Ultrastructural Analysis of Spores and Parasporal Crystals

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1982, p /82/ $02.00/0 Copyright C 1982, American Society for Microbiology Vol. 44, No. 6 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 and Department of Zoology, Arizona State University, Tempe, Arizona 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 isolated from dead mosquito larvae have been shown to produce a toxin(s) which is lethal when fed to healthy larvae (7, 13, 16). The toxic effect of the B. sphaericus toxin resembles that of the B. thuringiensis 8-endotoxin in that both rapidly affect the midguts of the intoxicated larvae (10, 12). In B. thuringiensis, the 8-endotoxin is contained within a parasporal body or crystal which is formed at the time of sporulation (1, 3, 8): Although the toxicity of B. sphaericus 1593 has also been shown to increase at the time of sporulation (15), initial studies did not detect any inclusion bodies within the cell. Subsequently, electron microscopy (5, 6) and light microscopy (14, 17) were used to demonstrate the presence of inclusions in some strains. Several strains, including some that are nontoxic, produce darkstaining elliptical or oval bodies that do not dissolve during passage through the larval gut (5, 6). Because of the failure to dissolve in the gut, they were judged to be unlikely sites of toxin. In addition to the oval and elliptical bodies, four of the most highly toxic strains (1593, , 1691, and 2297) also formed polyhedral inclusions. The polyhedral inclusion in strain 1593 was shown to have a crystal-like lattice structure. In this paper, this type of inclusion will be referred to as a parasporal crystal or simply as a paraspore. It has not been reported that the paraspore of strain 1593 dissolves in the larval gut. B. sphaericus 2297 produces a particularly large and easily detected inclusion, but it is unknown if the inclusion possesses the crystal-like lattice or if it dissolves in the larval gut. We examined the formation of this inclusion (a parasporal crystal) during the course of sporulation and the fate of the inclusion after ingestion by mosquito larvae. MATERIALS AND METHODS Bacteria. B. sphaericus 2297 was obtained from S. Singer, Western Illinois University, Macomb, Ill. This is the strain isolated by Wickremesinghe and Mendis and designated MR4 in their paper (17). Growth conditions. Spores of B. sphaericus 2297 to be used as inocula in growth curve experiments were produced by smearing the bacteria onto the surface of NYSM agar (nutrient agar [Difco Laboratories, Detroit, Mich.], 0.05% yeast extract, 5 x 10-5 M MnCl2, 7 x 10-4 M CaCl2, 10-3 M MgC12) and incubating the plates at 30 C for 48 h. The sporulated cells were washed off the plates with sterile distilled water and washed three times with sterile distilled water, and the final pellet was frozen and lyophilized. The spore powder was held at -20 C; it had an LC50 of 150 ng/ml (LC50 is the dry weight of bacterial cells that killed 50% of the test insect population in 3 days). Sporulated cells to be fed to larvae were prepared by growing the bacteria with shaking at 150 rpm in a model G

2 1450 YOUSTEN AND DAVIDSON incubator shaker (New Brunswick Scientific Co., New Brunswick, N.J.) at 28 C in NYSM broth. The bacteria were then washed free of medium and suspended in tap water. Growth curve experiments were begun by suspending dried spores in sterile distilled water (12 mg/ml) and boiling the suspensions for 15 min. This treatment either completely destroyed the toxicity of the spores or allowed a very small percentage of the original toxicity to remain. Also, approximately 99.99% of the spores lost viability after this treatment. These nonviable spores retained refractility and remained visible as free, nongerminated spores throughout the course of the experiment. A 3-ml portion of the spore suspension was added to 200 ml of NYSM broth in a 2-liter Erlenmeyer flask which was shaken (175 rpm) at 30 C on a model G25 incubator shaker. Growth was monitored by following absorbance with a Klett-Summerson photoelectric colorimeter and red filter. All total viable counts, spore counts, and the 11-, 12.5-, and 14- h samples for bioassay were taken from a single flask. The cells used for bioassay at 0, 3, 5, 7, and 9 h were recovered from 200 ml of broth taken from other flasks. The larger volumes were required in the early hours because of the low toxicity of the cells. The absorbance of the entire content of each flask did not differ by more than 0.03 from that of the flask from which total viable and spore counts were performed. Total viable cell counts were carried out by plating on NYSM agar. Spore counts were carried out by plating on NYSM agar after a 1.5-ml sample had been heated at 80 C for 12 min. Electron microscopy. Samples from flasks used for 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 microscopy. Second-instar Culex quinquefasciatus larvae were placed in a suspension of sporulated B. sphaericus 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 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 were rinsed, held overnight in 2% uranyl acetate, dehydrated, and embedded in Spurr resin. Thin sections were stained with uranyl acetate and alkaline lead citrate and observed in a Philips EM300 electron microscope. Bioassays. Bioassays were conducted with secondinstar C. quinquefasciatus larvae as previously described (15), except that final observations were made after 3 days rather than 4 days. Toxic activities are reported by LC50 values. Dry weights were determined in triplicate by drying 1-ml samples of the cell suspensions at 110 C for 24 h. RESULTS The data showing growth, sporulation, and toxicity development of B. sphaericus 2297 in a typical growth curve experiment are presented APPL. ENVIRON. MICROBIOL. in Fig. 1. After inoculation of the flask with boiled spores, approximately 90% of the spores which had survived boiling germinated (lost heat resistance) during the first 4 h of incubation. A small increase in total cell number was observed as early as 2 h after inoculation, and exponential growth occurred from 4 to 12.5 h. The toxicity of the inoculum was very low, and it changed little for the first 3 h of growth. However, after 3 h the toxicity of the cell mass began to increase at a rather steady rate. From 3 to 8 h, the vegetative population increased about 1,000-fold, the heatstable spore count failed to increase, and the toxicity increased about 30-fold. From 8 to 14 h, the vegetative population increased about 100- fold, the spore count increased about 1,000-fold, and the toxicity of the cell mass increased about 2,800-fold. Thin sections were prepared to determine the sporulation stage at which the inclusion first appeared. Examination of cells at stage II (forespore septum formation) did not reveal the presence of inclusions (Fig. 2A). At stage III, as the forespore septum engulfed the forespore, the inclusion became visible (Fig. 2B). The assembly of the inclusion appeared to be very rapid after the completion of the forespore septum and the beginning of forespore engulfment. In Fig. 2C, the inclusion is visible in a cell during stage III and in a cell in which spore cortex is being d.~~~~~~7 / / ~~~zs 106 /03J ot /\. =o 0 U10', \ / l HOURS FIG. 1. Growth, sporulation, and toxin production by B. sphaericus Symbols: 0, total colonyforming units; A, spores; *, LC50.

3 VOL. 44, 1982 A SPORE AND PARASPORE ULTRASTRUCTURE 1451 B formed at stage IV. The inclusion was separated from the outer spore coat by a halo of lowdensity-staining material (Fig. 3). The ultrastructure of the parasporal inclusion was visible in this section (Fig. 3), and the inclusion was confirmed as a parasporal crystal. Measurements made from enlargements of this photo- FIG. 2. B. sphaericus 2297 at different stages of sporulation. (A) Stage II. The forespore septum (arrows) 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 interested in determining the fate of spores and particularly paraspores after ingestion by the filter-feeding larvae. After 15 min of feeding on spores and paraspores, the only evidence of parasporal bodies in larval midguts was the presence of residual envelopes (Fig. 5A). The parasporal envelopes retained the shape of the paraspores and appeared to have a meshlike substructure (Fig. SB). In the partially digested

4 4 - e;.it i >., i..< > Xi7 X, Cs ^9> ja i;. waf ^t0 vs t 0vt 0 f..'s t* qr:ws xx.;s,: H. xw ' a0v vs d50! 1. i S * S. -ef ' E gi Wi. +. ; art rb; v YOUSTEN AND DAVIDSON.},.t.^' X B:ato a # Ss g '' q u! luli t1 s>o zilf _ vt ' *s, S ^ >;0 \S] iv7' 4;^ ud7env - :d-! '?_ APPL. ENVIRON. MICROBIOL. }. a irf, fj W 0i 0, Jlre,. SX xt, 092. wx n j Downloaded from 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 separated the paraspore from the spore (Fig. 5B). This branch of the exosporium crossed the cell between the paraspore and the halo of lowdensity-staining material. This halo of unidentified material was also observed in nonpathogenic strain 9602 by Holt et al. (9). Only a few paraspores found in the most anterior end of the larval midgut, near the stomodeal valve, retained their electron-dense appearance and presumably had not yet been dissolved by digestive fluids (Fig. 5C). In Fig. SC, the separation of the spore from the paraspore by a branch of the exosporium is visible. The spores did not appear to change significantly in structure for up to 1 h after ingestion, the longest this strain was observed. An earlier study with B. sphaericus 1593 showed that about 6 h were required before signs of spore germination and outgrowth appeared (5). However, signs of larval intoxication appeared shortly after the larvae were placed into the suspension of B. sphaericus DISCUSSION During B. sphaericus 2297 exponential growth, the toxicity per dry weight unit of the cells increased; i.e., the LC50 decreased. Although the synthesis of some toxin by vegetative cells cannot be excluded by these experiments, the decrease in the LC50 cannot be accounted for by the increase in numbers of vegetative cells. The reproduction of vegetative cells having the same amount of toxin would have resulted in an unchanged LC50. Rather, the increase in the number of spores in the culture seems to be directly correlated with the increasing toxicity. For B. sphaericus 9602, Holt et al. (9) found 6 to 7 h to be required from the time of the onset of on July 3, 2018 by guest

5 VOL. 44, 1982 FIG. 4. A parasporal inclusion (PI) present within the swollen end of the bacterial cell. Note the lack of a forespore septum. Bar = 0.4,um. sporulation to the development of mature spores in a medium similar to the one used in this study. This indicates that a small number of B. sphaericus 2297 cells probably initiated sporulation as early as 2 or 3 h after inoculation, and the early increase in toxicity could be attributed to the appearance of these forms in the primarily vegetative cell population. It is possible that the earliest appearing spores were the result of microcycle sporulation. Between 12.5 and 14 h, the vegetative cell population had stopped increasing, whereas the spores and sporulating cells constituted a higher percentage of the total population. The largest increase in toxicity took place in this time interval. The number of heatresistant spores continued to increase after 14 h (to 5.2 x 108/ml at 24 h), but the LC50 did not decline further. The large parasporal crystal was first observed during the engulfment of the forespore (stage III). This is the same as the time of parasporal appearance in B. thuringiensis (1). Whereas in most serovars of B. thuringiensis the paraspore is located outside the exosporium, the paraspore of B. sphaericus 2297 is partially enclosed by the exosporium and yet is separated from the spore by what appears to be an extension or branch of the exosporium across the cell. The parasporal envelope which remained after SPORE AND PARASPORE ULTRASTRUCTURE 1453 dissolution of the paraspore was often seen in contact with the cross-cell portion of the exosporium. 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 hexagonally arranged subunit structure shown by Holt et al. (9) on the exosporium of B. sphaericus The parasporal matrix was probably dissolved 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 exosporium and the parasporal envelope to the conditions in the larval gut which dissolved the parasporal 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 exosporium 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). Moreover, the distance observed was similar to the approximately 7-nm separation between the outer coat lamellar layers and the exosporium layers found in measurements made on the B. sphaericus 9602 photographs of Holt et al. (9). The dissolution of the parasporal matrix under conditions which could be expected to liberate a toxin from the ingested cells indicates that these inclusions could be a source of the toxin. However, even if these inclusions contain toxin, they are certainly not the only site of the B. sphaericus mosquito larval toxin. For example, some strains, such as SSII-1, do not form inclusions at all, and yet they possess some toxicity (6, 13). Also, purified cell walls of the highly toxic strain 1593 have been shown to be toxic, as have spores cleaned by sonication (14). There does appear to be a correlation between presence of the parasporal crystals and high toxicity. Davidson and Myers (6) found that strains 1593, 2297, , and 1691, which produced polyhedral bodies (which we now call parasporal crystals), have a lower LC50 than strains which produce no inclusions or which produce only the darkstaining elliptical or oval inclusions lacking a lattice substructure. The strains which produced the paraspores were found to be approximately equivalent in toxicity to one another. The toxicity of the strain 2297 paraspores can only be proven after their isolation from other cellular components. The rare appearance of the parasporal crystal in cells having terminal swelling but lacking a forespore septum seems to indicate an uncou-

6 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 paraspore. 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 terminal swelling is not invariably linked to engulfment of the forespore. However, terminal swelling 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 crystal (5). ACKNOWLEDGMENTS This research was supported by grant PCM 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 Ultrastructure, physiology,

7 VOL. 44, 1982 and biochemistry of Bacillus thuringiensis. Crit. Rev. Microbiol. 8: Charles, J.-F., and H. de Barjac Sporulation and cristallogenese de Bacillus thuringiensis var. israelensis en microscopie electronique. Ann. Microbiol. (Paris) 133A: Cooksey, K The protein crystal toxin of Bacillus thuringiensis: biochemistry and mode of action, In H. D. Burges and N. W. Hussey (ed.), Microbial control of insects and mites. Academic Press, Inc., London. 4. Dadd, R Alkalinity within the midgut of mosquito larvae with alkaline-active digestive enzymes. J. Insect Physiol. 21: Davidson, E A review of the pathology of bacilli infecting mosquitoes, including an ultrastructural study of larvae fed Bacillus sphaericus 1593 spores. Dev. Ind. Microbiol. 22: Davidson, E., and P. Myers Parasporal inclusions in Bacillus sphaericus. FEMS Microbiol. Lett. 10: Davidson, E., S. Singer, and J. Briggs Pathogenesis of Bacillus sphaericus strain SSII-1 infections in Culex pipiens quinquefasciatus larvae. J. Invertebr. Pathol. 25: Fast, P The crystal toxin of Bacillus thuringiensis, p In H. D. Burges (ed.), Microbial control of pests and plant diseases Academic Press, Inc., London. 9. Holt, S. C., J. J. Gauthier, and D. J. Tipper Ultrastructural studies of sporulation in Bacillus sphaericus. J. Bacteriol. 122: SPORE AND PARASPORE ULTRASTRUCTURE Huber, H., and P. Luthy Bacillus thuringiensis delta endotoxin: composition and activation, p In E. W. Davidson (ed.), Pathogenesis of invertebrate microbial diseases. Allanheld, Osmun and Co., Totowa, N.J. 11. Kellenberger, E., A. Ryter, and K. Sechaud Electron microscope study of DNA-containing plasms. II. Vegetative and mature phage DNA as compared with normal bacterial nucleoids in different physiological states. J. Cell Biol. 4: Luthy, P., and H. Ebersold Bacillus thuringiensis delta endotoxin: histopathology and molecular mode of action, p In E. W. Davidson (ed.), Pathogenesis of invertebrate microbial diseases. Allanheld, Osmun and Co., Totowa, N.J. 13. Myers, P., and A. Yousten Toxic activity of Bacillus sphaericus SSII-1 for mosquito larvae. Infect. Immun. 19: Myers, P., and A. Yousten Toxic activity of Bacillus sphaericus for mosquito larvae. Dev. Ind. Microbiol. 22: Myers, P., A. Yousten, and E. Davidson Comparative studies of the mosquito-larval toxin of Bacillus sphaericus SSII-1 and Can. J. Microbiol. 25: Singer, S Bacillus sphaericus for the control of mosquitoes. Biotechnol. Bioeng. 22: Wickremesinghe, R., and C. Mendis Bacillus sphaericus spore from Sri Lanka demonstrating rapid larvicidal activity on Culex quinquefasciatus. Mosq. News 40: Downloaded from on July 3, 2018 by guest