Co., Totowa, N.J. infecting mosquitoes, including an ultrastructural study of 13. Myers, P., and A. Yousten. 1978. Toxic activity ofBacillus larvae fed Bacillus sphaericus 1593 spores. Dev. Ind. sphaericus SSII-1 for mosquito larvae. Infect. Immun. Microbiol. 22:69-81. 19:1047-1053. 6. Davidson, E., and P. Myers. 1981. Parasporal inclusions in 14. Myers, P., and A. Yousten. 1981. Toxic activity ofBacillus Bacillus sphaericus. FEMS Microbiol. Lett. 10:261-265. sphaericus for mosquito larvae. Dev. Ind. Microbiol. 7. Davidson, E., S. Singer, and J. Briggs. 1975. Pathogenesis 22:41-52. of Bacillus sphaericus strain SSII-1 infections in Culex 15. Myers, P., A. Yousten, and E. Davidson. 1979. Compara- pipiens quinquefasciatus larvae. J. Invertebr. Pathol. tive studies of the mosquito-larval toxin of Bacillus 25:179-184. sphaericus SSII-1 and 1593. Can. J. Microbiol. 25:1227- 8. Fast, P. 1981. The crystal toxin of Bacillus thuringiensis, 1231. p. 223-248. In H. D. Burges (ed.), Microbial control of 16. Singer, S. 1980. Bacillus sphaericus for the control of pests and plant diseases 1970-1980. Academic Press, Inc., mosquitoes. Biotechnol. Bioeng. 22:1335-1355. London. 17. Wickremesinghe, R., and C. Mendis. 1980. Bacillus 9. Holt, S. C., J. J. Gauthier, and D. J. Tipper. 1975. Ultra- sphaericus spore from Sri Lanka demonstrating rapid structural studies of sporulation in Bacillus sphaericus. J. larvicidal activity on Culex quinquefasciatus. Mosq. Bacteriol. 122:1322-1338. News 40:387-389.