Vol. 155, No. 3JOURNAL OF BACTERIOLOGY, Sept. 1983, p. 1316-13230021-9193/83/091316-08$02.OO/OCopyright 0 1983, American Society for Microbiology

High Calcium Content in Streptomyces Spores and Its Releaseas an Early Event During Spore Germination

J. A. SALAS, J. A. GUIJARRO, AND C. HARDISSON*Departamento de Microbiologia, Universidad de Oviedo, Oviedo, Spain

Received 25 March 1983/Accepted 16 June 1983

The metal ion content of spores of five Streptomyces species was studied. Ageneral feature of this study was the finding of a very high calcium content (1.1 to2.1% of the dry weight). Accumulation of calcium occurred preferentially duringthe sporulation process. Spore calcium was located in the integument fraction,and more than 95% of the calcium was removed from intact spores by ethyleneglycol-bis(O-aminoethyl ether)-N,N-tetraacetic acid. Several divalent cations(Mg2+, Mn2+, Zn2+, and Fe2+) which induced darkening of spores and loss of heatresistance also caused the release of calcium from spores. In addition, darkeningof spores was blocked by metabolic inhibitors, whereas calcium excretion was notaffected. Two different categories of events in the initiation of germination may bedifferentiated; first, calcium release from spores which is not energy dependentand is a consequence of triggering of germination by some divalent cations, andsecond, some other events including loss of heat resistance, loss of sporerefractility, and a decrease in absorbance, with at least one energy-dependentstep.

Germination of Streptomyces spores, consid-ered as the morphogenesis of a resting structureinto a vegetative organism (4), is accompaniedby sequential morphological changes in thespores. In Streptomyces antibioticus, three dif-ferent stages have been described previously:darkening, swelling, and germ tube emergence(8). Many studies on Streptomyces spore germi-nation have been focused on the ultrastructuralchanges (8, 18) and physiological and biochemi-cal changes occurring during the process (2, 7-12, 15). The first stage of spore germination in S.antibioticus and Streptomyces viridochromoge-nes, i.e., darkening, is initiated by divalent cat-ions and is an energy-requiring process (6, 8).Differerices have been found among these spe-cies with respect to the ions which initiate sporegermination. In both Streptomyces species, thisstage is accompanied by changes in spore refrac-tility, a decrease in absorbance, a loss of heatresistance, and an increase in respiratory activi-ty. In addition, excretion of spore carbon andthe release of a germination inhibitor has beenreported to occur in S. viridochromogenesspores (11). However, it is not yet known howmetal ions interact with the spore or how thisinteraction is related to the subsequent releaseof dormancy.

In this paper we report a study of the contentof metal ions of spores in different Streptomycesspecies, showing that a high calcium content is ageneral feature of the Streptomyces species

studied. Furthermore, our data indicate thatmost of the spore calcium is located outside themembrane and is rapidly released into the medi-um as one of the earliest events in the germina-tion of S. antibioticus spores.

MATERIALS AND METHODS

Microorganism and culture conditions. SeveralStreptomyces species were used in this work: S.antibioticus ATCC 11891, Streptomyces griseusATCC 11429, Streptomyces scabies CMI 99049, S.viridochromogenes ATCC 14290, and Streptomycesaureofaciens ETH 13387. Spores were obtained aftersporulation in GAE (glucose, asparagine, yeast ex-tract) solid medium as previously described (8). Ger-mination was carried out at 35°C in a minimal syntheticmedium (7) in an orbital Gallenkamp incubator. Thegermination process was monitored both by followingthe absorbance at 580 nm and by phase-contrastmicroscope observations.

Metal ion measurements. The metal ion content ofspores was determined by atomic absorption spec-trophotometry with a Perkin-Elmer model 372 atomicabsorption spectrophotometer. Single-element hollowcathode light sources were used, and the analyticalwavelengths (in nanometers) selected for this studywere: Ca, 422.7; Mg, 285.2; Na, 589; K, 767.5; Fe,248.3; Cu, 324.7; Zn, 213.9; Co, 240.7. Metal contami-nation from glassware is a hazard for accurate ionanalyses, and for this reason, only plasticware wasused throughout these experiments. Removal of ioncontamination from this material was done by succes-sive washings in Radiacwash containing EDTA for 12h and in 1 N HCI for 12 h and, finally, extensive

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washings in double-distilled water. For measuremnentsofthe metal ion content, spores were dried at 160°C for5 h and then ashed at 500°C for 6 h. The ash wasdissolved in 100 ,ul of concentrated HCl and diluted to0.1 N HCI. Lanthanum chloride (1% [wt/vol]) wasadded to the samples before analysis for calcium tofree the analysis from interference by phosphates.Calibration curves were made for each experiment byusing standard metal ion solutions in 0.1 N HCI.

Calcium uptake assays. Portions (500 p.J) of sporesuspensions in distilled water (3 x 105 spores per ml)were incubated at 35°C in small glass vials for 5 min.Calcium uptake experiments were started by the addi-tion of 15CaCI2 (0.1 IuCi/ml). After 10 min at 35°C, 400-,ul samples were removed and carefully layered on thetop of a two-phase system in Eppendorf tubes contain-ing 40 RI of 20%o (wt/vol) Lubrol PX plus 0.25 Msucrose (lower layer) and 500 ,ul of silicone oil-dinonylphthalate (2:3 [vol/vol]) (upper layer). The sampleswere then centrifuged through the oil phase at 12,000x g for 30 s in an Eppendorf minifuge. In theseconditions, the entire spore population was quicklysedimented into the bottom of the centrifuge tube.Spores did not lope viability during this treatment asdetermined by viable unit counts. The upper aqueoussupemnatant layer and the oil layer were then removedwith a Pasteur pipette under vacuum, and the walls ofthe centrifuge tube were dried with tissue paper. Thelower layer of the tube containing the spore pellet wasresuspended by sonication with an Ultramet II soniccleaner water bath, and the radioactivity was countedafter the addition of 400 RI of scintillation fluid con-taining 5 g of PPO (2,5-diphenyloxazole) and 0.3 g ofPOPOP (1,4-bis-[2]-(5-phenyloxazolyl)benzene) per li-ter in 66% toluene-33% Triton X-100, using a Beck-man LS 100C scintillation spectrophotometer.Quenching corrections were made in these samples.

Labeling of spore calcium during sporulation. After 7to 9 days at 28°C, spores were obtained from GAEsolid medium plates supplemented with 0.1 pCi of'5CaCl2 per ml. The radioactivity present in sporesuspensions was measured, showing values between3,000 and 6,000 cpm per 10' spores. In this paper, wewill refer to these as 45Ca-spores.Calcium accumulation during sporulation. The

microorganisms were grown at 28°C on cellophaneifims placed on the surface of the agar in GAE solidmedium. Before the plates were inoculated with aspore suspension, 0.3 pCi of 45CaC12 per ml wasspread over the plates. The different phases of thegrowth and sporulation of S. antibioticus were differ-entiated on the basis of changes in the color of theplates and observations under the phase-contrast mi-croscope of semithin sections of whole colonies (C.Mendez, A. F. Brana, M. B. Manzanal, and C. Har-disson, manuscript in preparation). After differentperiods of incubation, the mycelium was gentlyscraped from the cellophane with a spatula and sus-

pended, in 5 ml of distilled water. After homogeniza-tion with a Sorvall Omnimixer for 5 min, samples (250p1) were counted for radioactivity, and 1.5-ml sampleswere removed for dry weight determinations. No morethan 15% of the initial added radioactivity was takenup during the growth of the microorganism.

Location of calcium within spores. '5Ca-spores were

broken by a Vortex mixer in the following way. A 1-mlspore suspension in distilled water (5 x 10P spores per

CALCIUM IN STREPTOMYCES SPORES 1317

ml) was mixed with 3 g of glass beads (0.10 to 0.11 mmdiameter) in a test tube and cooled on an ice waterbath. Breakage of the spores was done for 10 periodsof 30 s with intermittent cooling in ice water. After thistreatment, at least 95% of the spores were broken.Glass beads were removed by low-speed centrifuga-tion at 2,000 x g for 2 min and washed with 1 ml ofdistilled water. The spore integuments were sediment-ed at 12,000 x g for 5 min, and after resuspension bysonication in 200 ILI of distilled water, samples (50 pJ)were counted for radioactivity. The radioactivity pres-ent in the supernatant was also counted with 200-.lIsamples. Quenching corrections were applied in the

samples. In some experiments, spores were treatedwith 10 mM ethylene glycol-bis(P-aminoethyl ether)-N,N-tetraacetic acid (EGTA) before breakage to re-move all spore calcium and then washed five times indistilled water. In experiments to assess binding ofexogenous calcium, 0.1 ,uCi of 45CaC12 per ml wasadded to broken spores and incubated for 30 min atroom temperature before the integument fraction wasisolated as described before.Caldum releas studies. Calcium release from spores

was determined by measuring the Ca in the superna-tant after incubation of 45Ca-spores under differentconditions. Spore suspension (5 ml; 3 x 100 spores perml; 9,000 to 18,000 cpm/ml) were incubated in distilledwater at 35C in a water bath with shaking. Theexperiment was initiated by adding small volumes (10to 100 pll) of different compounds, and at subsequenttimes, samples (500 pll) were removed into centrifugetubes (1.5-ml capacity). The samples were centrifugedat 12,000 x g for 30 s in an Eppendorf minifuge, and400 p1 of the supernatant was counted for radioactivityin the scintillation fluid mentioned above. The totalradioactivity initially present in the spores was mea-sured by counting samples of the spore suspensiontaken immediately before staring the experiment.Values for radioactivity in the supernatants of thesamples at zero time were negligible (less than 3% ofthe total) and were always discounted from the timesamples.

Heat resistance studies. The loss of heat resistanceduring germination of spores was determined as fol-lows. Spores were incubated at 35C in various media,

TABLE 1. Metal content of Streptomyces sporesa% Dry wt of:

Species Na K Mg Ca Fe Zn Cu

S. aureo- 0.06 0.88 0.27 1.21 0.05 0.03 0.01faciens

S. griseus 0.02 1.06 0.20 1.10 0.01 0.04 0.01S. scabies 0.02 1.21 0.13 1.36 0.05 0.09 0.01S. anti- 0.16 0.80 0.40 2.10 0.05 0.09 0.01

bioticusS. virido- 0.04 0.92 0.54 1.52 0.04 0.06 0.01chromo-genes

a The metal content was determined by atomicabsorption spectrophotometry as described in the text.Each value is the average of three independent deter-minations. All the values obtained were in a range±10%o of the average.

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and at different time intervals, samples (0.5 ml) werewithdrawn and treated at 55C for 30 min. Aftercooling in ice water, the samples were diluted indistilled water, and spores surviving the treatmentwere enumerated by viable unit counts in GAE solidmediuip.Dry *e1ght determinations. Samples of spore sus-

pensions in distilled water were dried to a constantweight at 100NC on preweighed glass vials.

Chemicals. 45CaC12 (27.5 mCi/mg of calcium) wasobtained from Amersham International Ltd., Amer-sham, Great Britain. Lanthanum chloride, Lubrol PX,carbonyl cyanide m-chlorophenylhydrazone (CCCP),N,N-dicyclohexylcarbodiimide (DCCD), EGTA,MgSO4, FeSO4, NaCl, KCI, and CaCl2 were obtainedfrom Sigma Chemical Co., St. Louis, Mo. Dinonylphthalate was obtained from Fluka AG, Buchs, Swit-zerland. Pure copper and zinc were a gift of Ensidesa,Asturias, Spain. All other reagents were of analyticalgrade.

RESULTSMetal content of spores and changes during

germiation. The metal ion content of dormantspores of different Streptomyces species wasstudied by atomic absorption spectrophotome-try (Table 1). Spores showed a high potassiumcontent, 0.80 to 1.21% of the dry weight, and avery high calcium content, 1.10 to 2.10% of thedry weight. These characteristics were presentin the five species studied. Lower levels of Na,Mg, Fe, and Zn were found, and the sporecontent of Co and Cu was negligible (less than0.01% of the dry weight). The spore content offour of the metal ions (Ca, Mg, Na, and K) wasalso studied during germination of S. antibioti-cus spores in the minimal synthetic mediumsupplemented with the four metal ions cited(Fig. 1). The Na content remained constantduring the germination process, whereas the Kcontent increased slightly. However, the Mglevel of spores progressively increased duringthe darkening process and at the beginning ofspore swelling. Later, coinciding with the pres-ence of the highest proportion of swollen spores,the Mg level decreased to values slightly lowerthan those found in the dormant spore andremained constant throughout germ tube emer-gence. The calcium content of spores decreasedby about 55 to 65% in the first hour of germina-tion and then remained constant during theremainder of the germination process.Changes in spore calcium content as influenced

by environmental conditions. The finding of avery high calcium content in dormant spores ofseveral Streptomyces species prompted us tostudy the influence of different conditions on thecalcium content and the relative binding state ofthe ion to spore components. In this respect, thecalcium content of dormant spores increasedwhen calcium was added to the sporulationmedium. Thus, S. antibioticus spores harvested

to

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FIG. 1. Changes in metal content of S. antibioticusspores during germination. Spores were incubated at35C in a minimal synthetic medium containing thefour metal ions studied, and at different times, sampleswere removed and washed four times in distilledwater. The metal content of spores was then analyzedby atomic absorption spectrophotometry. Each valueis the average of three independent determinations.Symbols: 0, Na; *, K; A, Ca; A, Mg.

from GAE solid medium plates supplementedwith 0.2 and 1 mM CaCl2 had calcium levels ofabout 2.3 and 3.1% of the dry weight, respec-tively. The distribution of radioactivity in thesupernatants and in the pellets after each wash-ing during harvesting of the spores was deter-mined. The first washing of the spore released 5to 10% of the Ca from spores, but in fivesuccessive washings no more than 1% of theradioactive calcium was released. Furthermore,no calcium was lost from spores during storageat 4°C for at least a 2-month period. On thecontrary, calcium was completely extractedwith 0.1 N HCl and partially removed with 0.1 NNaOH, 5% (wt/vol) sodium dodecyl sulfate, or20% (wt/vol) trichloroacetic acid (Table 2).Treatment of spores with 10 mM EGTA re-moved more than 95% of the calcium. 45Caassociated with spores was not exchangeable byincubation of spores in 10 mM CaCl2 or CaSO4(Table 2). Taking advantage of the ability ofEGTA to remove spore calcium, we were able todetermine whether these calcium-deficientspores recovered their normal calcium contentby later incubation with calcium. The uptake ofcalcium in EGTA-treated and nontreated spores

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CALCIUM IN STREPTOMYCES SPORES 1319

TABLE 2. Influence of different treatments on the calcium content of S. antibioticus spores'

45Ca in spores ~ 'Ca in % 41CaTreatment (cpm) supenatant extracted(cpm)

Dormant spores 5,500 0 0After washing with distilled water at room temperature 5,225 440 8Storage at 4°C for 2 months 5,450 0 0Incubationb with:

10 mM CaCl2 or CaSO4 5,550 50 0.910 mM EGTA 120 5,400 980.1 N HCl 140 5,335 970.1 N Na OH 4,455 1,045 195% (wt/vol) sodium dodecyl sulfate 3,410 2,090 3820%o (wt/vol) trichloroacetic acid 3,300 2,225 40a 45Ca-spores (108 spores per ml) were incubated for 30 min at room temperature with the additions cited and

then centrifuged at 12,000 x g for 5 min. Samples of the supernatants and the pellets were then counted forradioactivity after resuspension of the pellets in distilled water. Each value is the average of three independentdeterminations.

b At room temperature.

was similar in a wide range of calcium concen-trations (0.01 to 40 mM), showing a high Km forbinding, 0.4 to 0.5 mM. The calcium content ofdormant Streptomyces spores was about 500nmol per mg of dry weight (about 2% of the dryweight). In the presence of 40 mM externalcalcium concentration, EGTA-treated and non-treated spores incorporated similar amounts ofcalcium, 160 nmol per mg of dry weight. Thiscalcium incorporation by calcium-free sporesonly represents 32% of the initial calcium con-tent of the spores. Furthermore, washing of bothEGTA-treated and nontreated spores with dis-tilled water after the incorporation experimentremoved nearly all the calcium incorporated.This result is in contrast to the fact that thecalcium content of mature spores (calcium accu-mulated during sporulation) was not eliminatedby extensive washing in distilled water (datamentioned above). In addition, these experi-ments proved that the content of tightly boundcalcium of spores cannot be increased by incu-bation with calcium once they are mature sincecalcium incorporated by mature spores is easilyremoved by simply washing in distilled water.Calcium accumulation during sporulation. The

incorporation of 45Ca during the growth andsporulation of S. antibioticus-vegetative myce-lium, aerial mycelium, and sporulation-isshown in Fig. 2. As can be seen, the amount of45Ca taken up per plate increased progressivelyat the beginning of the emission of the aerialmycelium. Then, a plateau was observed, fol-lowed by a sudden increase of 34% in the totalcalcium content of the mycelium, associatedwith the onset of sporulation. From this mo-ment, the level of calcium remained constantthrough the sporulation process. If we considerthe calcium accumulation per milligram of dryweight, again it is possible to observe how a net

increase in calcium during sporulation occurs.At the end of the process of spore maturation(about 5 days), levels of 9,200 cpm per mg of dryweight of the entire colonial growth were found.However, it must be pointed out that, in thisstage of the growth cycle, a great contribution tothe dry weight is due to cellular debris fromlysed vegetative mycelium and nonsporulatingaerial mycelium. Therefore, the content of calci-um per milligram of dry weight of spores mightbe significantly higher. Consequently, we har-vested spores from these plates and found thatthe proportion of dry weight accounted for bycalcium was about six times higher in sporesthan in the entire plate population (54,600 cpmper mg of dry weight of spores and 9.200 cpmper mg of dry weight of the whole plate).

Location of calcium within spores. To localizecalcium in dormant spores, "Ca-spores werebroken and the integument fraction was sedi-mented by centrifugation. Nearly all the radioac-tive calcium (80 to 85%) was found to be associ-ated with the integuments, and 95% of this Caremained firmly bound during repeated wash-ings with distilled water at room temperature. Itcould be argued that disintegration of the sporesmight have freed calcium from its original site(not in the integuments) and that redistributionof the element occurred with the consequentbinding to the integuments. However, the fol-lowing experiments allowed us to rule out thispossibility. (i) 45Ca-spores were broken in thepresence and in the absence of an excess ofnonlabeled calcium (10 mM), and the integumentfraction was isolated. If significant redistributionof calcium occurred during spore breakage, thenwhen this was performed in the presence ofexcess nonradioactive calcium there would be alarge reduction in the amount of 45Ca bound tothe integuments. This was not observed. (ii) In

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FIG. 2. Accumulation of 45Ca during growth and sporulation of S. antibioticus. The microorganisms weregrown on cellophane films placed on the surface of GAE solid medium plates containing 0.3 ,uCi of 45CaCl2 perml. At different times during growth, the growth on the cellophane was removed, the 45CaCl2 was incorporated,and the cell dry weight was determined as described in the text.

another experiment, nonradioactive spores werebroken with or without prior removal of calciumwith EGTA. Then, 45Ca was added and radioac-tivity associated with the integuments was mea-sured. This experiment assessed the ability offree calcium to bind to isolated iteguments.Moreover, if free calcium does bind, then wewould predict that integuments derived fromcalcium-depleted spores would contain moretightly bound calcium. It was observed that 32and 39% of the added calcium was bound to theinteguments of EGTA-treated and nontreatedspores, respectively. Furthermore, in both cas-es, 80% of the radioactive calcium was removedby washing the integuments once with distilledwater. Both of these experiments strongly sug-gest that calcium in S. antibioticus spores isfirmly bound to some structural component ofthe spore integuments.Calcium release during germination. Spore

germination in the minimal synthetic mediumwas accompanied by a progressive loss of 45Ca(Fig. 3). This release started within 5 min of theaddition of spores to the germination mediumand proceeded at a constant rate during the first10 min ofgermination (about 35% of the radioac-tive calcium was excreted in this time). Thenand during the following 50 min, the rate ofcalcium release diminished so that a maximumof about 60 to 70%o of the calcium was lost after60 min. A similar pattern of calcium release wasfound upon incubation of 45Ca-spores in thepresence of divalent cations (Mg2+, Fe2+, Mn2+,or Zn2+) (Fig. 3). In contrast, incubation of 45Ca-

spores in distilled water, glucose, asparagine,glucose plus asparagine, monovalent cations(Na+, K+, or Li') or some divalent cations(Ca2' or Ba2+) did not stimulate release ofsignificant amounts of calcium. In other experi-ments (data not shown), the release of radioac-tive calcium from 45Ca-spores during germina-tion was not affected by the presence of differentmetabolic inhibitors such as sodium azide, Ata-brine, 2,4-dinitrophenol, CCCP, DCCD, or po-tassium cyanide (all at 1 mM final concentra-tion).

Loss of heat resistance during germination.Dormant spores of S. antibioticus were resistantto heat shock for 30 min at 55°C. However, mostof the spores lost their viability when submittedto this treatment after 90 min of incubation in theminimal synthetic medium, by which time 60 to70% of the spores were phase dark. According-ly, we used the loss of heat resistance as aparameter to measure germination. When theheat resistance of spores was measured afterincubation under various nutritional conditions,it was found that loss of heat resistance wasaccompanied by release of calcium; conversely,there was no loss of heat resistance under condi-tions where no calcium was released (Table 3).These experiments support the hypothesis thatcalcium release is a germination-specific event,but do not prove that calcium contributes to thespore heat resistance. The response to heatshock (55°C for 30 min) of EGTA-treated spores(calcium-free spores) was also studied. The re-sults showed (data not shown) that these spores,

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00

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10 20 30 40 So 6 gTIME Cmin )

FIG. 3. Calcium release during germiination of S.antiiotcusspoes.45Ca-spores were incubated at

35°C in several nutritional conditions. At differenttimes during incubation, samples (0.5 ml) were with-drawn and centrifuged at 12,000 x g for 30 s. Theradioactivity present in 400 ,ul samples of the superna-tant was counted. Symbols: O, Ca ions; *, distilledwater; A, Zn or Fe ions; A, minimal synthetic medi-um; O, Mg ions; *, Mn ions; o, Na or K ions; V, Baions; O, Li ions.

which remain phase bright, had the same heatresistance to this treatment as nontreatedspores. These results suggest that the release ofcalcium is not sufficient for initiation of germina-tion and that other events which result in loss ofheat resistance must occur.

DISCUSSIONDescriptions of the metal ion content of Strep-

tomyces spores are scarce. In S. viridochromo-genes, the spore content of Mg, Ca, and K ionswas reported to be 0.17, 0.28 and 2.0%o of the dryweight, respectively (6). In this paper, we pre-sent a more complete study of the content ofeight metal ions in the spores of five Streptomy-ces species. Each of the ions was present inamounts typical of those usually found in bacte-rial endospores (13, 21), and a very high calciumcontent was found in the spores of all fivespecies, tightly bound to some structural compo-nent(s) of the spore integuments. After removalof calcium by EGTA, the spores did not recovertheir initial calcium content upon incubationwith exogenous added calcium. This experimentsuggests that a permeability barrier makes thecalcium binding sites in the spore inaccessible toexternally added calcium or that removal ofcalcium by EGTA modifies, in some way, the

calcium binding sites and this prevents the rein-sertion of exogenous added calcium, Our workdid not permit us to determine which compo-nent(s) of the spore integuments calcium islinked to. It has been reported that cell walls ofBacillus subtilis vegetative cells present a strongretention capacity for divalent cations includingcalcium and that cell walls possess selectivesites for different cations (3). Many potentialanionic sites in the walls of gram-positive bacte-ria are available to sequester cations (1). Theseinclude the teichoic acid phosphodiester groups,the free carboxyl groups of the peptidoglycan, orthe sugar hydroxyl groups of both wall poly-mers. Few studies have been made of the Strep-tomyces spore wall; hence, its composition andstructure are not well known. Teichoic acids area usual component of the cell walls of gram-positive bacteria (16), and cell walls of vegeta-tive mycelium of Streptomyces have been re-ported to contain teichoic acids (14, 19, 23).However, teichoic acids have not been reportedin the wall of Streptomyces spores (5). Theseauthors (5) did not find the typical componentsof teichoic acids (glycerol and ribitol), but theywere not able to explain the presence of organicphosphate in the spore walls. Because any lossof spore wall material could occur during prepa-ration for analysis, the existence of teichoicacids in the Streptomyces spore wall remains tobe clarified. Teichoic acids are negativelycharged molecules due to the high phosphatecontent and would be a candidate for bindingcalcium. Another possible source for sequestra-tion of calcium could be the free carboxyl groupsof the peptidoglycan. Aspartic acid was a majorcomponent of the Streptomyces spore wall and

TABLE 3. Relationship between calcium releaseand loss of heat resistance in S. antibioticus sporesa

%5a % LossAddition(s) released of heat

resistance

None (control) 2 01% glucose or 0.2% 3 0asparagine (or both)

1 mM Li+, Na+, K+, 2-5 1-5Ca2+, or Ba2+ ions

1 mM Mg2+ ions 63 701 mM Mn2+ ions 60 641 mM Zn2+ ions 64 681 mM Fe2+ ions 63 68Minimal synthetic medium 70 73

a 45Ca-spores were incubated at 35°C in distilledwater with the additions mentioned. After 90 min ofincubation, samples were removed for determinationof radioactive calcium and assays of heat resistance(SS°C for 30 min) as described in the text. Each value isthe average of four determinations.

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only a minor component in vegetative walls (5).This negatively charged dicarboxylic amino acidmight contribute to the existence of free carbox-yl groups in the spore wall, which could beneutralized by calcium ions. Moreover, regard-less of its binding site, no function can beassigned to calcium in Streptomyces spores atthis time. Based on experiments showing thatcalcium-free spores (EGTA-treated spores)were as heat resistant as nontreated spores to55°C for 30 min, a role for calcium in spore heatresistance cannot be proposed. It seems thatcalcium release from spores could be only one ofseveral events that take place during the initialstages of germination which are connected withthe loss of heat resistance.

Vegetative mycelial walls are sensitive to ly-sozyme, whereas spores are not (5, 19, 22). Alysozyme-resistant modified peptidoglycancould explain this insensitivity to the enzymeaction. The possibility that calcium would becontributing to this modification was ruled outsince lysozyme apparently did not affect thespores once calcium was removed by EGTA.Calcium release from spores during germina-

tion in the minimal synthetic medium was ob-served by either atomic absorption measure-ments or radioactive calcium determinations.Incubation of spores in the presence of somedivalent cations also induced the calcium excre-tion. Since the only compounds cited until nowas able to start the darkening process of Strepto-myces spores are divalent cations (6, 8), it maybe argued that calcium release from sporesmight be for exchange with the divalent cations.However, there are several reasons against thispossibility. (i) In general, free exchange betweenions occurs rapidly, but calcium release fromspores takes place with a lower rate than expect-ed if exchange were to occur. (ii) By definition,calcium must be the most effective exchangecation with itself. However, in the presence ofan excess of nonradioactive calcium, no morethan 3% of the 45Ca was released from 45Ca-spores. (iii) Studies on the specificity of calciumand magnesium uptake systems normally showthat magnesium is not a good competitor forcalcium (17, 20). In contrast, calcium excretionfrom Streptomyces spores was strongly stimu-lated by magnesium ions. (iv) 45Ca uptake bymature spores was strongly inhibited in thepresence of barium ions, and this divalent cationwas also readily exchangeable with Ca (data notshown). However, the barium ions were not ableto induce the release of calcium from 45Ca-spores.

Additional support for the view that calciumrelease is a germination event in Streptomycesspecies comes from studies of spore heat resist-ance. Darkening of spores occurred in experi-

ments in which calcium was excreted and heat-sensitive spores appeared (in the presence ofindividual Mg2', Mn2 , Zn2+, or Fe2+ ions).Therefore, it may be suggested that one of theearliest events occurring during germination ofS. antibioticus spores is the release of calciumfrom the spore integuments to the medium. Thiscalcium excretion would be a consequence ofthe interaction of the initiator cation and thedormant spore.Although darkening of spores is an energy-

requiring process (6, 8), calcium release was notaffected by different metabolic inhibitors. There-fore, we can distinguish two categories of eventsoccurring during initiation of germination. Oneis typified by the release of calcium, whichoccurs early in germination and is not energydependent. However, the other category is re-quired for the loss of heat resistance, which isassociated with darkening of spores and a de-crease in absorbance and includes at least oneenergy-requiring step. Removal of calcium fromspores by EGTA is not sufficient for the sporesto get through the second stage since the sporesremain in the dormant stage as monitored by thepresence of spore refractility and spore heatresistance. A possible explanation for this couldbe that for the initiation of germination to occur,the presence of the germinant ion would benecessary. Therefore, the treatment of sporeswith EGTA, although removing calcium fromspores, did not trigger the early events thatdivalent cations did, and consequently, thespores remain dormant. In S. viridochromo-genes, it has been proposed (6) that the site ofcalcium-initiated spore germination is externalto the cytoplasmic membrane and that calciumdoes not serve only as a trigger mechanism sinceit is required continuously during germination.At this stage, we do not know whether there is aspecific receptor for the metal ion in S. antibioti-cus spores or whether it exerts its effect in someother way (i.e., alteration of the transmembranepotential). Additional experimental work mustbe done to elucidate several points. What is thenature of the interaction between the initiationcation and the dormant spore? How does thisevent initiate spore germination and how is itconnected to the subsequent changes whichoccur in the spore?

ACKNOWLEDGMENTSWe thank David Ellar for helpful discussions, Eric Cundliffe

for reading a premanuscript, and Richard Skinner for helpingus in the corrections. We also thank the Departments ofQuimica T6cnica and Quimica InorgMnica, University of Ovie-do, for allowing us to use atomic absorption spectropho-tometry.

This work was partially supported by a grant from theFondo Nacional para el Desarrollo de la Investigaci6n Cienti-fica y T6cnica.

J. BACTERIOL.

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VOL. 155, 1983

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