Ultrastructural Analysis of Spores and Parasporal Crystals Formed by Bacillus Sphaericus 2297 ALLAN A
Total Page：16
File Type：pdf, Size：1020Kb
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.