L-ALANINE DEHYDROGENASE: A MECHANISM CONTROLLING THESPECIFICITY OF AMINO ACID-INDUCED GERMINATION

OF BACILLUS CEREUS SPORES

R. J. O'CONNOR AND HARLYN 0. HALVORSONDepartment of Bacteriology, University of Wisconsin, Madison, Wisconsin

Received for publication May 4, 1961

ABSTRACT

O'CONNOR, R. J. (University of Wisconsin,Madison), AND HARLYN 0. HALVORSON. L-Alanine dehydrogenase: A mechanism controllingthe specificity of amino acid-induced germinationof Bacillus cereus spores. J. Bacteriol. 82:706-713.1961.-A study has been undertaken of theproperties and specificity of germination ofspores of Bacillus cereus strain T. In the absenceof additional carbon sources, only L-alanine,L-a-NH2-n-butyric acid, and L-cysteine wereeffective germinating agents. The physicalproperties of germination, induced by L-alanineand L-a-NH2-n-butyric acid following extendedheat shock, were in close agreement with thoseof L-alanine dehydrogenase. The specificity of thegermination system, as well as amino aciddeamination in vivo, support the view thatL-alanine dehydrogenase activity is essential forgermination and that the enzyme serves as theinitial binding site for L-alanine in heat-shockedspores.

The triggering of aerobic spore germination byL-alanine (Harrell and Halvorson, 1955; Keynanet al., 1958) initially involves the binding ofL-alanine with a stereospecific spore site (Hills,1949; Woese, Morowitz, and Hutchinson, 1958).In recent years, an attempt to identify thealanine-binding site has been undertaken throughthe study of alanine metabolism during germina-tion. Since alanine utilization coincides with theactivation of spores (Murrell, 1952; Murty andHalvorson, 1957), the enzyme catalyzing itsutilization may be the alanine-binding site.The earliest detectable step in alanine metabolismis its deamination to pyruvate and NH3 (O'Con-nor and Halvorson, 1959), catalyzed by anL-alanine dehydrogenase (O'Connor and Halvor-son, 1960). This enzyme has been extracted fromspores, purified, and characterized. A description

of its catalytic site has been provided by ananalysis of its specificity (O'Connor and Hal-vorson, 1961). Our knowledge of the physicalproperties of the enzyme now permits the testingof the proposal that L-alanine dehydrogenase isthe alanine-binding spore site for germination.In an attempt to identify whether the enzyme

is the alanine-binding site, a comparable analysisof the structural requirements of L-alanineanalogues for the initiation of germination hasbeen undertaken. At least two steps are involvedin alanine-induced germination: (i) complexingwith a stereospecific binding site (Hills, 1949)and (ii) oxidative metabolism (Halvorson andChurch, 1957). For a study of the properties ofthe initial binding site, two systems may beutilized: (i) the "maximal system," where energyand carbon sources are supplied in the basalmedium, in which case germination is limited bythe suitability of the analogue for the bindingsite; (ii) the "minimal system," where supple-mentary compounds are not supplied. In thelatter system, a compound initiating germinationmust be metabolized to a utilizable carbon source,presumably pyruvate (Halvorson and Church,1957). The minimal system has been used hereto minimize the triggering of germination byalternative mechanisms. Presumably, all alanineanalogues serving as germination stimulants aremetabolized through the deaminating enzyme toprovide metabolizable carbon skeletons.The data presented below indicate that, with

the "minimal system," (i) the activity of alaninedehydrogenase is required for germination byalanine and its analogues, and (ii) the enzymeserves as the initial binding site for L-alanine inheat-shocked spores.

MATERIALS AND METHODS

Preparation and heat activation of spores.Spores of Bacillus cereus strain T were grown,harvested, and washed as described by Church,

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GERMINATION OF B. CEREUS SPORES

Halvorson, and Halvorson, (1954). Lyophilizedspores (stored at 20 C for 1 week to 6 months)were used for all experiments. Heat activationwas carried out by incubating spore suspensions(1 mg per ml) at 65 C in 0.067 M phosphatebuffer (pH 7.3), for various time intervals asindicated. Following heat activation, the sporeswere centrifuged, washed, and resuspended indistilled water.

Measurement of germination. For germinationexperiments, spores at a concentration of 0.1 mgper ml were suspended in 0.1 M carbonate bicarbo-nate buffer (pH 9.4) at 30 C. The decrease inrefractility (optical density decay) at 625 mu(Powell, 1950) was used as the criterion forgermination. The rate of germination was deter-mined by plotting the optical density decreaseas an exponential decay (Hachisuka et al., 1954;Woese et al., 1958) in the following manner:

01) -ODFlog

0) OFversus time,ODi - ODF

where OD is the optical density at time t, andOD, and ODF are the initial and final (limiting)optical densities, respectively. The germinationrate (K), the slope of the straight line obtainedby plotting the above function, is defined as the

1.4

.4

RATE OF ,.0GERMI NATION

0.8

LOG OD -ODF V.TO.6

0.4

0.2

E~~~~~IR

/-_., LA(z

decline in

OD - ODFlog

OD OFper 100 secOD, - ODF

W\hen this method was employed, the limitingvalue for optical density decline (ODF) variedwith the initial rate; germination media stimulat-ing a rapid initial rate gave a greater total opticaldensity decrease. The relationship between initialrate and limiting value is not understood at thistime. In calculating all rates, ODI - ODF wasaccepted as the optical density change achievedin a 1.0 M solution of L-alanine with spores pre-viously heat activated 4 hr at 65 C; under theseconditions, the entire spore population wasstainable within 30 min.

Chemical analyses. Amino acid deamination byintact spores was measured by the methodpreviously described (O'Connor and Halvorson,1959).

Chemicals. The amino acids used in theseexperiments were purchased from the CaliforniaCorporation for Biochemical Research, with theexception of D-alloisoleucine, which was obtainedfrom 1\Iann Research Laroatories, Inc., andD-cvsteine, a gift received from Bernard Krask,Fort Detrick, Frederick, Md.

ATE OF _ 200GER MINATION

_160LAG TIME(SECONDS)

_120

G TIME

I I

80

40

0 1 2 3 4 5

TIME AS (HRS.)

FIG. L. Effect of heat activation on lag time and rate of L-alanine-induced germzination. The heat-shockconditions are described in the Materials and Methods section. The germnination medinm consisted of 400,unmoles of L-alanine, 0.4 mg of spores (previously heatshocked at 65 C for the timze intervals indicated) in4 ml of carbonate-bicarbonate buffer at pH 9.4. The incubation temperature was 30 C. The abbreviation"v.t." appearing in the ordinate margin means "versus time." The symbol S = heat shock.

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RESULTS

Effect of prior heat activation on alanine-induced germination. 1) Effect on rate and lag:-To establish reproducible germination conditions,the effect of heat activation on germination wasanalyzed. As can be seen in Fig. 1, the rate ofgermination increases almost linearly with thetime of heat treatment up to 4 hr at 65 C. After4 hr heat shock, further heat activation results ina decreased rate of germination. The effect of heatactivation on the rate of alanine-induced germina-tion strongly resembles to effect of heat shock onalanine deamination by intact spores (O'Connorand Halvorson, 1959). In both cases, maximalrates were obtained after 4 hr heat shock at65 C with spores produced under similar condi-tions.Heat activation also reduced appreciably the

lag prior to germination. A lag time of 200 seefalls to a limiting value of 40 see after heatactivation for 5 hr. Although both lag and germi-nation rate are greatly influenced by heat shock,p)rior heat activation apparently has little effecton the total number of spores germinating.Complete germination was obtained in spite ofthe length of heat shock.

2) Effect on the saturation level of L-alanine:-When the rate of germination is plotted againstalanine concentration, a typical absorptionisotherm is observed (Woese et al., 1958). ALineweaver-Burk (1934) plot illustrating thisrelationship is shown in Fig. 2. Prior to heat acti-

O HEAT SHOCK

30-

HEAT SHOCK - 2 HRS., 65 C

22XA

v14 !

x 0(ALANINE)

FIG. 2. Lineweaver-Burk plot of the rate ofgermination as a function of L-alanine concentra-tion. See Fig. I for constituents of the germinationmedium. The letter "V" in the ordinate marginrefers to the germination rate (K).

TABLE 1. Effect of heat shock on the saturation ofL-alanine induced germination

Time heat shock at 65 C K8

hr X 103 M

0 601 202 104 1.45 1.85

See Fig. 2 for details.

vation, the concentration at which the alanine-binding component on the spore is half saturatedis 6 X 10-2 M (Table 1). As spores are heat acti-vated up to 4 hr, this value declines to 1.4 X10-3 M. Krask (personal communication) hasrecently observed a similar value for heat-shocked spores of this same organism. The KS forlpurified alanine dehydrogenase is 2.7 X 10-3 M.The close agreement between these two constantsindicate (i) that the alanine dehydrogenase isthe initial binding site in the fully heat-activatedspore, and (ii) the enzyme may be rate-limitingstep in germination under these conditions.

Effect of temperature on alanine-induced germina-tion. Since L-alanine-induced germination isconsidered to have an enzymic basis, the activa-tion energy for the process should be in theregion typical for enzymic reactions. The effectof temperature on alanine germination is shownin Fig. 3. The activation energy calculated fromthe Arrhenius plot is 19,400 cal per mole; Vasand Proszt (1957) have determined a value of21,000 cal per mole for B. cereus spores germinat-ing in a complex medium.The occurrence of a single temperature

optimum (30 C) for germination is in conflictwith the three distinct optima (23, 25, 43 C)observed by Wolf (1961) for this same organism.The explanation for this difference is not clear atthis time; however, in his experiment, Wolf usedsuboptimal concentrations (8 mM) of L-alanine,whereas L-alanine at 100 mm was used in theexperiment reported here. Only one temperatureoptimum has been observed for B. cereus (Vasand Proszt, 1957) and Bacillus licheniformis(Keynan, personal communication) spores.

Specificity of germination. To determine thespecificity of alanine-induced germination, 36compounds which are structurally related toalanine were screened as germinating agents.

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llJ

z0

z

wro.L

DEGREES TEMP. CENTIGRADE

FIG. 3. Effect of temperature on L-alanine-induced germination. See Fig. 1 for the constituentsof the germination medium. The spores were previ-ously heat shocked at 65 C for 4 hr.

This analysis was done in the "minimal system"where the germination medium contained onlythe analogue in carbonate-bicarbonate buffer atpH 9.4. A pH of 9.4 for the germination experi-ments was selected to provide conditions com-

parable to those used in determining the speci-ficity of spore alanine dehydrogenase (O'Connorand Halvorson, 1961). Since the K, of alaninefor the enzyme varied with pH (O'Connor andHalvorson, 1960), the use of a more physio-logical pH (7.0 to 8.0) for germination studieswould probably provide values not comparablewith those obtained at the higher pH.Of the compounds screened, only L-alanine

L-a-NH2-n-butyric acid and L-cysteine initiatedgermination (Table 2). L-Cysteine was originallyreported by Krask (1961) to be a germinationstimulant in spores of B. cereus strain T. As willbe seen below, several of the other compoundsare substrates of L-alanine dehydrogenase andare deaminated in vivo. However, their failure toyield pyruvate, a necessary intermediate forgermination (Halvorson and Church, 1957),apparently makes them ineffective as germinatingagents. In complex media containing additionalcarbon sources, many of the other amino acids in

Table 2 have been reported as germination stimu-lants (Hachisuka et al., 1955; Woese et al., 1958;Schmidt, 1958).

If alanine dehydrogenase is the initial bindingsite for L-alanine in germination after extendedheat shock, then the physical constants (K8 andVm) for germination induced by alanine analoguesshould reflect those of the enzyme. Table 3indicates that, in the case of L-alanine and L-ac-NH2-n-butyric acid, a close parallel existsbetween the K. and Vm for the two processes.The Vm of L-cysteine germination, however, ismuch greater than that of L-cysteine deaminationby the dehydrogenase. Apparently an alternativemechanism is available for L-cysteine metabolism.A cysteine desulfhydrase, yielding pyruvate,H2S, and NH3, has recently been discovered inspores (Krask, 1961), and may be operative inL-cysteine germination. This view is supportedby the fact that L-cysteine germination is 17times faster at pH 7.3 than at pH 9.4, the pHoptimum of alanine dehydrogenase. The optimalpH for cysteine desulfhydrase is 7.8 (MIetaxasand Delwiche, 1955).

Deamination of L-amino acids by intact spores.The above results support the view that L-alanine dehydrogenase is the initial step inL-alanine and L-a-NH2-n-butyric acid-induced

TABLE 2. Alanine analogues serving asgerminating agents for heat-activated

spores

Amino acid' Vm K,

X 102/ relative to X 103 M100 sec alanine 10m

L-Alanine 6.0 1.0 1.4L-a-NH2-n-Butyric acid 1.7 0.28 59L-Cystine 1.7 0.28 59Glycine, a-NH2-isobutyric 0

acidb

a Indicated amino acids (400,moles) were sub-stituted for L-alanine in the medium described inFig. 1. The spores were heat shocked at 65 C for 4hr prior to incubation.

b Negative results also obtained with the fol-lowing: DL-norvaline, L-norvaline, L-valine, L-iSO-leucine, DL-norleucine, L-leucine, L-serine,L-threonine, L-methionine, L-phenylalanine, DL-

f3-NH2-n-butyric acid, ,3-alanine, DL-a-NH2-iso-butyric acid, ,3-NH2-n-butyric acid, L-proline,L-hydroxyproline, DL-ornithine .HCI, L-histidineHCL-H20, L-aspartic acid, L-glutamic acid,D-alanine, L-asparagine.H20, taurine, DL-alanine-ethyl ester, alaninol, ethylamine, sarcosine, crea-

tine, acetate, 2,4-di-NH2-butyric acid, and DL-

allothreonine.

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TABLE 3. Comparison of physical constants asgerminating agents and as substrates of

alanine dehydrogenase

K, Vm relative toalanine

Ger- Sub- Ger- Sub-minat- strate minat- strateing of en- ing of en-

agenta zyme agenta zymeb

X 103 M X 103 M

L-Alanine 1.4 1.7 1.0 1.0L-a-NH2-n-Butyric 59 74 0.28 0.3

acidL-cysteine 59 72 0.28 0.01

a See Table 2 for details.b See O'Connor and Halvorson (1961).

ants of L-alanine dehydrogenase on L-alanine-in-duced germination is shown in Table 5. Nine com-plexants inhibited germination. These includecompetitive inhibitors of the extracted L-alaninedehydrogenase (glycine, D-alanine, and D-a-NH2-n-butyric acid) and substrates of the enzymewhich are not germinating agents. In the "maxi-mal system" of Woese et al. (1958), a-NHs-isobutyric acid, L-norvaline, L-valine, L-isoleucine,and L-cysteine were germinating agents forspores of B. subtilis. The competitive inhibitionof germination by D-amino acids, especiallyD-alanine, has been recognized for some time(Hills, 1949). The data of Tables 2 and 5 indicatethat the complexants of L-alanine dehydrogenasefall into two classes with respect to germination:

germination. One would expect, therefore, thatthe activity of this enzyme contributes to NH3liberation by intact heat-shocked spores andthat the deamination process should reflect thespecificity of the enzyme itself. An analysis ofdeamination specificity is complicated by thefinding that, during the deamination of exogenousL-alanine by intact heat-shocked spores, over 90%of the NH3 is derived from endogenous sources(O'Connor and Halvorson, 1959). A test wasundertaken in two ways. First, an attempt wasmade to recognize a superimposed NH3 contribu-tion from known substrates of the enzyme and,second, a survey was conducted to see if NH3liberation was inhibited by two nonsubstratecomplexants of the enzyme, D-alanine and glycine(O'Connor and Halvorson, 1961). The results areshown in Table 4. The measurements of NH3liberated were after a 2-hr incubation period andare not necessarily indicative of the initial rates.NH3 liberation by intact spores is high in thepresence of 7 substrates of the enzyme, low inthe presence of 4 nonsubstrates, and negative for24 other nonsubstrates. Deamination of thesubstrates of the enzyme is inhibited by non-substrate complexants of the enzyme. However,with the exception of L-alaninol, the deaminationof nonsubstrates of the enzyme is not affected byD-alanine or glycine.

Inhibition of L-alanine-induced germination bycomplexants of alanine dehydrogenase. If theactivity of the enzyme is essential to L-alanine-induced germination, then any complexant of theenzyme should inhibit L-alanine-induced germina-tion. A survey cf complexants and non-complex-

TABLE 4. Effect of analogues of L-alanine on theproduction of NH3 from activated,

intact spores

Amino acid

L-AlanineL-a-NH2-n-Butyric

acidL-NorvalineL-ValineL-IsoleucineL-CysteineL-SerineL-LeucineL-PhenylalanineL-AllothreonineL-OrnithineL-Alaninol24 Other amino

acidsd

En-zymecom-plex-ant'

±+

++++++

Deami-nationb

,umoles

3.067.90

1.735.805.27.027.362.181.741.190.661.720

Inhibition ofdeamination

byc

D-Ala- Gly-nine cine

61 6059 43

48241215280006

0

3678682160

0

0

a See O'Connor and Halvorson (1961).b For the deamination assay, each Conway unit

contained 200 ,umoles of the indicated amino acidand 25 mg of spores (heat-shocked 4 hrs at 65 C)in 2.0 ml of 0.1 M carbonate-bicarbonate buffer,pH 9.4. After 2 hr at 30 C, the liberated NH3 wascollected and measured as indicated in the Mate-rials and Methods section.

c Per cent inhibition when 200,umoles of the in-dicated amino acids added to the above reactionmixture.

d See Table 2 for other analogues tested.

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(i) L-alanine and L-a-NH2-n-butyric acid, whichcomplex with the enzyme, are deaminated, andprovide carbon skeletons supporting germination;(ii) the other complexants of the enzyme, whichare either inhibitory or are deaminated to carboncompounds not supporting germination. Thecompounds grouped in the latter class inhibitgermination initiated by L-alanine (Table 5).

Stimulation of L-alanine-induced germination byD-cysteine. Of the complexants with L-alaninedehydrogenase, D-cysteine has the highestaffinity (O'Connor and Halvorson, 1961). Onthis basis, D-cysteine would be presumed to bethe most effective inhibitor of L-alanine-inducedgermination. However, Krask (1961) reportedthat D-cysteine stimulates L-alanine germination,although D-cysteine itself is not a germinationstimulant. The data of Table 6 confirm theseobservations. In fact, nonheat-shocked spores

have an absolute requirement for D-cysteine togerminate in low levels of L-alanine (5 ,umolesper ml).The effect of D-cysteine on nonactivated spores

TABLE 5. Comparison of alanine analogues as

inhibitors of germination and complexantsof L-alanine dehydrogenase

Inhibi-Complexantsa tion of L-

Analogue inducedgermina-

K, Ki tion

X 103M X 1OJ M %b

D-Alanine 23 59D-a-NH2-n-Butyric acid 500 37L-Norvaline 941 37Glycine 42 36a-NH2-Isobutyric acid >3,400 36L-Cysteine 7.2 26L-Isoleucine 370 20L-Valine 29.4 18L-Threonine 13L-Serine 2.7 426 Other analoguesc 0 0 0

a See O'Connor and Halvorson (1961).b Per cent inhibition after 30 min incubation of

the indicated analogue at 0.1 M (final concentra-tion). The germination medium also contained 15Mmoles of L-alanine, 0.3 mg of spores (previouslyheat shocked at 65 C for 4 hr) in 3 ml of car-

bonate-bicarbonate buffer at pH 9.4. The percent inhibition was based on alanine germinationin the absence of the analogue.

c See Table 2 for other analogues tested.

TABLE 6. Stimulation of L-alanine inducedgermination by D-cysteine

Germination rate (K)with spores previously

Additions heat shocked

0 hr ½2 hr 3 hr

X 102/ X 102/ X7103/100 sec 100 sec 100 sec

L-Alanine (20 ,umoles) 0 0.09 3.75+ D-Cysteine (20 ,umoles) 0.06 0.56 5.13+ D-Cysteine (10 ,umoles) 0.06 1.06 5.07

D-Cysteine (20 ,umoles) 0 0 0

Additions were made to spore suspensions (0.1mg per ml) in 0.1 M phosphate buffer (pH 7.3);total volume was 4 ml.

is particularly interesting in that the germinationrate is independent of alanine concentration(0.005 to 0.1 M) in the presence of 0.005 MD-cysteine. Without D-cysteine, the germinationrate is highly dependent on L-alanine concentra-tion over this range (Fig. 2). In this respect,D-cysteine resembles heat shock in that muchlower concentrations of L-alanine are requiredwhen D-cysteine is added.Krask (1961) has postulated that this stimula-

tion is due to the inhibition of alanine racemaseby D-cysteine, thus prohibiting the conversion ofL-alanine to D-alanine, a germination inhibitor.This hypothesis is supported by the fact thatD-cysteine inhibits D-alanine germination, stimu-lates the inhibitory effect of D-alanine on L-alanine-induced germination, and inhibits ex-tracted alanine racemase activity (Krask,personal communication). Thus, alanine racemasemay serve as a regulatory system for controllingthe intracellular ratio of L- to D-alanine andthereby control the rate of L-alanine dehydro-genase activity and over-all germination.The rate of germination in the presence of

L-cysteine and L-alanine is slower than in thepresence of either germinating agent alone(Krask, 1961). These findings can be understoodsince L-cysteine is a complexant of L-alaninedehydrogenase and L-alanine is an inhibitor ofcysteine desulfhydrase (Fromageot and Grand,1943; Kallio and Porter, 1950).

DISCUSSION

The work reported here supports the hypothesisthat L-alanine dehydrogenase activity is essentialfor germination initiated by L-alanine. This

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conclusion is based on the following: (i) alaninedeamination is essential for germination, andthe enzyme accounts for all of the deaminatingcapacity of crude spore-extracts (O'Connor andHalvorson, 1960); (ii) complexants of the enzymeinhibit L-alanine deamination and germination;(iii) heat shock enhances both the expression ofthe enzyme in vivo (O'Connor and Halvorson,1959) and L-alanine-stimulated germination.The hypothesis that L-alanine dehydrogenase

is required for germination leads to the predictionthat (i) products of alanine deamination whichare not themselves germination stimulants (suchas NH4+) should inhibit L-alanine-inducedgermination; and (ii) spores lacking the enzymewould not be expected to germinate in responseto L-alanine. The inhibition of Bacillus anthracisspore germination by NH4+ has previously beenobserved by Hills (1950). The prediction involv-ing spores lacking the enzyme has not beentested; however, strains of B. subtilis devoid ofL-alanine dehydrogenase have been reported(Hong, Shen, and Braunstein, 1959). The pro-duction of spores from these strains wouldprovide a system in which the requirement ofthe enzyme for alanine-induced germination maybe tested.

After extended heat-shock treatment, theenzyme is apparently directly accessible to L-alanine placed in the medium and represents theinitial binding site for alanine in germination.Not only do the physical constants of L-alanineand L-a-NH2-n-butyric acid-induced germinationagree with those of the enzyme, but also thespecificity of the enzyme can be recognized inboth the specificity of amino acid deaminationand in the inhibition of L-alanine-inducedgermination.Heat shock leads to an increase in the maximal

rate and decrease in the L-alanine requirementfor germination. The biochemical alterationsunderlying these changes are poorly understood.Heat shock could influence L-alanine-inducedgermination by: (i) releasing an endogenousstimulant, (ii) altering the properties of L-alaninedehydrogenase, (iii) modifying the rate of reac-tion(s) linked with L-alanine dehydrogenase,(iv) altering permeability, or (v) removal of aninhibitor of germination. Of these, severalwould seem unlikely on the basis of the availableevidence. The release of endogenous germinationstimulants during heat activation should cause

spontaneous germination, which is not observedwith spores of B. cereus strain T; examples ofthis, however, have been reported with Bacillusmegaterium spores (Powell and Strange, 1953).Also, an alteration in the permeability of sporesof B. cereus strain T is probably negligible, sincegermination itself involves a change of only 18%in the diffusible spore volume (Black, 1961).An example of a reaction intimately linked

with alanine deamination which may conceivablybe affected by heat shock is reduced diphospho-pyridine nucleotide (DPNH) oxidation. Thisreaction, catalyzed by a flavin mononucleotide(FMN)-requiring DPNH oxidase, is stimulatedby dipicolinic acid (Doi and Halvorson, 1961).At rate-limiting diphosphopyridine nucleotide(DPN) concentrations, L-alanine deamination bycrude spore extracts is markedly stimulated bydipicolinic acid (O'Connor and Halvorson, 1961),apparently due to DPNH recycling. The changein the over-all K8 and Vm for L-alanine germina-tion with heat shock may, therefore, reflect thestimulation of the oxidase by the dipicolinic acidreleased during heat shock, which in turnpromotes alanine deamination.

ACKNOWLEDGMENTS

The authors are indebted to Bernard Krask fornumerous discussions and for the opportunity toinspect his unpublished experiments.

This research was supported in part by an insti-tutional research grant from the American CancerSociety and a grant-in-aid from the WisconsinAlumni Research Foundation.

LITERATURE CITEDBLACK, S. H. 1961. The physiology of spores of a

Bacillus: mass production, normal and"endotrophic" sporogenesis, and permea-bility. Ph.D. Thesis, University of Michigan,Ann Arbor.

CHURCH, B. D., H. HALVORSON, AND H. 0.HALVORSON. 1954. Studies on spore germina-tion: its independence from alanine racemaseactivity. J. Bacteriol. 68:393-399.

Doi, R. H., AND H. HALVORSON. 1961. The mech-anism of dipicolinic acid stimulation of thesoluble reduced diphosphopyridine nucleo-tide oxidase of spores. J. Bacteriol. 81:642-648.

FROMAGEOT, G., AND R. GRAND. 1943. The activegroups of cysteine desulfhydrase. Enzymo-logia 11:81-86.

HACHISUKA, Y., N. ASANO, N. KATO, AND T.

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HACHISUKA, Y., N. ASANo, N. KATO, M. OKAJIMAM. KITAORI, AND T. KUNO. 1955. Studies onspore germination. I. Effect of nitrogensources on spore germination. J. Bacteriol.69:399-412.

HALVORSON, H., AND B. D. CHURCH. 1957. Inter-mediate metabolism of aerobic spores. II.The relationship between oxidative me-tabolism and germination. J. Appl. Bacteriol.20:359-372.

HARRELL, W. K., AND H. 0. HALVORSON. 1955.Studies on the role of L-alanine in the germi-nation of spores of Bacillus terminalis. J.Bacteriol. 69:275-279.

HILLS, G. M. 1949. Chemical factors in the germi-nation of spore-bearing aerobes. Effect ofamino acids on the germination of Bacillusanthracis, with some observations on therelation of optical form to biological activity.Biochem. J. 45:363-370.

HILLS, G. M. 1950. Chemical factors in the germi-nation of spore-bearing aerobes. Observationson the influence of species, strain and con-dition of growth. J. Gen. Microbiol. 4:38-47.

HONG, M. M., S. C. SHEN, AND A. E. BRAUNSTEIN.1959. Distribution of L-alanine dehydrogenaseand L-glutamate dehydrogenase in Bacilli.Biochim. et Biophys. Acta 36:288-289.

KALLIO, R. E., AND J. R. PORTER. 1950. Themetabolism of cystine and cysteine by Proteusvulgaris and Proteus morganii. J. Bacteriol.60:607-615.

KEYNAN, A., M. HALMAN, AND Y. Avi-Dor. 1958.The influence of temperature on the differentstages of germination of Bacillus subtilisspores. 7th Intern. Congr. Microbiol., p. 37-38.

KRASK, B. J. 1961. Germination stimulants andenzyme systems in bacterial spores con-cerning their utilization. In H. 0. Halvorson[ed.], Spores II. Burgess Pub. Company (inpress).

LINEWEAVER, H., AND D. BURK. 1934. The de-termination of enzyme dissociation constants.J. Am. Chem. Soc. 56:658-666.

METAXAS, M. A., AND E. A. DELWICHE. 1955. TheL-cysteine desulfhydrase of Escherichia coli.J. Bacteriol. 70:735-737.

MURRELL, W. G. 1952. Metabolism of bacterialspores and thermophilic bacteria. Ph.D.Thesis, Oxford, England.

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