Germination ofBacillus cereus endospores: a proposed role for heat shock and nucleosides'

ALLAN A. YOUSTEN Biology Deporttnet~t, Virginia Polytecht~ic Institrite crnd State University, Blacksbrrrg, Virgit~ili 24061

Accepted April 10.1975

YOUSTEN, A. A. 1975. Germination ofBocill~rs cereus endospores: aproposed role for heat shock and nucleosides. Can. J . Microbiol. 21: 1192-1 197.

Spores of Bcrcilltrs cererrs T prepared in a glucose - yeast extract - salts broth germinated in L-alanine or more rapidly in L-alanine plus inosine or adenosine. The nucleosides alone were not germinative. lnosine was shown to produce no pregerminative changes in spores that prepared them for more rapid germination later in L-alanine. Experiments which measured the interaction of nucleosides, heat shock, and D-alanine on germination rates suggested that nucleosides may potentiate L-alanine-induced germination by causing discrimination against D-alanine at the L-alanine binding site(s) in the spore. D-Alanine is germinative when used with inosine probably because of L-alanine formation by alanine racemase. Heat shock, a prerequisite to D-alanine plus inosine-induced germination, may facilitate entry of inosine into the spore in amounts needed to discriminate against D-alanine.

YOUSTEN, A. A. 1975. GerminationofB~:cilllts cererrs endospores: aproposed role for heat shock and nucleosides. Can. J . Microbiol. 21: 1192-1197.

Des spores de Bacillrrs cererrs T , preparees dans un bouillon de glucose - extrait de levure - sels, germent dans L-alanine, mais plus rapidement dans L-alanine en prksence d'inosine ou d'adenosine. Les nucleosides employks seuls n'induisent pas lagermination. L'inosine ne semble pas produire des changements prkgerminatifs chez les spores ainsi traitees pour une germination plus rapide dans L-alanine. Des expkriences conduites pour mesurer I'effet des nuclCosides, d'un choc de chaleuret de D-alanine sur les taux de germination suggkrent que les nuclCosides peuvent accroitre le potentiel inductifde L-alanine en causant une discrimination B I'encontre de D-alanine au niveau du (ou des) site(s) L-alanine dans la spore. En combinaison avec I'inosine, D-alanine induit la germination probablement i cause de la formation de L-alanine par I'alanine ractmase. Un choc de chaleur, un pllrequis a la germination induite par D-alanine en presence d'inosine, peut faciliter I'entrke de I'inosine dans la spore en quantitk nkcessaire pourcauser une discrimina- tion B I'encontre de D-alanine. [Traduit par le journal]

Introduction A large number of metabolizable and non-

metabolizable compounds and several enzymatic and physical treatments are known to initiate germination of bacterial endospores. Among the metabolizable germinants, L-alanine is probably effective for the largest number of species and this compound and glucose have received the most attention. Despite many years of work, the mechanism of action of these compounds or their metabolites is still unknown. The study of ger- mination has often been complicated by the effect of growth conditions on the germination proper- ties of spores (9) and by possible interactions between germinants and inhibitors. In this study the interactions among the common germinant L-alanine, the germination potentiators inosine and adenosine, and the inhibitor of L-alanine- induced germination D-alanine have been ex-

'Received October 29, 1974.

amined in an effort to clarify the manner in which these materials bring about germination of Bacillus cereus T spores.

Materials and Methods Preparation of Spores

Spores of B, cereus T (culture obtained from R. S. Hanson, University of Wisconsin) were prepared by grow- ing the organism at 30C on a New Brunswick G25 gy- ratory shaker (200 rpm) for 20 h. The broth contained (4, w/v) 0.2 glucose, 0.2 (NH,),SO,, 0.2 yeast extract, 0.1 K,HPO,, 0.02 MgSO,, 0.008 CaC12. 2H20, and 0.005 MnS04 . H,O. Glucose, MgSO,, CaCl, .2H20, and MnS0, . H 2 0 were added separately as concentrated, sterile solutions. The free spores were harvested by centri- fugation, washed 10 X with sterile distilled water, lyo- philized, and stored at - 20C. Aged spores were prepared as described above but were held 6 months in aqueous suspension at 4C rather than being lyophilized.

Activation and Germination Lyophilized spores were suspended in 0.08 M tris-

(hydroxymethyl)aminomethane (Tris) buffer (pH 8.0) to an absorbance (440 nm) of 0.4W.45. Unless otherwise

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YOUSTEN: GERMINATION O F BACILLUS CEREUS 1193

indicated, the suspension was heat shocked at 65C for 120 min. Germination was initiated a t 22C by the addi- tion of compounds at the final concentrations indicated in the text. The fall in 440-nm absorbance was recorded continuously on a Coleman model 165 recorder attached to a Hitachi-Perkin Elmer 139 spectrophotometer. The germination rates reported are the maximum rates of absorbance decrease found under the particular germina- tion conditions and were repeatable through several trials. An indication of no germination means that no absorb- ance decrease occurred in 30 min. Points shown in Fig. 1 are values taken from the recorder tracings. Spores grown in the medium used in these experiments exhibited a lag in germination when exposed to germinants. The time of this lag was usually shorter for spores germinating at a high rate.

Alartine Racen~ase Assay A suspension of lyophilized spores was made in 0.08 M

Tris (pH 8.0) and where indicated, heat shocked at 65C for 120 min. The assay mixture contained 1.0 ml of spore suspension, 0.3 ml of L-alanine (60 mM), and Tris to 1.5 ml. A volume of 0.1 ml of nucleoside (1 mM) or D-cycloserine (20 mM) was added where indicated. The mixture was incubated for 2 min at 37C, chilled in an ice bath, and immediately centrifuged. The D-alanine formed was determined by oxidation with D-amino acid oxidase in a Gilford respirometer at 30C. Each cup contained 1.0 ml of the racemase reaction mixture, 0.1 ml of cata- lase, 0.1 ml of D-amino acid oxidase (0.85 units of a crystalline hog kidney preparation, Sigma Chemical Co., St. Louis, Missouri), and 0.8 ml of 0.08 M Tris (pH 8.0). Oxygen uptake was measured for 120 min by which time uptake had ceased. The D-amino acid oxidase was specific for D-alanine and the L-alanine used as substrate for the racemase contained no oxidizable D-alanine.

L-Alanine, D-alanine, inosine, adenosine, D-cycloserine, Tris buffer. mamino acid oxidasc (crystalline hog kidney preparation), and crude beef liver catalase were obtained from Sigma Chemical Co., St. Louis, Missouri.

Results EfSects of Heat Shock and Aging

To establish that the batch of spores to be used in this investigation responded typically to the activation effects of heat shock and aging (5 ) , lyophilized spores and spores aged 6 months at 4C in aqueous suspension were germinated with and without heat shock (65C, 120min). A relatively high level of L-alanine (50 mM) was used because preliminary experiments showed a very sluggish germination response at lower levels. Heat-shocked, lyophilized spores ger- minated more rapidly than unheated spores; however, the aged spores germinated at a higher rate than lyophilized spores whether heated or not (Fig. 1). Heating of aged spores retarded their germination rate slightly. These results indicated

FIG. 1. Effect of heat shock and inosine on germination of lyophilized and aged B. cereus T spores. (A) @, unheated, lyophilized spores plus 50 mM L-alanine; 0, heated, lyophilized spores plus 50mM L-alanine; A, unheated, agsd spores plus 50 mM L-alanine; A, heated aged spores plus 50 m M L-alanine. (B) @, unheated lyophilized spores plus 50 mM L-alanine and 1 mM inosine; 0, heated, lyophilized spores plus 50 mM L-alanine and 1 mM inosine.

that the spores to be used in later experiments responded typically to heat shock and aging, i.e. both of these treatments enhanced the rate of germination.

Inosine and Adenosine as Potentiators of Ger- mination

Heat-shocked, lyophilized spores did not germinate when supplied with 1.0 mM of either inosine or adenosine alone. The addition of 1.0 mM inosine to unheated spores germinated with 50 mM L-alanine enhanced their germina- tion rate considerably (from 0.014/min to 0.0581 min, see Fig. lA, B). Heat shock was not a prerequisite to the functioning of inosine when L-alanine was the germinant. However, heat shock further increased the germination rate in spores germinated with 50 mM L-alanine plus 1.0 mM inosine from 0.058/min to 0.085/min (Fig. 1B).

T o determine the minimum levels of inosine and adenosine required to produce an effect on L-alanine-induced germination, the nucleosides were diluted and used with 50 mM L-alanine to germinate heat-shocked, lyophilized spores. For both inosine and adenosine an increase in ger- mination rate was seen at a level of 1.0 pM. The rates increased as the levels were increased to 1.0 mM though the increase found when the levels were raised from 0.1 mM to 1.0 mM was small.

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1194 CAN. J. MICROBIOL. VOL. 21, 1975

Although 50 mM L-alanine was required to capable of germinating in inosine alone, found produce rapid germination when used alone, that D-alanine had no effect on germination much smaller amounts could be used when ino- induced by L-alanine plus inosine. When D-al- sine or adenosine was present. However, even anine was tested for its effect on L-alanine and L- with 1.0 mM inosine or adenosine, germination alanine plus nucleoside-induced germination of rates decreased as the level of L-alanine fell the spores used in these experiments, the results below 50 mM (Table 1). Inosine allowed ger- recorded in Table 3 were obtained. D-Alanine mination with a lower level of L-alanine than did inhibit L-alanine plus inosine-induced ger- did adenosine, a finding in confirmation of the mination but was more effective against ger- results of Powell and Hunter (6). mination caused by L-alanine plus adenosine. It

The mechanism by which nucleosides potenti- ate L-alanine-induced germination is unknown. The possibilities most often considered involve the nucleoside entering the spore and being metabolized, perhaps to produce some preger- minative structural modification, or being bound to an enzyme as an allosteric effector. In either case it might be possible to administer the non- germinative nucleoside before the alanine, re- move the unbound excess, and then germinate the spore with alanine alone at a rate character- istic of alanine pIus nucleoside. This was tested by incubating a heat-shocked spore suspension with 1.0 mM inosine for 5 min, removing the excess nucleoside by centrifugation and two washes with water, and germinating the spores by the addition of 50 mM L-alanine. The ger- mination rates in Table 2 reveal that spores pretreated with inosine germinated at a rate equal to spores which had not encountered inosine.

Eflect of Nucleosides on D-Alanine Inhibition of Germination

D-Alanine is an effective inhibitor of L-alanine- induced germination (3). Warren and Gould (9), using B. cereus T spores prepared in a supple- mented nutrient broth, i.e. spores which were

TABLE 1

Germination rates of B. cereris T spores in res- ponse to different levels of L-alanine and fixed

levels of inosine o r adenosine"

Concn. of Germination rate L-alanine,

mM 1 mM inosine 1 mM adenosine

OLyophilized spores were resuspended in O.OR M Tris (pH 8.0) and heat-shocked at 65C, 120 min.

was completely effective againit L-alanine alone. Although D-alanine is a completely effective

inhibitor of L-alanine-induced germination and a partially effective inhibitor of L-alanine plus inosine or adenosine-induced germination, it is itself an effective germinant when added with inosine to heat-shocked spores (Table 4). A requirement for heat shock for D-alanine plus inosine-induced germination was demonstrated and the lack of heat shock caused D-alanine to be far more inhibitory to L-alanine plus inosine- induced germination (Table 4). Unlike inosine, adenosine was not germinative when used with D-alanine and, in fact, it severely inhibited D-alanine plus inosine-germination. Adenosine inhibition of inosine-induced germination was also observed with B. cereus T spores prepared in nutrient broth (9), i.e. spores which could ger- minate in inosine alone. Adenosine did not, however, inhibit L-alanine plus inosine germina- tion. Aging of the spores did not allow adenosine to become germinative when used with D-alanine nor did it relax the inhibition caused by adeno- sine of D-alanine plus inosine-induced germina- tion (Table 4).

The time required to achieve adequate heat shock at 65C for D-alanine plus inosine ger- mination is re~orted in Table 5.

It seemed &ely that alanine racemase in the spores (1, 8) was involved in allowing germina- tion by D-alanine plus inosine, i.e. the production of L-alanine from D-alanine was actua!ly res- ponsible for germination. This concept was sup- ported by Jones and Gould (4), who found inhibition of D-alanine plus nucleoside germina- tion by 0-carbamyl-D-serine. It is further sup- ported by the observation (Table 6) that the alanine racemase inhibitor D-cycloserine inhibited D-alanine plus inosine-induced germination by 57% whereas the addition of L-alanine to the germination mixture reduced inhibition to about 14%. The effectiveness of D-cycloserine on alanine racemase was confirmed by direct assay of the

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YOUSTEN: GERMINATION O F BACILLUS CEREUS 1195

TABLE 2

Effect of introducing and removing inosine from B. cereus T spores before germination with L-alanine"

Treatment Germinantb Germination rate

1. Spores L-Alanine 0.046 2. Spores, washed 2 x w/HzO,

resuspended in p H 8 Tris L-Alanine 0.043 3. Spores, washed 2 x w/HzO,

resuspended in p H 8 Tris L-Alanine + inosine 0.081 4. Spores, incubated 5 min

wlinosine, washed 2 x w/HzO, resuspended in p H 8 Tris L-Alanine 0.045

'Lyophilized spores were resuspended in 0.08 M Tris (pH 8.0) and heat-shocked at 6512, 120 min. Spores were then ( 1 ) used for germination; (2 and 3) sedimented by centrifugation washed 2 x with water, and resuspended in 0.08 M Tris, pH 8.0, for germination; (4) incubated 5 min kith 1 mM inosine sedimented by centrifugation, washed 2 x with water, and resuspended in 0.08 M Tris, p H 8.0, fo; germination.

*Germinant concentrations were 50 m M L-alanine. I mM inosine.

TABLE 3

Effect of D-alanine on germination of B. cererrs T spores induced by L-alanine and nucleosides"

Germination Germinantb rate

L-Alanine 0.031 L-Alanine + D-alanine 0 L-Alanine + inosine 0.081 L-Alanine + inosine + D-alanine 0.047 L-Alanine + adenosine 0.081 L-Alanine + adenosine + D-alanine 0.007

'Lyophilized spores were resuspended in 0.08 M Tris (pH 8.0) and heat-shocked at 65C, 120 min.

*Germinant concentrations were 50 m M L-alanine, 50 m M D-alan- ine, 1 mM inosine. 1 m M adenosine.

enzyme. The addition of 20 mM D-cycloserine to a suspension of heat-shocked spores reduced the number of micromoles of alanine racemized per milligram of spores per 2 minutes from 1.01 to 0.62. The absolute requirement of heat shock for germination by D-alanine plus inosine might be explained by heat activation of alanine race- mase. However, when the racemase was assayed in heated and unheated spores, both caused racemization of 1.38 pmol of alanine per milli- gram of spores per 2 minutes.

It was possible that inosine aided germination by D-alanine by activating alanine racemase and that adenosine inhibited D-alanine plus inosine- induced germination by inhibiting the enzyme. The data in Table 7 do not support these possi- bilities. Neither inosine nor adenosine markedly stimulated the enzyme and the presence of adenosine with inosine was not inhibitory.

Discussion The effectiveness of nucleosides and heat shock

in enhancing the rate of spore germination was clearly seen in these experiments. In neither case, however, is the mechanism by which these factors affect germination understood.

The inhibitory effect of D-cycloserine (Table 6) and 0-carbamyl-D-serine (4) supports the con- cept that D-alanine functions as a germinant by virtue of conversion to L-alanine by alanine racemase. It is clear, however, that the mere formation of some L-alanine from D-alanine is not sufficient for germination since the residual D-alanine is an effective inhibitor of L-alanine- induced germination. The competitive nature (10) of D-alanine inhibition of L-alanine-induced germination suggests the existence of one or more specific binding sites for alanine in the spore. The results presented in this report showed that when inosine was present along with D- and L-alanine in the germination mixture, D-alanine inhibition was considerably lessened. One explanation for the effect of inosine might be that it inhibited the entry of D-alanine into the spore. This seems unlikely since inosine potentiated L-alanine germination in the absence of added D-alanine. Furthermore, for D-alanine itself to be germinative it must enter the spore and encounter the heat-stable, particle-bound alanine racemase (7). Inosine stimulation or inhibition of the racemase itself was not ob- served (Table 7). It seems clear from the data in Table 2 that inosine alone did not bring about a pregerminative structural change in spores that

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CAN. J . MICROBIOL. VOL. 21, 1975

TABLE 4

Germination of B. cereus T spores in response to combinations of D- and L-alanine, nucleosides, and heat shocka

Spore treatment Germinantb Germination rate

Heat-shocked D-Alanine 0 Heat-shocked D-Alanine + inosine 0.053 Unheated D-Alanine + inosine 0 Heat-shocked L-Alanine + inosine 0.066 Heat-shocked L-Alanine + D-alanine + inosine 0.041 Unheated L-Alanine + D-alanine + inosine 0.004 Heat-shocked D-Alanine + adenosine 0 Heat-shocked D-Alanine + inosine + adenosine 0.001 Heat-shocked L-Alanine + adenosine 0.066 Heat-shocked L-Alanine + adenosine + inosine 0.066 Heat-shocked, aged D-Alanine + inosine 0.029 Heat-shocked, aged D-Alanine + adenosine 0 Heat-shocked, aged D-Alanine + inosine + adenosine 0.003

'Lyophilized spores were resuspended in 0.08 M Tris (pH 8.0). If heat-shocked, this was done at 6SC. 120 min.

bGerminant concentrations were 50 mM D-alanine, 50 mM L-alanine, 1 m M inosine, 1 m M adenosine.

TABLE 5 binding since the enzyme or structural binding Time required to achieve adequate heat shock sites are unknown.

for germination of B. cereus T spores b y Although adenosine potentiated germination D-alanine plus inosinea by L-alanine to about the same extent as inosine,

Heat at 65C, Germination rate with 50 mM min D-alanine + 1 mM inosine

OLyophilized spores were resuspended in 0.08 M Tris (pH 8.0) and heat-shocked at 65C for the indicated times.

prepared them for more rapid germination with L-alanine. In addition, neither inosine nor possible metabolic by-products active in ger- mination were bound in the spore in a form that simple washing could not remove.

A possible role for inosine that is supported by these results is that it exerts its effect by causing discrimination against D-alanine at the L-alanine binding site(s). This effect would potentiate the germination potential of L-alanine in racemase-containing spores by preventing D-alanine interference with L-alanine binding. In addition this effect would allow germination by L-alanine produced from D-alanine when the latter was added to racemase-containing spores. Unfortunately, it is not possible at present to measure directly the effect of inosine on L-alanine-

i; was completely ineffective when used with D-alanine and inhibited germination initiated by D-alanine plus inosine. It is possible that adeno- sine is only effective in potentiating L-alanine- induced germination at a high ratio of L- to D-alanine. This ratio was present when L-alanine was the sole added germinant and as seen in Table 1, adenosine was as effective as inosine. When small amounts of L-alanine are used (Table 1) or when the ratio of L-alanine to D-alanine is low because the L-alanine is formed from D-alanine by racemase activity, adenosine would be less effective as was observed. The inhibition by adenosine of germination initiated by D-alanine plus inosine may be caused by competition between the less effective adenosine and-the more effective inosine for a common binding site. The failure of adenosine to inhibit L-alanine plus inosine germination may simply mean that either nucleoside is effective at a high L- to D-alanine ratio.

Heat shock was important in determining the effect of D-alanine on germination when D-

alanine plus inosine was the germinant combina- tion or when D-alanine was used with L-alanine plus inosine. In the first case heat shock was required for germination and in the second case heat shock lessened the inhibitory effect of D-alanine (Table 4). Both effects were probably

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YOUSTEN: GERMINATION OF BACILLUS CEREUS

TABLE 6

Effect of D-cycloserine on germinationa

Germinantb D - C y ~ l ~ ~ e r i n e ' Germination rate -

D-Alanine + inosine - 0.037 D-Alanine + inosine + 0.016 D-Alanine + inosine + L-alanine - 0.051 D-Alanine + inosine + L-alanine + 0.044

aLyophilized spores were resuspended in 0.08 M Tris (pH 8.0) and heat-shocked at 65C, 120 min. bGerminant concentrations were 50 mM D-alanine, 50 mM L-alanine, I mM inosine. CGerminant concentrations were 20 m M D-cycloserine.

TABLE 7 shock and postulated that denaturation or release Effect of inosine and adenosine on alanine racemase of coat components during heating might facili-

tate germinant entry. Micromoles L-alanine

converted to Acknowledgment D-alaninelmg

Nucleoside spores per 2 minutes The technical assistance of Alice Kite is gratefully acknowledged.

None 1.28 4 mM inosine 1.49 I . CHURCH, B. D., H. HALVORSON, and H. 0.

1 mM inosine 1.57 HALVORSON. 1954. Studies on spore germination: its

4 m M adenosine 1 .36 Independence from alanine racemase activity. J . Bac-

1 m M adenosine 1.14 teriol. 68: 393-399.

1 mM inosine + 1 m M adenosine 1.39 2. HASHIMOTO, T. , and S. F. CONTI. 1971. Ultrastruc- tural changes associated with activation and germina- tion of Bncilllts cererts T spores. J . Bacteriol. 105: 361-368. the of the same phenomenon but the 3 . HILLS, G. M. 1949. Chemical factors in the germina-

mechanism involved is The heat effect t ~ o n of spore-bearing aerobes. Effect of amino acids on did not seem to involve alanine racemase the eermination of Brrcilllrs n t ~ t l ~ r a c i s . with some ob- although this enzyme was probably involved in germination by D-alanine plus inosine. Since heat shock does not affect the inhibition of L-alanine germination by D-alanine when only those two compo~lnds are involved, it can be concluded that heat shock and inosine effects are interrelated. It seems possible that heat may act by facilitating the entry of inosine to the site of its activity in the spore. When large amounts of D-alanine are present because of its direct addition to the spore suspension, relatively large amounts of inosine may be needed to prevent its successful competition with L-alanine. Improving the entry of inosine into the spore would accom- plish this. On the other hand, when small amounts of D-alanine are involved as would arise from racemase activity, sufficient inosine to enhance germination may penetrate the spore without heat shock (see Fig. 1). Hashimoto and Conti (2) studied the effect of heat shock on physical structure of spore coats. They showed the production of striations in the coat by heat

servations on the relation of optical fo;m to biological activity. Biochem. J. 45: 363-370.

4. JONES, A,, and G. W. GOULD. 1968. Stimulation of germination of bacterial spores by analogues of D-alanine. J . Gen. Microbiol. 53: 383-394.

5. KEYNAN, A,, and 2. EVENCHIK. 1969. Activation. In The bacterial spore. Edited by G. W. Gould and A. Hurst. Academic Press Inc., New York. pp. 359-396.

6. POWELL, J. F., and J . R. HUNTER. 1955. Spore germi- nation in the genus Bncillrrs: the modification of ger- mination requirements as a result of preheating. J. Gen. Microbiol. 13: 59-67.

7. STEWART, B. T., and H . 0. HALVORSON. 1954. Na- ture and location of particle bound alanine racemase. Arch. Biochem. Biophys. 49: 168-178.

8. STEWART, B. T., and H. 0. HALVORSON. 1953. Studies on the spores of aerobic bacteria. I. The oc- currence of alanine racemase. J . Bacteriol. 65: 160-166.

9. WARREN, S. C., and G. W. GOULD. 1968. Bacillrts cereus spore germination: absolute requirement for an amino acid. Biochim. Biophys. Acta, 170: 341-350.

10. WATABE, K., T . NISHIHARA, and M. KONDO. 1974. Biochemical studies on germination of bacterial spores. 11. The inhibitory mechanism of D-alanine on L-alanine-induced germination of Bucill~ts thi- nminolytic~ts. Jap. J . Microbiol. 18: 181-184.

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