JOURNAL OF BACTERIOLOGY, June 1973, p. 1090-1098Copyright @ 1973 American Society for Microbiology

Vol. 114, No. 3Printed in U.S.A.

Glutamic Acid Decarboxylase in Spores ofBacillus megaterium and Its PossibleInvolvement in Spore Germination

CHARLENE WEHE FOERSTER AND HAROLD F. FOERSTER

Biology Department, Sam Houston State University, Huntsville, Texas 77340

Received for publication 19 February 1973

Spores of Bacillus megaterium were examined for glutamic acid decarboxylase(GAD). Although dormant spores showed no GAD activity, spores given sonictreatment and heat-activated spores had high activities when assayed for thisenzyme. Several parameters of GAD in heat-activated spores were examined.The effects of KCN, NaN8, 2,4-dinitrophenol, and KF on GAD activity were ex-amined. Only KCN was an effective inhibitor of GAD activity in heated sporesand was also shown to be the only effective inhibitor of GAD activity in vegeta-tive bacteria. Similar patterns of inhibition were obtained with GAD activity andwith spore germination, KCN being the only effective inhibitor of both, although atdifferent concentrations. Spore GAD activity in heat-activated spores showed aloss with storage at 4 C; on the other hand, storage at 25 C was not accompaniedby a loss, but, to the contrary, showed an increase in GAD activity of about 30%.A comparison of GAD activity at different times during germination, growth, andsporulation showed it to be highest in freshly germinated spores. Althoughvegetative cells contained GAD activity, the level in log-phase cells wasapproximately one-half the level obtained with freshly germinated spores.Heat-activated mutant spores with a requirement of y-aminobutyric acid forgermination gave no GAD activity. GAD activity appeared in mutant sporesafter germination and increased to levels comparable to parent spores after 9 minof germination.

Spores of several strains of Bacillus containlarge L-glutamic acid pools (16), and the conver-sion of L-glutamic acid to y-aminobutyric acidhas been described in bacteria (4, 9). Theconversion of L-glutamic acid to y-aminobutyricacid may play a key role in the germination ofbacterial spores. This was indicated by theisolation of mutants of B. megaterium QMB1551 which require y-aminobutyric acid forspore germination (7). Colorimetric and 14Canalyses showed that 1-glucose-initiated germi-nation of heated spores of the parent strain of B.megaterium was accompanied by a rapid dropin spore pool glutamate. On the other hand,glutamic acid pools of mutant spores requiring-y-aminobutyric acid remained unchanged dur-ing the first 9 min of germination (8).These findings suggest that bacterial endo-

spores may contain L-glutamic acid decarboxyl-ase (GAD). GAD has been reported in manymicroorganisms including numerous strains of

Escherichia and Proteus, as well as members ofthe spore-forming genera Clostridium and Ba-cillus (9). The enzyme has been obtained inpurified form from vegetative cells of C. perfrin-gens (4), and its presence has been demon-strated in all phases of vegetative growth andsporulation in B. thuringiensis (1). However, todate, no work has been presented on GAD inbacterial spores. Evidence is presented here forthe presence of GAD in spores and vegetativecells of B. megaterium. Comparative studies onGAD activities in spores and vegetative cells ofthe parent strain and in a germination mutantwith a requirement for y-aminobutyric acid arepresented.

MATERIALS AND METHODS

Bacteria. The organisms used included B. mega-terium QM B1551 (parent strain) and mutant strainswith a requirement of y-aminobutyric acid for sporegermination (7).

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GLUTAMIC ACID DECARBOXYLASE IN B. MEGATERIUM

Spores. The composition of the medium and themethods used to prepare and store spore suspensionshave been described (7). Spores were heat activatedas suspensions in demineralized water at concentra-tions of 9 x 10' viable spores per ml; samples were

freshly heated for each experiment.Sonic treatment. Spores were given sonic treat-

ment by using a Branson sonifier cell disruptor (modelW140D). Each sample contained 100 mg (dry wt) ofspores suspended in 10 ml of pyridine buffer, pH 4.6(23). Spores were given sonic treatment by using a

power density of 460 W/inch2 (maximum output) in a

cold shoulder cell containing 3 g of glass powder with amesh size of 5 Am. During operation, the cell wasimmersed in an ice bath. After sonic treatment, theglass powder was allowed to settle and the superna-tant liquid was decanted.GAD assay. Several techniques for demonstrating

GAD activity in microorganisms have been described.GAD activity can be shown by the production ofy-aminobutyric acid from L-glutamic acid (15). Sepa-ration of the enzyme(s) on polyacrylamide gel and itsdemonstration with specific color reagents has alsobeen used (1).

Because the pH optimum for GAD is stronglyacidic, conventional manometric techniques can beused to measure the CO2 produced from L-glutamicacid. Several buffer systems have been described foruse in manometric assays for GAD. These includeacetate buffer at pH 4.5 (10) and pyridine-pyridine-hydrochloride buffer at pH 4.6 (23). The pyridinebuffer of Shukuya and Schwert was used in thesestudies. A requirement of pyridoxal phosphate hasbeen demonstrated for GAD (28), and this coenzymewas routinely added to the assay.A standard assay was developed with commercially

prepared GAD (Nutritional Biochemical Corp., Cleve-land, Ohio) from Escherichia coli ATCC 11246. Op-timum GAD activity was obtained by employing 0.1M pyridine buffer (pH 4.6) containing 0.25 mg ofpyridoxal phosphate per ml (Sigma Chemical Co., St.Louis, Mo.) and 10 mM L-glutamic acid. All compo-nents are expressed as final concentration.

Manometric measurements were made at 36 C witha Gilson respirometer (model GRP 20). Each sample(10 mg), suspended in 2.7 ml of pH 4.6 pyridine buffercontaining pyridoxal phosphate, was placed in themain compartment of each manometric flask. L-Gluta-mic acid (0.3 ml), or any one of the other compoundstested, was added to the side arm of each flask. Theflasks in each experiment were equilibrated at 36 C for10 min with all valves (gassing manifold, operating,and disconnect) open. At the end of the equilibrationperiod, all valves, except the disconnect valves to theexperimental flasks on the operating manometers,were closed. The endogenous activity of each samplewas measured for 15 min, L-glutamic acid (or othertest compound) was then added, and the CO2 pro-duced was measured as increased manometric pres-sure.

Germinated, vegetative, and sporulating cells.Samples were cultured on spore medium in petridishes. Each petri dish was inoculated with 10 mg of

heat-activated (20 min at 80 C) spores; the sporeswere spread uniformly over the agar surface with asterile bent glass rod and incubated at 37 C. Cellswere collected by flooding each plate with demineral-ized water and scraping them from the agar surfacewith a glass rod. Cells were sedimented by centrifuga-tion at room temperature and suspended in deminer-alized water. Samples were used to determine cell dryweight and to measure GAD activity. Samples (10mg, dry wt) of germinating, vegetative and sporulat-ing cells were assayed for GAD in the manner de-scribed for spores, but without heat activation.Dry weight. Samples suspended in demineralized

water were dried in tared aluminum dishes at 85 C.Weights were determined with a Mettler H 16 bal-ance.

RESULTS

Clean, intact, unheated spores are metaboli-cally inactive as measured by conventionalmanometric techniques; respiratory quotientsof less than one have been obtained (5). Not-withstanding the high metabolic inertia, con-siderable evidence suggests that bacterialspores contain enzymes. The increase in meta-bolic activity of spores after heat activation (3),the rapid onset of metabolic activity observed ingerminating spore suspensions (6, 8), and thefailure of inhibitors of protein synthesis to affectthe onset of enzyme activity associated withgermination (25) indicate that enzymes exist indormant spores in an inactive state. Evidence

120

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FIG. 1. Effect of sonic treatment time on sporeGAD activity. Each sample contained the particulatefraction from 10 mg of spores (dry wt). Each pointrepresents the C02 produced after 30 min of incuba-tion in the presence of 10 mM L-glutamic acid.

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which indicates that GAD resides in spores of B.megaterium in an inactive form has been ob-tained. Sonic-treated and heat-activated prepa-

rations yielded high activities when assayed forthis enzyme.

Effect of sonic treatment on spore GADactivity. Figure 1 compares the CO2 producedin the presence of L-glutamic acid (10 mM) by10-mg samples of spores (dry wt) treated fordifferent periods of time. Each sample was

centrifuged at 15,000 x g for 15 min, the pelletwas suspended in pyridine buffer (pH 4.6), andthe supernatant liquid and particulate fractionswere assayed for GAD activity. Only the resultsobtained with the particulate fractions are pre-

sented; GAD activity was not obtained with anyof the supernatant fractions.

Although dormant spores gave no GAD activ-ity, sonic treatment of spores resulted in a rapidincrease in CO2 production when assayed forthis enzyme. Four to six minutes of treatmentgave maximum activity; periods longer than 6min resulted in a rapid decrease in CO2 produc-tion. Whether the decreases in activity obtainedwith extended periods of sonic treatment were

the result of inactivation of the enzyme after itsrelease from spores or were due to the loss ofstructural integrity of the spore or spore particlerequired for the decarboxylation of L-glutamicacid was not determined.Although maximum GAD activity was ob-

served after approximately 5 min of sonic treat-ment, examination of these samples with a

phase-contrast microscope showed that virtu-ally none of the spores was fragmented; indeed,the majority were still refractile. The speed withwhich GAD activity appeared in the sporessuggested that sonic treatment, like heat, mayactivate spore enzymes.

Effect of heat on spore GAD activity. Asearly as 1933, Tarr (26) demonstrated thatenzyme activity in bacterial spores can beincreased by heat treatment. Heating Bacillusspores at 80 C for 30 min both shortened the lagperiod of 02 uptake and accelerated the rate ofdehydrogenation of glucose. Church and Hal-vorson (3) found that spores of B. cereus var.

terminalis heated for 15 min at 65 C failed totake up oxygen. However, when the heat treat-ment was extended to 60 min, glucose, gluco-nate, 2 keto-gluconate and pyruvate were oxi-dized. Protease activity in spores of B. subtiliscan also be increased by heat treatment (2).That GAD activity in spores can be activated

by heat is shown in Fig. 2. The microliters ofCO2 produced in the presence of L-glutamic acidwere compared by using spores treated for 30

min at different temperatures. No GAD activitywas observed with spores held at room tempera-ture (25 C); however, treatment at 60, 70, and80 C gave increasingly higher activities, with a

maximum at 80 C. Treatment of spores attemperatures above 80 C resulted in rapid lossof GAD activity, with complete inactivation at100 C.Spore GAD specificity. Figure 3 compares

the CO2 produced by heated spores in thepresence of one of several compounds. Twotypes of compounds were tested: (i) structuralhomologues of L-glutamic acid, which includedD-glutamic acid and L-glutamine, the y-amideof L-glutamic acid, and (ii) one of severalcompounds germinative for heated spores of B.megaterium, namely, D-glucose, L-leucine, or

L-alanine. The specific nature of the assay was

emphasized by the fact that CO2 productionwas observed only in the sample containingL-glutamic acid.

Effect of metabolic inhibitors on spore

GAD activity and on germination. A survey

on the effects of metabolic inhibitors on GADactivity showed cyanide to be a potent inhibitorof this enzyme in vegetative bacteria. Potas-sium cyanide, at a concentration of 10-4 M,inhibited GAD in vegetative bacteria by more

than 90%. Sodium azide was a weak inhibitor,giving only 27% inhibition at a concentration of10-2 M. Although 2, 4-dinitrophenol (DNP) and

10

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FIG. 2. Effect of heat on spore GAD activity.Spores of Bacillus megaterium (parent strain) were

heated for 30 min. Each sample contained 10 mg ofspores (dry wt). CO2 production was measured byusing standard assay procedures.

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J. BACTERIOL.1092

GLUTAMIC ACID DECARBOXYLASE IN B. MEGATERIUM

KF are potent inhibitors of some enzymes, theydid not cause a reduction in the GAD activity ofvegetative bacteria (9).The effect of several inhibitors on GAD activ-

ity in heated spores was evaluated. Figure 4describes the results of this study. DNP and KFgave no inhibition and very weak inhibition,respectively. NaN3 gave 20% inhibition andKCN gave complete inhibition of spore GAD.The similar patterns of inhibition observed withspores and with vegetative cells of other orga-nisms (9) indicate that spore GAD shares theseproperties, at least qualitatively, with function-ally similar enzymes obtained from vegetativebacteria.The effect of metabolic inhibitors on spore

germination has been extensively studied. Theeffects of these compounds on spore germina-tion has particular significance here, since acomparison with their effects on a specificenzyme believed to be involved in germinationcan be made. Similar patterns of inhibition ingermination and GAD activity can be taken asadditional evidence for the direct involvementof GAD in spore germination.The effect of each of the metabolic inhibitors

on the germination of heated spores of B.megaterium is illustrated in Fig. 5. The spore

Compound100 a.L-Glutamic

b.D-GI u tam Icc.L-Glutamine ad.L- LOuc i no

s e.L- I aani ne~80 f.D-GI ucose00.v) U

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FIG. 3. Specificity of GAD in heated spores. Sporeswere heated at 80 C for 30 min and assayed for GAD.Each sample contained 10 mg of spores (dry wt). C02production was compared in samples containing L-glutamic, D-glutamic, L-glutamine, L-leucine, L-ala-nine, or D-glucose; each at a final concentration of 10mM.

Minutes

FIG. 4. Effect of metabolic inhibitors on GADactivity. Spores were heated at 80 C for 30 min, and10-mg samples were assayed manometrically. KCN,KF, and NaNs were tested at a final concentration of10 mM each; 2,4-dinitrophenol (DNP), 50 ,uM. L-Glutamic acid in all tests, 10 mM.

Minutes

FIG. 5. Effect of metabolic inhibitors on sporegermination. Spores were heated at 60 C for 60 min.Heated spores were germinated in 13- by 100-mmtubes at 40 C in solutions of KI (40 mM) andD-glucose (1 mM). Germination was measured as %reduction in optical density at 540 nm. KCN, KF, andNaN3 were each used at a final concentration of 100mM, and DNP at 50 uM.

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FOERSTER AND FOERSTER

germination and the GAD activity pattemsshowed a striking similarity. As in the GADstudies, KCN was the only effective inhibitor ofspore germination, NaN, and KF were onlyweakly inhibitory, whereas DNP was ineffectiveas an inhibitor of germination. However, adifference in sensitivity to KCN was observed;spore GAD required a lower concentration foressentially complete inhibition compared withgermination. Also, apparently not all bacterialspores are sensitive to cyanide. For example,spore germination of B. cereus var. terminalisand C. botulinum were not inhibited by 10-2 Mand 1.6 x 10-4 M KCN, respectively (14, 27).

Effect of storage temperature on sporeGAD and on selected spore properties.Shukuya and Schwert (24), working with puri-fied GAD from E. coli, found this enzyme to besensitive to cold temperature. The enzymeshowed a 50% decrease in decarboxylase activ-ity after storage at 0 C. This decrease wasattained after 1 h of storage but showed nofurther decrease up to 3 h. Storage of the E. colienzyme at 25 C resulted in no loss in activity. Ofinterest, though perhaps not strictly analogous,is the fact that the activities of some sporeenzymes rendered active by heat are similarlyaffected by storage temperatures.Church and Halvorson (3) noted that heat-

activated spores of B. cereus lost the capacity tooxidize glucose after storage for 24 h at 5 C. Asecond heat treatment restored the glucose-oxi-dizing capacity to the level obtained immedi-ately after the first heat treatment.The effects of different storage temperatures

on GAD activity in heat-activated spores of B.megaterium are described in Fig. 6. Samples (10mg) of freshly heat-activated spores produced102 ,uliters of CO2 after 30 min of incubation.Storage at 4 C resulted in a rapid drop in GADactivity; approximately 60% of the initial activ-ity was lost after 1 h of storage, and, after 4 h,essentially no activity remained. However, sim-ilar to the glucose-oxidizing enzyme (3), GADactivity was completely restored to its initialactivated level by a second heat treatment.Storage at 25 C resulted in no reduction, but, tothe contrary, gave an increase in GAD activityof almost 30% after 24 h. It is tempting tospeculate that continued storage at 25 C mightlead to an irreversible stage of activation, oreven germination. Reported cases of spontane-ous germination (18) can possibly be explainedon this basis.

Several spore properties can be used as cri-teria for evaluating the dormant state of bacte-rial spores. They include the retention of heat

resistance (29), refractility or phase brightness(20), optical density (17), and dipicolinic acid(DPA) (19). Compared with freshly heat-activated spores, heated spores stored for 24 hrat 4 C and 25 C showed no loss in viability afterpasteurization, retained over 95% of their initialrefractility and optical density, and lost lessthan 10% of the initial DPA.GAD activity during germination, growth,

and sporulation in B. megaterium. AlthoughGAD has been reported in many vegetativebacteria, including B. thuringiensis (1), itspresence in vegetative cells of B. megateriumhas not been demonstrated, nor has this enzymebeen observed in germinating spores. To obtainthis information, GAD activities were comparedin heat-activated spores and in cells at differenttimes during germination, growth, and sporula-tion. Examination at the time of each samplingshowed the following cultural characteristics: 90min, essentially all cells were germinated withsome vegetative cells present; 3 to 5 h, log phaseof growth; 6 h, many cells in early stages ofsporulation but refractile spores were absent;12 h, more than 95% of cells contained refrac-tile spores; 18 h, phase of autolysis with num-erous free spores.The result (Fig. 7) indicate that GAD activity

increased rapidly with spore germination; anincrease of 34% was obtained with the 90-min

140

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FIG. 6. Effect of storage temperature on GAD ac-

tivity of heated spores. Spores were heated at 80 C for30 min. Samples (10 mg each, dry wt) were assayedfreshly heated and after intervals of storage at 4 C and25 C. Each result represents the ,uliters of CO.produced after 30 min of incubation.

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4.

1094 J. BACTERIOL.

GLUTAMIC ACID DECARBOXYLASE IN B. MEGATERIUM

cultures compared with heat-activated spores.However, with the onset of vegetative growth,the level of GAD decreased rapidly to approxi-mately 50% of the maximum at the end of thelog phase of growth (5 h). During the next 12 h,the GAD activity continued to decline, althoughat a slower rate, so that only 17% of themaximum activity was obtained with cells from18-h cultures. Thus, vegetative cells of B. mega-terium gave GAD activity, but substantiallyless than freshly germinated spores based on adry weight comparison.GAD activity in a mutant strain with a

requirement of -y-aminobutyric acid for sporegermination. The presence of large glutamicacid pools in spores of B. megaterium (16) withhigh GAD activities and the availability ofmutant spores with a requirement of y-aminobutyric acid for germination (7) provide asubstantial case for the decarboxylation of en-dogenous glutamic acid as an important meta-bolic event in spore germination. Additionalsupport for this case is provided by the rapidconversion of endogenous L-glutamic acid-U- "Cto "C--y-aminobutyric acid during spore germi-nation in the parent spores, compared with thestable nature of glutamic acid- U- "C pools inmutant spores (8).These findings suggest that spore germina-

tion in the parent strain of B. megateriumdepends on the decarboxylation of endogenousL-glutamic acid and the generation of y-aminobutyric acid. Thus, although mutantspores accumulate glutamic acid pools equal toparent spores (8), germination in the mutantspores is not initiated by D-glucose or any of theother parent spore initiators but requires y-aminobutyric acid. This requirement possiblyresults from a lack of GAD activity in mutantspores. Mutant spores were therefore examinedfor the presence of active GAD.

Figure 8 compares GAD activities in heatedspores of the parent strain (QM B1551) and inthree mutant strains (-y020, y038, -y065). Sam-ples were prepared and assayed in the mannerdescribed. Samples of each of the strains wereheated at 60, 70, and 80 C for 30 min, and 10-mgsamples were assayed immediately for GADactivity. Parent spores gave GAD activity witheach of the three activation temperatures (seealso Fig. 2). On the other hand, none of thesamples which contained heat-activated mu-tant spores gave GAD activity.

Since vegetative cells of the parent strain ofB. megaterium contained GAD activity, itseemed of interest to examine vegetative cells ofa mutant strain for the presence of this enzyme.Vegetative cells obtained from 5-h cultures of

Hours Growth

FIG. 7. GAD activity obtained with Bacillus mega-terium at different stages of growth. Spores were heatactivated at 80 C for 30 min and assayed; germinatingspores and vegetative cells were assayed without heatactivation. Each result represents the ,iliters of CO2produced by 10 mg of cells (dry wt) after 30 min ofincubation.

Minutes

FIG. 8. GAD activities with heated parent andmutant spores of Bacillus megaterium. Parent (QMB1551) and mutant spores (yO2O, -y038, yO65) wereheated at 60, 70, and 80 C for 30 min and assayedimmediately for GAD.

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FOERSTER AND FOERSTER

strain 'y065 were compared with cells obtainedfrom similar cultures of the parent strain.Vegetative cells of both the parent and mutantstrains gave high and essentially equal GADactivities. Furthermore, yO65 cells showed GADactivity equal to parent cells after germinationfor 15 min on spore medium. Thus, while GADactivity was absent from heat-activated mutantspores, activity appeared rapidly in these cellswith germination.To determine the onset ofGAD activity in the

mutant cells, a comparison was made usingheat-activated and germinating cells of theparent and mutant strains. Heated spores ofeach strain were germinated at 40 C withcontinuous magnetic stirring. Spores of theparent strain were germinated in KI (40 mM)and D-glucose (1 mM); mutant spores weregerminated in KI (40 mM) and L-alanine andy-aminobutyric acid (1 mM each). Samples (10mg) were collected on 25-mm membrane filters(0.45-am pore size) and washed with deionizedwater. Filtration and washing of each samplerequired less than 15 s. Each membrane con-taining a spore sample was transferred to amanometric vessel and assayed for GAD activ-ity by using standard manometric procedures.

Figure 9 shows that heated parent spores(QM B1551) gave a high level of GAD activity.Exposure of the heated spores to germinants foronly a short time resulted in a 40% increase inGAD activity, and the activity remained essen-tially unchanged during 9 min of germination.

Seconds Germination

FIG. 9. GAD activities in heat-activated and ger-minating parent (QM B1551) and mutant ('yO65)spores of Bacillus megaterium. Results are expressedas uliters of CO, produced by 10 mg of spores (dry wt)after 30 min of incubation.

However, a striking difference was observedwith -yO65 spores. Heated spores of 7065 showedno GAD activity; however, the decarboxylationof glutamic acid commenced soon after theonset of germination. After 90 s of germinationapproximately 40% of the activity observed withthe parent cells was obtained, and the GADactivity increased steadily with germinationtime so that nearly equal activities were ob-tained with the parent and mutant cells after 9min of germination.

DISCUSSIONMany different compounds capable of initiat-

ing the germination of bacterial spores havebeen described (11), but the mode of action ofnone of these initiators is known. The diversityof compounds with germinative properties forbacterial spores has thus far eluded the demon-stration of any unifying mechanism of sporegermination.

Although chemically diverse, germinativecompounds such as D-glucose, L-alanine, andinosine could each provide both an enzymaticbasis for germination and potential energy forthe germinating spore. An exogenous require-ment of energy for spore germination may,however, be questioned on the grounds thatgermination in B. megaterium appeared not todepend on an external energy source sinceheated spore suspensions could be germinatedin solutions of one of a number of inorganic salts(21). Although this does not preclude an en-zymatic basis for germination, it does raise animportant question with regard to the metabo-lite role of organic initiators. These observationscould mean that germination of heated spores ofB. megaterium may not depend on an exoge-nous energy source, and that the primary func-tion of germinative compounds, both organicand inorganic, may be to initiate an endogenousmechanism, itself responsible for spore germi-nation.

Preliminary evidence consistent with an en-dogenous mechanism for germination in spores,which can be activated by heat and set inmotion by specific initiators added exogenously,has been obtained. The basis for such a mecha-nism in Bacillus derives from (i) the presence inspores of large glutamic acid pools (8, 16), (ii)the rapid metabolism of spore pool glutamicacid with D-glucose-initiated germination in B.megaterium and with L-alanine-initiated germi-nation in B. licheniformis (8), and (iii) theavailability of germination mutants of B. mega-terium which require 'y-aminobutyric acid forgermination (7).

1096 J . BACTrERIOL .

GLUTAMIC ACID DECARBOXYLASE IN B. MEGATERIUM

Studies on GAD provide additional supportfor an endogenous mechanism and indicate thatthis enzyme may be involved in spore germina-tion. In summary, these results showed thatalthough unheated (dormant) spores of B. me-gaterium gave no activity when assayed forGAD, heated spores gave high activities, whichincreased markedly with germination. A com-parison of the effects of metabolic inhibitors,including KCN, NaN3, DNP, and KF, on sporeGAD activity and on spore germination gavesimilar patterns of inhibition. KCN was theonly effective inhibitor of both spore GADactivity and spore germination, but only atrelatively high concentrations; concentrationsof 10 and 100 mM, respectively, were requiredfor essentially complete inhibition. Deactiva-tion of GAD activity in heat-activated sporesafter storage at 4 C was observed-a propertycommonly seen in germination studies. Likereactivation of germination activity in cold-deactivated spores (3), GAD activity was com-pletely restored by a second heat treatment. Itis of interest, and may be more than a coinci-dence, that highly purified GAD from E. coliwas found to lose activity with storage at lowtemperature (24).

Additional support for the involvement ofGAD in spore germination can be inferred fromstudies with -y-aminobutyric acid-dependentmutant spores. The absence of GAD activity inheat-activated mutant spores (Fig. 8) coupledwith their dependence on y-aminobutyric acidfor germination (7) suggest that this enzymemay be required for germination by spores ofthe parent strain. Since the mutant sporesresisted germination by D-glucose, L-leucine, orL-proline in solutions of KI, each of which is aneffective germinant for the parent spores (7),initiation of germination by each of these com-pounds may depend on a single endogenousmechanism.Two additional properties of GAD could re-

late to this discussion. In the first place, consid-ering the high concentrations of acidic com-pounds such as dipicolinic acid and glutamicacid present in spores, acidophilic enzymes maybe the best suited for triggering spore germina-tion. The low pH optimum (3.8 for purified E.coli GAD) demonstrated for bacterial GAD (23)lends itself favorably to such a role. Secondly,anaerobic (fermentative) and aerobic pathwaysfor glutamate metabolism via -y-aminobutyricacid have been described in bacteria (12). Ifpresent in Bacillus spores, these pathways couldprovide a temporary bypass of aerobic metabo-lism in the early stages of germination in thesespores (13, 22).

Although the reported results are consistentwith a GAD-mediated mechanism for sporegermination, they do not rule out alternativemechanisms. Attention is directed to a pointthat projects as a possible inconsistency. Thestable nature of "4C-glutamate pools in mutantspores during 9 min of germination (8) contrastswith the rapid increase in GAD activity ob-served in these spores during germination. Inview of the rapid increase in GAD activity in themutant spores during germination, one mightexpect an early, at least partial, drop in pool"4C-glutamate. The fact that a drop was notobserved suggests several possibilities. It couldmean that GAD is not absent from mutantspores and that these spores contain this en-zyme in an inactive form, which becomes activeonly when the spores germinate, perhaps aresult of increased permeability. The criticalrole of y-aminobutyric acid in the germinationof mutant spores may be to increase sporepermeability, and permit germination by L-ala-nine, or another suitable germinative compound(e.g., L-valine). This would help to explain thedual requirement of y-aminobutyric acid andL-alanine for the germination of the mutantspores.

Alternatively, it could mean that spores con-tain more than one form of GAD; one, missingor defective in the mutant spores and involvedin germination, another, fully active in themutant spores but involved in postgerminativemetabolism. Evidence for two types of GAD inBacillus has been reported (1). Clarification ofthese points must await the availability ofactive, preferably pure, preparations of GADfrom spores and vegetative cells of parent andmutant strains.

ACKNOWLEDGMENTSWe thank Andrew Dewees for helpful discussions. We

gratefully acknowledge the generous help of Frances Blountand Darrell Hall.

LITERATURE CITED

1. Akers, E., and J. N. Aronson. 1971. Detection on polya-crylamide gels of L-glutamic acid decarboxylase activi-ties from Bacillus thuringiensis. Anal. Biochem.39:535-538.

2. Bishop, H. L., and R. H. Doi. 1966. Isolation andcharacterization of ribosomes from Bacillus subtilisspores. J. Bacteriol. 91:695-701.

3. Church, B. D., and H. 0. Halvorson. 1957. Intermediatemetabolism of aerobic spores. I. Activation of glucoseoxidation in spores of Bacillus cereus var. terminalis. J.Bacteriol. 73:470-476.

4. Cozanni, I., A. Misuri, and C. Santoni. 1970. Purificationand general properties of glutamic decarboxylase fromClostridium perfringens. Biochem. J. 118:135-141.

5. Crook, P. G. 1952. The effect of heat and glucose onendogenous endospore respiration utilizing a modified

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FOERSTER AND FOERSTER

Scholander microrespirometer. J. Bacteriol.63:193-198.

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