AN-ALYSIS OF ACTION OF L-ALANINE ANALOGUES IN SPORE GERMINATION

CARL R. WOESE, HAROLD J. MOROWITZ, AND CLYDJE A. HUTCHISON III

Biophysics Departmtlent, Yale University, New Haven, Connecticut

Received for publication Jtune 1, 1958

The initiation of spoIre germination appearsto be a particularly clear case of a "trigger"mechanism in biology. The first stage, consistingin the uptake of water and release of dipieolinicacid and other substances, may in the limitingcase take place in the presence of water and asingle amino acid (Woese and Morowitz, 1958).In such simple media the process ceases after thefirst stage and additional metabolites must beadded to initiate subsequent development (Hyattand Levinson, 1957). These first steps in themorphogenesis of bacilli can, therefore, be studiedin relative isolation.

Since a single amino acid can initiate the gei-mination process, it has been of interest to studythe specificity of this reaction. A study of thisspecificity should prove helpful in elucidatingthe mechanism of dipicolinate release and thesites in the spore where the amino acid reacts.

Tlhe experiments reported here were carriedout on a strain of a bacillus which initiated ger-mination very rapidly in the presence of L-ala-nine. A series of L-alanine analogues were foundeither to initiate germination, inhibit L-alanine,or to be inactive. An extensive series of experi-ments on these analogues and the riesultantinterpretations are presented in this paper.

Various spores have differing requirements interms of germination initiators (Halvorson andChurch, 1957), so that the existing publisheddata give little information about the specificityof the initiators. However, there have been a fewreports in which the action of L-alanine analoguescan be compared (Hachisuka et al., 1955; Wvnne,1957). In general, these reports give the impres-sion that amino acids closely related to L-alaninestructurally as well as some non-amino acidsstructurally related to L-alanine can cause ger-mination, whereas the corresponding D-analoguesof these compounds (if such exist) may inhibitgermination (Wynne, 1957).

MATERIALS AND METHODS

The spores used in these experiments werefrom Bacillus sutbtilis (.Marburg strain). The

spores were prepared by seeding potato agarpetri plates, pH 7.0, with a growing culture of B.subtilis. The plates were incubated at 37 C for5 days; the fraction of non-spore forms wasnegligible by this time. The growth was thenscraped from the plates and purified by 2 cyclesof centrifugation in distilled water. A 10 per centsolution of egg white in phosphate buffer, pH7.0, was then used to destroy any remainingvegetative cells. After 2 more cycles of centrifu-gation and washing in distilled water, the spoIesuspension was lyophilized. At the time of thisexperiment the spore stock was about one y-earold, having been stored at room temperatureduring this time.The techniques for measuring germination

by drop in optical density of the culture, as wellas the germination media used, have beeni (le-scribed (WVoese and Morowitz, 1958) (germinationoccurred at 38 C in a pH 7.0 phosphate buffercontaining glucose and Mn++ ion, supplemenitedby L-alanine anialogues' in concentrations indli-cated in the text, and germination was measuredby optical density drop of the culture measuredlat 6250 A in a Bausch and Lomb "Spectronicno. 20" colorimeter).Each compound was tested to deterimine

whether it initiated germination as measured bythe optical density method. If a compoundcaused ger-mination, the germination rate con-stant, ko was (letermined as a function of con-centration of that compound. It has been shownpreviously (Hachisuka et al., 1955; Woese andMorowitz, 1958) that when L-alanine (or otherinitiators) are added to a spore suspension theoptical densitY of the culture remains constant

I All compouinds used in this study were puir-chased from California Foundation for Biochem-ical Research with the following exceptions:D(-)soditum lactate was purchased from M\IannResearch Labs; L-alanine, D-alanine, tauirine,hydroxy-L-proline, and L-serine were purchasedfrom Ntutritional Biochemicals Corp. Dr. JesseGreenstein of the National Cancer Instittute verykindly supplied the L-alloisoleucine.

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L-ALANINE ANALOGUES IN SPORE GERMINATION

for a short period of time (5 min under optimalconditions at 38 C). After the initial lag theoptical density begins to drop and eventuallvreaches a limiting value. If the drop in opticaldensity with time is plotted as the followingfunction on a semilog plot, a straight line is ob-tained (after the initial lag); the slope of thisstraight line is the germination rate constant, ko(Woese and Morowitz, 1958). The plotting func-tion is as shown in (1), where OD is the opticaldensitv at time t, and ODi and ODf are theinitial and final (limiting) optical densities, re-

spectively (Woese and Morowitz, 1958).

log OD ODf versus time (1)

RESULTS

For all compounds which cause germination,.the rate of germination can be related to theconcentration of the initiator in the manner as

indicated in equation (2), where ko is the rate of

ko = kC/(C + K) (2)

germination, k and K are constants characteristicof the initiator, and C is the concentration of theinitiator. Figure 1 shows a graph of ko versus Cfor L-alanine. From equation 2 it is apparentthat for each initiator we can measure the maxi-mum rate of germination as well as the concen-

tration required to give 50 per cent of the maxi-mum rate (the latter being the constant K).This allows accurate quantitative comparisonsamong various initiators.At concentrations of initiators which produce

maximal germination rates, inhibition is a func-tion of the ratio of initiator to inhibitor concen-trations and does not depend upon the absolutevalue of either, i. e., inhibition in this systemfollows the kinetics of a competitive inhibitionreaction. This result holds for all the inhibitorsused in this study. (Detailed theoretical andexperimental studies on the kinetics of initiationand inhibition of germination have been carriedout. These are in preparation for publication, andwe present here only the pertinent results.) It isthus possible to characterize initiator-inhibitorsystems by the ratio of inhibitor to initiator whenthe rate of germination has decreased to 50 percent of its maximum value.The data on initiators and inhibitors are pre-

sented in the following manner. Table 1 sum-

iparizes the data on initiators; the structure ofeach compound is compared to its relative ef-fectiveness as an initiator in terms of both themaximum rate attainable and the concentrationto give 50 per cent maximum rate; and the rela-tive ease with which its action can be inhibitedby D-alanine is characterized in terms of therelative concentration of D-alanine to reduce theuninhibited germination rate to 50 per cent. Thedata in table 1 are for L-compounds only, as no

D-a-amino acids or other D-compounds are ini-tiators. The inhibition data are summarized intable 2, where the relative concentration of an

inhibitor necessary to reduce the rate of an

initiator to 50 per cent of its value is given.Inhibition studies were done mainly in terms ofinhibition of L-alanine and L-valine. It can beseen that, although D-a-amino acids mainly are

inhibitors, some L-forms do inhibit also.

100- 90-

cr

80 >z

° 70Z 60_9QC

50uJ

w 40I,<30-

w 20.1

10

0 10 20 30 40 50 60 70 s e 90 100

CONCENTRATION of L-ALANINE in MICROGRAMS per ml

Figure 1. Germination rate constant as a function of L-alanine concentration

1958] 579

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WOESE, MOROWITZ, AND HUTCHISON

TABLE 1Parameters characterizing initiation of germination caused by L-alanine analogues

Relative Conc ofMax Conc to Give 50% Max (mg/L) D-Alanine to Re-Gemax duce Rate to 50%

Germi- ( 0tStructure ~~~nation (X 10-3)Compound RStructureate

(X 10- 3/ In in-min) K In L-alanine verSe D-mm) K ~~~~units alanine

units

H

HOOC-C-H

NH2

H

HOOC C-CH3

NH2

CH3

HOOC-C-CH3

NUW

H

HOOC-C-CH2-CH3

NH2

H

HOOC-C-CH -CH2-CH3

NH2

H CH3

HOOC C-CH

NH2 CH3

H CH2,CH3

HOOC-C-CH

NH, CH:3

H CH3

HOOC C-CH

NH2 CH. CH3

>100,000

100

65

65

70

65

60

8

120

50

500

500

1500

>15,000

>12,500

1 270

15

6.3 20

1

18

13

63 1.3 210

63

190

>1900

1.5 1180

0.7 390

580 [VOL. 76

Glycine

L-Alanine

L-a-NH2-isobutvricacid

L-ca-NH2-n-butyricacid

L-Norvaline

L-Valine

L-Isoleu-cine

r.-Alloiso-leuicine

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TABLE 1-Continued

Relative Conc ofMax Conc to Give 50% Max (mg/L) duAlRanie to Re-dcRaeto 50%Germi- (X 10-3)

Compound Structure nationCompound ~~~~~~~~~~~Rate-_____(X 10'/ In in-min) K In L-alanine verse D-mm)Kunits alanine

units

L-Norleu- H 50 700 87 1.8 150cineI

HOOC-C-CH2-CH2-CHx-CHs

NH2

L-Leucine H CHI| 10 5000 630 - -

HOOC-C-CH2-CH

NH2 CHs

L-Cysteine H 35 1200 150 1 270

HOOC-C-CH2-SH

NH2

L-Serine H - >40,000 >5000 - -

HOOC-C-CH2-OH

NH2

L-Threo- H CH-O- >40,000 >5000nine

HOOC-C-CH-OH

NH2

D,L-Allo- HOOC-CH-CH-OH- >40,000 >500"threonineI

NH, CII,

D, L-Phos - H >40,000 >5000phoserine

HOOC-C-CH2-O-POsH,

NH,

L-Phenyl- H > 40, 000 >5000alanine

HOOC-C-CH,/2

NH2

L-Methio- H 12 2000 250 - -nine

HOOC-C-CH,-CH2-S--CH3

NH,

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TABLE 1-Continued

Relative Conc of

10Mx(m/L) -Alanine to Re-Max Conc to Give 50% Max (mg/L) duce Rate to 50%10Germi- (X 108)Compound Structure Riation

Rate-(X l0"I Inimin) K In L-alanine verse D-

units alanineunits

S-Alanine H 65 900 110 1.5 180

HOOC-C-CH2-NH2

H

D, L-0-NH2- H 75 150 19 9 30n-butyricacid HOOC-C-CH-CH3

H NH2

D, L-13-NH2- CH3 65 7000 875 - -isobutyricacid HOOC-C-CH2

H NH2

,-NH2- H H 50 60,000 7500butyricacid HOOC-C-C-CH2-NH2

H H

L-Proline CH2-CH2/ \ 45 8,000 1000 0.07 3800

HOOC-C CH2

NH2 CH2

L-OH-pro- CH2-CHOH - >120,000 >15,000 - -

line/HOOC-C OH2

",1

NH2 OH2

D,L-Orni- H >20,000 >2500thine

HOOC-u-CH2-CH2-NH2

NH2

L-Histidine H - >20,000 >2500 -

HOOC-uC-H2-C==C-HNH2 N NH

C

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TABLE 1-Continued

Relative Conc ofMax Conc to Give 50% Max (mg/L) duAlanine to Re-Germi- duce Rate to ~50%

Gertion (X 10-3)Compound Structure nati

Rate(X 10-3/ In in-min) K In L-alanine verse D-mm) K units alanine

units

L-Aspartic H -_ >20,000 >2500acidI

HOOC-C-CH2-COOH

NH2

L-Glutamic H >20, 000 >2500acid

HOOC-C-CH2-CH2-COOH

NH2

L-Gluta- H > 20, 000 > 2500mineI

HOOC-C-CH2-CH2-CONH2

NH2L-Aspara- H >20,000 >2500gineI

HOOC-C-CH2-CONH2

Pyruvate 0 >80, 000 > 10, 000

HOOC-C-CH2

D(,L-Lactate H - >80,000 >10,000

HOOC-C-CH3

OH

D(-)-Lac- H - >80,000 >10,00tate

HOOC-C-CH3

OHD,L-N-ace- H >20, 000? >2500tylalanineI

HOOC-C-CH3

esterNH2

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WOESE, MOROWITZ, AND HUTCHISON

TABLE 1-Continued

Relative Conc ofMax Conc to give 50%0 Max (mg/L) duce Rate to 50%

germi- (X 10-8)Compound Structure nation

(X 10' In in-min K In L-alanine verse D-

units alanineunits

L-2,4-di- H > 120,000 > 15,000 _NH2-n-butyric HOOC-C-CH2-CH2-NH2acid

NH2

Taurine H - >78,000 >10,000 -

HO3S-C-CH2-NH2

H

DISCUSSION

In our discussion of germination initiationcaused by analogues of L-alanine we assume thatall the analogues act at the same sites on thespore. The kinetics of inhibition by D-alanine andother D-amino acids and the fact that D-alanineinhibits all of the initiators argues that D-aminoacid inhibitors are capable of reacting at all siteswhere initiators act. This implies that probablyL-alanine and other initiators act at all thesesites also. The fact (not reported above) thatlarge amounts of a-amino-isobutyric acid, an

initiator whose maximum rate is 65 per cent thatof L-alanine, can slow the rate of L-alanine causedgermination down to its characteristic rate, alsoimplies common sites of action.

Before comparing the effects of varying thestructure of the alanine molecule, it is well toemphasize that (in the analysis used) germinationis measured as a function of two parameters. Oneis the "fit" of the molecule into the spore struc-ture, in other words, the binding of the moleculeto the spore, characterized by the constant K.The other is the rate of reaction, ko, as measuredby optical density change, subsequent to binding.If a molecule will bind to the spore, it is eitheran initiator or an inhibitor; if it will not bind, it

is neither. For inhibitors one studies only binding;in studying initiators both binding and rate ofgermination are involved. The two can be sep-arated by a concentration versus rate study, forat high enough concentrations, the binding sites

are saturated and a maximum rate is obtained.The binding can then be characterized by theconcentration at which the rate of reaction ishalf maximal or, in terms of inhibitors, the rela-tive concentration necessary to reduce the rateof an initiator to half its value, providing concen-trations of initiator and inhibitor sufficient tosaturate the binding sites are used. This argu-ment on the separation of the binding "effect"from the rate "effect" is possible since the samesites are in common between all inhibitors andall initiators.

In attempting to understand the mechanism ofaction of L-alanine in germination we shall con-sider the effect of substitution or replacement oneach of the four groups bonded to the centralcarbon atom.The effects of varying the COOH group are

these: (a) esterification reduces its effectivenessbut by no means eliminates it (glycine andD ,L-alanine esters inhibit rather well); (b) reduc-tion to an alcohol or removal destroys activity;(c) replacement by sulfonic acid group destroysactivity (taurine versus ,3-alanine). The fact thatesters are effective whereas alcohols are not indi-cates that a certain degree of electronegativity isrequired, and also, that ionization of the groupis not required. The degree of steric specificityfor this group is uncertain as yet.The NH2 group has a rather rigid steric re-

quirement, as N-methyl glycine (sarcosine) isineffective as an inhibitor; the charge on the

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1908] L-ALANINE ANALOGUES IN SPORE GERAIINATION 585

TABLE 2Parameters characterizing inhibition of germination by analogues of z-alanine

Relative Conc to Inhibit Relative Conc to InhibitCompound L-Alanine to 50% Max L-Valine to 50%0 Max

D-Alanine units D-Alanine units

Glycine ............................. 2.4 9 0.10 67N-methyl-glycine .................... >800 >3000 > 240 > 160,000N-acetyl-glycine ............ ......... >330 > 1200 - -

Glycine-methyl-ester................. 18 67Glycine-ethyl-ester . ................. 40 150 -

D-Alanine ............................ 0.27 1 0.0015 1D, L-N-acetyl-alanine ........ ......... > 170 >630D, L-Alanine-methyl-ester ...... ...... 1.8 6.6 0.018 12D-a-NH2-n-butyric acid.............. 12 45 0.11 78D-Norvaline......................... > 760 >2800 15 10,000D-Valine ............................. > 1000 >3700 >33 >22,000D-Isoleucine ......................... >760 >2800 >52 >35,000D-Alloisoleucine -......................_ >68 >45,000D-Norleucine ......................... 760 2800 >56 > 37,000D-Leucine ........................... 1000 3700 >80 > 53,000D-Cysteine ........................... 10 37 -1 660D-Serine ............................. 1.5 5.5 0.006 4L-Serine ............................. 30 110 1 660D-Threonine ......................... 41500 4.5 3000L-Threonine ......................... > 1600 >5900 30 20,000D, L-Allothreonine -.................... > 120 >80,000D, L-Phosphoserine .......... ......... > 170 >630 - -

D-Histidine .......................... > 1000 >3700 _D-Methionine ........................ > 760 >2800 -

D-Phenylalanine ............ ......... > 1600 > 5900 > 48 >32,000L-Phenylalanine ..................... >1600? >5900 -13 -8,500Pyruvate ............................ > 1600 > 160 -

D, L-Lactate ............ ............. > 1600 > 160 -

Betaine ............................. > 160 -

Creatine ............................. > 160 -

Citrate ............................. > 160 - -

Succinate ............................ > 160Fumarate ............................ > 160 _Maleate ............................. > 160 -Oxaloacetate ........................ > 160Aconitate ............................ > 160 _Glycerophosphate ............ ........ > 160 -

Chloroacetate ....................... > 160 - -

Mercaptoacetate ............ ......... > 160 - - -

Levulinate ........................... > 160 - - -

Valarate ............................. > 160 - - -

Propionate .......................... >1000 - -

Ethanolamine....................... >1000 >70 -

Taurine ............................. > 160 - - -

Ethylamine .......................... > 160 - _

-NH-CH3 group is the same as that on the attached to the N, is an initiator at very high-NH2 group as both groups have almost the concentrations. This difference could conceivablysame pK for ionization (Cohn and Edsall, 1943). be explained by the fact that the free rotation ofHowever, L-proline, which also has a carbon the carbon in question is extremely restricted by- on June 14, 2020 by guest

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WOESE, MOROWITZ, ADN HUTCHISON

its being part of a five membered ring in proline.Replacement of the amino group of glycine by H,OH, SH, or Cl, or replacement of the amino groupof alanine by OH leads to loss of activity. We cansay in general of the -NH2 portion of the mole-cule that any steric change or change in electro-negativity results in a drastic change in itsability to bind to the spore.

Turning next to the H attached to the centralcarbon, we see that its presence is not essentialfor initiation, as a-amino-isobutyric acid is aninitiator; but, all compounds which inhibit dohave at least oneH attached to the central carbon.However, the latter statement should be furtherinvestigated using the D- and L-isomers of iso-valine, etc.The steric requirements for the remaining CH3

group are reflected in the initiation caused by theamino-butyric acid, valine, leucine series. L-a-Amino-n-butyric acid initiates with }E the ef-fectiveness of L-alanine. Addition of a second"methyl" group in this series gives either valineor norvaline, which are equally effective as ini-tiators, but bind X10 as well as does L-a-amino-n-butyric acid. If still another carbon is addedto this side chain, its effect depends upon itsposition; norleucine, a straight chain addition,is almost (within 25 per cent on a molar basis)as effective as the valine group, while isoleucine,a branched chain compound, is only % as effec-tive as the valine group. However, L-alloisoleu-cine is ineffective as an initiator and cannotinhibit L-valine caused initiation either. L-Leucineis also practically ineffective as an initiator dueto a reduction in binding by a factor of 3 overL-isoleucine and a reduction in rate to 14o thatof L-alanine. Straight chain a-amino acids oflonger chain length than norleucine are sparinglysoluble in aqueous solutions; consequently, thisinteresting cffect of steric considerations cannotbe completely studied.The effect of charged group substitutions on

the CH3 portion of the L-alanine molecule canbe seen in 2,4-diamino-butyric acid, ornithine,and aspartic acid. The L-forms of the two com-pounds containing a second amino group (and,therefore, positively charged under these condi-tions) are completely inactive in the system. (Itwill be noted that 2-amino-n-butyric acid and4-amino-butyric acid are initiators at concentra-tions at which the 2 ,4-diamino compound is not.)The negatively charged L-aspartic acid is in-

active, though this could equally well be ascriibedto its size or to its charge.

It can be seen from table 1 that, when centersof electronegativity are introduced into the CH3portion of the molecule, the rate constant, k3,tends to be reduced (see cysteine and serine intable 1).The requirements on the CH3 portion of the

molecule, then, seem to be that additions can bemade to the CH3 group, but each extra carbonatom added reduces the effectiveness as an initia-tor by about a factor of 10, up to 2 added carbons;for 3 added carbons there are strict steric require -ments as to relative placement of the carbons,straight chain being preferable. Positive charge onthis portion of the molecule renders it completelyineffective, as probably does net negative charge,but weak charge displacements such as occur forcysteine do not destroy initiator activity, al-though the stronger electronegative displacementin serine renders what could conceivably bo aninitiator into an inhibitor by reducing the con-stant, ko, to a negligible value.Another way of looking at some of the substi-

tutions studied is that they form a class of sub-stitutions in which each of the four groups ismoved in turn by one or more carbon atomsdistant from its position relative to the centralcarbon atom in the alanine molecule; for ex-ample, replacement of the CH3 group by CH2CH3(a-amino-n-butyric acid) lowers the binding bya factor of 6. In effect, this operation can be doneon the H group approximately by introducing amethyl group in its stead (a-amino-isobutyricacid), which lowers the binding by a factor of 15.One of the isomers of D ,L-3-amino-n-butyric acidperforms this for the COOH group, and thebinding is reduced by a factor of 19. One of theisomers of D, L-f3-amino-isobutyric acid performsthis for the NH2 group, and the binding is re-duced by a factor of over 500. To date we havebeen able to obtain only racemic mixtures of,B-amino acids.

,B-Alanine is an initiator, though only yE aseffective in binding as is D,L-/3-amino-n-butyricacid (N 2 if only one of the isomers of the latteris active in initiation). The relative position ofCOOH to NH2 in the two is the same, so thatthe greater effectiveness of the latter is un-doubtedly due to the added CH3 group. Why3-alanine is an initiator at all is an interesting

question. If molecular models of glycine, a-

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alanine, fl-alanine, and 3-amino-n-butyric acidare constructed, it is seen that all four have con-figurations in which the relation of COOH toNH2 is practically the same; in configurations inwhich the COOH and NH2 are close together,one fact is strikingly apparent, that all of themolecules have a decidedly greater "protrusion"of a portion of the molecule above the line be-tween the centers of the COOH group and theNH2 group than does glycine.The relationship among the four groups about

the central carbon in alanine is obviously of thegreatest importance, as interchanging any twotransforms L-alanine, the most potent initiator,into its mirror image, D-alanine, the most potentinhibitor. Inhibitors and initiators can best becompared by their binding constants; the bind-ing constants of initiators are given in table 1,those of the inhibitors can be calculated fromthe data of table 2 from the formula J = (B/A)1/2x K, where J and K are the binding constantsof the inhibitor and the initiator respectively,and (B/A)1/2 iS the ratio of concentration ofinhibitor to initiator at 50 per cent maximumrate under conditions of saturation of bindingsites (Morowitz and Woese, unpublished data).Table 3 gives the binding constants for the D-and L-isomers of several amino acids; K is thebinding constant for the L-isomer obtained fromthe initiation studies presented in table 1, exceptwhere the L-isomer is an inhibitor, in whichcase K is calculated by the above formula fromthe data of tables 1 and 2; J is the bindingconstant of the D-isomer calculated by theabove formula from the data of tables 1 and 2.When J can be calculated from data on inhibi-tion of several L-amino acids by a particular D-amino acid, the range of values so obtained isas given in table 3. The final column of thetable gives the concentration ratios of D-formto its own L-isomer for 50 per cent inhibitionof the L-isomer initiation rate. In the last lineof the table the binding constant for a-amino-isobutyric acid (for which no optical isomersexist) calculated from initiation rate data(table 1) is compared to the binding constantfor this amino acid calculated from inhibitiondata. L-Alanine has a maximum rate of ger-mination of 100 on a relative scale, whereasthe maximum rate for a-amino-isobutyric acid isonly 65, which allows one to study the slowingof the rate of the former by the lattei, a 50 per

TABLE 3Comparison of the binding constants for analogues

of L- and D-alanine which vary the CH3portion of the molecule

(B/A)1Amino Acid K(L- J(D-ISO- JK [D-

Isomer) mer) J/K VS. L-Isomer]

Alanine......... 8 0.7-2.2 0.09-0.27 0.27Serine .......... 240 3-12 0.05 -

Cysteine .......... 1200 80 0.07 -

a-NH2-n-buty-ric acid.... 50 55-100 1.1-2.0 1.2

Threonine ...... 15,000 2250-3200 0.20 -

Norvaline.... 500 7,500 15 -

Valine...... 500 >15,000 >30 >8.3

a-NH2-isobuty-ric acid....... 120 140 1.2 -

cent inhibition occurring at a germination rateof about 82. The agreement of the binding con-stants in these two cases confirms experimentallythat the comparison of binding constants calcu-lated from two different types of experiments isa valid comparison.

Several things are obvious from the data pre-sented in table 3. First, the compound whichbinds best to the spore is D-alanine; L-alanineand D-serine are next in that order. Second, theeffect on binding of adding additional carbons tothe CH3 portion of the molecule (leaving serineand threonine out of consideration for the mo-ment) is far more drastic when the additions aremade to the D-amino acids than to the L-aminoacids. This effect can also be seen in the finalcolumn of table 3.

Turning now to the data on serine and threo-nine and considering only the D-isomers, we seethat D-alanine, the smallest molecule givinginhibition (except for glycine, which does not con-tain a CH3 group) binds the best; serine, some-what larger, binds next best; cysteine and a-amino-n-butyric acid, somewhat larger again,next best; threonine, still larger, next best, andso on. In the case of the D-amino acids, then,binding seems to be mainly a function of thesize of the group substituted for the CH3 group.However, the L-isomers of serine, cysteine, andthreonine are not in accord with an analogouspattern in terms of the L-amino acids. Apparentlythe binding of the L-forms of serine, cysteine, and

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WOESE, MOROWITZ, AND HUTCHISON

threonine is such that the small amount ofelectronegativity centered about the oxygen orsulfur atoms is able to affect the binding drasti-cally, although it cannot do so for the D-forms.The fact that the spore can distinguish the

D- and L-isomers of alanine argues that theremust be at least a three point specificity involvedin binding the alanine molecule. It is obviousthat one point of specificity is shown in connec-tion with the CH3 group and another with theNH2 group (as acetylated D, L-alanine is inactive).A third point of specificity is connected with theH group; otherwise the spore would confusea-amino-isobutyric acid with both L-alanine andD-alanine. (Since D-alanine binds better thandoes L-alanine, a-amino-isobutyric acid wouldact primarily as an inhibitor; however, it initiates,but less than N4o as well as does L-alanine.Therefore, specific restrictions on the H groupof the alanine molecule exist.) There are alsocharge, if not steric restrictions on the COOHgroup. Consequently, all the groups about thecentral carbon of the alanine molecule are speci-fied to some degree as to net charge, the posses-sion of polar groups, or steric factors; four pointspecificity is, then, involved.

It is important to determine which of thegroups on the L-alanine molecule possibly bindat the same "points" on the spore site as do thecorresponding groups on the D-alanine molecule.The fact that L-alloisoleucine does not bindwhereas L-isoleucine does means that there aresome very specific requirements as to the CH3portion of the molecule. This high degree ofspecificity manifests itself in connection with thefour isomers of threonine; D- and L-threonine (aswell as a mixture of the twvo) are both inhibitors,whereas D- and L-allothreonine are both inactivecompounds (because D,L-allothreonine is inac-tive). Therefore, the same high degree of speci-ficity is shown for the CH3 portion of the mole-cule whether the D- or L-form is involved. Thiscan mean either that two of the four attachment''points" on the spore site have nearlv identicalspecificities, or that the CH3 portion of themolecule fits into the same position whether themolecule is in the D- or L-configuration. Thelatter alternative seems a likely possibility.

SUMMARY

The initiation of spore germination by L-alanine and analogues of L-alanine and the in-hibition of this germination by D-alanine and itsanalogues have been studied. Each compoundwhich is active in the germination system can becharacterized by a binding constant; each initi-ator can be further characterized by the maxi-mum rate of germination attainable. These datashow that any theory of L-alanine action mustaccount for the following facts: first, that thebinding of D-alanine is stronger than that ofL-alanine. Second, that the restrictions onmodifications of the CH3 group of D-alanine aregreater than those on modifications of the CH3group of L-alanine. Third, the addition of anelectronegative element to the CH3 portion ofthe L-alanine molecule has a drastic effect onboth binding of the molecule and upon the maxi-mum germinationi rate, whereas such an addi-tion to the CH3 group of D-alanine has little ef-fect other than acting to decrease binding mainlyin proportion to the increase in size it produces.Fourth, it is likely that the CH3 group of theL-alanine molecule fits into the spore site at thesame "point" as does the CH3 group of theD-alanine molecule.

REFERENCESCOHN, E. AND EDSALL, J. 1943 Proteins, amnino

acids, and peptides. Reinhold PublishingCorp., New York.

HACHISUKA, Y., ASANO, N., KATO, N., KAJIMA,Al., KITAORI, M., AND KUNO, T. 1955 Stud-ies on spore germination. I. Effect of nitrogensource on spore germination. J. Bacteriol.,69, 399-406.

HALVORSON, H. AND CHURCH, B. 1957 Biochem-istry of spores of aerobic bacilli with specialreference to germination. Bacteriol. Revs.,21, 112-131.

HYATT, M. AND LEVINSON, H. 1957 Sulfur re-quirement for postgermination developmentof Bacillus inegaterium spores. J. Bacteriol.,74, 87-93.

WOESE, C. AND NIOROWITZ, H. 1958 Kinetics ofthe release of dipicolinic acid from spores ofBacillus subtilis. J. Bacteriol., 76, 81-83.

WYNNE, E. S., convener. 1957 Symposium onbacterial spore germination. Bacteriol.Revs., 21, 259-262.

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