REVERSIBLE ACTIVATION FOR GERMINATION AND SUBSEQUENTCHANGES IN BACTERIAL SPORES'

W. H. LEE2 AND Z. JOHN ORDALDepartment of Food Technology, University of Illinois, Urbana, Illinois

Received for publication 15 August 1962

ABSTRACT

LEE, W. H. (University of Illinois, Urbana)AND Z. JOHN ORDAL. Reversible activation forgermination and subsequent changes in bacterialspores. J. Bacteriol. 85:207-217. 1963.-It waspossible to isolate refractile spores of Bacillusmegaterium, from a calcium dipicolinate germina-tion solution, that were activated and wouldgerminate spontaneously in distilled water. Someof the characteristics of the initial phases ofbacterial spore germination were determined bystudying these unstable activated spores. Acti-vated spores of B. megaterium were resistant tostains and possessed a heat resistance intermedi-ate between that of dormant and of germinatedspores. The spontaneous germination of activatedspores was inhibited by copper, iron, silver, ormercury salts, saturated o-phenanthroline, orsolutions having a low pH value, but not by manycommon inhibitors. These inhibitions could bepartially or completely reversed by the additionof sodium dipicolinate. The activated sporescould be deactivated and made similar to dormantspores by treatment with acid. Analyses of theexudates from the variously treated spore suspen-sions revealed that whatever inhibited the germi-nation of activated spores also inhibited the re-

lease of spore material. The composition of thegermination exudates was different than that ofextracts of dormant spores. Although heavysuspensions of activated spores gradually becameswollen and dark when suspended in solutions ofo-phenanthroline or at pH 4, the materials re-leased resembled those found in extracts of dor-mant spores rather than those of normal germina-tion exudates.

'From a thesis submitted to the Departmentof Food Technology, in partial fulfillment of therequirements for the Ph.D. degree, University ofIllinois, Urbana.

2 Present address: Merck and Co., Danville,Pa.

Since the demonstration by Powell (1953) thatbacterial spores contained dipicolinic acid (DPA),there has been much interest in its biological role.Several investigators have sought a correlation be-tween the heat resistance of bacterial spores andtheir DPA content (Church and Halvorson,1959; Black et al., 1960a; Pelcher, 1961). Doi andHalvorson (1961) presented evidence that DPAcould be involved in an electron-transport system.Riemann and Ordal (1961) reported that calciumdipicolinate (CaDPA) caused the germination ofspores of many species of the genera Bacillus andClostridium. This investigation further character-izes the role of CaDPA in the germination ofbacterial spores.

In many bacterial spore-germination systems,the changes occur so rapidly that they are con-sidered simultaneous and irreversible. Little isknown about the initial reaction or phases in-volved when a refractile dormant spore is changedto a germinated spore. One difficulty in studyingthe early phases of germination has been to finda suitable procedure to delay and to separatethese changes. Riemann and Ordal (1961) demon-strated that the rate of germination was decreasedby a reduction in temperature. They (Riemannand Ordal, unpublished data) also demonstratedthat spores of Putrefactive Anaerobe 3679, ex-posed to CaDPA or ethylenediaminetetraaceticacid (EDTA) at low temperatures, were refractilebut would germinate spontaneously when thespores were separated from the CaDPA or EDTAsolutions and resuspended in distilled water.

This report describes the preparation of ac-tivated refractile spore suspensions of Bacillusmegaterium. The suspensions were prepared bytreating them in a solution of CaDPA at tempera-tures of 7 to 10 C. For convenience, these un-stable spores, which will germinate in distilledwater, are called activated spores. Likewise,CaDPA-activated spores stabilized by acidtreatment are referred to as deactivated spores

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to distinguish them from dormant spores. Some ofthe germinal changes which occurred in sporesuspensions receiving various treatments are re-ported and discussed. In particular, the changes inthermal resistance and the release of DPA,amino acids, and peptides are correlated withother criteria of germination.

MATERIALS AND METHODS

Spore preparation. B. megaterium (ATCC13632) was cultured on nutrient agar that con-tained 0.05% MnSO4 and 0.1% CaCl2 and wasmade up with tap water. After 2 days of incuba-tion at 30 C, the spore suspension was washed andfreed of vegetative cells and debris by the methodof Long and Williams (1958). The cleaned sporeswere stored in distilled water at 3 C. Sporesstored in water at 3 C for 3 months or more re-mained refractile but responded more slowly toCaDPA activation than did fresh spores. Be-cause of this change, new spore crops were grownat least every 2 months.

Criteria for germination. The germination ofactivated spores was indicated by an increase inthe per cent transmission of the spore suspensionat 650 m,u in a Coleman Junior spectrophotom-eter (model 6A). Spore refractility was deter-mined microscopically by use of the agar slidemethod (Riemann, 1961).

Preparation of the spore exudates and extracts.Exudates for analysis were collected from treatedspore suspensions (5 ml of 5 X 109 spores/ml or67 mg of spores) after the spores had been ex-posed to the given conditions for 24 hr. The sporesolids were separated from the exudate solutionby centrifugation at 16,000 X g for 10 min. Thespore solids were rinsed twice with 2 ml of water,and the rinsings were added to the supernatantsolution. Extracts of dormant spores were ob-tained by boiling them in water for 90 min or bydisruption in 50% ethanol with a Mickle disinte-grator and Ballantoni glass beads in the room at3 C. All samples were stored at -20 C untilanalyzed.

Paper chromatography of amino acids andDPA. Spore exudates were concentrated andapplied to Whatman no. 1 paper for chromatog-raphy. The chromatograph was developed over-night in an ascending system by using n-butanol,ethanol, water, HCI (80:20:20:2; v/v) at 24C. The chromatogram was air-dried and theresidual HCl removed by steaming in an auto-

clave for 1 to 2 min. Amino acids were detectedby spraying with 0.2% ninhydrin in n-butanol.After the amino acid color had formed at 25 C,the same chromatogram was sprayed with 0.5%thiourea, Fe(NH4)2(SO4)2, and acetic acid solu-tion for the detection of DPA. The FeDPAcomplex color could be further intensified byspraying the chromatogram with 4% collidinein acetone.

Estimation of free amino acids by paper electro-phoresis and scanning. Free amino acids wereseparated by high-voltage paper electrophoresisby using the method of Michl (1959), modified intwo respects. First, instead of using cellophanemembrane tabs to prevent oversaturation, tworows of 16 holes (6-mm diam) were punched inthe paper 3 in. from each end. Second, the paperwas cooled in a chlorobenzene bath in a room at10 C, instead of with cooled plates. Samples wereapplied in 1-in. bands, 6 in. from the cathode, onWhatman no. 1 paper (5.5 by 22 in.) The solventused was 12% acetic and 2.65% formic acid (v/v)in water. Separation was complete after 90 min at0.5 ma per cm width and 30 v per cm, suppliedby a Beckman Spinco Constat power unit. Theamino acids were developed by dipping the stripsin 0.2% ninhydrin in n-butanol. The density ofboth standard and unknown amino acids wasdetermined by using a Beckman Spinco Analytrolequipped with a B-5 cam at 500 m,.Paper electrophoresis of spore protein and pep-

tide and their dinitrophenyl derivatives. The proteinand peptide components of spore germinationexudates from 1 to 5 mg of spores were separatedand characterized by the paper electrophoresisprocedure of Fynn and De Mayo (1951) whichwas used by Strange and Powell (1954) for spore-peptide identification. A better resolution of thepeptide was obtained when the paper electro-phoresis was performed at 20 ma and 500 to 600v per cell for 1 hr at 25 C instead of 120 v for 20hr. The equipment used consisted of a BeckmanSpinco Constat power unit, a Durrum Cell, B-2buffer (pH 8.6 barbital, 0.075 ionic strength), andSchleicher & Schuell 2043 A paper strips (Block,Durrum, and Zweig, 1958). After development,the paper strips were dried in an oven (100 C) for10 min and then stained overnight in a saturatedsolution (1:200 dilution) of Buffalo Black NBR(National Aniline) or Amidoschwartz 10 B in10% acetic acid methanolic solution. The stripswere then rinsed in fresh 10% acetic acid metha-

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FIG. 1. Dark-phase contrast photomicrographs of activated, germinated, and deactivated spores of Bacillusmegaterium. A = spores immediately after activation and washing. B = germinated spores. Spores wereheld in distilled water at 24 C for 1 day following activation and washing. C = deactivated spores. Washedactivated spores were suspended in 12 mm H3PO4 for 1 hr at 3 C, washed in 10 mM phosphate buffer (pH 7),and resuspended in 10 mM MgSO4. Photo taken after 20 days at 3 C.

nolic solution for 0.5 hr and air-dried. In someexperiments, the exudates were also reacted withfluorodinitrobenzene to make the yellow dinitro-phenyl derivatives by the method of Levy (1954),and the samples were dialyzed with four to fivechanges of distilled water in a 24-hr period. Thedialyzed dinitrophenyl derivatives of protein andpeptide were separated by the electrophoresismethod described above.

Analytical methods. Ammonia was determinedby the microdiffusion method of Conway (1950).Reducing sugars were determined by the methodof Somogyi (1945) before and after hydrolysiswith 6 N HCl at 106 C for 3 hr. The ninhydrin-positive materials were determined by the colori-metric method of Moore and Stein (1948). Thefree amino acids of two samples were quantita-tively determined in a Spinco Model 120 aminoacid analyzer patterned after the method ofSpackman, Stein, and Moore (1958).

RESULTS

Activation of spores. Spore activation wasaccomplished by placing dormant spores in asolution of 40 mm CaDPA (pH 7.0). The neces-sary conditions (time and temperature) had tobe established for each spore crop. The firstthree spore crops were activated after 70 min at

10 C, whereas two later crops were activatedafter 70 min at 7 C. The activation solution wasprepared immediately before use by mixingsolutions of CaCl2 and NaDPA to yield thedesired concentration in 1.6 mm tris(hydroxy-methyl)aminomethane (tris) buffer (Riemannand Ordal, 1961). Activation was stopped byadding 2 to 3 volumes of crushed ice and water,and the preparation was immediately centri-fuged. The spore pellet was resuspended incrushed ice and water, recentrifuged, and finallyresuspended in cold distilled water. Followingactivation, the spores were refractile (Fig. 1A)and were resistant to staining by crystal violet.The activated spores would germinate (Fig. 1B)rapidly in water at ambient temperature butmore slowly if the spore suspension was kept inan ice bath. For this reason, the spores werestudied immediately after activation. Figure 2 isa schematic outline of the activation procedureand subsequent treatments used to characterizethe activated spores. Included also is the percent-age of refractile spores in the variously treatedpreparations.The properties of an activated spore suspension

depended on the extent of activation. Sporesactivated at a higher temperature or for a longerperiod of time germinated more rapidly than

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2LEE AND ORDAL

Stock suspension of dormant spores (98 to 100)1I

Suspend spores in 40 mm CaCl2, Na2, DPA, and 1.6 mMtris buffer (pH 7.0) at 7 or 10 C for 70 min. Sporesuspension diluted and washed with ice and water.

IActivated spores (90 to 98)

//

Suspend in distilled Suspend in soltwater at 3 or 24 C per sulfate, fer

\ ~~~~nanthroline or

\\~~ution of inhibitor (cop-rrous gluconate, o-phe-pH 3 to 5.0 buffer)

1ISuspend in 12 mM H3PO4 for 1 hr at3 C, then wash in cold 10 mm phos-phate buffer (pH 7.0) and resuspendin 10 mM MgSO4 (pH 7.0)

Deactivated spores (90 after 30 daysat 3 C)'

\\

Inhibited activated spores l\Nl Suspend in 40 mm CaCl2 and DPA

Add DPA\M //

Germinated spores (5 to 10)1FIG. 2. General schema for the production and characterization of activated and deactivated spores.

1 Percent refractile spores in suspension after indicated treatment.

those receiving a reduced activation treatment.It was also noted that the increase in the percent-age of light transmission of some partially in-hibited activated-spore suspensions was due to auniform graying or reduction in refractility ratherthan to more complete germination of some sporesin the suspension.

Cation inhibition of the germination of activatedspores. Murty and Halvorson (1957) reportedthat various cations inhibited the germination ofdormant spores of B. cereus, and that the inhibi-tions could be reversed by chelating agents.Germinal changes of activated spores were con-sistently inhibited by the addition of cation saltsof Cu+, Fe++, Ag+, and Hg++ in concentrationsof 1 to 5 mM at neutral pH (pH 6.8 to 7.0). AsAg+ and Hg+ were toxic and did not permitsubsequent outgrowth, they were not extensivelyinvestigated. Figure 3 demonstrates the effect ofCut and Fe++ on the germination rate of ac-tivated spores. The activated spore control was

over 90% germinated after 90 min, whereas 1mM CuSO4 completely inhibited the germinationof all preparations tested for at least 24 hr. Resultswith ferrous gluconate were variable. It onlypartially inhibited the activated spore prepara-tion used to provide the data for Fig. 3, but com-pletely inhibited germination of other activatedspore preparations. When these cation-inhibitedspore suspensions were examined after severaldays at 24 C, it was noted that the spore coats ofsuch spores began to show signs of disintegration.The inhibition by copper was partially counter-acted by the addition of 50 mm DPA but not by50 mm EDTA. The inhibition by ferrous ion wascounteracted by DPA or EDTA. The addition of10 mM L-alanine had no demonstrable effect incounteracting these inhibitions. Cation saltswhich were tested but did not inhibit the germi-nation of activated spores at pH 7 were 5 mmCaCl2, MnCl2, NiSO4, and CoCl2, and 10 mMMgSO4.

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0° 40

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0 30 60 90 6 12 24MIN HR

TIME

FTG. 3. Inhibitory effect of copper and ferrous ion

solutions on the germination of activated spores ofBacillus megaterium and its reversal by DPA or

EDTA. All solutions in 50 mM acetate buffer (pH6.8) at 24 C. Ordinate: scale units increase in %transmission. All suspensions adjusted to 50%transmission at zero time. A, control; 0, 1 mM

CuS04;@*, 1 mM CuS04 plus 10 mM DPA; O, 1

mM ferrous gluconate; 1 mM ferrous gluconateplus 10 mM DPA, A, 1 mM ferrous gluconate plus10 mM EDTA.

Inhibition of the germination of activated spores

by o-phenanthroline. Although numerous com-

pounds have been reported to inhibit the germi-nation of dormant spores, of the many com-

pounds tested only a saturated solution ofo-phenanthroline (300 ppm) inhibited the germi-nation of activated spores (Fig. 4). o-Phenanthro-line is a strong chelating agent and has been usedto bind cations in the study of the relationshipof metals to enzymatic activity (Valle, 1960). Sur-prisingly, the o-phenanthroline inhibition couldbe counteracted by the addition of 10 mm DPAor 25 mm phosphate buffer at pH 7. The inhibi-tory effect of o-phenanthroline was variable fordifferent spore harvests and occasionally onlylasted for about 6 to 8 hr. Heavy spore suspen-

sions of 5 X 109 spores/ml also counteracted theinhibitory effect of o-phenanthroline. Inhibitorsthat failed to inhibit the germination of activatedspores at pH 7 were: 5 MM D-alanine, NaCN,NaF, NaAsO3, NaNO2, atebrine, dipyridyl, 2,4-dinitrophenol, and pyridine, and 10 mM NaN3,NH20H, iodoacetate, and ethyloxamate.

50

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TIME. Min)

FIG. 4. Inhibitory effect of o-phenanthrolineon the germination of activated spores of Bacillusmegaterium and its reversal by DPA or phosphate.Temperature, 24 C. Ordinate same as in Fig. S.A\, control activated spores in distilled water; O,saturated solution of o-phenanthroline; *, satu-rated solution of o-phenanthroline in 50 mM phos-phate buffer (pH 7); O, saturated solution ofo-phenanthroline plus 10 mM DPA.

Inhibition of the germination of activated s3poresby low pH. With many dormant spores, germina-tion does not occur at pH 4 or below (Vas andProszt, 1957). Figure 5 demonstrates that germi-nation of an activated spore suspension was re-lated to pH. At pH 7.0, over 90%7 of the activatedspores were germinated after 120 min, at pH 5.0there was some darkening, and at pH 3.5 thespores were still refractile. Inhibition due to lowpH was partially counteracted by the addition of10 rmM DPA. The response of activated spores toinhibition by low pH was dependent on the degreeof the CaDPA activation treatment. For example,when a sample of the same crop of dormant sporeswas activated at 7.6 C for 70 min instead of at7 C as in Fig. 5, the spores activated at the highertemperature germinated at pH 5 but not at pH 4.Heavy spore suspensions (5 X 109 spores/ml)were less affected at pH 4 and gradually darkened(germinated).

Stabilization of activated spores, deactivation.Activation was reversible in the sense that bothspontaneous germination and further germinalchanges could be arrested and the activatedspores stabilized. Deactivation was discovered

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Eo 40

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S 2

z0V5U)30z

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0zz 10C,

0

0

TIME (Min)

FIG. 5. Inhibitory effects of acetate buffers (H+)at low pH on the germination of activated spores ofBacillus megaterium and its partial reversal byDPA. Ordinate same as in Fig. S. A, controlspores in water or acetate buffer (pH 6.8 to 7.0);0, 50 mM acetate buffer (pH 3.5); 0, 50 mM acetatebuffer (pH 3.5) plus 10 mM DPA; 0, 50 mM acetatebuffer (pH 5.0); *, 50 mM acetate buffer (pH 5.0)plus 10 mMDPA.

when an activated spore suspension was sus-

pended in 8 mm acetic acid for 1 day. These spores

retained their refractility even after the aceticacid was removed and they were resuspended inneutral buffer. Additional study demonstratedthat various acid treatments served to deactivatean activated spore suspension. The followingprocedure was adopted to produce deactivatedspores for further study. Freshly activated spores

were immediately suspended in 0.1% H3PO4 for1 hr at 3 C. The acid was removed by centrifuga-tion, and the deactivated spores were washed with10 mm phosphate buffer (pH 7) at 3 C. The de-activated spores were then resuspended in 10 mMMgSO4 (pH 7), and stored at 3 C. Deactivatedspores were stored in MgSO4 solution because a

comparative study indicated that the spores

retained their refractility better in 10 mm CaCl2,MgSO4, or MnCl2 than in 10 mM CUS04, CoCl2,ZnCl2, ferrous gluconate, or water at pH 7. FigureIC is a photomicrograph of a deactivated spore

suspension that had been stored for 20 days at3 C. The control activated spore suspension was

87% germinated after a storage period of 1 dayat 3 C. Some DPA was lost during activation anddeactivation. In one test, the control (dormant)

C,,0

coe0

z

0

4

30 1 0 20 30 4 0 50

HEATING TIME (Min)

FIG. 6. Survival at 84 C of dormant, activated,and deactivated spores of Bacillus megaterium in25 mM barbital buffer (pH 7). 0, dormant spores;A, activated spores; O, deactivated spores; 0,deactivated spores stored in 50 mM MgSO4; +,deactivated spores stored in 10 mM CUS04.

spores contained 9.0% DPA, whereas after deac-tivation comparable spores contained 8.1% DPA.Heat resistance of treated spores. The effect of

the several treatments on the thermal resistanceof the treated spores is presented in Fig. 6. Thesesurvivor curves indicate that the various treat-ments only slightly affected the thermal resistanceof the treated spores. Although there was a smallloss in thermal resistance during activation andsome additional loss during storage, all prepara-tions had thermal-resistance properties character-istic of dormant spores rather than of germinatedspores or vegetative cells. The survivor curves fordeactivated spores treated in 10 mm CaCl2, CoCl2,MnSO4, or ZnCl2 were similar to those of the de-activated spores treated with CuS04 or MgSO4.The various treatments had no effect on the vi-ability of the spores, as the zero-heating-timecounts on comparable samples were almost thesame.

Germination requirements of deactivated spores.The germination requirements for deactivatedand dormant spores were similar. Both typesgerminated in a 40 mm solution of CaCl2 andDPA, but did not when the concentration wasreduced to 20 mM. It was, therefore, possible tostart the germination process by the CaDPAactivation at low temperature, stop it by the

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TABLE 1. Estimation* of free amino acids released by activatedt and dormant sporesGluta- Leucine AAnneG rgnistiine,

Condition mate Leup Alani.neGlycine hisnine, Lysinegroup gou

Activated spores suspended inWater at 24C........................... 10 10 5 4 5 6Acetate, 50 mM (pH 3) ....... .......... 1 0 0.1 0 1 0Acetate, 50 mM(pH 4) .................. 1 0 0 0.5 2 0CuS04, 9 mM (pH 6.8) .................. 2 0 0 0 0 0Saturated o-phenanthroline ...... ....... 7 0.1 0 0 1.5 0.5Water at 3 C ........................... 5 1.5 1.5 2 3 0.5

Deactivated spores (supernatant solution)... 0 0 4 0 ±4 iActivation solution (40 mM CaDPA) ....... +4 0 0 0 0 0Deactivation solution (12 mm H3PO4) ....... 0 0 0 0 0 0Dormant spores (extract)

Boiled in water for 90 min.............. 7 0 0 0 3 0Broken in 50% ethanol.................. 5 0 0 0 3 0.5Stored in water at 3 C for 40 days, thenboiled in water ....................... 5 3 1.5 1.5 5 1.5

* Based on paper electrophoresis of solutions; see text.t Spores activated at 7.6 C for 70 min and the exudates collected after 24 hr at 24 C, unless otherwise

indicated.I Values in table expressed as mmoles of amino acid per 100 g of dry spores.

acid deactivation treatment, and start germina-tion again by a second exposure to CaDPA. Thedeactivated spores were like dormant spores inthat 10 mM L-alanine, glucose, or adenosine, 20mM CaDPA, or 40 mm NaDPA or EDTA did notstimulate germination even after 24 hr at 24 C.The similarity in germination requirements forthe deactivated and dormant spores indicatedthat the activation effect was removed by theacid treatment and that the deactivated sporeswere physiologically similar to dormant spores.Exudate analyses. To further characterize bio-

chemical changes during activation, germination,and inhibition, exudates were collected and ana-lyzed. As very little material was released frominhibited or deactivated spores, supernatantswere collected after the activated spores (7.6 Cfor 70 min) had been subjected to a particularcondition for 24 hr. The long treatment periodand the heavy (5 X 109 spores/ml) suspensionused caused some preparations to lose their re-fractility; spores in water at either 3 or 24 C, inacetate at pH 4, or in o-phenanthroline lost theirrefractility by the time the supernatant was re-moved, whereas spores suspended in 5 mM CuS04or acetic acid were still refractile. The deactivatedspore suspension which provided the superna-tant for analysis contained 17% partially refrac-

tile spores, but the germination changes had beenarrested as the refractility did not change oncontinued storage.The estimations of free amino acids found in

the samples analyzed are presented in Table 1.The data show that glutamate is the major com-ponent of extracts of fresh dormant spores, butthat extracts of refractile dormant spores aged at3 C demonstrated an accumulation of additionalfree amino acids. To substantiate the findingsobtained by paper chromatography and electro-phoresis, the extract from fresh dormant sporesand the exudate from activated spores allowed togerminate were quantitatively analyzed (Table2). In contrast to dormant spores, the germina-tion exudate of activated spores contained 14 freeamino acids and 5 unknown ninhydrin-positivematerials. Powell and Strange (1953) identifiedthe same amino acids except phenylalanine inthe germination exudate of the B. megateriumspores they studied.The quantitative analysis of DPA and pep-

tides, and the electrophoretic identification ofpeptide are presented in Table 3. As Powell andStrange (1953) demonstrated the hexosamine andreducing-sugar values of various germinationexudates to be within a few per cent of each other,the reducing-sugar value of the hydrolyzed sam-

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TABLE 2. Free amino acid* composition of ethanolicextract of broken dormant spores and ofthe germination exudate of activated spores

germinated in water

ActivatedAmino acids Dormant germi-sporesf nated

spore4t

Aspartic acid.................. 0 1.58Glutamic acid ................. 7 7.76Methionine .................... 0 2.02Threonine ..................... 0.192 2.54Serine......................... 1.11Leucine ....................... 0 2.68Isoleucine ..................... 0 2.01Valine ........................ 0 2.88Tyrosine ...................... 0 1.01Phenylalanine ................. 0 1.31Alanine ........................ 0 5.32Glycine ........................ 0 3.86Histidine ...................... 0 0.84Arginine ....................... 0.253 0Lysine ........................ 0 6.65Ammonia......................±z t9.1

* Determined by Spinco model 120 amino acidanalyzer. Results expressed as mmoles of aminoacid per 100 g of spores.

t Dormant spores broken in and extracted with50% ethanol by using Mickle disintegrator.

$ Activated spores suspended in water. Super-natant collected after 24 hr at 24 C. No apparentspore lysis as indicated by phase-contrast micro-scopic examination, or change in per cent trans-mission of the suspension.

ple was used as a measure of the amount of pep-tide in the sample. The peptide material extractedfrom dormant spores under conditions whichexcluded enzymatic activity did not have anelectrophoretic mobility similar to that of pep-tides obtained from germination exudates. Theexudates of the activated spores that germinatedat 3 C contained only 20% of the peptide and 10to 50% of the free amino acids found in the normalgermination exudate collected at 24 C. The resultsshow that the breakdown and release of materialswere retarded at 3 C. Exudates from inhibitedactivated spores and deactivated spores weredifferent in composition than the normal germi-nation exudate. The exudates collected fromactivated spores inhibited by CUSO4 and aceticacid contained only small amounts of glutamateand DPA. When dormant spores were incubatedin 50 mm acetic acid for 24 hr at 24 C, only a

small amount of DPA was detected. The peptideand free amino acid composition of exudates,collected from activated spores suspended inacetate solution at pH 4 or in o-phenanthroline,was more like that of an extract of dormantspores than a normal germination exudate.These two solutions did not stop the release ofDPA and peptide material, but the productionor release of NH3 and the other 12 amino acidswas retarded. Under certain adverse conditions,such as low temperature or pH, activated sporesappeared to be germinated and swollen whenexamined microscopically, but their germinationexudates were not normal.Very small amounts of material were released

from the spores by the activation and deactiva-tion treatments. This may be the reason why theactivation and deactivation treatments weresuccessful. The small amounts of materials re-leased from the deactivated spores and the factthat they remained refractile for long periodsindicated that the release of material started byactivation had been arrested. In these analyses,it was not possible to determine whether thesmall amount of material found originated fromthe activated spores or primarily from the 5 to10% partially germinated spores present in thesepreparations. Generally, whatever inhibited thegermination of activated spores also inhibitedthe release of exudate material.

DISCUSSION

Calcium dipicolinate-activated bacterial sporesrepresent an early phase of germination, and someof their properties are quite different from thoseof germinated or dormant spores. The significanceof the findings presented above will be discussed.The most distinctive characteristic of activated

spores was that their rate of germination could becontrolled or stopped altogether. The rate ofgermination could be regulated by adjusting thepH or temperature as well as the concentration ofactivated spores. A stronger acid treatment de-activated the activated spores and made themstable. The exact role of the H+ cannot beevaluated at this time, but it is known that acidextracts DPA from dormant spores (Foster,1960). The acid treatment may have caused theremoval of DPA that was attached to particularsites of the activated spore, with the result thatstability was regained and such spores were madesimilar to dormant spores.

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TABLE 3. Composition of exudates and extracts from activated and dormant spores

Reducing sugart Paper electro-

Condition Ninhydrin* t phoresis DPAtvalue NHfUnhydro- Hydro- Poen Pep-lyzed lyzed Protein tide

Activated spores suspended in:Water at 24 C .................... 59 26.4 0.75 16.2 + + 47.5Acetate, 50 mM (pH 3) ...... ..... 5.9 0 0 1.5 i 13.8Acetate, 50 mM (pH 4) ...... ..... 15.8 0 0 7.7 + + 35.4CuSO4, 9mM (pH 6.8)............ 3.4 0 i i 0 0 2.8Saturated o-phenanthroline.... ... 16.9 0 0 8.1 0 + 39.6Water at 3C ..................... 28.6 14.7 0 3 0 i 44.4

Deactivated spores (supernatant so-lution) ......................... 5.6 0 0 1.5 0 0 5.1

Activation solution (40 mM CaDPA). 2.8 1.9 0 i 0 0 -

Deactivation solution (12 mM H3PO4) 1.8 0 0.3 1.8 0 0 0.6Dormant spores (extract)Boiled in water for 90 min........ 12.9 0 0 9.2 ++ 14 42.5Broken in 50% ethanol........... 15.4 0 ± 0 45

* Monosodium glutamate used as reference start Expressed as mmoles per 100 g of spores.t Spores were activated at 7.6 C for 70 min and

Germination of activated spores was inhibitedby various cations (H+, Ag+, Cu+, Fe++, andHg+) and by the chelating agent o-phenanthro-line. Although the nature and action of theseinhibitors are divergent, their inhibitions werepartially reversed by NaDPA. This suggestedthat these inhibitors were competitive withNaDPA. Curran, Brunstetter, and Myers (1943)and others have demonstrated that spores con-tain more cations than do vegetative cells. Rie-mann (1961) proposed that exogenous CaDPAcould be chelated to spore material and coulddisrupt a normal stereostructure. Vinter (1961)reported that bacterial spore coats were rich in*cysteine. Perhaps, like ion-exchange resin, dor-mant spores could provide an insoluble matrixfor the chelation of CaDPA and other solublematerials which would make them "insoluble" inwater.

Other studies have indicated that the dormantspore is anhydrous and yet permeable to water.Ross and Billing (1957) concluded that dormantspores were anhydrous, on the basis of their re-fractive index measurements. Murrell and Scott(1958) demonstrated that D20 exchanged freelywith 99%O of the spore water, and Black et al.(1960) demonstrated that small molecules pene-trated 40% of the spore volume. Powell (1957)as well as Rode and Foster (1960) stated that

the exudates were collected after 24 hr at 24 C.

there is a marked permeability change duringgermination. The data presented here emphasizethat dormant spores are impermeable to theextent that they release only a limited amount ofsoluble constituents. The analysis of the exudatesof the various activated spore suspensions indi-cated that the permeability (as reflected by therelease of spore material) was altered duringactivation. The increased permeability of ac-tivated spores and the effect of the several inhibi-tors in reducing this increase indicated thatexogenous DPA affected the permeability of suchspores. When Cut , Fe++, or H+ was added tothe activated spore suspensions, the release ofspore material was decreased but could be par-tially counteracted by the addition of NaDPA.Riemann (1961) postulated that the DPA of anormal dormant spore, if released from thenormal internal complex, was enough to germinatebacterial spores. It is possible that heat shock,mechanical abrasion, and other germinationstimuli may directly or indirectly upset the DPAstability of the dormant spore and initiate apermeability change that would result in germi-nation. The relationship of the release of DPA tochanges in spore permeability as a result of theaction and metabolism of other germinationagents has yet to be studied.The exudate analyses demonstrated that the

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LEE AND ORDAL

free amino acid composition and the electropho-retic mobility of the peptide material extractedfrom newly harvested dormant spores was differ-ent from that present in a normal germinationexudate. In contrast to dormant spores, thegermination exudate of activated spores con-tained 14 amino acids and additional unknownninhydrin-positive material. Under adverse ger-mination conditions, such as low temperature orpH, dense suspensions of activated spores ap-peared to be germinated (dark) or swollen whenexamined microscopically, but the exudates con-tained a reduced amount of spore material. Like-wise, although glutamate was the major free aminoacid found in the extracts of newly harvesteddormant spores, extracts made from refractile dor-mant spores aged for 40 days or more at 3 C con-tained significant amounts of additional aminoacids. During aging, free amino acids accumulatedin dormant spores, without any apparent changesin refractility. Conversely, activated spores ger-minated at 3 C released less than the normalamount of spore material, and those germinatedin the presence of o-phenanthroline had an exu-date with a reduced amino acid content. Theseobservations imply that biochemical changesgradually occur in refractile dormant spores keptin the cold, and that physical changes (loss of re-refractility) can occur to activated spores in aninhibitory solution without normal biochemicalchanges. Changes in the spore coat undoubtedlyoccur during germination; Strange and Dark(1957) described a lytic enzyme which solubilizesa peptide from isolated spore coats, and Levinsonand Wrigley (1960) recorded, by means of elec-tron micrographs, the disintegration of the outercoat of B. megaterium. The exact role of lytic andproteolytic enzymes during spore germination inrelation to physical changes is not yet known.Further studies on the breakdown of spore ma-terials during germination as related to physicalchanges induced by different germinating systemsare needed to clarify this point.

LITERATURE CITEDBLACK, S. H., T. HASHIMOTO, AND P. GERHARDT.

1960. Calcium reversal of the heat suscepti-bility and dipicolinate deficiency of sporesformed "endotropically" in water. Can. J.Microbiol. 6:213-224.

BLACK, S. H., R. E. MACDONALD, T. HASHIMOTO,AND P. GERHARDT. 1960. Permeability ofdormant bacterial spores. Nature 185:782-783.

BLOCK, R. J., E. L. DURRUM, AND G. ZWEIG.1958. A manual of paper chromatography andpaper electrophoresis, 2nd ed. AcademicPress, Inc., New York.

CHURCH, B. O., AND H. HALVORSON. 1959. De-pendence of the heat resistance of bacterialendospores on their dipicolinic acid content.Nature 183:124-125.

CONWAY, E. J. 1950. Microdiffusion analysis andvolumetric error. Crosley Lockwood & Son,Ltd., London.

CURRAN, H. R., B. C. BRUNSTETTER, AND A. T.MYERS. 1943. Spectrochemical analysis ofvegetative cells and spores of bacteria. J.Bacteriol. 45:485-494.

Doi, R. H., AND H. HALVORSON. 1961. Comparisonof electron transport systems in vegetativecells and spores of Bacillus cereus. J.Bacteriol. 81:51-58.

FLYNN, F. V., AND 0. P. DEMAYO. 1951. Micro-electrophoresis of protein on filter paper.Lancet 261:235-239.

FOSTER, J. W. 1960. Dipicolinic acid and bac-terial spores, p. 1-38. Lectures on thetheoretical and applied aspects of modernmicrobiology. University of Maryland,College Park.

LEVINSON, H. S., AND A. S. WRIGLEY. 1960. Sporegermination and emergence of Bacillusmegaterium. Science 131:1382.

LEVY, A. L. 1954. A paper chromatographicmethod for the quantitative estimation ofamino acids. Nature 174:126-127.

LONG, S. K., AND 0. B. WILLIAMS. 1958. Methodfor removal of vegetative cells from bacterialspore preparations. J. Bacteriol. 76:332.

MICHL, H. 1959. High voltage electrophoresis.Chromatog. Rev. 1:9-38.

MOORE, S., AND W. H. STEIN. 1948. Photometricninhydrin method for use in the chroma-tography of amino acids. J. Biol. Chem.176:367-388.

MURRELL, W. G., AND W. J. SCOTT. 1958. Thepermeability of bacterial spores to water.Intern. Congr. Microbiol., 7th, Stockholm,p. 26.

MURTY, G. G. K., AND H. 0. HALVORSON. 1957.Effect of enzyme inhibitors on the germina-tion and respiration of and growth fromBacillus cereus var. terminalis spores. J.Bacteriol. 73:230-234.

PELCHER, E. A. 1961. Some characteristics ofspores of Bacillus cereus produced by a re-placement technique. M.S. thesis. Universityof Illinois, Urbana.

POWELL, J. F. 1953. Isolation of dipicolinic acid(pyridine-2,6-dicarboxylic acid) from spores

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of Bacillus megaterium. Biochem. J. 54:210-211.

POWELL, J. F. 1957. Biochemical changes occur-ring during spore germination in Bacillusspecies. J. Appl. Bacteriol. 20:349-358.

POWELL, J. F., AND R. E. STRANGE. 1953. Bio-chemical changes occurring in the germina-tion of bacterial spores. Biochem. J. 54:205-209.

RIEMANN, H., AND Z. J. ORDAL. 1961. Germi-nation of bacterial endospores with calciumand dipicolinic acid. Science 133:1703-1704.

RIEMANN, H. 1961. Germination of bacteria bychelating agents, p. 24-48. In H. 0. Halvorson[ed.], Spores II. Burgess Publishing Co.,Minneapolis.

RODE, L. J., AND J. W. FOSTER. 1960. Mechanicalgermination of bacterial spores. Proc. Natl.Acad. Sci. U.S. 46:118-128.

Ross, K. F. A., AND E. BILLING. 1957. The waterand solid content of living bacterial sporesand vegetative cells as indicated by refractiveindex measurements. J. Gen. Microbiol.16:418-425.

SOMOGYI, M. 1945. The determination of bloodsugar. J. Biol. Chem. 160:61-73.

SPACKMAN, D. H., W. H. STEIN, AND S. MOORE.1958. Automatic recording apparatus for usein the chromatography of amino acids. Anal.Chem. 30:1190-1206.

STRANGE, R. E., AND J. F. POWELL. 1954. Hexosa-mine-containing peptides in spores of Bacillussubtilis, B. megaterium and B. cereus. Biochem.J. 58:80-85.

STRANGE, R. E., AND F. A. DARK. 1957. A cell-wall lytic enzyme associated with Bacillusspecies. J. Gen. Microbiol. 16:236-249.

VALLE, B. L. 1960. Metal and enzyme interaction:correlation of composition, function, andstructures, p. 225-276. In P. D. Boyer, H.Lardy, and K. Myrback [ed.], The enzymes,vol. 3, 2nd ed. Academic Press, Inc., NewYork.

VAS, K., AND G. PROSZT. 1957. Effect of tempera-ture and hydrogen ion concentration in thegermination of spores of Bacillus cereus.Nature 179:1301-1302.

VINTNER, V. 1961. The formation of cysteine-richstructure in sporulating cells and its possiblerole in the resistance of spores, p. 127-141.In H. 0. Halvorson [ed.], Spores II. BurgessPublishing Co., Minneapolis.

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