JOURNAL OF BACTERIOLOGY, Mar., 1966Copyright © 1966 American Society for Microbiology

Vol. 91, No. 3Printed in U.S.A.

Endotrophic Calcium, Strontium, and Barium Sporesof Bacillus megaterium and Bacillus cereus1

HAROLD F. FOERSTER2 AND J. W. FOSTERThe University of Texas, Austin, Texas

Received for publication 15 November 1965

ABSTRACT

FOERSTER, HAROLD F. (The University of Texas, Austin), AND J. W. FOSTER.Endotrophic calcium, strontium, and barium spores of Bacillus megaterium andBacillus cereus. J. Bacteriol. 91:1333-1345. 1966.-Spores were produced bywashed vegetative cells suspended in deionized water supplemented with CaCl2,SrCl2, or BaC12. Normal, refractile spores were produced in each case; a portion ofthe barium spores lost refractility and darkened. Thin-section electron micrographsrevealed no apparent anatomical differences among the three types of spores.Analyses revealed that the different spore types were enriched specifically in themetal to which they were exposed during sporogenesis. The calcium content of thestrontium and the barium spores was very small. From binary equimolar mixturesof the metal salts, endotrophic spores accumulated both metals to nearly the sameextent. Viability of the barium spores was considerably less than that of the othertwo types. Strontium and barium spores were heat-resistant; however, calciumwas essential for maximal heat resistance. Significant differences existed in the ratesof germination; calcium spores germinated fastest, strontium spores were slower,and barium spores were slowest. Calcium-barium and calcium-strontium sporesgerminated readily. Endotrophic calcium and strontium spores germinated withoutthe prior heat activation essential for growth spores. Chemical germination of thedifferent metal-type spores with n-dodecylamine took place at the same relativerates as physiological germination. Heat-induced release of dipicolinic acid oc-curred much faster with barium and strontium spores than with calcium spores.The washed "coat fraction" from disrupted spores contained little of the sporecalcium but most of the spore barium. The metal in this fraction was released bydilute acid. The demineralized coats reabsorbed calcium and barium at neutral pH.

An important sector of spore science hasemerged from the special significance of ions inthese resistant cells. Bacterial spores are en-riched in metallic ions, notably calcium (6); theheat resistance of spores is dependent on a rela-tively high metallic ion content, notably calcium(3, 6, 48); and exogenous strong electrolytes areessential for physiological germination of thegreat majority of spore strains and for maximalgermination of the remaining minority (12, 43,44, 45). [In earlier papers (43, 44, 45), germina-tion compounds were classified as ionic (varioussalts) or nonionic (L-alanine, inosine, glucose).

I Based on a thesis submitted by H. F. Foerster toThe University of Texas, 1963, in partial fulfillmentof requirements for the Ph.D. degree.

2Present address: Department of Biology, SamHouston State College, Huntsville, Tex.

More suitable designations are "strong electro-lytes" and "weak electrolytes" (alanine, inosine)or "nonelectrolytes" (glucose), and the newterms will be used in this paper.]A determination of how spores are affected by

modifications of the above may help to elucidatethe role of metal ions in these unique cells. Thispaper deals with the substitution of strontiumand barium for calcium in spore formation, andthe properties, particularly germinative, of thevariously constituted spores. To ensure that theeffects obtained are concerned with spore forma-tion per se, and not merely with the capacity ofvegetative cells to sporulate subsequently, theendotrophic sporulation technique was employed(20, 33).Strontium and barium spores have been pre-

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FOERSTER AND FOSTER

pared previously and their heat resistance hasbeen studied (3, 19, 32, 48).

MATERIALS AND METHODS

Bacteria. Bacillus megateriulm QM Bi551 and B.cereus T were studied comparatively because theirspores differ anatomically (37) and germinatively.The former can be germinated with inorganic ions orglucose, or both, the latter with strong electrolytesand a mixture of alanine and inosine (21, 43, 45).

Growth spores. The following medium was used:glucose, 1.0 g; KH2PO4, 5.0 g; (NH4)2HP04, 1.0 g;MgSO4.7H20, 0.2 g; NaCl, 0.1 g; CaCI2, 5.0 mg;MnSO4 5H20, 7.0 mg; ZnSO4 .7H20, 10 mg; FeSO4-5H20, 10 mg; sodium L-glutamate, 1.33 g; yeastautolysate (Basamine, Anheuser-Busch, St. Louis,Mo.), 0.5 g; Difco agar, 20 g; water, 1 liter. Deion-ized distilled water was used throughout this work. Abacterial suspension (0.1 ml) from a 12-hr nutrientagar slant was spread uniformly over the dry agarsurface in petri plates. After incubation for 3 days at30 C, the spores were rinsed off the agar, washed sixto eight times by centrifugation in 25 volumes of coldwater, and finally suspended in water. They remainedstable indefinitely at 4 C.

Enidotrophic spores. Vegetative cells were grown inthe following medium which contained no addedcalcium: glucose, 2.0 g; sodium L-glutamate, 5.2 g;yeast autolysate (Basamine), 0.5 g; NaH2PO4, 1.0 g;MgSO4.7H20, 0.2 g; NaCI, 5.0 mg; CuSO4.5H20, 0.1mg; MnSO4 *5H20, 10 mg; ZnSO4 5H20, 10 mg;FeSO4, 10 mg; water, 1 liter. A 3-liter Fernbachflask containing 1 liter of medium was inoculatedwith 1 ml of an 8-hr nutrient broth culture and incu-bated at 30 C with continuous shaking. At the earlymaximal stationary phase of growth (12 to 14 hr), thecells were centrifuged, washed once in 50 ml of sterilewater, and then suspended in 500 ml of sterile 1.0mM CaC12, SrCl2, or BaCI2. These suspensions wereshaken at 30 C until sporulation was maximal,usually for 24 to 48 hr. The final spore suspensionswere prepared as described above.

Determination of viability. Colony-forming capacityof spores was determined with Difco nutrient agarsupplemented with 0.1% soluble starch (14), bymeans of conventional spread plate procedures.

Heat resistance. Deionized water suspensions con-taining approximately 2,000 viable spores per milliliterwere dispensed in sterile test tubes. One suspensionwas heated at each desired temperature and platedfor viability.

Heat activationz. The spore suspensions were heatedfor 30 min at 60 C prior to each germination test (10,43).

Optical denisity (OD) reduction. Germination wasfollowed by Powell's (35) method. Suspensions ofspores which microscopically have all lost theirrefractility display a 50 to 80% reduction in OD (41,42). Intermediate OD levels reflect the proportion offully germinated (nonrefractile) versus ungerminated(refractile) spores in the suspension. A Klett-Sum-merson and a Bausch & Lomb photoelectric colorim-eter were used. The spore suspensions were adjusted

initially to a reading of approximately 100 Klettunits.

Electroni microscopy. Spores were fixed for 2 hr in2% aqueous, buffered KMnO4, and then were cen-trifuged and washed three times in water. They weredehydrated by successive 10-min exposures to 30, 50,70, 95, and 100%o ethyl alcohol. This was followed bya 15-min exposure to 100%7 propylene oxide. Thefixed spores were embedded for 1 to 2 hr in 25' (l, thenin 506X0, epoxy resin dissolved in propylene oxide,then overnight in 75% epoxy resin, followed by 100%epoxy resin. After 6 hr, the samples were embeddedin no. 5 gelatin capsules and allowed to harden at60 C. Sectioning was performed with a Porter-Blummicrotome at thicknesses ranging from 300 to 600 A.Sections were floated on grids and examined andphotographed with an RCA model EMU 3-D electronmicroscope.

Preparation of spore coat fractionzs. The Nossaldisintegrator cylinder was loaded with 10 g of no. 12Ballotini beads and approximately 1 g of washedspores in 8 ml of water. The cap of the cylinder wassealed with masking tape, and the container waschilled in an acetone-Dry Ice bath between therepeated 25-sec shaking periods. These were continueduntil microscopic examination showed the suspensionto contain only broken spores. The turbid liquidcontaining the coat fragments was decanted, andbead washings were added to it. It was chilled andcentrifuged at 18,000 X g for 30 min at 0 C. Thepellet was suspended in 30 ml of cold water, and thecentrifugation procedure was repeated eight timesbefore lyophilization and storage over P205. Approxi-mately 50% of the initial spore dry weight was re-covered as the coat fraction. Membranes probablywere included, but light and electron microscopicexamination indicated that the material recoveredconsisted predominantly of pieces of coats.To determine metals, samples of spore suspensions

were ashed in porcelain microcrucibles in an electricfurnace at 650 to 700 C for 1 hr. The ash was dis-solved in a hot mixture of 0.05 ml of concentratedHCl and 0.5 ml of 30% H202, and made to 10.0 mlwith washings and water. Strontium was determinedin a sample by the method of Chow and Thompson(4) by use of a Beckman DU flame spectrophotometerequipped with a photomultiplier. Transmission andbackground were measured at 460 and 454 mA,respectively, with the use of a slit width of 0.03. Cal-cium was determined in a similar fashion but withtransmission measured at 422.7 m,u and backgroundat 418 m,u (5, 8). Barium was measured gravimetricallyas BaSO4 (24).

Dipicolinic acid (DPA) was determined spectro-photometrically (30) or colorimetrically (22) incentrifuged extracts made by autoclaving spores for15 min at 120 C (38). Barium ions interfered with theassay; the barium spore extracts were treated withexcess (NH4)2SO4, and the resulting BaSO4 wasremoved by filtration.

Isotope counting techniques. Samples of the speci-mens were evaporated to dryness in planchets. Radio-activity of the infinitely thin samples was measured

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SPORES OF B. MEGATERIUM AND B. CEREUS

in a Tracerlab "64" scaler equipped with a thin-window Geiger-Muller tube.

Hexosamine and phosphorus were determined bythe colorimetric methods of Elson and Morgan (9)and Fiske and SubbaRow (11).

tESULTS

Microscopic appearance. Endotrophic sporeformation took place regardless of which of thethree metals tested was present. Prespore polarbodies indicative of imminent sporulation ap-peared simultaneously in all three cultures. Inthe presence of Ca+ and Sr++, B. megateriumQM B1551 began to exhibit refractility in 6 to 8hr; sporulation was practically completed within1 hr after the appearance of the first fully re-

fractile spores, which became completely freefrom their sporangia in 24 to 48 hr. Endotrophiccalcium and strontium spores appeared identicalin size and refractility (Fig. 1). Refractility de-veloped more slowly in the barium cultures; theyrequired more than twice the time required forthe calcium and the strontium cultures. Thebarium spores contained a refractile central regionsurrounded by a dark rim of variable thickness(Fig. 1).Calcium and strontium spores remained per-

manently refractile, whereas a substantial frac-tion of the barium spores darkened during andafter sporangolysis (Fig. 1). Similar behaviorwas noted with endotrophic magnesium spores,and it has also been reported for endotrophicmanganese spores (32).

B. cereus T behaved similarly, though a muchsmaller fraction of the barium spores darkened(Fig. 1).Metal andDPA contents. Calcium usually is the

predominant cation in spores harvested fromconventional media (6), though not always (1,48, 52). This may be merely the consequence ofthe relative abundance of metals in the sporula-tion environment. The preponderance of sporecalcium often is not great, and metals other thancalcium can be made to accumulate to a strikingdegree (48).

Endotrophic spores became strongly and selec-tively enriched in the particular metal added tothe cultures (Table 1). The strontium and bariumspores were essentially devoid of calcium. Thus,typical endotrophic spores can be formed in theabsence of added calcium, provided a suitablealternative bivalent metal is present (see also 3,32).The percentages of accumulated strontium

and barium were appreciably greater than thatof calcium (Table 1), but the differences parallelthe atomic weights of the respective metals so

that, in terms of milliatoms, the three metals donot differ much. They may be occupying the sameavailable sites.

B. megaterium QM B1551 did not discriminatebetween members of metal pairs furnished inequivalent concentrations (Table 2). This be-havior resembles that of growth spores (1, 27, 31,48, 52). The calcium uptake was only slightly re-duced below what it was when furnished singly;the strontium and barium uptake, while also less,were nevertheless coaccumulated with calcium toa striking degree. From pairs of equimolar con-centrations, approximately 2 Ca-1 Sr and 1Ca-1 Ba were coaccumulated, respectively.Strontium and barium uptake seemed to be in-dependent of calcium uptake since the calcium-strontium and calcium-barium totals were de-cidedly greater than any single metal.The calcium-strontiu n and the calcium-

barium spores were fully refractile. Since bariumspores were not, the copresence of calcium evi-dently was responsible for normal refractility.Strontium spores contained nearly the same con-tent of DPA as calcium spores, but barium sporescontained markedly less; this confirms otherfindings (3, 32).Whether accumulated barium or strontium is

a cause of efficient DPA biosynthesis (3, 49) ora result of it (19) is still a conjecture. If metal ionsstimulate DPA formation, barium did so ineffi-ciently; the concomitant presence of calcium,however, did stimulate it nearly to the maximum,so barium per se obviously was not inhibitory.

Likewise, as shown later, the high strontium orbarium content did not interfere with subsequentgermination, provided calcium was also present.In these spores, it appears that sites nonfunctionalin germination bind strontium and barium.

Thin-section electron micrographs. The copiousquantities of strontium and barium in sporessuggested that their presence might be detecteddirectly by electron microscopy or that mani-festations of their presence would be revealed byalterations in the spore anatomy. If metals areconcentrated in the cortex where their electrondensity might be expected to reveal them, theymay be lost during the fixation procedures (46).Although not instructive regarding location ofthe metals, the electron micrographs did never-theless indicate that the regular anatomicalfeatures in spores were not conspicuously altered.

Viability and germination rates. Most of thecalcium and strontium cells were viable in thetest employed but only approximately one-quarter of the barium cells were. Microscopicexamination established that the calcium sporesgerminated fully in 30 min and that the strontium

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1336 FOERSTER AND FOSTER J. BACTERIOL.

:I

ii

FIG. 1. Enidotrophic spores. Left columrn, Bacillus megaterium QM B1551. Right columrn, B. cereus T. Top,calcium spores; middle, strontium spores; bottom, barium spores. X 1,700.

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SPORES OF B. MEGATERIUM AND B. CEREUS

TABLE 1. Analysis of enriched endotrophic spores of Bacillus megaterium QM B1551 andB. cereus T

Calcium Strontium Barium DPA Matoms of

enriched in Organism metal/Per cent Matoms* Per cent Matoms* Per cent Matoms* Per cent Mmoles* DPAX 102 0 X 102 ecntX 102 Pexcn lo,0

Calcium B. megaterium 1.74 4.35 10.8 6.47 0.67B. cereus 1.45 3.63 9.8 5.87 0.62

Strontium B. megaterium 0.004 0.01 2.57 2.94 8.6 5.15 0.57B. cereus 0.003 0.01 2.28 2.60 8.2 4.91 0.53

Barium B. megaterium 0.002 0.005 5.74 4.18 2.0 1.20 3.5B. cereus 0.002 0.005 6.20 4.51 5.0 2.90 1.6

* Per 100 mg of dry spores.

TABLE 2. Metal and DPA contents of endotrophicspores of Bacillus megaterium QM B1551formed in equimolar mixtures of ions

Metals (matoms)*

Sporetype ~~~DPA Metal/Sporetyperon- (mmoles)* DPACalcium Stron- Barium

Calcium .... 43.5 70.8 0.62Strontium 34.3 61.7 0.56Barium- 41.1 20.0 2.05Calcium-

strontium. 38.0 17.2 65.2 0.85Calcium-barium..... 36.0 30.5 67.1 0.99

* Per 100 g of dry spores.

spores did so much more slowly; even after 90min, the strontium spores of B. megaterium andB. cereus were only 83 and 1% germinated, re-spectively.Heat resistance. Endotrophic strontium spores

were reported to be more heat-resistant than cal-cium spores; barium, zinc, or nickel growthspores, though less so, were nevertheless decidedlyresistant (27, 32, 48). Endotrophic spores of thetwo species used in this work had a high degreeof heat resistance, which is emphasized by therapid killing of the nonresistant, germinated cellsused as controls (Fig. 2). Though less so thancalcium spores, the strontium and barium sporesof both species unquestionably were heat-re-sistant. Differences between the metal spores were,of course, magnified at the higher temperature.Also noteworthy were the observations thatstrontium spores of B. megaterium were fully asresistant as calcium spores at 65 C, barium sporesof B. cereus were much more resistant than thoseof B. megaterium at 75 C, and a large fraction ofthe strontium and barium spores had heat re-

sistance little different from calcium spores atboth temperatures.The strontium and barium populations of both

organisms consisted of two fractions, one killedvery quickly and the other fully as resistant inthis test as calcium spores (Fig. 2). At 75 C, three-quarters of the B. megaterium strontium sporeswere as resistant as the calcium spores; the samewas true of half of the B. cereus strontium andbarium spores. This type of heterogeneity inspore populations has been observed previously(3, 48).

Germination. Conditions in which spores areproduced are known to affect the subsequentgermination response (17, 26, 27). The germina-tion response of growth and endotrophic sporesto strong electrolytes (43) bears that out (Table3). The main conclusions are that endotrophiccalcium spores and growth spores were compar-able in their germinative response to the potas-sium halides, that endotrophic strontium andbarium spores germinated poorly compared tocalcium spores, and that growth spores germi-nated efficiently in the chlorides of the alkaliearth cations (Mg, Ca, Sr, Ba), whereas endo-trophic calcium spores did not.The OD reduction values for barium spores of

B. megaterium QM B1551 are not directly com-parable to those for the calcium and the bariumspores, since a sizable fraction of the populationwas not refractile to begin with. The conclusionsfrom OD measurements were corroborated bymicroscopic inspection of refractile versus non-refractile cells. The correlation between low ODreduction and poor germination of barium sporeswas particularly obvious in the case of B. cereusT, where practically all of the spores were refrac-tile (Fig. 1). Here, also, the microscopic checksverified the conclusion from the OD reductiondata (see later section).

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1338 FOERSTER AND FOSTER J. BACTERIOL.

650 C 750 CI~~~~~~~~~~~~~~~~~~~~~~~~~~

00 g X > Stron t lSCoCalciumSpores

90 Calcium Spores

z50W| t X miumSpStrontiuU)~~~~~~~~~~~~~~~~~~~~ \

Spores80

70

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30 Germinated Calcium Spores

20 *

10Barium Spores

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A Stron~~~~tium90 Spores

80 ~~~~~~BariumSpores

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M N U T E SFIG. 2. Tlhermal killing of endotrophic spores. Top pair, Bacillus megaterium QM B1551. Bottom pair, B.

cereus T.

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SPORES OF B. MEGATERIUM AND B. CEREUS

TABLE 3. Ionic germination of metal-enrichedendotrophic spores of Bacillus megaterium

QM B1551*

Optical density decrease (%)

Germination ions Endotrophic sporesGrowth

sporesCalcium Strontium Barium

None.............. 1 4 3 1Tris buffer, 50 mM.. 54 53 4 4Phosphate buffer,30mM............ 3 4 4 7

KF, 20mM......... 4 8 4 4KCl, 20mM.37 53 9 9KBr, 20mM ........ 51 56 10 10KI, 20mM.......... 53 54 7 8

MgCl2, 10mM.27 8 5 4CaCi2, 10 mM ....... 51 13 3 3SrCl2, 10 m M.38 10 3 7BaCl2, 10mM ....... 45 9 4 3

* Spores were preheated in deionized water at60 C for 30 min. Germination period, 30 min at40 C.

The striking difference between germinationresponses of the growth spores and the endo-trophic calcium spores to the potassium halideson the one hand and the alkali earth metal chlo-rides on the other highlights the importance ofstrong electrolytes for germination (43, 44, 45); italso demonstrates a significant difference betweenthe two kinds of spores.

Potassium dipicolinate (2 mM), when addedwith the alkali earth metal chlorides, which bythemselves had only weak germination activityfor the endotrophic calcium spores (Table 3),caused full germination in 20 min (data not in-cluded; see also 12, 36, 42). The activity of thischelating agent suggests that ionic factors mayinfluence or govern the germinative behavior ofendotrophic spores.

Bypassing of heat activation. Growth spores ofB. megaterium QM B1551, like many Bacillusspecies (7), require heat activation for optimalphysiological germination (43). Germination ofendotrophic calciu n and strontium spores,however, took place very well, although some-what slower, without heat activation (Fig. 3). Agermination mixture consisting of a strong elec-trolyte (Nal) and the organic substances foundbest for growth spores of aerobic bacilli, i.e.,alanine-inosine mixture (12), also sufficed for theendotrophic spores. Strontium spores wereslower than calcium spores, and the bariumspores germinated negligibly. Keynan, Murrell,

and Halvorson (23) reported a similar bypassingof the heat-activation requirement for the alaninegermination of low-DPA growth spores of B.cereus T.

Endotrophic spores of B. cereus T. Ratesof germination of the calcium, strontium, andbarium spores of this organism were similarto those just described for B. megaterium. Aswith growth spores (45), the alanine-inosine mix-ture was inactive unless strong electrolytes werepresent. The activity of Nat and the inactivityof K+ for germination of growth spores were alsocharacteristic of endotrophic spores.

n-Dodecylamine. Relative germination rateswith this surfactant (41) for the different metalspores were similar (Fig. 4) to the physiologicalgermination rates already described.Are spore strontium and barium inhibitory to

germination? The germinative sluggishness ofthese spores probably is not attributable to aninhibitory effect of their strontium and bariumper se. This is the conclusion from an experimentcomparing spore populations produced in solu-tions containing calcium, strontium, or bariumindividually, with spores produced in equimolarbinary mixtures. Calcium-strontium spores andcalcium-barium spores germinated at rates equalto calcium spores, whereas the strontium andbarium spores were typically sluggish (Fig. 5).Spore calcium is evidently required for highestgerminative competence, and the copresence of asecond metal does not interfere. This suggestsagain that strontium and barium are bound atsites nonspecific for germination.

Germination ofendotrophic binary metal spores.When the Ca-Sr and Ca-Ba ratios in the sporula-tion medium were less than 1, the rate and theextent of germination of the spores producedwere decreased proportionately (Fig. 6). Therapid reduction in OD to intermediate levels thatchanged little thereafter is characteristic of frac-tional germination (12, 43). It indicates a strik-ing heterogeneity in the spore population inducedby the ions present in different ratios, and thisheterogeneity is expressed in the step-like maximaof germinations (see Materials and Methods).The calcium-second metal ratio determined theproportions of these populations which wouldgerminate. Whether the same effect would obtainat higher absolute concentrations of metal ionswas not determined. Possibly, under the condi-tions used, the kinds of spores obtained were tosome extent a consequence of limiting amounts ofone or the other metal ions.

Heat-induced release of DPA. A variety oftreatments release calcium and DPA from spores(13, 38). Other evidence (1, 3. 50, 53, 54) also

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FOERSTER AND FOSTER

UN HEATED HEATED

30 60 90 120 30 60 90 120MINUTES

FIG. 3. Germination of unheated and heated spores of Bacillus megaterium QM B1551. Germination solution:Difco Casamino Acids, 40 pg/ml; glucose, 0.5 mM; L-alanine, 0.5 mM; inosine, 0.5 mM; NaCi, 10 mM; NaBr, 10mM; CaCl2 , 10 mM; NaDPA, 10 mM; Na propionate, 10 mM. Heated spores were heldfor 60 min at 60 C. Germi-nation temperature, 40 C.

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MINUTESFIG. 4. Germination of unheated endotrophic spores

of Bacillus megaterium QM B1551 by n-dodecylamine,0.02 mM in 13.0 mM phosphate buffer (pH 8.0). Tem-perature, 40 C.

provides a basis for the view that DPA and Caand other metals may be at least partially che-lated in the dormant spore.DPA was released by heat much more readily

from barium and strontium spores than fromcalcium spores (Fig. 7). As the various metalspore types had no definite anatomical differ-ences, the heat-release patterns may be deter-mined by the stabilities of the bound forms ofmetal and DPA in spores. The release was in-versely related to the stability constants of theDPA chelates (Riemann, Ph.D. Thesis, Univ.of Copenhagen, Copenhagen, Denmark, 1963).Autoclaving at 120 C for 15 min released allthe DPA from each of the spore types.Metal distribution. A marked disparity was

found in the extent to which calcium and bariumwere retained by the particulate fraction of auto-claved or broken spores (Table 4). Spore cal-cium was mostly soluble and spore barium wasmostly particle-bound. The physiological dif-ferences between the calcium and barium sporesmay prove to be related to that difference. Thedistribution of soluble versus particle-bound totalhexosamine and phosphate was not appreciablydifferent in these spores.

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SPORES OF B. MEGATERIUM AND B. CEREUS

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M N U TE SFIG. 5. Germination of preheated endotrophic

spores ofBacillus megaterium QM BJSSJ. Spores wereproduced in the presence of 0.5 mM chlorides of theindicated cations and heated 60 min at 60 C. Thegermination solution was glucose (3.0 mm) and KNO3(40 mM). Temperature, 40 C.

Bound barium. Barium-133 spores were pro-duced endotrophically and disrupted (see Mate-rials and Methods); no intact spores were visiblemicroscopically. Samples were dried in planchets,and the Ball' was counted. Practically all of theparticle-bound Bal33 was released by 0.5 N HCl,and only a minor amount by 0.5 N NaOH overthat of water (Table 5). Endotrophic Ca45-labeled spores behaved similarly.The coat fractions from endotrophic barium

and calcium spores were demineralized with acidaccording to the procedure just described. Eachthen bound more than 2% (w/w) of Bal13 (Table6).

DIscussIoN

As a morphogenetic entity with typical anat-omy and physiology, but demonstrably differentfrom growth spores, endotrophic spores are theproduct of the minimal nutritional conditionssufficient for sporogenesis. The vegetative cell'spool of metal ions, a function of strain and me-dium composition, may suffice to provide thehigh metal content of spores (20, 33). If the poolis inadequate for either reason, the endotrophicneed can be supplied exogenously (3, 32; thispaper).

Also depending on the strain and conditions,

lysis of some cells has been reported to occur(for literature, see 2), and the question has beenraised whether lysate nutrients support multi-plication of the survivors in an endotrophic sys-tem. Apart from the obvious metal requirement,the endotrophic process implies an organicnutritional support of the metamorphosis with-out vegetative multiplication (see also 51). Theextent to which growth may occur in some casesmay become a semantic matter; whether at theexpense of strictly endogenous metabolites or ofsome released by lysis, spore synthesis is "sup-ported exclusively by the pre-existing makeup ofthe vegetative cell" (15). In the final analysis,growth and endotrophic spores are different and,as originally visualized (20), the endotrophictechnique permits the study of controlled sporesynthesis uncomplicated by growth in a completemedium.

It is possible to produce anatomically typicalspores whose major metal components differquantitatively, if not strictly qualitatively. Theyare dormant and resistant to various stresses(3, 48), including moderate temperatures (thiswork). If substitute cations are present, addedcalcium is not essential for the endotrophic meta-morphosis. But, in confirmation of numerousprevious reports, only when adequate calciumor strontium is present does heat resistance of thespores develop to the maximum.Some types of endotrophic spores undergo

prompt germination without heat activation,whereas growth spores of the same organismneed the heat activation. This provides an oppor-tunity to study physiological germination with-out unknown consequences devolving from theusual heat treatment (e.g., solubilization, chem-ical reactions, tertiary structure changes).

Spores contain large amounts of metal ionsother than calcium (Tables 1 and 2; references1, 48, 52, 53). Most of the metals form chelateswith DPA (47) and appear to be absorbed moreor less concomitantly fro -n the medium. Themetal composition of the spore undoubtedlyrepresents an empirical consequence of the par-ticular mineral makeup of the medium. Sinceno one knows which, if any, of the metals arechelated with DPA in the intact spore, the 1:1ratio of Ca-DPA sometimes found in spores(for literature, see 31) may be fortuitous and ofdoubtful significance when taken out of contextof the other cations. However, our data confirmthat calcium is necessary for maximal thermo-stability of spores (18).The distinctive accumulation of various metals

in spores, as compared with the correspondingvegetative cells, is usually ascribed to the DPA;yet, there are many examples where the metal-

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FOERSTER AND FOSTER

A B

10 30 60 10 30 60

MINUTESFIG. 6. Germination ofendotrophic spores ofBacillus megaterium QM B1551 produced in solutions with differ-

ent Ca-Sr and Ca-Ba ratios. The spores were produced in the presence of 0.5 mM total chlorides in the indicatedcation proportions. Spores were heated for 60 min at 60 C and germinated at 40 C in 3.0 mM glucose and 40 mMKNO3.

DPA ratio is significantly greater than 1.0,exceeding 3.0 in some instances (Tables 1 and 2;references 25, 28, 31, 52, 53). One must concludethat metal-binding sites other than DPA existin spores, which may be functional even in casesexhibiting a 1:1 Ca-DPA ratio. Conversely, it isnot known whether all the DPA in spores binds amolar equivalent of metals; in some cases, themolar DPA content greatly exceeds the calciumcontent (e.g., 34).

Regulation of metal cation ratios in sporulationmedia is a way of artificially securing controlledpopulation heterogeneity among spores in re-spect to their germination (see 48). This tech-nique may become a useful experimental tool.The parallelism in degrees of germinationbetween the strontium and the barium series(Fig. 6) suggests that the level of Ca++ appar-ently is the critical factor. The germinating por-tions may have a higher content of calcium thanthe ungerminated portions. They may differintrinsically from the others in their ability,during sporulation, to accumulate calcium selec-tively in the presence of the second metal (see 16).Alternatively, all the spores may be genotypically

identical, only the rates of absorption of thevarious ions being different. In either case, theearliest sporulating members of the populationwould preferentially consume most of the avail-able calcium. Whereas the residual strontiumand barium serve for spore formation in theremaining cells, only calcium, as we have seen,confers prompt and full germinative competence.The strikingly different rates of germination of

the endotrophic calcium, strontium, and bariumspores is a clear indication that interchange-ability of the metals in the synthesis and themaintenance of the dormant spore does notapply to their function in subsequent germina-tion (Fig. 3). Strontium and barium are not in-hibitory, since prompt germination occurs whenthey are paired with calcium in the spore (Fig.5). The spore metal itself is thus an importantfactor in the prime germination event.The chemical germination with n-dodecyla-

mine is of interest in this connection (Fig. 4). Amost efficient germinative compound with endo-trophic calcium as well as growth spores (39, 41),this ionic surfactant was less effective on stron-tium and barium spores, and to the same relative

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SPORES OF B. MEGATERIUM AND B. CEREUS

B. CEREUS T

1 2 4

HOURS

FIG. 7. Thermal release of dipicoliniic acid fromenidotrophic spores. The spore suspensions were heatedin a water bath at 80 C. Thte initial DPA contents ofthe spores were determined on supernatant liquids ofspores autoclaved at 120 C for 15 min.

TABLE 4. Distributionl of metals in fractions ofendotrophic calcium and barium spores of

Bacillus megaterium QM B1551*

Ca45 Ba

TreatmentParticulate Soluble Particulate Solublefractiont fractiont fractiont fractiont

15 min atlOO C....... 11 89 82 18

Mechanicaldisruption. 14 86 60 40

* Figures represent per cent of total Ca45 or Bapresent in the respective Ca and Ba spores. Endo-trophic spores were produced in 0.5 mm CaCl2 orBaC12. The Ca culture was labeled with 0.02 jAcCa4'Cl2 per ml.

t Sedimentable by centrifugation for 15 min at18,000 X g.

$ Supernatant liquid from particulate fraction.

degree as physiological compounds (Fig. 3). Areaction at the metal sites in both physiologicaland surfactant germination may account for thisrelation, the relative reactivities of the respectivespore ions determining the efficacy of the twokinds of germinative compounds. Only ionic

TABLE 5. Release of Ba'33 from "coat fractiont" ofdisrupted endotrophic spores of Bacillus

megaterium QM B1551

Treatment*Fraction Ba"' Ba"'3 retainedTreatment FractBa B' in coatcontaining Ba fraction

coumnt/min /O

Water Soluble t 187Insolublet 1,252 100

HCl, 0.5 N Soluble 1,222Insoluble 8 0.7

NaOH, 0.5 N Soluble 290Insoluble 1,091 87

* For 2 hr at 40 C. Samples (1 mg) of Ba'33-coatfraction used in each case. Ba'33 spores were pro-

duced in 0.5 mM BaCl, labeled with 0.02 4c ofBa"33C12 per ml.

4- t Supernatant liquid after centrifuging for 15min at 18,000 X g.

t Sedimentable by centrifugation for 15 min at_ 18,000 X g.

TABLE 6. Binding of Ba133 by demineralized coat8 fractions of endotrophic spores of Bacillus

megaterium QM B1551*

Ba content (v,'w)Coat fraction source Ba'33 bound

Per cent Amt

colintl/nin mmoles/g

Calcium spores..... 1,194 2.3 0.167Barium spores. 1,114 2.2 0.167

* Portions (1 mg) of the washed, demineralized(see text) coat fraction were suspended in 1 ml of0.25 mm Ba"33CI2, containing 0.04,c (1,731 counts/min). After 2 hr at 25 C, the particulate coatfractions were centrifuged and washed three timesin deionized water before counting.

surfactants germinate bacterial spores; nonionicsurfactants do not (40). Whereas one tends toascribe their germination properties to theirsurfactant character, the action of these com-pounds could prove to be prime examples ofionic germination involving strong electrolytes(43, 44).Our data suggest that the metals bound in the

spore coat fractions are interchangeable, that thebinding sites of calcium and barium (and stron-tium) may be identical, and that the affinity ofbarium and calcium for the coat sites is in in-verse relation to their efficacy in germination ofthe respective metal spores. The germinativebehavior of endotrophic calcium, strontium, andbarium spores thus seems to reflect the relativeaffinities of the spore metals for DPA (chelation)and for other components (salt formation).

Vol. 91, 1966 1343

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FOERSTER AND FOSTER

ACKNOWLEDGMENTSWe are grateful to L. J. Rode for his interest and

helpful discussions.This investigation was supported by Public Health

Service grant AI-03564 from the National Instituteof Allergy and Infectious Diseases, grant 375 (12)from the Office of Naval Research, and grant G14568from the National Science Foundation.

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