levels of h+ and other monovalent cations in dormant and

JOURNAL OF BACTERIOLOGY, Oct. 1981, p. 20-290021-9193/81/100020-10$02.00/0

Vol. 148, No. 1

Levels of H+ and Other Monovalent Cations in Dormant andGerminating Spores of Bacillus megaterium

BONNIE MASSEY SWERDLOW, BARBARA SETLOW, AND PETER SETLOW*

Department of Biochemistry, University of Connecticut Health Center, Farmington, Connecticut 06032

Received 6 April 1981/Accepted 25 June 1981

Previous investigators using the extent ofuptake of the weak base methylamineto measure internal pH have shown that the pH in the core region of dormantspores of Bacillus megaterium is 6.3 to 6.5. Elevation of the internal pH of sporesby 1.6 U had no significant effect on their degree of dormancy or their heat orultraviolet light resistance. Surprisingly, the rate of methylamine uptake intodormant spores was slow (time for half-maximal uptake, 2.5 h at 24°C). Most ofthe methylamine taken up by dormant spores was rapidly (time for half-maximaluptake, <3 min) released during spore germination as the internal pH of sporesrose to -7.5. This rise in internal spore pH took place before dipicolinic acidrelease, was not abolished by inhibition of energy metabolism, and during ger-mination at pH 8.0 was accompanied by a decrease in the pH of the germinationmedium. Also accompanying the rise in internal spore pH during germinationwas the release of >80% of the spore K+ and Na+. The K+ was subsequentlyreabsorbed in an energy-dependent process. These data indicate (i) that betweenpH 6.2 and 7.8 internal spore pH has little effect on dormant spore properties, (ii)that there is a strong permeability barrier in dormant spores to movement ofcharged molecules and small uncharged molecules, and (iii) that extremely earlyin spore germination this permeability barrier is breached, allowing rapid releaseof internal monovalent cations (H+, Na+, and K+).

Despite extensive study, several key featuresof sporulation and germination in bacteria arenot understood at the molecular level. In partic-ular, the mechanisms whereby developing sporesattain and maintain metabolic dormancy andheat and radiation resistance, yet lose theseproperties in the first minutes of spore germi-nation, have not been clarified. Various factors,including decreased water content (9), bindingof unique proteins to spore DNA (20), decreasedlevels of free divalent cations (26), and a highlevel of dipicolinic acid (DPA) (9), may be in-volved in the dormancy or resistance or both ofspores. However, the significance and possiblemechanism of action of these various factors arenot understood.

Elevated heat and radiation resistance at oneparticular stage in the life cycle of an organismis seen in an extreme form in bacterial spores.However, many other types of organisms alsohave as part of their life cycle a stage of growthin which rates of metabolism and macromolec-ular synthesis are slowed. These dormant ormetabolically quiescent stages of growth oftenshow no significant increase in heat and radia-tion resistance. Two examples of this are unfer-

tilized sea urchin eggs and dormant fungal spores(7, 10). Strikingly, in both of these systems thedormant or quiescent stage of growth may havea significantly lower intemal pH than the ac-tively growing form (2, 12). Furthermore, in un-fertilized sea urchin eggs, artificial elevation ofthe internal pH results in large increases in therates of endogenous metabolism and macromo-lecular synthesis (R. A. Steinhardt and M. M.Winkler, Fed. Proc. 38:465, 1979). This findingsuggests that a decreased intemal pH may beone cause of the metabolic quiescence of unfer-tilized eggs.

In common with the fungal (yeast) and seaurchin systems, dormant bacterial spores alsohave a pH within the spore core which is sig-nificantly lower (6.3 to 6.5) than that in germi-nated spores (-7.5) (21). This finding suggestedthat a low internal pH might be involved in thedormancy or resistance or both of bacterialspores. Consequently, we undertook furtherstudy of this system to determine (i) whetherthe low internal spore pH contributes to sporedormancy or resistance or both and (ii) how andwhen during spore germination the internal pHrises to the value of -7.5.

20

MONOVALENT CATIONS IN B. MEGATERIUM SPORES

MATERIALS AND METHODSChemicals, reagents, and bacteria. Sources of

labeled compounds were as described previously (21).HCl, double distilled from Vycor, was obtained fromthe G. Frederick Smith Chemical Co., Columbus, Ohio;KNO3 and NaNO3 standard solutions were purchasedfrom Aldrich Chemical Co., Milwaukee, Wis.Most of the work described was carried out using

Bacillus megaterium QMB1551 (originally obtainedfrom H. S. Levinson, U.S. Army Natick Laboratories,Natick, Mass.). Spores of this organism were grown,harvested, and washed as previously described (23).Cleaned spores were stored at 4°C in distilled water ata concentration of -30 mg (dry weight) per ml. Thespores were washed with distilled water every 24 to 48h and were used only while free of germinated sporesor bacterial contamination or both. Only with sporesstored in this manner did we get rates of methylamineuptake which were reproducible from one spore cropto another. Lyophilization and dry storage of sporesoften resulted in spore crops which gave variable re-sults. Spores of Bacillus cereus T (originally obtainedfrom H. 0. Halvorson, Brandeis University, Waltham,Mass.) were also used for a few experiments and wereprepared as described above.Measurements of methylamine incorporation

and internal pH. Measurement of methylamine in-corporation was carried out as described previously(21). Unless otherwise noted, spores (12 to 15 mg [dryweight] per ml) were incubated in 200 mM Tris-hy-drochloride buffer containing 20,uM [14C]methylamineand, in some cases, a small amount of 3H20. At varioustimes, samples (1.0 ml) were centrifuged (30 s) in anEppendorf microcentrifuge, and the supernatant andpellet fractions were processed and counted as de-scribed previously (21). As noted previously (21), therewas no detectable methylamine catabolism during anyexperiments reported in this work. The internal watervolume of dormant or germinated spores was deter-mined with ['4C]sorbitol as described previously ex-cept that 50 ,uM sorbitol was used (21). The internalpH of spores was calculated from the methylamineuptake and the values for the sorbitol-impermeablevolume as described previously (21). Note that allvalues for internal spore pH are only calculated values.Measurements of methylamine or ion release

during germination. Spores (15 to 25 mg/ml) wereheat shocked (15 min, 60°C) in water, cooled in ice,centrifuged (10 min, 15,000 x g), washed with an equalvolume of cold water, and suspended in an equalvolume of cold 200 mM Tris-hydrochloride (pH -8.5at 4°C) unless otherwise noted, and [14C]methylaminewas added. After incubation for 12 to 16 h at 40C, thespores were added to a prewarmed (30°C) vessel con-taining germinants and any other compounds to betested. The final spore concentration during germina-tion was about 10 mg/ml, and the buffer concentrationwas 200 mM in Tris-hydrochloride unless otherwisenoted. Samples were taken as described above, andthe pellet fraction was analyzed for methylamine afterprocessing as described above; the supernatant frac-tion was used for analysis of K+, Na+, and DPA. Incases in which only methylamine release was to beanalyzed, a calibrated amount of NaOH was present

in the prewarmed vessel to ensure that the pH duringgermination in Tris-hydrochloride at 30°C was thesame as during methylamine uptake into dormantspores in Tris-hydrochloride at 4°C.Other methods. K+ and Na+ were measured using

a model 56 atomic absorption spectrometer (The Per-kin-Elmer Corp., Norwalk, Conn.) with an HGA-2200graphite furnace. All dilutions made for these deter-minations used milli-Q-water (Millipore Corp., Bed-ford, Mass.), and all pipettes and tubes used wereplastic and were prewashed with HCI (double distilledfrom Vycor) and then milli-Q-water. Measurement ofK+ and Na+ in dormant spores was carried out onsamples which were washed with 200 mM Tris-hydro-chloride (20 min, 4°C), washed with water, and thendigested with HCI (30 min, 1000C).

All values for Na+ and K+ in germination superna-tant fluid have been corrected for the Na+ or K+contributed by the components of the germinationmedium alone. This correction was small (<4% of totalspore level) for K+, but was more significant (-30% oftotal spore level) for Na+. Because Tris buffers wereused, all pH values were measured at the temperatureof the experiment. Conductivity was measured with aRadiometer conductivity meter. DPA was measuredcolorimetrically (17).

RESULTSExtent and rate of uptake of methyla-

mine by dormant spores. Measurement of theextent of the uptake of the weak base methyla-mine is a simple way for measurement of the pHwithin a membrane which is permeable to un-protonated methylamine but not the protonatedspecies (13, 14); this method has been used suc-cessfully for measurement of the pH within bothcells and organelles (13, 14). With this technique,we calculated that the pH in an internal regionof dormant spores of B. megaterium was 6.2(Table 1), similar to the value obtained previ-ously (21). The rate of uptake of methylamineby dormant spores was quite slow at pH 8.5 to8.9; the time for half-maximal uptake was 2.5and 7 h at 24 and 4°C, respectively (Fig. 1A). Incontrast, the rate of uptake of methylamine bygerminated spores was too fast to measure (timefor half-maximal uptake, <2 min; data notshown) as has been the observation with othercells and organelles tested (13, 14).Most of the methylamine taken up by spores

at 4°C remained in the spores over a long periodof time. In contrast, at 24°C much of the meth-ylamine taken up was slowly lost (Fig. 1A).Because there was neither methylamine catab-olism nor a change in the external pH underthese conditions, there must have been somechange in the spores themselves, presumably aslow increase in their intemal pH. However, thelatter phenomenon was reversible. If spores werepreincubated at pH 8.7 and 24°C for 5 days, they

21VOL. 148, 1981

22 SWERDLOW, SETLOW, AND SETLOW

took up hittle methylamine. However, subse-quent washing with water and incubation at

TABLE 1. Alterations in internalpH ofdornantspores of B. megaterium

Calcu-Treatment lated in-teral

pHUntreateda.... 6.2Incubated with 20 mM (NH4)2SO4 presentb 7.8Incubated with 20 mM (NH4)2SO4 (12 h,400) and then washed.6.4a Spores were incubated at 12 mg/ml in 200 mM

Tris-hydrochloride (pH 8.6), and the internal pH wascalculated from measurements of the extent of incor-poration of [14C]methylamine at 4°C over an 18-hperiod as described in the text.

b Spores were treated as described in footnote a, butwith 20 mM (NH4)2S04 present.

'Spores were incubated for 16 h as in footnote b,but without methylamine. They were then centri-fuged, suspended in cold water for 3 h, and centrifuged;this procedure was repeated three times. The internalpH was then measured as described in footnote a.

0

S

2

240C in water for 4 days resulted in spores whichtook up an amount of methylamine similar tothat of the original spores (Fig. 1A).Methylamine uptake at 40C with added KCI

also was slow, but less methylamine was takenup than in Tris alone, and it was then slowly lost(Fig. 1B). LiCl and NaCl gave similar results(data not shown). It seems likely that the effectof KCI (and presumably other alkali metal ions)was due to a K+-H+ exchange, resulting in ele-vation of internal spore pH, since the KCI effectwas reversible (Fig. 1B). Also, addition of 0.25M KCI to dormant spores incubating at 4°C indilute (10 mM) Tris-hydrochloride (pH 8.7) ledto a sevenfold increase in the rate at which thepH of the medium fell (from 0.01 to 0.07 perday) (data not shown).

Effect of manipulation of the internal pHof spores on spore properties. The findingthat the internal pH (as measured by methyla-mine incorporation) of the spores could changeslowly in response to their environment sug-gested that it might be possible to manipulatethe internal pH in a simpler way. One method

Time in hours

FIG. 1. Uptake of[14C]methylamine by dormant spores of B. megaterium at (A) different temperatures or(B) with and without KCI. (A) Spores (12 mg/ml) were incubated with ['4C]methylamine in 200 mM Tris-hydrochloride at the indicatedpH and temperature and sampled at various times for methylamine uptake.The values for methylamine uptake represent the percentage of total methylamine in the culture which wastaken uip by the spores. The sample represented by A was incubated without methylamine at 240C in Tris (pH8.7) for 54 h..Methylamine was added to a portion of the culture, and its incorporation was measured after 5h at 24°C. The remainder of the culture was centrifuged, incubated in water at 240C, and washed bycentrifugation (once every 24 h for 4 days). The spores were then returned to Tris-hydrochloride atpH 8.7 at24°C, and at the point represented by A methylamine incorporation was measured after 5 h. (B) Spores wereincubated at 40C in Tris-hydrochloride buffer (pH 8.8) with or without 250 mM KCI, and methylamineincorporation was measured. The point represented by A represents spores incubated 104 h in Tris-hydro-chloride plus KCI alone, followed by methylamine addition and measurement of its incorporation after anadditional 14 h. The sample represented by A was treated as was the sample described above, but before themethylamine addition it was washed with water by centrifugation, resuspended in water, and rewashed onceevery 24 h for 170 h. The spores were then suspended in Tris-hydrochloride at 4°C, and methylamineincorporation was measured after 14 h.

J. BACTERIOL.

MONOVALENT CATIONS IN B. MEGATERIUM SPORES 23

for manipulation of internal pH which has beensuccessful in other systems (Steinhardt andWinkler, Fed. Proc. 38:465, 1979) is the use ofhigh levels of a weak base (such as NH3) whichcan enter cells and act as an internal buffer.Indeed, soaking spores in (NH4)2SO4 at pH 8.6greatly reduced methylamine incorporation,raising the calculated internal pH by 1.6 U;again, this phenomenon was reversible (Table1). A similar decrease in methylamine incorpo-ration was seen when spores were soaked in(NH4)2SO4 before methylamine addition (datanot shown) or when (NH4)2SO4 and methyla-mine were added simultaneously (Table 1).However, 10 ,uM (NH4)2SO4, a concentrationgiving a level of weak base equal to that of themethylamine normally used for our measure-ments, had no effect on methylamine uptake(data not shown). Similarly, spores incubatedwith 20 mM methylamine exhibited a very lowmethylamine uptake and an internal pH of -8.0(data not shown).With spores having an elevated internal pH,

we examined several spore properties to deter-mine whether there was some variation withinternal pH. However, spores with calculatedinternal pH's of 6.2 or 7.8 had identical levels ofDPA, showed no detectable utilization of their

100- A ,o B

0w'1 glucose

r00

I /

E 5' 1 t1E. izoEE

EI

E

0 25 ! A

pool of 3-phosphoglyceric acid upon extended(6 days at 400) storage, germinated identicallyas measured by loss of DPA or optical densityof the culture, and had essentially identicalcurves for killing by either a temperature of900C or by UV irradiation (data not shown).Changes in internal pH upon spore ger-

mination. Although the exact value of the in-ternal pH (at least between 6.2 and 7.8) of sporeshad no detectable effect on spore properties, itwas still of interest to learn the time course andmechanism of how the internal pH of sporesrises to 7.5 during spore germination (21). Mostof the methylamine accumulated by dormantspores was released in the first minutes of ger-mination in glucose (Fig. 2A). This rapid releaseof methylamine took place only in spores whichwere germinating; spores without germinants orspores which were not heat shocked showedlittle methylamine or DPA release (Fig. 2A andB). However, rapid methylamine release wasseen with spores germinating in either a meta-bolizable (glucose) or non-metabolizable (KBr)germinant (Fig. 2A and B). The slower rate ofrelease of methylamine during germination inKBr compared with glucose was almost certainlydue to a slower rate of initiation of germinationin KBr as measured either by DPA release (Fig.

Time in minutes

FIG. 2. Release ofmethylamine and DPA during germination of B. megaterium spores (A) with or withoutglucose, (B) in KBr or with no germinants and no heat shock, or (C) with glucose and DCCD. After prior heatshock (unless otherwise noted), 13 mg of spores per ml was incubated in 200 mM Tris-hydrochloride (pH 8.8)plus methylamine for 16 h at 4°C, resulting in uptake of about 60% of the methylamine in the culture. Thespores were then germinated at 30°C, and 10 mg of spores per ml with 100 mM glucose, 50 mM KBr, 100mMglucose plus 1 mM DCCD, or no added germinant as described in the text. The maximum release ofmethylamine in these experiments amounted to more than 90%s of the total originally present in the dormantspore. Symbols: A, methylamine; 0, DPA.

VOL. 148, 1981


2A and B) or the fall in optical density at 600nm of the culture (data not shown). Rapid meth-ylamine release was also seen during germina-tion in glucose plus 3 mM Tris-hydrochloride(pH 8.5) or with B. cereus spores (data notshown). Surprisingly, it appeared that methyla-mine release preceded DPA release by 1 to 3min depending on the experiment (Fig. 2A andB). This was shown even more dramatically bygerminating spores with glucose plus the inhib-itor dicyclohexylcarbodiimide (DCCD) (Fig.2C). Under these conditions, methylaminerelease was relatively unaffected, whereas DPArelease was significantly retarded. Similar re-sults were obtained when 1 mM HgCl2 was usedinstead ofDCCD or when B. cereus spores weregerminated with DCCD (data not shown).One prediction from the experiments noted

above is that before the excretion of DPA byspores germinating at pH values of >6.5 thereshould be a significant release of H+. Indeed,when spores were germinated in dilute buffer(pH 8) with the non-metabolizable glucose ana-log a-methylglucoside (to eliminate generationof acid by catabolism of the germinant), therewas a significant fall in the pH of the mediumbefore a significant DPA release (Fig. 3A andB). This was most obvious when DCCD was alsopresent during germination to block oxidativephosphorylation (Fig. 3B). Similar data werealso obtained for spores germinated in KBr (datanot shown). The rapid fall in the pH of the

7.1 A

73 0-1

75 /6DPA

I ZS S I /A/

medium early in germination was not due tohydrolysis and release of spore cortex fragments,since the latter process follows DPA release (11).

Earlier studies by Vary (29) and Rossignoland Vary (16) also have shown a release ofprotons early in the germination of the samestrain of B. megaterium by constantly recordingthe pH of a germinating culture. The rate of acidrelease was significantly greater with the meta-bolizable germinant glucose than with the non-metabolizable analog a-methylglucoside. How-ever, there was significant acid production dur-ing germination with a-methylglucoside (29).Addition of DCCD to spores germinating inglucose reduced the rate of acid production to~25% of that of spores germinating in a-meth-ylglucoside. However, the values reported forthe rates of acid production during germinationin a-methylglucoside or with DCCD must beconsidered approximate numbers, since a highbackground rate of acid release (almost equal tothe value reported for acid release due to ger-mination in a-methylglucoside) was subtractedfrom all of the data (29). Our conditions differsomewhat from those of Vary in that we used athree- to fourfold-higher spore concentrationand a slightly lower buffer concentration. Con-sequently, the pH changes that we observedwere much higher than those seen by Vary (29)and Rossignol and Vary (16). Comparison of thedata in Fig. 3A and B show that DCCD additiondid indeed decrease the rate of fall in the pH of

B +OCCD

_ l~~~~~~~so@SI

CPA _o.--

I3conductWU

Thuw in minutes

FIG. 3. Release ofDPA and change in mediumpH and conductivity during germination of B. megateriumspores (A) without or (B) with DCCD. Spores were heat shocked, incubated in 200mM Tris-hydrochloride (pH8.5) for 14 h at 4°C, centrifuged, washed three times with cold water, and suspended at 13 mg/ml in cold 4mM Tris-hydrochloride (pH 8.0 at 30°C). The spores were germinated at 10mg/ml by addition to aprewarmed(30°C) flask containing a-methylglucoside (final concentration, 100mM) (A) without or (B) with DCCD (finalconcentration, 1 mM). One-milliliter samples were taken at various times and centrifuged; the supernatantfluid was analyzed for DPA and conductivity, andpH was measured. The relative conductivity of the mediumon our instrument at zero time was 20 pS (,umho).

J. BACTERIOL.

MONOVALENT CATIONS IN B. MEGATERIUM SPORES 25

the medium slightly more than twofold. Al-though it is possible that this may reflect a directeffect of DCCD on acid release, it is more likelythat this reflects only a significant slowing of theearly steps in spore germination by DCCD asevidenced by the much slower DPA release withDCCD (compare Fig. 3A and B) in this experi-ment.Release of other monovalent ions during

germination. During germination of spores ina-methylglucoside with or without DCCD, wenoticed that in addition to the early H+ releasethere was an increase in the conductivity of thegermination medium (Fig. 3A and B). Althoughthe increase in conductivity of the medium dur-ing germination is due to several factors, includ-ing DPA and divalent cation release and excre-tion of metabolic end products (4, 21, 26), sporesgerminating with DCCD in particular showed asignificant increase in the conductivity of themedium before DPA release (Fig. 3B). Becausedivalent cations are released only in parallel withDPA and because detectable metabolism duringgermination begins only after DPA release (8,19, 24), there must then be a significant releaseof ions early in germination. However, it wasimpossible from this experiment to quantitatethis early ion release.

0

a

to

S0

00

cZ

a

0Ea0

00

0

0

0.a

A. No additions

/ *-DPA

/

To test the latter point directly, we measuredlevels of K+ and Na+ in dormant spores and therelease of these ions during germination. Dor-mant spores of B. megaterium contained 880 ,ug/g (dry weight) of K+ and 78 ,ug/g (dry weight) ofNa+, levels which are similar to those reportedby others for the spores of B. megaterium andB. cereus (15, 28, 30). Strikingly, >80% of the K+of the spores was released during germination inglucose, but there was no K+ release when thespores did not germinate (Fig. 4A). K+ releasewas followed by rapid K+ uptake (Fig. 4A); theK+ uptake was presumably energy dependent,because it was abolished by DCCD (Fig. 4B).Under conditions in which K+ reabsorption wasblocked, K+ release clearly preceded DPArelease (Fig. 4B). Greater than 80% of the K+ ofthe spores was also rapidly released when thespores were germinated with glucose in either 2mM Tris-hydrochloride (pH 8.5) or 0.1 M Tris-hydrochloride (pH 7.2) (data not shown). Thekinetics of K+ release during spore germinationwere similar to those of methylamine release(Fig. 5A), and Na+ was also released with kinet-ics similar to those for K+ (Fig. 5B). Greaterthan 85% of the Na+ of the spores was releasedin this process (data not shown). The release ofK+ before DPA release was also seen during

B. iDCCD

/4DPA

10 20

Time in minutes

5 10 20

FIG. 4. Release ofDPA and K+ by B. megaterium spores germinating in glucose (A) without or (B) withDCCD. Spores were incubated in 200mM Tris-hydrochloride (pH 8.7) for 16 h at 4°C after a prior heat'shockunless otherwise noted. The spores were centrifuged, resuspended in an equal volume of cold 200 mM Tris-hydrochloride (pH 8.6 at 4°C), and germinated in 100 mM glucose (A) without or (B) with 1 mM DCCD,samples were taken at various times and centrifuged, and the supernatant fluid was analyzed for DPA andKV. The sample which was not heat shocked was added to aprewarmed (30°C) container without glucose andanalyzed as described above.

VOL. 148, 1981


0aa

0

E

E0

00

+

0D

EU

10

E

a.

z0

I

I

0

3

Ii

00

Tine in minutes

FIG. 5. Excretion ofK+, Na+, methylamine, and DPA during germination ofB. megaterium spores. Spores(13 mg/ml) were heat shocked, washed with water, and incubated at 4°C in 200mM Tris-hydrochloride (pH8.8). After I h, the spores were centrifuged, resuspended in fresh 200 mM Tris-hydrochloride (A) with or (B)without [14C]methylamine, and incubated for an additional 16 h at 4°C. The spores were added to glucose(final concentration, 100 mM) plus DCCD (final concentration, 1 mM) at 30°C as described in the text.Samples of the germinating culture were centrifuged, the supernatant fluid was analyzed for DPA, K+, andNa4., and the pellet fraction was analyzed for methylamine. For the spores containing methylamine, acalibrated amount ofNaOH was added to the germination mix to ensure that thepH was the same at 30°Cas it had been at 4°C. Symbols: 0, DPA; A, K4; E4 methylamine; A, Na4.

germination of B. cereus spores (data notshown); again, >70% of the total spore K+ (780,ug/g [dry weight]) was released followed by K+reabsorption (data not shown).

DISCUSSIONOf obvious importance to the interpretation of

the data presented in this communication is thatour measurements of the extent of methylamineuptake indeed give accurate estimates of theinternal pH of dormant bacterial spores. Al-though the accuracy of this method has beenverified in other systems, one can easily imaginepotential artifacts in our system, especially inview ofthe long incubation times required. How-ever, we believe that our measurements do giveaccurate estimates of the internal pH of thespores for the following reasons. (i) Fragmentsof spore coats, cortex, or membranes absorblittle methylamine (21). (ii) Removal of sporecoats from intact spores by detergent treatmentat high pH alters neither the kinetics nor theextent of methylamine uptake (reference 21 andsee below). These two findings argue stronglyagainst nonspecific methylamine binding tospore layers external to the spore core. (iii) Thepercentage of total methylamine in a culturetaken up by a constant amount of spores at an

exogenous pH of 8.5 was constant with total'methylamine concentrations in the culture from5 to 300 ,uM (B. M. Swerdlow, B. Setlow, and P.Setlow, unpublished data). At 300,M methyla-mine in the culture, this resulted in a methyla-mine concentration in spores of -30 mM, againarguing against a nonspecific binding. (iv) Theconcentration of methylamine calculated to beinside spores varied inversely with the exoge-nous hydrogen ion concentration, at least be-tween pH 7.1 and 8.4 (21). (v) In all cases inwhich spore methylamine levels fell (during ger-mination or in dormant spores with KCI pres-ent), methylamine release was, as predicted, ac-companied by a release of protons and presum-ably an increase in internal pH. Findings iii to vabove are most consistent with the uptake ofmethylamine by dormant spores being driven bya pH gradient. In addition to the findings di-rectly related to methylamine uptake by spores,there is also one independent measurement ofinternal spore pH which is in good agreementwith our calculated values. Thus, the internalpH of dormant B. subtilis spores determined ina preliminary study with 31P nuclear magneticresonance was 6.3 (2), essentially identical to thevalues that we determined for B. cereus and B.megaterium spores (21). A more difficult ques-

J. BACTERIOL.


tion to answer is whether the long incubationused for the measurements for dormant sporesalters them in some way. However, we do knowthat their germination properties are not af-fected by the incubation. Similarly, the stabilityof spore methylamine levels for 40 to 50 h at 40Cargues that any changes which do take place arevery slow. It seems unlikely that large amountsof Tris enter spores during our routine 16- to 20-h incubation at 40C in Tris, since this wouldhave resulted in elevation of spore pH to valuescomparable to those generated by (NH4)2SO4.Similarly, preliminary analysis of the incubationsupernatants revealed that <10% of the DPA,K+, Na+, or Pi of the spores was released duringa 16- to 20-h incubation at 40C (B. M. Swerdlowand P. Setlow, unpublished data). It is of coursealmost impossible to rule out subtle changes inspores during our measuring periods or thatfactors other than internal pH may play a partialrole in spore methylamine uptake. However, wefeel that the findings given above strongly sug-gest that the spores are not significantly alteredduring our measuring period and that the greatmajority of the methylamine uptake of spores atpH values from 7.5 to 8.8 is in response to a pHgradient.Given the conclusions noted above, a third

significant conclusion from work in this report isthat the internal pH within dormant bacterialspores can vary significantly. Thus, soakingspores in (NH4)2SO4 elevated the internal pH by1.6 U. Similarly, we interpret the methylamineexcretion during extended incubation of dor-mant spores with KCI as due to a rise in theinternal pH, possibly caused by a K+-H+ ex-change reaction. It is well documented that bac-terial spores exhibit reversible K+-H+ exchange(as well as H+ exchange with other cations)when more drastic procedures are used (1; R. E.Marquis, E. L. Carstensen, G. R. Bender, and S.Z. Child, 8th Int. Spores Conf., abstr. no. 80,1980). Thus, soaking spores at low pH can resultin exchange of spore cations for H+ with littlechange in viability (1; Marquis et al., 8th Int.Spores Conf., abstr. no. 80,1980). This treatmentshould lower the internal pH of spores, which itdoes in B. megaterium spores by at least 1.5 U(B. Swerdlow and P. Setlow, unpublished data).Both the latter H+ loading of spores and theincrease in pH of up to 1.6 U are reversible,suggesting that the spores behave in this regardas cation-exchange resins.Although we could vary the intemal pH of the

spores between 6.2 and 7.8, within these limitsthe intemal pH of the spores had no detectableeffect on their ability to gerninate or their dor-mancy or resistance or both. Although spores

which have been H+ loaded often have greatlydecreased resistance properties (1; Marquis etal., 8th Int. Spores Conf., abstr. no. 80, 1980), itis not clear whether this is due to the loweredinternal pH or to the loss of spore cations orboth. It is, of course, possible that a fall in pHwithin developing spores during sporulation(from 7.5 to 6.2) plays a role in the acquisition ofsome spore property such as dormancy, but thatsubsequently some other change in spores (e.g.,dehydration) is the primary mechanism formaintaining dormancy. However, it is also pos-sible that a fall in the intrasporular pH may beonly an effect of the exhaustion of the ATP poolof the developing spores during sporulation (25)rather than a cause of such a phenomenon.One striking result in the present work was

that the rate ofmethylamine uptake by dormantspores was slow, in contrast to results from othersystems. This suggests that there is a permea-bility barrier in dormant spores which not onlyblocks passage of charged species (CH3NH3+) (8)but also retards passage of uncharged lipid-sol-uble species (CH3NH2). Because previous workhas suggested that most of the methylamineenters the spore core (21), a likely candidate forthe permeability barrier is the inner spore mem-brane. More external spore layers (coats, cortex,and outer spore membrane) are thought not tobe permeability barriers to small hydrophiliccompounds, whereas the inner membrane is (8).Indeed, spores whose coats (and presumablymuch outer membrane) were removed by deter-gent treatment at high pH and then soaked inwater also took up methylamine slowly (B. Set-low and P. Setlow, unpublished data). The innerdormant spore membrane may well have prop-erties resulting in a low permeation rate forsmall lipophilic molecules, since the phospholip-ids in this membrane may be tightly packed andin a crystalline structure. However, this struc-ture is disrupted early in spore germination (6,27).Whatever the permeability barrier resulting

in slow uptake of methylamine by dormantspores, this barrier is rapidly destroyed uponspore germination as most of the methylaminetaken up was released along with H+, Na+, andK+. In contrast, dormant spores released theseions slowly. This permeability change accom-panying germination took place before DPArelease and is an extremely early event. Al-though no other biochemical events have beencharacterized which precede DPA release duringspore germination, loss in spore heat resistanceprecedes DPA release (4).

In the case of K+, the ions released early ingermination were reabsorbed in a DCCD-sensi-

27VOL. 148, 1981


tive process, and an energy-requiring K+ trans-port system has been identified in dormantspores of B. subtilis (5). Although the K+ reab-sorption required energy, we have no evidencethat the release of H+, K+, or Na+ early ingermination required energy. Indeed, the ionrelease took place during germination in DCCDand during a period before the accumulation ofdetectable ATP or NADH or both (18, 19, 24).Although the release of monovalent ions early

in germination was studied most extensivelywith B. megaterium spores, methylamine andK+ release also preceded DPA release duringgermination of B. cereus spores. B. subtilisspores containing high K+ levels also lose muchK+ early in germination (3). However, it wasunclear in the latter report whether K+ was lostby release or by exchange with exogenous K+.The present work indicates that monovalentions are simply released, presumably in conjunc-tion with some anion(s). The finding of thiscomparable phenomenon with spores of threedifferent species strongly suggests that mono-valent ion release and the permeability changethat allows the ion release are common featuresof the first minutes of bacterial spore germina-tion. An obvious question then is whether theK+ or Na+ release or both is a process essentialfor early steps in spore germination or only theresult of other essential processes. At presentthis question cannot be answered definitively,because ion release has been observed under allgermination conditions and with all speciestested. However, it seems unlikely that Na+ andK+ release generates energy by dissipation of aconcentration gradient. Indeed, it seems morelikely that the net release of Na+ and K+ earlyin our germination experiments is only the resultof an increased permeability at this time plusthe low level of Na+ or K+ in the germinationmedium. This net Na+ and K+ release would bedriven solely by the concentration gradient be-tween the inside and the outside of the spore.However, it appears unlikely that this is essen-tial for spore germination, because spores of ourstrain of B. megaterium germinate well in 0.4MK+ or Na+ (P. Setlow, unpublished data), con-centrations well above that of free K+ of Na+within spores.The location of most of the methylamine

within dormant spores (and thus the region oflow pH) is thought to be the spore core (21).However, this assignment is based on indirectmeasurements, and direct measurement seemsimpossible. However, there is direct evidencethat the K+ excreted during germination comesfrom the spore core. Thus, in spores of B. cereuscontaining K+ levels similar to those in ourexperiments, >85% of the spore K+ was shown

J. BACTERIOL.

by electron probe microanalysis to be in thespore core, with at most 15% in the cortex pluscoat regions (28). Given the high percentage oftotal spore K+ released in the first minutes ofgermination, most of the K+ must come fromthe core. This finding further supports the con-cept presented previously by Vary (29) that alarge change, possibly a transient change, inperneability in the inner spore membrane is oneof the first events in bacterial spore germination.

ACKNOWLEDGMENTISWe are extremely grateful to William Bradbury and Jay A.

Glasel for their advice and for the use of the atomic absorptionspectrometer.

This work was supported by a grant from the Army Re-search Office.

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heat resistance in bacterial spores. Biochem. Biophys.Res. Commun. 10:139-143.

2. Barton, J. K., J. A. den Hollander, T. M. Lee, A.MacLaughlin, and R. G. Shulman. 1980. Measure-ment of the internal pH of yeast spores by 31p nuclearmagnetic resonance. Proc. Natl. Acad. Sci. U.S.A. 77:2470-2473.

3. Dring, G. J., and G. W. Gould. 1971. Movement ofpotassium during L-alanine-initiated germination ofBacillus subtilis spores, p. 133-142. In A. N. Barker, G.W. Gould, and J. Wolf (ed.), Spore research 1971. Aca-demic Press, London.

4. Dring, G. J., and G. W. Gould. 1971. Sequence of eventsduring rapid germination of spores of Bacillus cereus.J. Gen. Microbiol. 66:101-104.

5. Eisenstadt, E., and S. Silver. 1972. Restoration ofcationtransport during germination, p. 443-448. In H. 0. Hal-vorson, R. Hanson, and L. L. Campbell (ed.), Spores V.American Society for Microbiology, Washington, D.C.

6. Ellar, D. J. 1978. Spore specific structures and theirfunction. Symp. Soc. Gen. Microb. 28:296-325.

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8. Gerhardt, P., and S. H. Black. 1961. Permeability Qfbacterial spores. III. Molecular variables affecting Sol-ute permeation. J. Bacteriol. 82:750-760.

9. Gould, G. W. 1969. Gernination, p. 397-444. In G. W.Gould and A. Hurst (ed.), The bacterial spore. Aca-demic Press, Inc., New York.

10. Halvorson, H. O., and A. S. Susnman. 1966. Spores:their dormancy and germination. Harper & Row, Pub-lishers, New York.

11. Hsieh, L. K., and J. C. Vary. 1975. Peptidoglycan hy-drolysis during initiation of spore germination in Bacil-lus megaterium, p. 465-471. In P. Gerhardt, R. N.,Costilow, and H. L. Sadoff (ed.), Spores VI. AmericanSociety for Microbiology, Washington, D.C.

12. Johnson, J. D., D. Epel, and M. PauL 1976. IntracellularpH and activation of sea urchin eggs after fertilization.Nature (London) 262:661-664.

13. Johnson, R. G., and A. Scarpa. 1976. Ion permeabilityof isolated chromaffin granules. J. Gen. Physiol. 68:601-631.

14. Kashket, E. R., and T. H. Wilson. 1973. Proton-coupledaccumulation of galactoside in Streptococcus lactis7692. Proc. Natl. Acad. Sci. U.S.A. 70:2866-2869.

15. Nishihara, T., T. Ichikawa, and M. Kondo. 1980. Lo-cation of elements in ashed spores of Bacillus megate-


rium. Microbiol. Immunol. 24:495-506.16. Rossignol, D. P., and J. C. Vary. 1979. Biochemistry of

L-proline-triggered germination ofBaciUus megateriumspores. J. Bacterio. 138:431-441.

17. Rotman, Y., and M. L. Fields. 1967. A modified reagentfor dipicolonic acid analyses. Anal. Biochem. 22:168.

18. Scott, I. R., G. S. A. B. Stewart, M. A. Koncewicz, D.J. Ellar, and A. Crafts-Lighty. 1978. Sequence ofbiochemical events during germination of Bacillus meg-aterium spores, p. 95-103. In G. Chambliss and J. C.Vary (ed.), Spores VII. American Society for Microbi-ology, Washington, D.C.

19. Setlow, B., and P. Setlow. 1977. Levels of oxidized andreduced pyridine nucleotides in dormant spores andduring growth, sporulation, and spore germination ofBacillus megaterium. J. Bacteriol. 129:857-865.

20. Setlow, B., and P. Setlow. 1979. Localization of low-molecular-weight basic proteins in Bacillus megate-rium spores by cross-linking with ultraviolet light. J.Bacteriol. 139:486-494.

21. Setlow, B., and P. Setlow. 1980. Measurements of thepH within dormant and germinated spores of Bacillusmegaterium. Proc. Natl. Acad. Sci. U.S.A. 77:2744-2746.

22. Setlow, B., L K. Shay, J. C. Vary, and P. Setlow.1977. Production of large amounts of acetate duringgermination of Bacillus megaterium spores in the ab-sence of exogenous carbon sources. J. Bacteriol. 132:744-746.

23. Setlow, P., and A. Keornberg. 1969. Biochemical studiesof bacterial sporulat ion and germination. XVII. Sulffly-dryl and disulfide lE vels in dormancy and germination.

J. Bacteriol. 100:1155-1160.24. Setlow, P., and A. Kornberg. 1970. Biochemical

changes during bacterial sporulation and germination.XXII. Energy metabolism in early stages ofgerminationof Bacillus megaterium. J. Biol. Chem. 245:3637-3644.

25. Singh, R. P., B. Setlow, and P. Setlow. 1977. Levels ofsmall molecules and enzymes in the mother cell com-partment and the forespore of sporulating Bacillusmegaterium. J. Bacteriol. 130:1130-1138.

26. Singh, R. P., and P. Setlow. 1979. Regulation of phos-phoglycerate phosphomutase in developing foresporesand dormant and germinated spores of Bacillus mega-terium by the level of free manganous ions. J. Bacteriol.139:889-8.

27. Stewart, G. S. A. B., M. W. Eaton, K. Johnstone, M.D. Barratt, and D. J. Ellar. 1980. An investigation ofmembrane fluidity changes during sporulation and ger-

mination of Bacillus megaterium KM measured byelectron spin and nuclear magnetic resonance spectros-copy. Biochim. Biophys. Acta 600:270-290.

28. Stewart, M., A. P. Somlyo, A. V. Somlyo, E. Shuman,J. A. Lindsay, and W. G. Murrell. 1980. Distributionofcalcium and other elements in cryosectioned Bacilluscereus T spores, determined by high-resolution scan-ning electron probe X-ray microanalysis. J. Bacteriol.143:481491.

29. Vary, J. C. 1978. Glucose-initiated germination in Bacil-lus megaterium spores, p. 104-108. In G. Chambliss andJ. C. Vary (ed.), Spores VII. American Society forMicrobiology, Washington, D.C.

30. Warth, A. D. 1979. Molecular structure of the bacterialspore. Adv. Microb. Physiol. 17:145.

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levels of h+ and other monovalent cations in dormant and

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