ALTERATIONS IN METAL CONTENT OF SPORES OF BACILLUS

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1 ALTERATIONS IN METAL CONTENT OF SPORES OF BACILLUS MEGATERIUM AND THE EFFECT ON SOME SPORE PROPERTIES' RALPH SLEPECKY2 AND J. W. FOSTER Department of Bacteriology, The University of Texas, Austin, Texas The relation of metal ions to bacterial spores has almost invariably been studied from the standpoint of the influence of their presence or absence during bacterial growth upon the degree of subsequent sporulation of the cultures (for literature see Curran, 1957; Grelet, 1957). Information on the participation of metal ions in the sporulation process and on their incorporation in spores deals mainly with calcium (Powell and Strange, 1956; Vintner, 1956, 1957), originating with the Curran et al. (1943) discovery of the high concentration of calcium in the spores of various aerobic bacilli as compared to the vegetative cells. Vintner's (1956, 1957) experiments with "defective" spores suggest that a high calcium content is essential for formation of normal spores. deficiency leads to reduced spore formation and to a lowered heat resistance of the spores formed (Grelet, 1952; Sugiyama, 1951). This information, together with the high calcium content of normal spores and the excretion of calcium dipicolinate during germination (Powell and Strange, 1953), has led to the belief that calcium is rather specifically associated with the acquisition of heat resistance in bacterial spores. The metal chelating properties of dipicolinic acid (Powell, 1957) might account for the exceptional accumulation of calcium by spores. However, in view of the fact that dipicolinic acid chelates bivalent cations nonspecifically 1 This work was supported in part by the Microbiology Branch of the Office of Naval Research, by the Division of Biology and Medicine of the Atomic Energy Commission, and by a grant-in-aid from the American Cancer Society. This work was presented in part at the 58th Annual Meeting of the Society of American Bacteriologists, May, 1958, Chicago, Illinois (Bacteriol. Proc., 1958, 43). 2 Present address: Department of Biological Sciences, Northwestern University, Evanston, Illinois. Received for publication January 12, 1959 (Slepecky and Foster, 1959, unpublished data) the question arises as to whether calcium is absorbed selectively or nonspecifically by a sporulating cell. The preponderance of calcium in the spore might then be simply a reflection of the relative abundance of calcium in the medium. Expressed differently, (a) is calcium enrichment requisite for formation of a spore? and (b) is calcium enrichment essential for resistance of those spores to various imposed stresses? This work contributes to the answers to these questions by showing how the metal cation content of spores of Bacillus megaterium becomes altered according to the content of metals in the medium. EXPERIMENTAL METHODS Spores were harvested from a chemically defined, liquid medium, the normal composition of which was as follows: sucrose, 1.0 g; KH2PO4, 5.0 g; (NH4)2HP04, 1.0 g; MgSO4, 0.2 g; NaCl, 1.0 g; CaCl2, 5 mg; MnSO4-H2O, 7.0 mg; ZnSO4, 10 mg; FeSO4, 10 mg; deionized water, 1 L. Trace elements were added or subtracted as indicated for particular experiments. The phosphates were adjusted to ph 6.8 with NaOH and sterilized separately prior to addition to the medium. Fifty-ml portions of medium in 250-ml Erlenmeyer flasks were inoculated with 0.5 ml of a 24-hr culture of B. megaterium grown in one-half strength nutrient broth. Flasks were incubated at 30 C on a reciprocating shaking machine operating at seventy-five 7.5-in strokes per min. Maximal sporulation, about 90 per cent as determined by a direct count in a Petroff- Hauser chamber, ordinarily occurred by the 60th hr. Cells were then centrifuged in a Servall SS-1 angle centrifuge at 25,000 x G for 5 min and washed 3 times with deionized water. The last wash water contained negligible amounts of the metals under analysis. Examination of the finally resuspended spores under the phase contrast of the light microscope indicated the spores were 117

2 118 SLEPECKY AND FOSTER [VOL. 78 "clean" for the purposes of this study. The entire harvesting process was carried out at 4 C to retard germination of the spores. Dry weight determinations were made on aliquots of the final spore suspensions; they were dried overnight at 100 C and weighed after cooling in a desiccator containing anhydrous CaCl2. In some instances, the highest levels of a metal salt resulted in development of a precipitate of the metal phosphate during incubation. This was dissolved prior to centrifugation of the spores by addition of 0.2 ml of 7.5 N HCl per 50 ml of culture. Preparatory to metal analyses, the organic matter of the spores was eliminated by a wet oxidation procedure. Dried spores in a 30-ml micro-kjeldahl flask were treated with 0.5 ml of 69 per cent HNO3, 0.2 ml of 95 per cent H2SO4, and 0.2 ml water. After mixing, the contents of the flask were slowly digested over a flame to fuming; more water (0.2 ml) was then added and the contents were further digested to fuming a second time. After cooling, the volume was adjusted to 25 ml with deionized water. These solutions were used for the various metal analyses, mostly by colorimetric procedures. Cobalt was determined by the nitroso-r salt method of McNaught (1939). Manganese was measured as KMnO4 after oxidation with K104 (Coleman and Gilbert, 1939). The dimethylglyoxime method was used for nickel (Sandell, 1950). Dithizone extractions were employed for the analyses for zinc and copper (Sandell, 1950). was precipitated and determined as the phosphate (Roe and Kahn, 1929). RESULTS 1letal content of spores as influenced by metal content of the medium. Each of the chelating metals listed in table 1 was quantitatively measured in spores produced under the two extremes of nutrition with respect to the individual metals, namely, the minimal and the maximal. By "minimal" and "maximal" is meant the lowest and the highest concentrations, respectively, of an individual metal salt in the chemically defined medium described under Experimental Methods, which reproducibly allowed complete sporulation. Each metal salt was tested in a series of concentrations differing by a factor of 2. Of the metals tested in relation to sporulation, an absolute requirement was observed only for TABLE 1 Metal content of spores of Bacillus megaterium formed in media containing, respectively, minimal and maximal levels of individual metals* Metal Ion Added to Metal Ion Metal Ion 50 ml of Medium Content of Dry Spores mg matoms % Zinc Nickel 0 0 < Copper 0 0 < Cobalt Manganese Min * "Maximal" (max) and "minimal" (min) refer to the lowest and the highest concentrations, respectively, of the individual metal salts allowing essentially complete sporulation of the culture. In the case of manganese, levels higher than mg allowed sporulation, but it was this level that resulted in maximal incorporation of manganese in the spores. See text for composition of basal medium. manganese; nonaddition of manganese to the growth medium led to nonsporulating cells, as discovered by Charney et al. (1951). Nonaddition of zinc or calcium resulted in diminished sporulation. Table 1 shows that normal spore crops were formed over extremely wide ranges of concentrations of individual metal ions. For calcium the range was 1805-fold, and for zinc, 2270-fold. Inasmuch as normal sporulation occurred without addition of nickel, copper, or cobalt the ranges in these cases could not be calculated. Table 1 also shows that, with the exception of copper which was very toxic, there was significant incorporation in the spores of each of the metals studied. The incorporation was greater the more metal the medium contained. However, it is clear

3 1959] METAL CONTENT OF SPORES OF B. MEGATERIUM 119 that a concentration mechanism was at work, to account for the high accumulation of calcium, nickel, zinc, and manganese in the respective treatments. Although dipicolinic acid in the spores might accumulate these metals through chelation, one would expect cobalt to be accumulated similarly, but its accumulation was relatively minor. The data in table 1 establish that the content of individual metals in bacterial spores can be varied over a wide range, and that under the appropriate conditions metals other than calcium may be accumulated to a striking degree. This nonspecificity is illustrated by a nickel content of 1.81 per cent, compared to 3.04 per cent calcium in the corresponding maximal treatment. The interchangeability of certain ions in sporulation is also illustrated by the induction of spore formation in Bacillus coagulans by the addition of 1 ppm of sulfates of manganese, nickel, or cobalt (Amaha et al., 1956). Extracts of the various types of spores obtained with a Mickle tissue disintegrator had ultraviolet absorption spectra quite similar to the authentic metal chelates of dipicolinic acid (Slepecky and Foster, 1959, unpublished data) in the cases of calcium, zinc, nickel, and manganese enriched spores, but not with the copper and cobalt enriched spores. If the metals do not exist in the spores as chelates of dipicolinic acid, they are at least available for chelation with dipicolinic acid when the spores are disrupted. content of spores as influenced by the content of other metals. Table 2 lists the calcium contents of each of the kinds of spores described in table 1. The most significant conclusion from table 2 is that maximal levels of other ions effectively competed with calcium for accumulation in spores. In the presence of these metals the absorption of calcium by the spores was markedly diminished as compared to calcium absorption in their absence. For example, maximal zinc, nickel, or manganese reduced the calcium content to a small fraction of what it was in the minimal treatments. Although the zinc, nickel, and manganese contents of the spores was substantially higher in these instances, the increments were not equivalent to the drop in calcium content. Thus, a substitution definitely occurred, though not quantitatively. The calcium content of spores was not materially influenced by the addition of maximal copper or cobalt, a finding consistent with the TABLE 2 content of spores formed in media containning, respectively, minimal and maximal levels of individual metals* Metal Ion Zinc Nickel Copper Cobalt Manganese Metal Ion Added to 50 ml of Medium?ng * See footnote to table 1. Ion Added to 50 ml of Media mg Content of Dry Spores earlier discovery that these two metals are accumulated only poorly, in comparison with the others. The low calcium content of the minimal copper, cobalt, and manganese spores is ascribed to the interference of calcium uptake by the other ions in the respective basal media (Mn++, 0.11 mg; Zn++, 0.20 mg; Fe++, 0.18 mg). These levels are considerably above the minimal concentrations supporting full sporulation, and their effectiveness in blocking calcium accumulation (maximal zinc and manganese, table 2) indicate this is the likely explanation for the above-mentioned low calcium values. It will be noted from the maximal manganese culture that spores containing an extremely low calcium content can be obtained. Properties of the spores with different metal contents. (1) Thermal resistance:-this was examined at different temperatures for a given time, and for different times at a given temperature, using deionized water suspensions of washed spores of the various types described in tables 1 and 2. The tubes were shaken frequently during heating in the water baths; they were

4 120 SLEPECKY AND FOSTER [VOL. 78 then immersed in ice water. Counts of survivors were made by spreading 0.10 ml of appropriately diluted suspensions on the surface of quadruplicate nutrient agar plates. Survivors were judged by the development of typical colonies after 24 hr incubation at 30 C. A different experiment in which killing was followed as a function of time of heating at 70 C gave results permitting the same conclusions as figure 1. It is evident from figure 1 that spores with higher accumulations of zinc, manganese, or nickel were decidedly less tolerant to heat than spores with the minimal contents of those metals. The reverse was true of calcium. Since high zinc, manganese, or nickel content of the spores results in lowered calcium (table 2), it is possible that the resistance of such spores is simply a reflection of their calcium levels. However, the situation is probably more complex than that, and suggests that heat tolerance to some extent is an attribute of the metal itself, since similar results were obtained 4tt'---'--- sjr ~---@ 80 A Min Zn Mn Mn r so ~ I Mon Zn Mkom Mn' taie imax iu 60~~~~~~~~~~~8 MimnCoMoal Mco Min Max-Ni0 20~~~~~~~~~~~ FIT S TEMKPRATURE Figure 1. Suirvival curves for the different metal types of spores of Bacillus megateriutm described in table 1. R.T. denotes room temperature. The broken line (Min) in the lower right quadrant refers to spores obtained in the normal medium (see Experimental Methods). with cobalt spores not displaying a significant change in calcium content. Also, the maximal manganese spores contained only Kj the calcium content of the minimal calcium spores, yet were not more heat sensitive. Conversely, the minimal manganese spores, with about the same calcium content as minimal calcium spores, were appreciably more heat resistant. The slopes of the killing curves in figure 1 are essentially the same for the various types of spores. The distinguishing characteristic of the more resistant suspensions appears to be that the critical temperature for killing in 10 min lies above 70 C, whereas it is below that temperature for the counterpart suspensions. In the sensitive series about half the spores were killed when there was no significant killing in the resistant series. The different experiments on heat resistance of the spores indicate the presence of two types of spores in each of the more sensitive suspensions, i. e., the maximal zinc, the maximal manganese, and the minimal calcium spores. About one-half the spores were killed in 10 to 15 min at 70 C, but there was no further reduction in viable count for 40 min, the longest time tested. Thus, the extremes of metal nutrition which reduced heat resistance did so by a conspicuous lowering of the threshold killing temperature of one-half the spores, but without appreciably affecting the sensitivity of the remaining half. A possible interpretation of this phenomenon is presented under Discussion. (2) Ultraviolet irradiation resistance:-a separate killing curve for each of the various types of spores was obtained in conventional fashion, in a room with yellow light to exclude photoreactivation. The ultraviolet source was a 15-watt General Electric germicidal lamp which at 38 cm from the surface of the bacterial suspension had an output of 4 ergs per mm2 between 2450 and 2800 A. Curves depicting the survivors at intervals up to 7 min, when killing was practically 100 per cent, were virtually superimposable, regardless of the metal content of the spores. Thus, extremes of metal content in the spores did not appreciably alter the susceptibility of the spores to killing by ultraviolet irradiation. (3) Resistance to desiccation:-about 100 spores in 2 ml of water were collected on 50-mm membrane filters (Bac-T-Flex filters, purchased from Carl Schleicher and Schuell Company, Keene, New Hampshire), in duplicate. One filter

5 1959] 15IETAL CONTENT OF SPORES OF B. MEGATERIUM1121 was immediately placed on nutrient soft agar (0.75 per cent agar) and incubated 24 hr at 30 C. This furnished the zero time viable count of the suspension. The second membrane was stored in vacuo in a desiccator containing P205 for 7 days, then placed on nutrient soft agar. No significant reduction in viable count following desiccation was found for any of the different kinds of spores listed in table 1. (4) Phenol resistance :-Preliminary control experiments established that about 50 per cent of the spores failed to produce colonies on nutrient agar after being suspended in 5 per cent aqueous phenol for 5 min. Sufficient spores were used to obtain 50 to 100 colonies per 0.1 ml after a 105 dilution of the phenol suspension, to stop the action of phenol. There was no significant difference found in phenol resistance among the different kinds of spores listed in table 1. DISCUSSION The data presented here demonstrate convincingly that (a) the spore content of individual metal ions may vary within very wide limits, (b) the metal content is dependent on the concentration and probably on the relative balance of metals in the medium during sporulation, and (c) spores concentrate certain metals. In each case studied, the spores were typical with respect to the rate of their formation, the percentage of the vegetative cells which sporulated, refractility in phase microscopy, nonstainability with stains that are readily taken by vegetative cells, and resistance to heat, phenol, desiccation, or ultraviolet irradiation. Thus, these are true spores, and the metal composition of a spore is not rigidly circumscribed, but is dependent on the environment prevailing at sporulation. The high concentration of calcium obtainable in spores (Curran et al., 1943; Powell and Strange, 1956; Vintner, 1957) has been confirmed. However, it is evident that a high content of calcium is not requisite since spores normal in appearance and properties were obtained containing as low as 0.1 per cent calcium. Dipicolinic acid analyses were not performed on the various types of spores described here; but if the levels are in the range reported for aerobic spores (Powell and Strange, 1953; Perry and Foster, 1955b; Janssen et al., 1958; Krishna Murty and Halvorson, 1957), the calcium content would be considerably less than equivalent. This was the case in endotrophic spores of Bacillus cereus var. mycoides (Perry and Foster, 1955a). Although some calcium is essential for spore formation, it would appear that the bulk of the dipicolinic acid, if chelated with metal cations, need not be complexed with calcium, in which case other metal ions can substitute. The extraordinary accumulation of calcium in spores reported by different investigators may well be a fortuitous consequence of its presence in the media in relatively large concentrations, especially in relation to other chelating metals. In media containing complex organic extracts the calcium contents of these ingredients (Difco Manual, 1953) would provide an ample supply of that ion. Where the calcium content of the medium can be regulated, as in chemically defined media, this cation can be shown to lose its uniqueness. The toxicity of other metal cations ordinarily precludes their presence in media in amounts equivalent to calcium which, of course, is nontoxic at relatively high levels. Indeed, the nickel content might well have exceeded the maximal calcium content had nickel not been so toxic to the sporulation process that its level in the medium had to be restricted to 1/5 that of calcium ( matoms Ni++ vs matoms Ca++). Similar possibilities apply to the other metals, particularly zinc (0.35 matoms vs matoms) and manganese ( matoms vs matoms). Although an exceptionally high calcium content appears not to be uniquely associated with spore formation, refractility, or resistance to desiccation, phenol, or ultraviolet irradiation, there is clearly an involvement of this ion in the thermal resistance of spores. Our experiments confirm the conclusions of others on this point (Grelet, 1952; Sugiyama, 1951). However, our data are not decisive as to whether the lower thermal tolerance of calcium-deficient spores is due to the deficiency, or to the presence of replacement metals. In any event, the survival of such organisms in nature is not dependent on high content of calcium for the formation of cation-rich, desiccation-resistant spores. It is extremely unlikely that thermal resistance is a selective force in the vast majority of natural habitats, thus reducing the role of calcium in the evolution of sporeforming bacteria. In each case where the heat sensitivity was

6 122 SLEPECKY AND FOSTER [VOL. 78 increased as a result of metal ion nutrition (figure 1) the spore suspension in reality consisted of two populations, one sensitive and one resistant to the test (70 C for 10 min). Aberrations in killing curves attributable to inhomogeneity among members of spore populations is well known (Church et al., 1956); it remains to determine how metal nutrition could predispose this con(lition. One could visualize this occurring automatically during sporulation of a culture, particularly in the minimal metal treatments. Cells sporulating first would have an opportunity to absorb the metal from a relatively large volume of medium. Their high capacity for absorption might result not only in a marked reduction in the amount of metal available to the fraction of cells sporulating later, but they themselves would tend to resemble the maximal metal spores. Analytical values for metals (tables 1 and 2) may, therefore, represent the mean of metal-rich and metal-poor spores in any one culture. If this is so, then true maximal metal contents probably are higher and the true minimal metal contents much lower than indicated by the data in this paper. SUMMARY Spores of Bacillus megaterium were harvested from media containing individually high and low levels of calcium, zinc, manganese, nickel, cobalt, and copper, respectively. The various crops of washed spores were analyzed for the content of the corresponding metal and also for calcium. The results showed that the content of individual metals in spores is flexible within a very wide range and is dependent on the relative concentration of the particular metal in the growth medium. The accumulation by spores of calcium and of nickel was especially striking. It was also marked in the case of zinc and manganese. The accumulation of the other metals studied seemed to be accompanied by a reduction in the amount of calcium taken up. Thus, the various cations in spores seem to be interchangeable to a certain extent. The various maximal and minimal metal spores were not different in morphologv, staining characteristics, refractility, and resistance to killing by desiccation, phenol, and ultraviolet irradiation. About one-half the maximal zinc, the maximal manganese, and the minimal calcium spores were thermosensitive to 70 C for 10 min, suggesting that calcium is essential for highest thermal resistance of spores. REFERENCES AMAHA, M., ORDAL, Z. J., AND TOUBA, A Sporulation requirements of Bacillus coagulans var. thermoacidurans in complex media. J. Bacteriol., 72, CHARNEY, J., FISHER, W. P., AND HEGARTY, C. P Manganese as an essential element for sporulation in the genus Bacillus. J. Bacteriol., 62, CHURCH, B. D., HALVORSON, H., RAMSEY, D. S., AND HARTMAN, R. S Population heterogeneity in the resistance of aerobic spores to ethylene oxide. J. Bacteriol., 72, COLEMAN, D. R. K. AND GILBERT, F. C Manganese and caffeine contents of some teas and coffees. Analyst, 64, CURRAN, H. R The mineral requirements for sporulation. In Spores. Edited by H. 0. Halvorson. Publ. No. 5, pp American Institute Biological Sciences, Washington, D.C. CURRAN, H. R., BRUNSTETTER, B. C., AND MYERS, A. T Spectrochemical analysis of vegetative cells and spores of bacteria. J. Bacteriol., 45, Difco manual of dehydrated culture media and reagents for microbiological and chemtical laboratory procedures th ed., p Difco Laboratories, Inc., Detroit, Michigan. GRELET, N Le determinisme de la sporulation de Bacillus megatherium. IV. Constituants mineraux du milieu synth6tique necessaires a la sporulation. Ann. inst. Pasteur, 83, GRELET, N Growth limitation and sporulation. J. Appl. Bacteriol., 20, JANSSEN, F. W., LUND, A. J., AND ANDERSON, L. E Colorimetric assay for dipicolinic acid in bacterial spores. Science, 127, KRISHNA MURTY, G. G. AND HALVORSON, H Effect of duration of heating, L-alanine, and spore concentration on the oxidation of glucose by spores of Bacillus cereus var. termninalis. J. Bacteriol., 73, McNAUGHT, K. J The determination of cobalt in animal tissues. Analyst, 64, PERRY, J. J. AND FOSTER, J. W. 1955a content of spores of Bacillus cereus var. mtycoides in relation to heat resistance and content of dipicolinic acid. Texas Repts. Biol. and Med., 13,

7 1959] METAL CONTENT OF SPORES OF B. M1EGATERIUM 123 PERRY, J. J. AND FOSTER, J. W. 1955b Studies on the biosynthesis of dipicolinic acid in spores of Bacillus cereus var. mycoides. J. Bacteriol., 69, POWELL, J. F Biochemical changes occurring during spore germination in Bacillus species. J. Appl. Bacteriol., 20, POWELL J. F. AND STRANGE R. E Biochemical changes occurring during the germination of bacterial spores. Biochem. J. 54, POWELL, J. F. AND STRANGE, R. E Biochemical changes occurring during sporuilation in Bacillus species. Biochem. J., 63, ROE, J. H. AND KAHN, B. S The colorimetric determination of blood calcium. J. Biol. Chem., 81, 1-8. SANDELL, E. B Colorimetric determination of traces of metals, 2nd ed. Interscience Publishers, Inc., New York. SUGIYAMA, H Studies on factors affecting the heat resistance of spores of Clostridium botulinum. J. Bacteriol., 62, VINTNER, V Sporulation of bacilli. III. Transference of calcium to cells and decrease in proteolytic activity in the medium in the process of sporulation of Bacillus megaterium. Ceskoslov. mikrobiol., 1, VINTNER, V The effect of cystine upon spore formation by Bacillus megateriuim. J. Appl. Bacteriol., 20,