BACILLUS. The second and third groups are comprised of organisms whose spores possess no exosporium. Those in the second were received as strains of

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1 COMPARISON OF SPECIES AND VARIETIES OF THE GENUS BACILLUS STRUCTURE AND NUCLEIC ACID CONTENT OF SPORES' PHILIP C. FITZ-JAMES2 AND I. ELIZABETH YOUNG3 Department of Bacteriology and Immunology, and Department of Biochemistry, University of Western Ontario, London, Canada Received for publication May 10, 1959 A preliminary study of the nucleic acid content of the spores of Bacillus cereus and of crystalbearing organisms related to B. cereus revealed that although the medium could affect the spore composition, certain fractions, particularly the deoxyribonucleic acid, were remarkably constant when expressed on a per spore rather than a dry weight basis (Fitz-James, 1957). The characteristic content of deoxyribonucleic acid in spores of certain species prompted further analyses of this group of organisms and also of spores of other species in the genus Bacillus. This comparison is the subject of the present communication; the results of some comparisons of the structure of spores which arose from this work are presented separately. MATERIALS AND METHODS Details of the organisms studied are given in table 1. To facilitate this comparison they were grouped as outlined. Those assigned to the first group form spores which possess an exosporium. This structure, recognized by de Bary in 1884 and so named and further described by Lewis in 1934, can be readily seen on spores in water mounts and air-mounted nigrosin smears when observed with the oil immersion objective. Observations with the electron microscope, however, have greatly facilitated the recognition and characterization of this saclike appendage (Robinow, 1951) (figures 10 and 11). It covers the entire spore but is usually closely applied only around the sides of the spore. Thus, it remains attached to the outer spore coat when the latter is separated from the spore body by disruption 1 Supported by a grant-in-aid from the National Research Council of Canada. 2 Medical Research Associate, National Research Council of Canada. 3Present address: Department of Bacteriology, University of Alberta, Edmonton, Canada. or is cast off following germination (figure 11). Its formation during spore development as a new and distinct layer within the sporangial cytoplasm can be seen in ultrathin sections of sporulating cells (Hannay, 1956). The second and third groups are comprised of organisms whose spores possess no exosporium. Those in the second were received as strains of Bacillus megaterium, whereas those in the third are two rapidly motile, sporeforming rods. The classification and nomenclature of Smith et al. (1952) have been followed where possible. Methods of spore production. The agar medium of Howie and Cruickshank (1940) supplemented with 0.5 per cent casamino acids (H and C) and a beef papain digest nutrient agar (Asheshov, 1941) were the two solid media used in most of the experiments to maintain cultures and to grow spore crops. Sporulated cultures were harvested and washed as described previously (Fitz-James, 1955a). When the spores or sporecrystal mixtures were freed of detectable vegetative debris, sufficient 0.1 N NaOH was added at room temperature to raise the ph to 10.8 to The spores could then be separated under any swollen parasporal material by centrifugation. The spores were resuspended and rewashed, twice with 0.5 N NaOH, once with 0.1 N HCl, and 4 or 5 times with water or saline. Any debris on the surface of the pellet was removed after each centrifugation. The final pellet of spores was made into a thick suspension (1010 per ml) in water and dispensed for dry weight determinations, counting, and analysis. The purity of the spore suspension was further checked in the electron microscope during the measurements of spore volume. For reasons of safety, B. cereus var. anthracis, and for comparison B. cereus strain N, were grown in an aerated liquid medium developed for studies of sporulation (Young, 1958). Spores 743

2 744 FITZ-JAMES AND YOUNG [VOL. 78 TABLE 1 Description of organisms studied Name of Organism Strain Source and Characteristics Reference Group 1 1. Bacillus cereus 2. Bacillus Berliner thuringiensis 3. B. thuringiensis Berliner 4. B. thuringiensis Berliner 5. Bacillus sotto Ishiwata 6. B. sotto Ishiwata 7. Bacillus cereus var. alesti Toumanoff and Vago 8. B. cereus var. alesti Toumanoff and Vago 9. B. cereus var. alesti Toumanoff and Vago 10. B. cereus var. alesti Toumanoff and Vago 11. B. cereus 12. B. cereus N B A31-9 A30 B30-1 Dr. C. F. Robinow. This organism showed a typical smooth colony type of growth on agar. Dr. T. Angus, Sault Ste. Marie, Canada. This insect pathogen forms a spore and a parasporal crystal in each cell (figure 3). Isolated from platings of heat-activated spores which had been diluted in formaldehyde-saline. These colonies appeared identical to the T+ strain but only one half of the cells produced a crystal (figure 4). Six separate isolates have been made. The spores did not lie obliquely in those cells which did not contain a crystal. Isolated in the same way as the T± strain. Cells of this isolate formed only spores (figure 5). The spores did not lie obliquely within the sporangium. Dr. T. Angus, Sault Ste. Marie, Canada. This insect pathogen forms both a spore and parasporal crystal in each cell (figure 1). Isolated from platings of heat-activated spores of strain S+ which had been diluted in formaldehyde-saline. Cells in these isolates produce only spores (figure 2). Dr. C. Toumanoff, Pasteur Institute. Isolated from diseased silkworm larvae, this organism forms both a spore and parasporal crystal in each cell. Isolated from a plating of the A+ strain, this organism resembled the parent strain in regard to size of spores and colonial form but the cells did not form crystals. Dr. C. Toumanoff, Pasteur Institute. This organism formed uniformly large crystals. Dr. C. Toumanoff, Pasteur Institute. After culturing strain A for 31 passages on alkaline medium, ph 9.0, this strain which had lost its crystal-forming ability was isolated. Dr. C. Toumanoff, Pasteur Institute. This organism is classified at the Pasteur Institute as a typical B. cereus. Dr. C. Toumanoff, Pasteur Institute. Isolated following 8 passages through the body cavity of the larvae of Galleria mellonella of strain A30. Each cell forms a spore, a small crystal and a larger, triangular-shaped inclusion (figure 6). Heimpel and Angus (1958) Heimpel and Angus (1958) Toumanoff and Vago (1951) Toumanoff (1956)

3 -1959] COMPARISONS OF SPORES OF BACILLUS TABLE 1-Continued 745 Name of Organism Strain Source and Characteristics Reference -13. B. cereus 14. B. cereus -15. Bacillus cereus var. anthracis 16. Bacillus cereus var. mycoides 17. B. cereus var. mycoides 18. Bacillus medusa Group 2 1. Bacillus megaterium 2. B. megaterium 3. B. megaterium 4. Bacillus 350 Group 3 1. Bacillus subtilis 2. Bacillus apiarius B30-2 B-1 w y L KM Penn Marburg Dr. C. Toumanoff, Pasteur Institute. Isolated following 2 passages of strain B30-1 per os through larvae of Galleria mellonella. This isolate resembles B. thuringiensis. Dr. C. Toumanoff, Pasteur Institute. Isolated from larvae of Galleria mellonella into which an asporogenous culture of a Bacillus species had been previously injected. Morphologically this isolate resembles B. thuringiensis but has a mesenteric type of growth on agar. Isolated in 1954 from a fatal case of bovine anthrax. Dr. C. F. Robinow. Typical whorls of white growth on agar. Dr. C. F. Robinow. Typical whorls of growth on agar; produces a yellow pigment during vegetative growth. Dr. C. F. Robinow. Isolated from cow dung, this organism produces a large spore and an ellipsoidal or spherical parasporal body in each cell (figure 7). Dr. C. F. Robinow. Isolated from boiled sliced carrots by the method of Koch (1888). Isolated following repeated platings on papain-digest agar of the asporogenous strain received from Dr. S. Spiegelman, University of Illinois. Dr. E. D. DeLamater, University of Pennsylvania Dr. John Risbeth, Botany School, Cambridge, England. Isolated from a sample of African soil and classified by Dr. Risbeth as a B. megaterium (figure 8). Vegetative cells are sensitive to lysozyme (figure 9) but do not grow on glucosenitrate agar. Dr. Ruth Gordon, New Jersey Experimental Station, New Brunswick, New Jersey. Dr. H. Katznelson, Science Service Laboratory, Ottawa, Canada. Hannay (1956) Katznelson (1955) were harvested and washed free of debris by cycles of centrifugation and resuspension. Another fluid medium developed for the study of spore and crystal formation in B. cereus var. alesti (Young, 1958) was also used for the production of spores of this and closely related strains. Analytical methods. (1) Dry weights:-dry weights to the nearest 0.1 mg were determined in duplicate on 0.5 or 1.0 ml of the final spore suspension. After 10 to 20 hr at 60 C, they were dried to a constant weight over phosphorus pentoxide. Duplicates were within 1 per cent of the mean weight.

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5 ~~~~ l to Downloaded from FIGS Figures 1 to 6 Water mounts of sporulated cells from 40- to 48hr cultures on agar. Figure 1. Bactillus sotto Ishiwata in final stages of sporulation forming a spore and small crystal inclusion in each cell. Figure 2. Two chains of a nonerystal-forming strain isolated from Bacillus sotto. Except for the absence of the crystals the morphology of growth and sporulation was very similar to the parent strain. Figure 3. Bacillus thur-ingiensis Berliner. Figure 4. A varient isolated from the parent culture of B. thuringiensis, which, during sporulation, forms slightly smaller crystals in some cells and none in others. Figure 6. Another variant isolated from B. thuringiensis which forms only spores. Figure 6. Bacillus sp. B30-1 which forms a triangular (t) and diamond shaped (c) inclusion during sporulation. Figure 7. Bacillus medusa showing the refractile parasporal bodies in a 22-hr fluid culture. Figure 8. A water mount of Bacillus 350 treated for 5 mi in 0.15 per cent KMnO4and stained 4 mmn in 0.25 per cent thionine. The lightly stained spores focused below the plane of refractility show the coat wrinkles characteristic of this large spore former. Photograph courtesy of Dr. C. IF'. Robinow. Figure 9. Bacillus 350 protoplasts formed 10 min after the addition of lysozyme to vegetative cells grown in 2 per cent peptone and stabilized in sucrose (0.3 m). All, with the exception of figure 8, which is bright field, are dark phase contrast photomicrographs. Magnification is approximately 4000X as indicated by the 5 ja marker. Figure 10. An electron micrograph of an unshadowed spore of Bacillus cereus var. alesti illustrating the characteristic exosporium found with minor variations of the spores of B. cereus and varieties thereof (X39,000). Figure 11. Carbon replica of spores and spore coat of B. cereus var. alesti, after the method of Bradley and Williams (1957). 747 on December 6, 2018 by guest

6 748 FITZ-JAMES AND YOUNG [VOL. 78 TABLE 2 Comparison of the size, weight, and composition of the spores of Bacillus cereus and those of related Bacillus species harvested from H and C agar Organism and Straina Comparison of Spores B. cereus B. B.sl soto B..tuigess thuringiensi. cereus B. B. cereus var. mycoides N S+ S- T+ Tt T- var m edusa_ alesti (A+) W y Spore volume (u3 X 102 i SEM) i17.0 i 17.4 i ±44.4 ± ± 71.5 ± 72.5 i Avg wt per spore (g X Nitrogen per spore (g X Phosphorus fractionsb: RNA-PC DNA_Pd Residue-P... e RNA-P/DNA-P a + Normal crystal-former; +, partial crystal-former; -, noncrystal-former; RNA, ribonucleic acid; DNA, deoxyribonucleic acid; SEM, standard error of mean. 'Values expressed as g P per spore. c By the orcinol reaction on trichloroacetic acid extracts (Schneider, 1945). d By the diphenylamine reaction on trichloroacetic acid extracts of the DNA separated by the Schmidt and Thannhauser (1945) procedure as in Fitz-James (1955a). e The spore residue remaining after N KOH extraction (Schmidt and Thannhauser, 1945) plus hot trichloracetic acid extraction; composed of spore coat. TABLE 3 Comparison of the size, weight, and composition of the spores of Bacillus cereus and those of two related organisms harvested from nutrient agar Comparison of Spores Organism and Straina B. cereus B. solto B. thurin- (N) (S+) gsens(s Spore volume (,u3 X 102 ±4 SEM) ± ±t19.0 ± Avg wt per spore (g X 10-12) Nitrogen per spore (g X 10-16) Phosphorus fractions: RNA-P DNA-P Residue-P RNA-P/DNA-P a Strain designation symbols, abbreviations, and description of phosphorus fractions as in footnotes of table 2. (2) Spore counts:-counts were made in the Petroff-Hausser counting chamber using the phase-contrast condenser and 40 X phase-contrast objective (Zeiss). Spore suspensions were diluted in either saline or formaldehyde-saline (0.9 per cent NaCl containing 1 per cent formaldehyde) until the count fell between 20 and 80 spores per large square of the chamber. Six or more large squares were counted for each of at least four applications or until the average number of spores was within 5 per cent of the mean of all applications. (3) Phosphorus-containing compounds:-the compounds were fractionated and analyzed according to methods already described (Fitz- James, 1955a). To begin the fractionation, spores were disrupted in methanol. Cold trichloroacetic acid extraction followed removal of the lipid fraction. Total nitrogen (N) was estimated by micro- Kjeldahl procedure using a microadaptation of the ashing procedure of Beet (1955). When possible, the chemical data were calcu-

7 1959] COMPARISONS OF SPORES OF BACILLUS 749 TABLE 4 Comparison of the weight, volume, and phosphorus fractions of the spores of Bacillus cereus and closely related Bacillus species (varieties) harvested from aerated fluid cultures" Comparison of Spores B. cereus and Variants in Medium A B. cereus var. alesti and Related Strains in Medium B B cereus B. cereus cereus B. B. cereus B. cereus B. cereus B. cereus (N) var. var. var. alesti var. alesti var. alesti var. alesti B.cereus anthrax mycoides (A+) (A-) (B C')) (31-9()) (-i) Wt per spore (1012 g) Volume per spore (M3 X 102 :1: SEM) i 24.8 :i i : Phosphorus fractions: RNA-P DNA-P Residue-P RNA-P/DNA-P a Strain designation symbols, abbreviations, and description of phosphorus fractions as in footnotes of table 2. lated both as a function of dry weight and of spore count (g X per spore). Only the latter values have been included in the tables; those for the per cent dry weight can be calculated by dividing the amount of the fraction per spore (10-16 g) by the average weight per spore (10-12 g) and adjusting the decimal. Morphological methods. Spore volumes were calculated from measurements taken directly from the electron microscope screen and from electron micrographs of shadowed and unshadowed material. The screen measurements were made at a magnification of 20,000 X with reference to the 1,u marker of the microscope (Philips E.M. 100A) and the measurements from the micrographs were similarly made after projection from an enlarger. As the majority of the spores were elliptical in outline, the formula 7rab2/6 (where a is the length and b the width of a prolate spheroid) was applied to 10 or more spores in each group. The standard error for the mean volume was calculated. However, as the variation due to the magnification on the screen of the microscope was found to vary at different times of measurement by about 10 per cent, only the major differences in spore volume can be considered significant. Methods of fixation, hydrolysis, and staining smears for light microscopy have been described in detail elsewhere (Robinow, 1951; Fitz-James, 1955a). RESULTS Analysis of spores in group 1 (table 1). (1) Ribonucleic acid content and spore volume.-the analysis of the spores in group 1 are summarized in tables 2 to 5. Of the three crystal-forming organisms, B. cereus var. alesti forms the largest and Bacillus sotto the smallest spores. The spores of Bacillus medusa, like those of B. cereus var. alesti are approximately twice the size of B. cereus strain N whereas those of B. cereus var. mycoides are approximately three times larger. The content of ribonucleic acid (RNA-P) varied with the spore volume, larger spores containing up to twice as much RNA as the smaller ones (table 2). However, a change in medium could affect both the size and RNA-P content of the spores; for instance, those of the crystal-forming organisms were smaller and possessed a considerably reduced content of RNA when grown on nutrient agar (table 3) or in aerated fluid medium (table 4). While spores of B. cereus strain N appeared unaffected by such changes in media (tables 2 to 4) those of B. cereus var. mycoides were also decreased in RNA-P content and spore volume (table 2 and 4). It has also been a consistent observation that spores of the crystal-forming organisms formed on nutrient agar are prone to spontaneous germination and lysis when kept in aqueous suspension. These spores, although possessing the decreased amount of RNA are enriched in

8 750 TABLE 5 Comparison of the size, weight, and amounts of the phosphorus fractions of the spores of Bacillus cereus ASO and of the spores of two different crystal-forming bacilli isolated from Galleria mellonella following injection and feeding of culture ASO Comparison of Sporesa FITZ-JAMES AND YOUNG B. cereus Strain A30 B30-1 B30-2 Spore volume (ju3 X 102 itsem) ±t46.7 it Ob Wt per spore (10-12 g) Nitrogen per spore (10-16 g) Phosphorus fractions: RNA-P DNA-P Residue-P RNA-P/DNA-P a Spores for this comparison were grown on papain digest agar. Abbreviations and description of phosphorus fractions as in footnotes of table 2. b Of the same width as the other two organisms but slightly longer. nitrogen (tables 2 and 3). It should be emphasized, however, that the crystals formed on either medium were indistinguishable. (2) Deoxyribonucleic acid content:-it became apparent from these various analyses that the quantity of one component, deoxyribonucleic acid (DNA-P) remained remarkably constant for each species regardless of the growth medium or attendant changes in volume of RNA-P content of the spores. For example, spores of B. cereus strain N contain a similar DNA-P content on all media so far employed for their production. Separate studies have further revealed that the rough strain of this organism has this same amount of DNA-P which is also possessed by B. cereus var. anthracis. However, this amount of DNA-P per spore is not common to all members of group 1 (table 1) for B. cereus var. alesti and B. medusa contain approximately twice this amount, but Bacillus thuringiensis and B. cereus var. mycoides contain an amount slightly greater than B. cereus strain N. It should be noted, furthermore, that the non- and partial-crystalforming organisms isolated from crystal-formers possess the same DNA-P content as the parent organisms (tables 2, 4, and 5). (3) Residue phosphorus:-the amount of phosphorus in the residue fraction was also useful for distinguishing certain members of this group. Associated with the spore coat remnants, this residue-p was similar in amount in B. cereus strain N, B. sotto and B. cereus var. alesti. However, spores of B. thuringiensis possessed almost 10 times this quantity. Similarly, those strains of B. thuringiensis which had lost partially or completely the crystal-forming ability still contained this same high content of phosphorus in the residue fraction (table 2). It is also of interest to note the level of residue- P in the spores of the two crystal-forming organisms isolated from Galleria mellonella following injection and feeding of B. cereus strain A30 (Toumanoff, 1956). The organism B30-1, which formed two parasporal inclusions formed spores which contained four times the amount of residue-p as did B. cereus strain A30, whereas the second isolate (B. cereus strain B30-2) produced spores which contained a further 10-fold increase in this fraction (table 5). Analysis of spores in group 2 (table 1). The three strains of B. megaterium, L, KM, and Penn, all possessed similar amounts of DNA-P per spore (table 6). The larger and heavier spores of TABLE 6 Comparison of the size, weight, and amounts of the phosphorus fractions of the spores of varieties of Bacillus megaterium (grown on H and C agar except Bacillus 350, potato agar) Comparison of Sporesa Spore volume Culture Designation [VOL. 78 L KM Penn Bacillus 350 (113 X 102 ± SEM) ±t it Wt per spore (107-2 g) Nitrogen per spore (10-1 6g) Phosphorus fractions: RNA-P DNA-P Residue-P RNA-P/DNA-P a Abbreviations and description of phosphorus fractions as in footnotes of table 2.

9 1959] COMPARISONS OF SPORES OF BACILLUS 751 TABLE 7 Comparison of the phosphorus fractions of the spores of two highly motile bacilli grown on H and C agar Comparison of Sporesa Bacillus subtilis Organism Bacillus apiarius Spore volume (0A X 102 ± SEM) i 2.0 Avg wt per spore (10-'2 g) Phosphorus fractions: RNA-P DNA-P Residue-P RNA-P/DNA-P a Abbreviations and description of phosphorus fractions as in footnotes of table 2. Bacillus 350, on the other hand, contained about twice as much DNA-P as the others and were richer in N and RNA-P. A comparison of these spores with those of B. megaterium strain L grown on the same medium (potato agar) indicated that these differences were not due to the medium. In a previous comparison of spores, the large quantity of residue-p associated with the spore coat in B. megaterium compared with that in B. cereus strain N was emphasized (Fitz-James, 1955a). From table 6 it is obvious that a large content of phosphorus in this residue is not a characteristic of all strains of B. megaterium. In two strains, the residue phosphorus is over one per cent of the dry weight of the spores, whereas in the other it is practically nil. This apparent difference in spore coat composition prompted a further structural comparison (Fitz-James and Young, 1959). Analysis of spores in group 3 (table 1). Spores of Bacillus subtilis as those of Bacillus laterosporus (Fitz-Jamesand Young, 1958) havea much smaller volume than those just described (table 7). Spores of Bacillus apiarius, although they appear large, have a remarkably thick outer coat which gives an erroneously large estimate of the true volume of the spore protoplast. Thus, in keeping with the smaller volume of the actual spore protoplast, the spores of both these organisms contain an average amount of DNA-P which is half that found in the smaller spores in group 1 (figure 12) and an amount of RNA which is 20 I.*t BOcllus cersus vaalestl crystal-former e non crystal-former o Bgcillus 350 V Bacillus medusoa 15 w ~~~~~~~~~white strain w.[,, Y D Bacillus cereus vor mycoldes e strain YI yellow strdnv o full crystol- forrmer e * C Bacilus thuringlensis portiol crystal-former 2 non crystol-formor O AV a Bacillus corsus N tx B Bacillus megaterium strs L, K M and Penn. V 5I to x VA Bocillus solto crystol- former A '-a---' non crystol-former a X. Bacillus cereus vor anthrocls + z Bacillus subtilis. 2 6 Bacillus oplarius 2 5. I 4 A Bacillus lsntimorbus 3 ' ' Bacillus popilliae 4 Bodllus laterosporus 5 Figure 12. A compilation of the average deoxyribonucleic acid (DNA-P) content of the spores of some 22 species, subspecies, and variants of the genus Bacillus taken from this and companion publications. Except for Bacillus thuringiensis, the DNA-P/spore tends to be a multiple of the amount found in the smallest spores (group A). also much lower. In this respect they are again similar to the spores of B. laterosporus (Fitz- James and Young, 1958) and those of Bacillus lentimorbus and Bacillus popilliae Dutky (Fitz- James, unpublished data); the DNA content of these organisms has been included in figure 12). As would be expected, the amount of DNA-P in the spores was reflected in the size of the nuclear bodies seen after hydrolysis and staining; those with the smallest DNA-P content also possessed the smallest nuclear bodies. DISCUSSION As a criterion for taxonomy, the presence of an exosporium about the spore can be of considerable value. Such a covering is not present on spores of B. megaterium or B. subtilis but is present on those of B. cereus and other organisms believed to be varieties or close relatives of it. It must not, however, be confused with the cellwall remnant which remains attached to the ripe spores of some other species. Within this B. cereus group, the organisms can now be further identified according to the average content of DNA within the spores. The average amount of

10 752 FITZ-JAMES AND YOUNG this compound is constant and characteristic of each organism and strain derived from it and remains unaltered during nutritionally induced changes in spore size and RNA content. Three subgroups are thus formed each containing both crystal and noncrystal-forming organisms. That subgroup of which B. thuringiensis is the prototype can be further identified by the characteristically high content of residue phosphorus in the spores. Thus, from these criteria, i. e., spore content of DNA and residue phosphorus, it appears that B. cereus var. alesti and B. sotto may not be as closely related to B. thuringiensis as previously thought by others (Delaporte and Beguin, 1955; Heimpel and Angus, 1958). For convenience in comparisons of this type we consider all these crystal-bearing and derived noncrystal-bearing organisms as variants of B. cereus as already suggested by Smith et al. (1952) for B. thuringiensis. In addition to possessing an exosporium, as B. cereus, cultures of these organisms on egg yolk agar showed the positive reaction due to lecithinase production (Colmer, 1948). The spores of the three strains of B. megaterium studied possess similar amounts of DNA. The strains of this species, however, can be further separated by the presence or absence of a phosphorus-containing spore coat residue. On the other hand, Bacillus 350, in keeping with its nutritional requirements, is distinguishable from typical strains of B. megaterium by its doubled amount of DNA. Recently, Woese (1958) reported that spores of B. subtilis, Bacillus mesentericus, and Bacillus brevis exhibited "single hit" inactivation parameters on exposure to X-rays whereas spores of B. cereus, B. cereus var. mycoides, and three species of B. megaterium showed "multiple hit" inactivation curves. To explain these differences, the author suggested that the genetic material in the multiple hit spores is doubled in comparison to the single hit spores. It is interesting that of these spores, B. subtilis belongs in the group with the lowest content of DNA and that this amount is indeed doubled in B. cereus and in the three B. megaterium species analyzed, but tripled in B. cereus var. mycoides. An interpretation of the present results, in the light of Woese's data, is that 5 X 1016 g DNA-P represents a single chromosome set and those spores with multiples of this amount of DNA possess 2, 3, and 4 similar units. [VOL. 78 It must be emphasized, however, that no increase in number of "chromosome sets" can be observed cytologically when spores with these increasing amounts of DNA are compared. All of these spores, regardless of DNA content, contained one nuclear body, i. e., a single continuum of DNA as discerned microscopically, and all of the vegetative cells contained two chromatin bodies in some stage of division (Fitz-James, 1955b; Young and Fitz-James, 1959). However, the content of DNA was reflected by the size of the stained nuclear bodies. Thus, the increased amount of DNA between organisms represents an increase in DNA per chromatin body and not an increase in number of chromatin bodies per cell. It is interesting to note that a similar increase in DNA per discrete nuclear body was encountered by Ogg and Zelle (1957) during the derivation of a strain of giant cells of Escherichia coli by treatment with camphor. With this increase Zelle and Ogg (1957) observed a shift from a single to a multiple hit irradiation survival curve. Further studies on other species may reveal that the DNA content of the spores of this genus form a more closely integrated scale of values than the stepwise pattern encountered so far. The shorter step from B. cereus strain N to B. thuringiensis (groups B to C, figure 12) may be so explained. Nevertheless, the grouping of the various organisms in figure 12 suggests that a changing DNA content is part of the process of variation in the evolution of this genus. ACKNOWLEDGMENTS The authors wish to thank Dr. C. F. Robinow for his interest, encouragement, and helpful discussions throughout the course of these studies. We are grateful to MIrs. Sheila Newton for her careful technical assistance. SUMMARY The size and phosphorus fractions of spores of an assortment of Bacillus species have been compared. Noncrystal-forming mutants were isolated from and compared to parent crystalforming strains of Bacillus sotto, Bacillus thuringiensis, and Bacillus cereus var. alesti. These, like B. cereus, B. cereus var. mycoides, and B. cereus var. anthracis and others, all possess a distinctive exosporium, on which basis they were grouped. The B. cereus group could be further divided into 4 subgroups depending on the

11 1959] COMPARISONS OF SPORES OF BACILLUS 753 spore content of deoxyribonucleic acid and, in some instances, of residue phosphorus. The medium used and the presence or absence of crystal-forming ability did not alter these two parameters, but on a limiting sporulation medium the ribonucleic acid content of spores of crystalformers was lower. Spores of three Bacillus megaterium strains possessed identical contents of deoxyribonucleic acid, but the quantity of residue phosphorus varied markedly. The spores of Bacillus subtilis and the heavy, square-coated spores of Bacillus apiarius had a deoxyribonucleic acid content half that found in B. cereus. When the values for the average deoxyribonucleic acid per spore of all the cultures studied were assembled, except for one group, all were near multiples of the amount found in the srnallest spores. REFERENCES ASHESHOV, I. N Papain digest media and standardization of the media in general. Can. Public Health J., 32, BEET, A. E Potassium permanganate in Kjeldahl method for determination of nitrogen in organic substances. Nature, 175, 513. BRADLEY, D. E. AND WILLIAMS, D. J An electron microscope study of the spores of some species of the genus Bacillus using carbon replicas. J. Gen. Microbiol., 17, 75. COLMER, A. R The action of Bacillus cereus and related species on the lecithinase complex of egg yolk. J. Bacteriol., 55, DE BARY, A Vergleichende Morphologie und Biologie der Pilze, Mycetozoen und Bacterien, pp Verlag. Wm. Engelmann, Liepzig. DELAPORTE, B. AND BGJGUIN, S Etude d'une souche de Bacillus pathogbne pour certains insects indentifiable a Bacillus thuringiensis Berliner. Ann. inst. Pasteur, 89, FITZ-JAMES, P. C. 1955a The phosphorus fractions of B. cereus and B. megaterium. I. A comparison of spores and vegetative cells. Can. J. Microbiol., 1, FITZ-JAMES, P. C. 1955b The phosphorus fractions of Bacillus cereus and Bacillus megaterium. II. A correlation of the chemical with the cytological changes occurring during spore germination. Can. J. Microbiol., 1, FITZ-JAMES, P. C Discussion in Spores, A Symposium held at Allerton Park, Illinois. Edited by H. 0. Halverson. Am. Inst. Biol. Sci. Publ. no. 5, pp FITZ-JAMES, P. C. AND YOUNG, I. E Morphological and chemical studies of the spores and parasporal bodies of Bacillus laterosporus. J. Biophys. Biochem. Cytol., 4, HANNAY, C. L Inclusions in bacteria. In Bacterial anatomy, pp , Vol. VI. Edited by E. T. C. Spooner and B. A. D. Stocker. Symposium Soc. Gen. Microbiol. Cambridge University Press, Cambridge, England. HEIMPEL, A. M. AND ANGUS, T. A The taxonomy of insect pathogens related to Bacillus cereus Frankland and Frankland. Can. J. Microbiol., 4, HOWIE, J. W. AND CRUICKSHANK, J Bacterial spores as antigens. J. Pathol. Bacteriol., 2, KATZNELSON, H Bacillus apiarius n. sp. an aerobic spore forming organism isolated from honeybee larvae. J. Bacteriol., 70, KOCH, A Ueber Morphologie und Entwickelungsgeschichte einiger endosporer Bacterienformen. Botan. Zeitung, 46, LEWIS, I. M Cell inclusions and endospore formation in Bacillus mycoides. J. Bacteriol., 28, OGG, J. E. AND ZELLE, M. R Isolation and characterization of a large cell possibly polyploid strain of Escherichia coli. J. Bacteriol., 74, RoBINOW, C. F Observations on the structure of Bacillus spores. J. Gen. Microbiol., 5, SCHMIDT, G. AND THANNHAUSER, S. J A method for the determination of desoxyribonucleic acid, ribonucleic acid and phosphoproteins in animal tissues. J. Biol. Chem., 161, SCHNEIDER, W. C Phosphorus compounds in animal tissues. J. Biol. Chem., 161, SMITH, N. R., GORDON, R. E., AND CLARK, F. E Aerobic spore forming bacteria. U. S. Dept. Agr. Agr. Monograph No. 16. p. 7. TOUMANOFF, C Virulence experimentale d'une souche banale de Bacillus cereus Frank et Frank pour les chenilles de Galleria mellonella et Pierris brassicae. Ann. inst. Pasteur, 90, TOUMANOFF, C. AND VAGO, C L'agent pathogene de la flacherie des vers A soie endemique dans la r6gion des Cev6nnes; Bacillus cereus var. alesti var. nov. Compt rend., 233, WOESE, C. R Comparison of the X-Ray sensitivity of bacterial spores. 75, 5-8. J. Bacteriol.,

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