THE JOURNAL OF BIOLOGICAL CNE~~TRY Vol. 247, So. 6, Issue of March 25, pp. 1861-1868, 1972

Printed in U.S.A.

Germination of Bacterial Spores by Calcium Chelates of

Dipicolinic Acid Analogues

(Received for publication, June 28, 1971)

JAMES C. LEWIS

From the Western Regional Research Laboratory, Agricultural Research Service, United States Department of Agriculture, Berkeley, California 94710

SUMMARY

Calcium salts of analogues of dipicolinic acid were sub- stituted for calcium dipicolinate in tests of germination of spores of Bacillus megaferium ATCC 10778. Only 4H- pyran-2,6-dicarboxylic acid was as active as dipicolinic acid. 3-Methyldipicolinic acid and 4-methyl-4H-pyran-2, B-dicar- boxylic acid were active but the germination proceeded less rapidly. In the presence of a threshold concentration (0.020 M) of calcium dipicolinate, calcium salts of pyrimidine- 2,4-dicarboxylate, pyrazine-2,6-dicarboxylate, 4-hydroxydi- picolinate, and furan- ,5-dicarboxylate also showed activity. A hypothesis is proposed for mobilization of native calcium dipicolinate of dormant spores during germination, by way of a dimerization like that exhibited in crystals of calcium dipicolinate trihydrate and the isostructural calcium pyrandi- carboxylate trihydrate.

The compounds 4-bis(2-hydroxyethyl)iminopyridine-2,6- dicarboxylic acid and ethyl ester, 3-methyl-2,6-distyryl- pyridine, 3-methylpyridine-2,6-dicarboxylic acid, and pyrimi- dine-2,4-dicarboxylic acid are reported for the first time. Calcium and proton association constants are given for various ligands.

A bewildering variety of chemicals induce germination of bacterial spores. Some tend to be specific for certain strains whereas others are active on broad classes of spores. Among the latter calcium dipicolinate is of particular interest because both moieties of the chelate compound occur in substantial, roughly stoichiometric amounts in dormant spores and are liberated early in the course of germination. A more detailed knowledge of CaDPA*-induced germination may aid in under- standing the role of Ca2+ and DPA2- in the development, maintenance, and release of dormancy in the bacterial spore. The available knowledge has been summarized recently (1).

Until this study no structural analogue had been found to substitute for DPA in CaDPA-induced germination. 4H- Pyran-2,6-dicarboxylate was found to do so (a), and also to

1 The abbreviations used are: CaDPA, calcium dipicolinate; DPA, dipicolinic acid; PDC, 4H-pyran-2,6-dicarboxylic acid; CaPDC, calcium 4H-pyran-2,6-dicarboxylic acid; NMR, nuclear magnetic resonance.

replace exogenous DPA for sporulation of a DPA-less mutant of BaciZZus megaterium (3). This paper details the complete effectiveness of CaPDC in simulating CaDPA-induced germi- nation, and the lesser effectiveness of other closely related analogues. The structures of these analogues have been il- lustrated (3). The requirements for activity are discussed in terms of association constants, steric factors, electroneutrality, and solubility. The occurrence of a strong dimeric association in the crystalline trihydrates of CaDPA (4) and CaPDC (5) may be significant.

EXPERIMENTAL PROCEDURE

Spores-Most of the experiments were carried out with lyophilized spores of B. megatekm ATCC 10778 (NRRL B-938) from a lot grown and harvested as described previously (6), and stored at 5”. On the morning of use a weighed portion was dispersed (7) in water and kept at room temperature. The spores were not heat-activated.

Cation-stripped spores (H-spores) were prepared from a similar lot of B. megaterium by exposure for 8 hours at 25” and pH 3.2 maintained by automated addition of HNOB (8). The spores were washed thoroughly and lyophilized. Ca2f- loaded spores (Ca-spores) were prepared by exposure of un- modified spores to 0.02 M calcium acetate at pH 5.7 for 17 hours at 50”, washing, and lyophilizing (8).

This strain of B. megaterium has the prominent equatorial ridge described by Rode (9) for type GN. I am indebted to W. H. Ward for scanning electron micrographs. The DPA content is 10.2% (10).

iMeasurements-Absorbance at 600 nm was recorded with a Cary2 model 14 spectrophotometer for l-cm cells held at 25.0”. An automatic changer permitted sequential observation of 13 cells with a 2-min cycle.3 Continuous observation of critical periods was achieved manually.

Spore suspension, buffer, neutralized (Na+) ligand solution, and water were mixed in a cell, and the chelate formed by addition of a stoichiometric amount of CaCl2 (usually) solution to give a volume of 3.5 ml. The mixture was stirred with bubbling air and inserted in the spectrophotometer chamber. Recording started 20 s after addition of CaC12. The germina-

2 Reference to a company or product name does not imply ap- proval or recommendation of the product by the United States Department of Agriculture to the exclusion of others that may be suitable.

3 I am indebted to Glen Bailey for design of the cell changer.

1861

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1862 Spore Germination by Calcium Chelates Vol. 247, No. G

\

0.020 M CaDPA

0.060 M CaDPA

MAX SLOPE4 \

\ \ \

I I rn A^ I ” L”

MINUTES

FIG. 1. Effect of CaDPA concentration on germination re- sponse. Values of the parameters ti,,tz and max slope are 6.0 and 0.103 for 0.040 M CaDPA, and 5.0 and 0.107 for 0.060 M CaDPA.

tion suspension ordinarily contained 0.10 mg of spores per ml which gave an initial absorbance of 0.8. The usual control contained 0.040 M CaDPA, 0.010 JI Tris (Cl-, pH 8.05), and 0.080 M NaCl.

Although recording continued for 4 to 5 hours, the record became unreliable earlier in many cases through crystallization on the faces of the cells. For this reason parameters based on the ratio of final to initial absorbance (11) could not be used. After a lag, absorbance decreases as the spore cortex lyses and the refractive index of the residual body decreases (1). It sufficed to estimate graphically the steepest slope of the nor- malized sigmoidal absorbance to t)ime curve

max slope (min-I) = (AA/A~),,,/A;,~~~,~

and the elapsed period between addition of CaClz to the spore suspension and the inflection of the sigmoidal curve (Fig. 1; tintI). pH and phase-microscopic observations were made after about 5 hours. The pH usually was within 0.1 of the initial buffer value. Dormant spores are phase-bright and germinated spores are phase-dark (1).

Association Constants-All determinations were made at 25.0”. Thermodynamic acidity constants (pKT) were computed for titrations performed as recommended by Albert and Serjeant (12) by means of a Fortran program using activity coefficients (13). In a few cases titrations were performed also in 0.070 M KCl, and computed for ionic strength (cl> 0.1.

Ca2f associations were determined in the standard germina- tion ion solution (0.080 M NaCl, 0.010 M Tris, 0.005 M HCI) by use of the Orion model 92-20 calcium electrode, 9041 reference, and 801 pH meter. The logarithm of the concentration con- stant for p = 0.1 (pK:i) was computed from Ca2+ concentra- tions obtained by calibration of the electrode response in the standard germination solution. The total concentration of ligand was about 0.01 M and the ratio of total calcium to ligand varied from 1 to 0.75 to 0.5 as permitt#ed by the range of the calcium electrode. Small corrections of measured pCa and of

the association constants to p = 0.1 were obtained by use of the Davies formula (14) for activity coefficients. For those cases in which pKb,’ varied significantly with calcium to ligand ratio, constants for binding of the second as well as the initial ligand molecule were computed by successive approximations.

With DPA and ligands of similar Ca2+ binding strength the Orion electrode method failed because of inability to determine low Ca2f concentrations, although the binding strengths could be ordered. In these cases calcium constants were computed from pH titrations performed at calcium to ligand ratios of 0.5 and 1.0 in the presence of 0.070 M KCl. Here also the constants were computed for p = 0.1 by use of the Davies formula.

Chemicals-The following chemicals were obtained: from hl- drich, 2,3,6-collidine, dipicolinic acid, dipicolinic acid N-oxide, 6-hydroxy-2,4-dimethylpyrimidine, 2-methylquinoxaline, mu& acid, pyridine-2,4- and -3,5-dicarboxylic acids, sodium imino- diacetate; K and K, chelidamic acid, chelidonic acid, dilactic acid, 2-hydroxyethyliminodiacetic acid ; Eastman, 2,4,6-col- lidine, diglycolic acid, isophthalic acid; Calbiochem, Tris and thiodiglycolic acid.

Pyridine-3,5-dicarboxylic acid was recrystallized from 95”/; ethanol after carbon treatment. White crystals of ii-hydroxy- pyridine-2,6-dicarboxylic acid (chelidamic acid) monohydrate were obtained after carbon treatment of a 0.4% solution in hot, water. The dipicolinic acid was of high purity (10).

N-(2,3-Dihydroxypropyl)iminodiacetic acid was a gift of Farbenfabriken I3ayer AG.

The remaining analogues were synthesized. I am indebted to L. IM. White and A. L. Potter for carbon, hydrogen, nitrogen, and calcium analyses on materials dried in vacuum at 100“ ex- cept as shown and to R. E. Lundin for NMR evaluation. The preparation of 4H-pyran-2,6-dicarboxylic acid4 has been de- scribed (3). I am indebted to R. M. Seifert for this and other preparations.

S-Methylpyricline-d, 6-dicarboxylic Acid-2,3,6-Collidine was oxidized via the distyryl derivative (15) to avoid unwatlted isomers. For 5 days 24.2 g of 2,3,6-collidine (0.20 mole1 + 42.4 g of benzaldehyde (0.40 mole) + 60 g of acetic anhydride (0.6 mole) were refluxed. The mixture was distilled in vacuum to a thick black tar, which was dissolved in hot 95?$ ethanol and refrigerated. Recrystallization from hot 955; ethanol after carbon treatment gave 24 g (4Oc/ yield) of pale yellow 3-methyl- 2,6-distyrylpyridine; m.p. 106’; C 89.0, H 6.40, N 4.555; (C22H19N requires 88.85, 6.44, 4.71ch) ; NMR confirmed the monomet.hyl substitution. This compound and 3-methylpyri- dine-2,6-dicarboxylic acid have not been reported previously.

The methyldistyrylpyridine, 17.8 g (0.060 mole), was dissolved in 250 ml of acetone, the temperature lowered to j-10”, and 50.6 g (0.32 mole, the stoichiometric ratio) of powdered KhIrlOd added incrementally over a 7-hour period to the well stirred suspension. The acetone was filtered off, the Mn02 precipitate extracted with 400 ml of hot 95tid ethanol (which removes much of the potassium benzoate), and then extracted with 400 ml of boiling water three times. The water extracts were passed through polystyrene sulfonate resin (200 ml of Donex 50-H+), and the effluent was dried on the steam bath and purified by vacuum sublimation. At 0.5 mm the residual benzoic acid sub- limed at 50-70”; 3-methylpyridine-2,6-dicarboxylic acid at 115- 130”. The product melted at 183”; NMR gave a ratio of 2 ring to 3 methyl protons; C 53.3, 11 3.94, S 7.369; (CsHiNO4 requires

4 R. M. Seifcrt preparation.

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Issue of March 25, 1072 J. C. Lewis 1863

53.0, 3.89, 7.73%). The yield from methyldistyrylpyridinc was 61:;. The calcium chelate after vacuum drying at 80” gave 16.5’; calcium (CaCsH&04.HZ0 requires 16.890/,).

4-ll~cthylpyridine-d,6-dicarboxylic Acid4--The method of Koe- nigs and Jaeschke (16) was followed. 4-Chloropyridine-2,6- dicarboxyethyl ester was prepared from chelidamic acid with PC&,; m.p. 90-91” (literature 92-94” corrected), 80% yield. This tras converted to 4-bis(carbethoxy)methylpyridine-2,6-dicar- boxy rthyl ester by reaction with sodium malonic ester; m.p. 74-75” (literature 70-72” corrected), 43% yield. The tetraester ~L~OIL hydrolytic decarboxylation gave the desired 4-methyl- DPA; m.p. 247-248 decomposition (literature 245” decomposi- tion); C 52.9, H 3.879; (CSI-I~NO~ requires 53.0, 3.900!); NMR satisfactory. The calcium chclatc: gave 17.7% calcium (CaCs- H&Ol requires 18.29;).

Pyridine-2,4,6&icarboxylic Acid4 --The acid was prepared l'rorn 2,4,6-collidine (0.10 mole) by oxidation with stoichiomet- ric K&In04 (0.60 mole) in wat,er at 100”. The monopotassium salt crystallized from t,hta water extjr:tcts upon concentration and acidification to pH 3. Recrystallized material was used to pre- I)arc: t,he triethyl ester; m.p. 127” (lit,erature 127.5O) ; NMR &owcd 2 ring H per 3 ethyl groups. The free acid was crystal- lized from 1.5 N HCl.

4-(Moropyridine-2,6-dicarbozylic Acid-This compound was l)rc:pnred from recrystallized chelidamic acid with phenylphos- phallic dichloride (17). The yield was 40%; m.p. 210-211” (lit- eraturc 208.5“); NMR showed the single type of ring proton.

4-A Gnopyridine-S,6-dicarboxylic AcidP-4-Chloro-2,6-dicar- hoxylic acid was treated with aqueous NH,OH at 150” (1.8) ; m.p. 29%~-302” (literature 299” corrected).

/,-Bis(2-hydroxyethyl)iminopyridinc-d,6-dicarboxylic Acid-4- Gh toropyridine-2,6-dicarbnxylic acid was heated gradually over a I -hour period to 140” with a 4 M ratio of redistilled diethanola- rninc~. :2fter extraction with acetone, precipitation from the kJWW phase with 959; cthallol gave an impure product melting at I!11 -192.5". The dicthyl ester crystallized twice from acetone- r:ther gave m.p. 135-138.5” and a satisfactory NMR pattern. The csalcium chelate, recrystallized from 2500 parts of hot water, gave ?; 8.53, Ca 12..57; (CuC11T-112N206.Hz0 8.60, 12.287;). l’hcsc compounds have Ilot, been reported previously.

.G-I-I-/,-i~ethylpyran-d,6-di~arDoxljlic Acid-This analogue was prcl)ared by a schema I)arall(>l t,o t,hat used for 4%pyrarl-2,6- dicarboxylic acid (3). Control of spontaneous heating in the rcactinn of aqueous acetaldchydc with oxaloacetic ester in the viscous mixture is difficult, unlike the reaction with formalde- hydr, and crystallizat,ion of ovcrhcatctl reaction mixture may not mew c‘ven after seeding.

Fine white needles of the final I)roduct, melt with decomposition at ahnut 264” (literature 260”). They sllhlime slowly at 140” and 10 p. The sublimed product, gave C .52.1, H 4.41’3, (CgHsOs rcquircs 52.17, 4.389;). l’hc calcium chelate gave 16.5yfi cal- cium (CaC~H~O~ ‘Hz0 requires IG.fiX~~), a,nd the crystals were stable in dry air unlike CaI’lX.

Pgrimidine-9, ..&dicarhoxylic A cid- -6-J Jydroxy-2,4-dimethyl- pyrimidine was converted to the R-chloro compound in 50°! yield by use of POCl, (19); t,hcnce Ilear quantitatively to 2,4-d& methplpyrimidine by tlehalogcnatinn in water with 112 (1 atm), palladium on carbon, and excess MgO at, 25” (20). After ex- t,raction with CHCla and removal of CHCla and Hz0 by distilla- tiorl t,hrough a Vigreaux column with a low vacuum the crude productj (v”,” 1.4878; literature I .48X0) was convcrtcd (507;

yield) to 2,4-distyrylpyrimidine by refluxing with benzaldehyde and ZnClz (21) ; m.p. 147” (literature 145-146”) ; C 84.4, H 5.51, N 9.875& (C20H16N2 requires 84.5, 5.63, 9.86%). The mixture was held at 140” for 2 hours to effect monostyrylation of the dimethylpyrimidine (b.p. 146”); then at 160” for 10 hours to complete the conversion to distyrylpyrimidine. This compound in acetone at 5-10” was oxidized with KMn04 (stoichiometric: 5.33 M ratio) added dropwise over a 5-hour period. The acetone solubles were discarded; the MnOn extracted with warm water; K+ removed by batchwise addition of Dowex 50-H+; the precipi- tated benzoic and pyrimidine dicarboxylic acids and warm water washes were extracted with ether to remove benzoic acid. A 65% yield of pyrimidine-2,4-dicarboxylic acid was obtained by chilling a 0.8c/, solution of the solids. The fine white crystals gave C 43.1, H 2.42, N 16.6Y0,; CeH4N204 requires 42.9, 2.40, and 16.68%i. The compound has not been reported previously.

Pyrazinetricarboxylic Acid-2-Methylquinoxaline was oxidized in vigorously agitated aqueous 0.5% KOH at 90-95” by drop- wise addition of the stoichiometric 75 M ratio of KMnOa over a 3-hour period. The solubles and hot water extract of the MnOz were passed through Dowex 50-H+ after preliminary batch treatment to remove COS. Pyrazinetricarboxylic acid dihydrate was recovered from the concentrated washings after carbon treatment in 850/, yield (22, 23). It should be noted that Jones et al. (24) obtained a 71y0 yield of 2-methylpyrazine-5,6-dicar- boxylic acid under conditions similar except that KOH was not added. The dried crystals gave C 39.7, II 1.99, N 13.3% ; C7H4N206 requires 39.6, 1.90, 13.2%.

Pyrazine-2,6-dicarboxylic Acid-The tricarboxylic acid is decarboxylated preferentially at position 3 in hot water (23). Autoclaving a 2% solution at 122” for 15 hours gave complete conversion, but 8 hours did not, nor did 72 hours of refluxing at atmospheric pressure. The yield was 58a/,; the loss was largely due to complete decarboxylation. After drying the crystals gave C 42.8, H 2.39, N 16.5%; CsH4NzOd requires 42.9, 2.40, 16.66%.

Furan-2,5-dicarboxylic Acid (Dehydromucic Acid)-This acid was prepared in 209;, yield by the dehydrative cyclization of mucic acid in 48’9; HBr at 140” for 20 hours (25). The NMR spectrum and neutralization equivalent were satisfactory.

RESULTS

General Conditions for 1 tlduction of Germination of Spores of B. megaterium 1077s by CaDPA-The germination treatment dis- covered by Riemann and Ordal (26) involves mixing solutions of NazDPA and CaCb with a spore suspension buffered by 0.01 M Tris, Cl-, pH 7 or 8, to give a 0.040 M highly supersaturated solution of CaDP-1. It 25’ in the presence of 0.08 M NaCl (formed by the above procedure), the solubility of CaDPA is 0.011 M. Nevertheless, the 0.04 M solutions of CaDPA usually did not crystallize for several hours nor overnight on occasion. Preliminary trials confirmed the susceptibility of the chosen strain and lot of spores and the suitability of the Riemann-Ordal conditions. The titration capacity of the dormant spores (0.7 meq per g for pH 4.0 to 9.5 (6)) corresponds to less than 10e4 N for the 0.1 mg of spores per ml used. Omission of buffer gave less reproducible results.

The shape of the absorbance to time curves, the nature of the parameters tinfl and max slope, and the effect of concentration of CaDPA arc shown in Fig. 1. The curves are sharply sig- moidal; 0.04 hI ~-a:: required for maximal effect, and 0.020 M gave

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TASLE I

by CaDFAa

TABLE II

Sernin neqateri

Variable cont. tinfl

hi min -

8.1

7.5-8.9

0.020,0.020

0.030,0.030 12.0

0.040,0.040 a.0

0.050,0.050 7.0

0.060.0.060 7.0

0.080,0.080 6.0

0.060,0.040

0.060,0.060 8.8

0.120,0.080 10.5

0.24 14.8

0.60 la.5

2.4 25

11.4

1.0 12.4

30% v/v 10.0

a.0 11 .o

6.0 20.5

10% v/v 14.0

1 O,% ” 11.0

30% * 21.0

10% w/v 10.2

10% ” 11.2

46.6% w/w 65

60.3% - 210

0.6% w 9.5

“The spore suspensions contained 0.01 E Trlr and (except as noted) 0.040 y CaDPA and 0.01

max slope

tieplaceabi I ity

Ligand

‘r ,d salts

max slope

contra I idea” (10 expts)

min-’

0.078

.072-0.093

no gerrr’n

0.047

0.075

o.oeo

0.065

0.062

no germ”n

0.034

0.017

0.022

0.010

0.013

0.050

0.038

3FA (H-spores)

PJC ”

DPA (Ca-spores)

FSC II

II

1,

Ca Iir -

tinfl

mfn

8.5

15.5

5.5

8.6 10.0

” I excess Na2PDC

3-Methyl-UPA

II

4-Methyl-3PA

4-Methyl-PDC

II

contra I b

Urea

Preexposed 1 hour at 250 in ethanol

” in urea

at 650 ”

Ethano I

Glycol

II

0.061

0.035

0.017

0.032

0.067

0.022

0.057

0.065

0.016

0.0035

0.064

:I-, pH 8.0) ! NaCI.

DPA, FDC

1, , 3-methyl-DPA

41 II

1, I,

DFA, 4-methyl-PDC

II II

DPA, pyrimidine- dicarboxylate

,I II

n II

DPA. pyrazirie- dicarboxylate

11 11

II ”

DPA, 4-hydroxy-DPAb

II ,I

DPA, furan- dicarboxylate

II II

11 II

3-Methyl-DFA, 4-methyl-PDC

II n 3-Methyl-DPA, pyraz-

tnedicarboxylate

0.040,0.040

0.040

0.060

0.080

0.040

0.040

0.060

0.060

0.020,0.020

0.020,0.020

0.020,0.040

0.040,0.020

0.020,0.020

0.020,0.040

11 .o

7.8

7.7

50

26.5

12.0

9.6

11 .o

6.5

5.8

5.2

8.6

6.6

min -1

0.044

0.032

0.060

0.046

0.040

no germ’n

0.043

0.057

0.047

0.004a

0.026

0.046

0.055

0.052

0.080

0.080

0.092

O.ObO

0.092

0.020,0.020 45 0.008

0.020,0.040 27 0.018

0.020,0.080 18.4 0.026

Glucose

Sucrose

11

GI ycerol

Methoce I

0.020,0.020 67 0.0046

0.020,0.040 36 0.012

0.020,0.060 21 0.023

0.020,0.040 51 0.009

0.020,0.060 25 0.020

.020,0.02-.O’i 60-l 1C 0.003-0.004

.020,0.06-,12 50-37 0.014-0.021

.020,0.16-.24 28-21 0.026-0.033

bThls and the test suspensions given below were at PH 8.3- 8.4, somewhat above the optimum.

little or no response. Partial germination sometimes was seen the following day. The range of effective concentrations ex- tended to 0.08 M but solutions with 0.06 and 0.08 M CaDPA began to crystallize within 15 mm. An excess of Na*DPA is inhibitory (Table I), even when the concentration of CaDPA re- mains in the optimal range. Inhibition has been attributed to Ca(DPA)k (26). Computation of ion concentrations suggests that an excess of DPA2- (>O.OOl M) or a deficiency of Ca*+ ( <O.OOl M) might contribute to the inhibition as well as an excess of Ca(DPA)t- (>0.015 M). DPA*- promotes release of spore cortex-lysing enzyme from disrupted spores (27).

The period and temperature (2, 4, and 20 hours at 4, 25, and 40”) of pre-exposure of the spore suspension to each of the re- agents (CaC&, Na2DPA, and Tris (Cl-, pH 8) buffer) and the order of addition were varied for both unmodified and Ca-spores.

0.020,0.020

0.040,0.040

0.020,0.020

16.0

8.3

0.042

0.064

very slow Nut corn0 lete

aHalf of the spores were phase-dark at 6 and 85% at 24 hr.

bpH suboptimal (6.89,6.36 through partial Ionization of the 4-hydroxyl upon assoc ation of the Iigand with Ca2+.

At most a moderate increase of the lag period or slowing of the lysis, or both, was observed with the longer periods or the es- treme temperatures.

Ionic strength and the nature of the ions in the germination mixture are important. A low ionic strength is inhibitory. A germination mixture free of NaCl was obtained bv dissolvina

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CaDPA. 3Hz0 crystals~ in hot water and filtering through a 0.8-p 0.08 M CaClz in excess of that required to form CaDPA was membrane filter to inhibit crystallization. A cooled 0.040 M slightly stimulatory; 0.36 M was slightly and 1.16 M CaClt was

CaDPA solution gave a slow partial (-30% at 7 hours) germi- completely inhibitory. At high concentrations divalent cations nation. The presence of 0.010 M Tris, Cl-, increased the speed of were more inhibitory than monovalent cations. The orders phase-darkening only moderately but -75% germinated. The were Mg 2+ > Sr2f > Ca2f (0.5 M) and K+ = Rbf > Cs+ > Na+ presence of 0.08 M NaCl plus 0.01 M Tris, Cl-, gave rapid and > Li+ (1.0 M).

complete germination. The pH was 7.7 in these three cases. Viscous solutions of permeating (28) solutes (sucrose and glyc- The concentration of NaCl (0.08 M) that arises from the reac- erol; -10 x the viscosity of water) slowed but did not stop the

tion of NazDPA with CaClz under the standard condition is germination. Nonpenetrating Methocel was without effect. nearly optimal; even 2.4 M NaCl gave complete and fairly rapid Germination did not proceed in 2.0, 4.0, or 8.0 M urea, in 1.5 or germination (Table I). NH&l or NaN03 replace NaCl as does 3.0 M guanidinium chloride, or in 30% ethanol. However, 1.0 Tris (0.080 M;Tris’ + 0.080 M Tris+, Cl-). M urea, or 10% ethanol, glycol, sucrose, or glucose had little effect.

TABLE III

Proton and Ca2+ association constants (logarithmic) for dipicolinic acid analoguesa

I pKa I PC

LI gand t EXDerlmenta I. Literature Expt’ I

Olplcol inate

4-Pmlnodlplcol inate

4-Chlofodipicol inate

4-Hydroxydipicollnate

3-Methyldipicol inate

4-Methyldipicolinate

pyrldine-2,4-dlcarboxylate

pyridine-3,5-dlcarboxylate

Pyrldlne-2,3,6-tricarboxylate

Pyridlne-2,4,6-tricarboxyiate

Dlpicol lnate N-oxide

4H-Pyran-2,6-dicarboxylate

4-Methyl-4H-pyran-2,6- dlcarboxylate

4-Pyrone-2,6-dicarboxylate

Pyrlmidine-2,4-dicarboxylate

Pyrazine-2,6-dicarboxylate

Pyrazinetricarboxylate

Phthalate

Isophtha late

Furan-2,5-dicarboxylate

aiglycolate

Thtodiglycolate

)J=o

5.11,2.42

jA = 0.1 F JJ as noted

4.7,2.2 (0.1)

9.19.1.8 (o.l)e

3.75,1.7 (O.lle

10.9,3.1,1.4(0.1

u = 0.1

4.63,1 .98 4.61,2.71b

1

Lit.

/J = 0.1

4.6.2.6

5.2ae

3.61e

5.6 (1.9)’

5.36,2.15

5.67,2.10

5.19,2.30

4.64,2.00

5.25,3.30,1.9

5.06,3.12,2.4

4.64,2.02

4.05,2.85

4.00,2.78

2.32,1.88

3.43,2.14

3.53,2.35

4.21,2.76,<<2

5.46,3.02

4.72,3.63

3.57,2.41

4.34,3.02

4.52,3.36

4.86,1.67

4.58,2.88,1.6

4.37,2.71,2.2

5.17,2.17 toIf

4.62,2.72 (Olf

5.41,2.95 (0) h

4.7,3.7 (0)

3.55,2.60 d

4.37,2.97 (0) j

4.56,3.24 (O)j

4.08.2.23

&.3,3d

insoluble

0.88

=5

6,’

3.27.1.95

3.15,1.94

1.51

3.68.2.23

3.39.2.19

3.67,1.70

1.59

0.93

0.88

3.41,1.67

1.02

2.1

2.4 (0)

2.0 (0)

3.4

1.4

‘The constants are corrected to 0 and 0.1 p for Ii+ and to 0.1 for Ca2+ by use of the Davies (14) formula. The rounded Ilterature values are from Sillen and Martell (44); others as noted.

bBy PH titration. CFor formation of Ca(ligand)- and CaH(ligand), respectively; see text and (29).

d Lack of mareriai precluded an accurate determination. eReference 45. fReference 46.

‘The assoclatlon with Ca2+ appeared to be more complex than just Ca(ligand)- and Ca(llgand)z-.

hRererence 47. iReference 4e. jReference 49.

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Spore Germination by Calcium Chelates Vol. 247, No. 6

Pre-exposure (followed by washing) of the spores for 1 hour at 25” to 30% ethanol had no effect; with 8 M urea at 25” lysis was slowed moderately, and at 65’ germination was delayed and lysis slowed markedly but all spores became phase-dark. Exposure to a 50% solution of LiBr for many days had no effect.

Non-heat-activated spores germinated slowly but almost com- pletely in 0.5% tryptone + 0.25% glucose + 0.01 M Tris (Cl-, pH 7.4); tinfl25 min, max slope 0.017 min-I. Part of the spores responded slowly to 0.020 M glucose + 0.020 M KN03 at pH 5.2, and none at pH 7.2. They did not respond to 0.001 M L-alanine + 0.001 M inosine at pH 7.1 or to 0.1% pentanol-1 at pH 6.1 or 7.1. They responded at pH 7.1 to 0.0001 M n-dodecylamine (tinfl 3;. min, max slope 0.022 mine’) but the spores remained phase-bright and AA was about two-thirds that obtained under good lytic conditions (about 55%).

Replacement of DPA by Other Ligands-The action of DPA in CaDPB-like germination was simulated best by PDC. As with DPA, addition of 0.040 M excess NazPDC prevented ger- mination (Table II). 0.04 M BaPDC did not crystallize for 25 min whereas BaDPA crystallized at once. The spores remained phase-bright and absorbance unchanged, demonstrating that Ca2+ is not replaceable by Ba2+.

Three other analogues gave CaDPA-like germination, al- t,hough much more slowly t’han 0.040 M CaDPA. The most active is 3-methyl-DPA (Table II). The response with 0.060 and 0.080 M solutions was only slightly slower than that with 0.040 IM CaDPA. Crystallization from the 0.080 M solution was delayed for many hours. Ca-4-methyl-PDC was less active than Ca-3-methyl-DPA. No crystallization occurred. Ca-4- methyl-DPA gave slow phase-darkening of part of the spores. A 0.040 M solution crystallized at 90 min; a 0.080 M solution at once. Ca2+ salts of other analogues did not induce germination by themselves. Some crystallized too quickly for evaluation (0.04 M 4-amino-DPA and the tricarboxylates). With others crystallization was delayed sufficiently that they were judged to be inert (0.08 M 4-hydroxy-DPA, and 0.04 M 4-chloro-DPA, DPA N-oxide, pyridine-2,4-dicarboxylate, 4-pyronedicarbox- ylat’e) . 0.04 M Ca-pyrazinedicarboxylate crystallized after 7 min and induced no germination. The more soluble Ca2+ salts were tested at 0.040 M (thiodiglycolate and the iminodiacetates) or a range of concentrations up to 0.08 M (diglycolate, pyrimi- dinedicarboxylate), 0.12 1% (dilactate, isophthalate), or 0.16 M

(furandicarboxylate, pyridine-3,5-dicarboxylate). Addition of 0.040 M excess CaCl2 failed t’o overcome the inac-

tivity of 0.040 M solutions of Cal-hydroxy-DPA, Ca-pyridine- 3,5-dicarboxylate, Ca-isophthalate, Ca-diglycolate, Ca-dilac- tate, or Ca-iminodiacetate.

Combinations of ligands were tested since a marginal activity might be expressed in the presence of 0.020 M CaDPA, and since chelates that crystallized too quickly at 0.040 M could be tested at lower concentrations. 0.020 M CaPDC + 0.020 M CaDPA was fully active (Table II), as might be expected from the ac- tivity of 0.040 M CaPDC or CaDPA. Combinations of Ca-3- methyl-DPA and Ca-4-methyl-PDC with each other and with CaDPA had the expected activity. In the presence of 0.020 M

CaDPa4, Capyrimidinedicarboxylate and Ca-pyrazinedicarbox- ylate were active although 0.080 and 0.060 M, respectively, were subopt,imal. Less activity was found with Cal-hydroxy-DPA and Ca-furandicarboxylate. No activity was found with Ca- diglycolate. Slight stimulations observed with high concentra-

tions of other (weak) chelators is consistent with a slight effect from CaC&.

Germination by 0.040 M CaDPA was inhibited by Na2 ligands strong enough to compete with DP,42- for Ca2f. Thus, addition of 0.040 M diglycolate or hydroxyethyliminodiacetate (log pK:i 3.4 and 4.7) resulted in no phase-darkening. Isophthalate, pyridine-3,5-dicarboxylate, or 0.08 M acetate (log pK:i = 0.9) had little effect. 4-Pyronedicarboxylate (log pKk,’ 1.5) slowed germination markedly (tin*1 12.4, max slope 0.018). Addition of Ca-diglycolate or Ca-pyridine-3,5-dicarboxylate had little effect. The lack of effect of supplemental calcium chelate is also shown by the broad range of effective CaDPA concentration.

Association Constants-Constants for the equilibrium

Na2 ligand + CaClz $ Ca ligand + 2 NaCl

and for binding of a second ligand

Naz ligand + Ca ligand + Ca(ligand$ + 2 Na+

where appropriate, are given in Table III together with proton constants. Values have not been reported before for half of the compounds.

Ionization of t’he hydroxyl of 4-hydroxy-DPA and equilibra- tion with the pyridinone tautomer has been described (29) by 3 proton constants and 2 calcium constants (for CaH ligand and Ca ligand-). The Ca2+ of the anionic complex is bound much more strongly than the Ca2f of the uncharged complex so that a 1:l mixture of Cazf and 4-hydroxy-DPA appears to give a pro- ton dissociation at about pH 7.4 (29).

DISCUSSION

The following evidence (1) indicates that DPA is chelated with divalent metal ions (mainly Ca2+) in dormant spores: (a) approximately equal molar contents of divalent metals and DPA, (b) ultraviolet and infrared spectra characteristic of che- lated DPA, (c) electron spin resonance characteristic of chelated Mn(II), and (d) inability to isolate significant amounts of cova- lently bonded DPA. Various functions related to resistance and dormancy have been suggested (1) for CaDPA: (a) associa- tion with or a masking of individual sensitive macromolecules to achieve resistance or repression of activity, and (b) a struc- ture-stabilizing role that achieves resistance and dormancy- endowing functions in a less direct manner. With both hypothe- ses the problem remains of providing for rapid mobilization and excretion of CaDPA during germination. The properties of CaDPA involved in this phase of natural germination may also be necessary for CaDPA-like germination by analogues of CaDPA.

One may distinguish some chelator, DPA, and DPA analogue- induced germinations from CaDPA-like germination. Some uncomplexed chelating agents germinate spores of the putrefac- tive anaerobe 3679 (30). Spores of Bacillus stearothermophilus M are germinated by 0.004 M Na2DPA, with a sharp optimum near pH 5.5 (31). Ca2+ and other divalent cations inhibit the germination; EDTA is ineffective. The partial germination of heat-activated spores of B. megaterium Texas in the presence of 0.020 M CaC12 and 1c4 M L-alanine is accelerated markedly by 0.020 M NazDPA (32). Pyridine - 2 - carbinol - 6 - carboxylate, pyridine monocarboxylates, DL-pipecolate, and other less closely related compounds are less active. The other pyridinedicar- boxylates are inactive. Omission of Ca2f or other divalent metal ions greatly reduces the activity.

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Issue of March 25, 1972 J. C. Lewis 1867

Herein the concept of a “CaDPA-like germination” is re- stricted as follows: (a) Ca*+ plus ligand at about 0.04 M is active, (b) excess ligand anion is inhibitory, (c) an intermediate range of ionic strength is necessary, (d) Sr2+ substitutes for Ca2f, al- though a higher concentration may be required (33), (e) heat- activation or other prior treatment of the spores is unnecessary, (f) a wide variety of spores respond. Riemann and Ordal found three Clostridium and five Bacillus species to be germinated; however, Bacillus mycoides germinated only at a low tempera- ture, Bacillus subtilis and Bacillus coagulans gave incomplete ger- mination, and the response of several species was dependent upon cultural conditions (30). Spores of Sarcina ureae are germinated (38). Unresponsive mutants have been found for some strains of B. megaterium with spores that respond to CaDPA (34, 35), and responsive mutant spores have been produced for an unre- sponsive strain (3). Exposure to 0.040 M CaDPA at 7” gives an acid reversible activation of spores of B. megaterium for subse- quent spontaneous lysis at a higher temperature in the absence of CaDPA and enzyme inhibitors (36). Non-pretreated spores of a DPA-less mutant B. megaterium formed with exogenous DPA lyse on addition of CaDPA without a lag phase, unlike the wild type which resists CaDPA germination (3).

Phase-darkening and AA reflect complex processes (37). The lag and several lytic stages are separably affected by pH, specific ions, and non-ionic chemicals; e.g. at low pH AA is reduced and the spores show a gray central area rather than a uniformly phase- dark appearance. The lag phase with Sarcina ureae is reduced with increasing NaCl concentration (0.080 to 0.28 M), but max slope is little affected (38).

Less specificity of the organic moiety is required for CaDPA- like germination of spores of B. megaterium 10778 than for sporu- lation of a DPA-less mutant of another strain of B. megaterium where only PDC is incorporated into the developing spore in place of exogenous DPA (3). For germination CaPDC is as effective as CaDPA. Ca-3-methyl-DPA and Ca-4-methyl-PDC are active but less effective. Ca-4-methyl-DPA is slightly active and Ca-pyrazine-2,6-dicarboxylate is inactive but the super- saturated solutions crystallize rapidly.

The inactivity of calcium salts of 4-hydroxy-DPA, 4-chloro- DPA, 4-pyrone-2,6-dicarboxylate, pyrimidine-2,4-dicarboxylate, and furan-2,5-dicarboxylate is noteworthy. More CaC12 does not confer activity. Ligand anions inhibit germination by 0.04 hl CaDPA only where its concentration is reduced by competi- tion. The inactivity of Ca-pyridine-3,5-dicarboxylate and of Ca-isophthalate agrees with earlier observation (26, 39).

With threshold CaDPA (0.020 M) activity is shown with cer- tain chelates that are inactive alone even at high concentrations. Pyrimidine-2,4-diearboxylate is most active, followed by pyra- zine-2,6-dicarboxylate, 4-hydroxy-DPA, and furan-2,5-dicar- boxylate. Diglycolate is inactive. Weak Ca2+-binding ligands could not be evaluated since CaC12 gave a slight stimulation with the threshold concentration of CaDPA.

CaDPA germination might be caused by a mechanical ger- mination (40) arising from crystallization of CaDPA, or of a re- lated composition with spore components, from the highly CaDPA-supersaturated spore suspension. The most favorable site might be within the spore, the growing crystal puncturing the spore coat. The activity of the highly soluble CaPDC makes this explanation implausible.

Neither gross steric factors nor CR 2+ binding strength individ- ually or cooperatively control activity; PDC with a substantially

lower association constant (log Kk,’ = 3.3 rirsus 4.6 for DPA) is fully active, whereas 3-methyl-DPA or diglycolate with con- stants almost identical with that of DPA have reduced or no activity. Pyrimidine-2,4-dicarboxylate (log K:i = 3.7) and pyrazine-2,6-dicarboxylate (log Kki = 3.4) with Ca2f binding strengths similar to PDC and geometry idemical with DPA and PDC have marginal activity (with threshold CaDPA). Pyri- dine-3,5-dicarboxylate and isophthalate with steric similarity to DPA but with small association constams have little or no activity.

More is involved than ability to penetrate t,o the site of action. Thus, inactive diglycolate can be looked on as possessing a frag- ment of the active PDC structure. Furan-2,5-dicarboxylate with a 5- instead of a 6-membered ring is inactive by itself, but its calcium association constant is small. Insolubility of Ca-4- carboxy-, Ca-4-amino-, and Ca-4-bis(2- hydroxyethyl)imino- DPA’s prevented evaluation of the effect of charge on activity of these compounds. Ca-4-hydroxy-DPA at pH 6.4, where the hydroxy proton is largely associated, gave marginal activity (with threshold CaPDA).

One must look to more subtle properties of DPA and PDC than those discussed above to account for the effectiveness of these ligands in sporulation (3) and germination, and the inef- fectiveness of other analogues (especially of pyrimidine-2,4- dicarboxylate and pyrazine-2,6-dicarboxylate) . The strong di- merit bond in crystalline CaDPA’3HzO (4) and CaPDC.3Hz0 (5) may indicate a property important in CaDPA-like and pos- sibly in natural germination. il hypothesis is proposed for mobilization of structural CaDPa via formation of a dimeric link- age with exogenous CaDPA and release of the dimers into solu- tion where the equilibrium is so strongly in favor of monomer that dimer is not detected spectrophotometrically (10). Thus,

Spore structure.CaDP-4 + CaDPA (in solution)

TI IL

Spore structure.CaDPA.CaDPA

TI I

Spore structure + CaDP-&.CaDPA tin solution)

Spore structure + 2 CaDP-4 (in solution)

CaDPA may be embedded with spore macromolecules in such a way that formation of dimer is facilitated with coincident weak- ening of bonds to spore structure. The net effect could be to activate a lytic enzyme, expose a structural substrate to lytic attack, alter a permeability, or other possibilities. To explain activity of analogues and st’rontium chelates one postulates formation of mixed dimers between structural CaDPA and the heterologous chelate salt.

The less active SrDPA possesses in crystalline SrDP,4’4Hz0 a polymeric linkage of metal coordination polyhedra with the following pattern (41):

0 0’ 0” y - \J - \sr,,/ \

y \,/ ‘.o,,/ \ Inactive Ca-furan-2,5-dicarboxylatje.3Hz0, Ca-diglycolate~6H~0,

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1868 Spore Germination by Calcium Chelates Vol. 247, No. 6

and Ca-pyridine-3,5-dicarboxylate.H20 do not show dimeriza- 22. SCHAAF, K. H., AND SPOERRI, P. E. (1949) J. Amer. Chem. Sot.

tion tendencies expressed by oxygen doubly-linked metal ions in 71, 2043

the crystals.5 Such structures are observed in inorganic com- 23. MAOER, H. I. X., AND BERENDS, W. (1958) Rec. Trav. Chim.

pounds; P-CazP87 (4% CazcrOGl, and C%Po&I (43). x-ray Pays-Bas 77, 827

crystallography of other organic chelates is under investigation. 24. JONES, R. G., KORNFELD, E. C., AND MCLAUQHLIN, K. c.

(1950) J. Amer. Chem. Sot. 72,3539 Structures of the pyrimidine and pyrazinedicarboxylate calcium 25. PHELPS, I. K., AND HALE, W. J. (1901) Amer. Chem. J. 26,445

salts have not been solved but powder data show that the crys- 26. RIEMANN, H., AND ORDAL, Z. J. (1961) Science 133, 1703

tals are not isostructural with CaDPA.3HzO and CaPDC- 27. GOULD, G., AND KING, W. L. (1969) in L. L. CAMPBELL (Edi-

tor), Spores IV, p. 276, American Society for Microbiology, 3HzO (3). Ann Arbor, Mich.

1.

2. 3.

4.

5.

6.

7. 8.

9. 10. 11. 12.

13.

14.

15.

16. 17. 18.

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James C. LewisAnalogues

Germination of Bacterial Spores by Calcium Chelates of Dipicolinic Acid

1972, 247:1861-1868.J. Biol. Chem. 

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