JOURNAL Op BACTERIOLOGYVol. 88, No. 4, p. 821-830 October, 1964Copyright © 1964 American Society for Microbiology

Printed in U.S.A.

TRACE METAL REQUIREMENTS FOR SPORULATIONOF BACILLUS MEGATERIUM'

BRUNO J. KOLODZIEJ2 AND RALPH A. SLEPECKY3Department of Biological Sciences, Northwestern University, Evanston, Illinois

Received for publication 4 March 1964

ABSTRACT

KOLODZIEJ, BRUNO J. (Northwestern Univer-sity, Evanston, Ill.), AND RALPH A. SLEPECKY.Trace metal requirements for sporulation ofBacillus megaterium. J. Bacteriol. 88:821-830.1964.-A sucrose-mineral salts medium was puri-fied for the purpose of definitively re-examiningthe trace metal cations associated with the sporu-lation of Bacillus megaterium. Two heretoforeunknown requirements for copper (0.013 1sog/ml ofcupric ion) and molybdenum (27.2,ug/ml of molyb-date ion) were uncovered. In the purified copper-supplemented medium, sporulation levels of othermetals were determined as follows: iron, 0.5,ug/mlof ferrous ion; zinc, 1.1 ,Ag/ml of zinc ion; man-ganese, 0.037 pg/ml of manganous ion; and cal-cium, 0.9 ,g/ml of calcium ion. The approximatetime during which the various metals were re-quired was determined with iron, zinc, calcium,and manganese. A molybdenum substitution forcopper, iron, or zinc was noted. The copper re-quirement was shown for the sporulation of B.cereus var. mycoides and var. albolactis, suggestingthat this may be a general requirement for sporu-lation. The specific functions of metal ions insporulation are not known, but they probably actas activators of the various enzyme systems neces-sary for sporulation.

Previous reports, as reviewed by Curran(1957) and Murrell (1961), indicated that traceamounts of certain metal ions were indispensablefor the sporulation of various sporeformingbacteria. Although important metal associationsare known, the presence and effect of unsuspected

' This report was taken in part from a disserta-tion submitted by the senior author in partialfulfillment of the requirements for the Ph.D.degree, Northwestern University.

2 Present address: Department of Microbiology,University of Chicago, Chicago, Ill.

I Present address: Biological Research Labora-tory, Department of Bacteriology and Botany,Syracuse University, Syracuse, N.Y.

trace amounts of metallic ions have not beeninvestigated extensively.The present study demonstrates the effect of

various cations on sporulation of Bacillus mega-terium in a medium consisting of highly purifiedchemicals.

MATERIALS AND METHODS

Organisns. B. megaterium and B. cereus var.mycoides, obtained from the Department ofMicrobiology of the University of Texas, and B.cereus var. albolactis ATCC 7004, were used inthis study.

Chemically defined medium. A minimal syn-thetic medium, modified from that of Slepeckyand Foster (1959) to eliminate an adjustment ofpH, was utilized. The unpurified sucrose-mineralsalts medium (SS) consisted of either chemi-cally pure or reagent-grade chemicals in distilled-deionized water: sucrose, 0.1%; NaCl, 0.1%;MgS04 7H20, 0.02%; KH2P04 ,0.24%; (NH4)2.HP04, 1.1%; FeS04-7H20, 0.001%; ZnSO4-7H20, 0.001%; MnSO4, 0.0007%; CaCl2,0.0005%. The pH of the medium after auto-claving was 7.1.Medium purification. Purification of the SS

medium was accomplished by replacing mostcomponents with spectrographically pure stand-ardized chemicals (spec-pure; obtained fromJohnson-Matthey and Co., London, England,through the Jarrell Ash Co., Newtonville, Mass.).Chemicals not available as spec-pure [sucroseand (NH4)2HP04] were purified in the labora-tory.Each spec-pure chemical was accompanied

with a spectrographic examination report givinga quantitative analysis of each salt, indicatingthe purity and exact trace-metal contaminantcontent. Sucrose was purified by four passagesof a 0.1% solution through a Crystalab de-eminite L-10 column; the solution was alwayscollected in a specially cleaned 1-liter polyethyl-

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ene bottle. Elimination of trace-metal contamina-tion was followed by measuring the decrease iniron content by the orthophenanthroline method(Fortune and Mellon, 1938). Three extractionseach with 5 mg of the metal-chelating agent,8-hydroxyquinoline, per 150 ml of solution(Waring and Werkman, 1942) were used topurify (NH4)2HPO4. After each extraction, themetal quinolates and residual 8-hydroxyquinolinewere removed with three 5-nd chloroform treat-ments, each of 1-min duration. The purified(NH4)2H2PO4 was delivered into a speciallycleaned 500-ml polypropylene flask, and to itwas added spec-pure KH2PO4. This solutionwas autoclaved at 121 C for 30 min for steriliza-tion and for vaporization of residual chloroform.This buffer solution was added to 80 ml of sucroseplus spec-pure NaCl, MgSO4 7H20, and tracemetals as indicated for each experiment, givinga total of 100 ml of medium. All water utilizedwas first distilled and then deionized by passagethrough a Crystalab deeminizer.

Cleaning of materials. All materials used werecleaned scrupulously according to acceptedprocedures for the removal of trace contaminantsadsorbed to their surface (Waring and Werkman,1942; Theirs, 1957). Polyethylene and poly-propylene ware were substituted for glasswarewhenever possible, since Theirs (1957) showedthe advantage of these in trace-metal studies.The equipment consisted mainly of polyethyleneor polypropylene Erlenmeyer flasks, beakers,pipettes, graduate cylinders, and solution storagebottles. Glassware used included a few Erlen-meyer flasks, centrifuge, and Klett tubes. Bothglass and polypropylene ware were washed in ahot alconox solution, followed immediately withfive rinses in tap water and five rinses in distilledwater. Glassware cleaning was extended ac-cording to the procedure of Waring and Werkman(1942). Glass vehicles were soaked in 40%alcoholic KOH and then in aqua-regia, followedby 15 rinses with distilled-deionized water aftereach treatment. The polyethylene and poly-propylene ware were treated with a concentrated(1:1) H2SO4-HNO mixture for 15 min (Theirs,1957), followed by ten rinses in distilled waterand five rinses with distilled-deionized water.Finally, all flasks containing 100 ml of distilled-deionized water were autoclaved for 30 min at15 psi and 121 C to remove any residual acid,and were then inverted and allowed to air-dry.

Cotton plugs were wrapped in Saran wrap as aprecaution to prevent any extraneous ion con-tamination by the cotton itself.

Details of cultivation. Spores of B. megateriumharvested from a 60-hr culture grown on theunpurified SS medium were used as the inoculum.Spores were collected by centrifugation with aServall SS-1 angle centrifuge at 1,200 X g for15 min at 6 C. The spores were stored at -20 Cuntil used. Prior to use they were defrosted at 6C, washed twice, and standardized with dis-tilled-deionized water. A 1-nl inoculum stand-ardized to 500 Klett units (measured in a Klett-Summerson photoelectric colorimeter with a*50 filter) and consisting of about 2.0 X 108spores (dry weight, 1.9 mg) was used per 100 mlof medium in 350-ml Erlenmeyer glass flasks or500-ml Erlenmeyer polypropylene flasks. Flaskswere incubated at 30 C on a reciprocating shakeroperating at 78 4-in. strokes per min. Samples(5 ml) were removed at 0, 14, 18, 24, 28, 38, 48,62, or 66 hr for turbidimetric growth determina-tions (Klett-Summerson photoelectric colorimeterwith a # 50 filter) and for direct counts with aPetroff-Hausser chamber. Cell types were dis-tinguished by dark-contrast phase microscopy.Nonrefractile rod-shaped cells were scored asvegetative cells; rod-shaped cells containingdistinct refractile bodies were considered sporan-gia; and oval or spherical highly refractilebodies were counted as free spores. A completegrowth curve and pattern of sporulation showingthe various cell types was determined for eachexperiment; however, with many results onlythe 66-hr readings are reported.

Spectrographic analysis. A spectrographicqualitative analysis of the four-salt mixture ofCaCl2, MnSO4, FeSO4 7H20 was undertakenby Accurate Metal Laboratories, Chicago, Ill.

Dipicolinic acid analysis (DPA). Spores wereanalyzed for DPA content by the colorimetricassay of Janssen, Lund, and Anderson (1958).

RESULTSB. megaterium exhibited good growth and

sporulation (greater than 90%) when grown inthe unpurified sucrose-mineral salts medium inglass or polypropylene flasks. Maximal growthwas reached by 28 hr (Fig. 1), with 80% asporangium maximum noted at this time fol-lowed by complete sporulation by 38 hr. Over90% spores were recorded. The results from either

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( e ._. The purified (NH4) 2HP04 was without effect on

\/--- \3 sporulation; the purified sucrose reduced it to/. 160 69%; the spec-pure salt mixture replacement led

\1'/.k,/OD to a complete loss of sporulation. When chemi-60- V 0 9) '/ 120 cally pure or reagent (NH4) 2HP04, sucrose, or

four salt mixture was added separately to the4\_80 purified medium, only trace salt mixture in-

/ \ / \ / \duced sporulation to about 50%, suggesting thatco /2 2 40 W the missing component(s) for sporulation wasa. (' Wd4 z probably a trace contaminant from the fourn B r-- 3 F- salts FeSO4 7H20, ZnSO4 .7H20, MnSO4, and_w.D\/CaC12. The trace salt mixture used in the me-0

8C \ Z vi r O.D - 160Y dium was subjected to spectrographic qualitative\/--. g\ / M O analysis (done by Accurate Metal Laboratory,6C\/88 \ / _ 120 Chicago, Ill.), and the metals found as trace

contaminants were Al, 0.001 to 0.01%; Cu,40C /\sioip\80 0.003 to 0.03%; Sr, 0.005 to 0.05%; Mg, 0.005 to

0.05%; Si, 0.0001 to 0.001%; Co, 0.001%; and20 /,' W / U \ 1 - 40 Mo, 0.001%. Besides these, other metals, shown

/ *s -.*---2 *--. to be good absorbents to glassware (Theirs,042~~60 1957), were also considered because of the

TIME IN HOURS

FIG. 1. Growth and sporulation patterns of ABacillus megaterium in unpurified sucrose-mineralsalts medium in (A) 850-ml Erlenmeyer glass flasks \60and (B) 500-ml Erlenmeyer polypropylene flasks.Per cent cell types at 0, 14, 18, 24, 38, 48, and 62 hr: 0/ -__.-0OD 120(1) per cent vegetative cells; (2) percent sporangia;(3) per cent spores. Total growth indicated by optical K so80density (OD).

l2) 40 _

flask type were comparable, indicating that a>-polypropylene ware had no effect on growth or B-sporulation. , \The growth and sporulation patterns of B. I60s

megaterium in the purified medium contained \in either glass or polypropylene Erlenmeyer 0 a 120flasks (Fig. 2) differed somewhat from those iobserved in the unpurified medium. In both 4{ so

flask types, growth was reduced by about 15%and no sporulation was observed by 62 hr. This 2response suggested that the purified medium 2was deficient of some important component(s) 3vital for sporulation, and that the deficiency was TIME IN HOURSprobably due to the purification and substitutionprocdur.Ocasona]y, omespoulaionwas FIG. 2. Growth and sporulation patterns ofprocedure. Occasionally, some sporulation was Bacillus megaterium in purified sucrose-mineral

observed in glass flasks, but only rarely in poly- salts medium in (A) Erlenmeyer glass flasks andpropylene containers. (B) Erlenmeyer polypropylene flasks. Per cent cellTo determine the component deficiency, types at 0, 14, 18, 24, 28, 88, 48, and 62 hr: (1) per

purified (NH4)2HP04, sucrose, and the four cent vegetative cells; (2) per cent sporangia; (3) pertrace salt mixture (spec-pure) were added one at a cent spores. Total growth indicated by optical densitytime as a replacement to the unpurified medium. (OD).

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CO)w'a.I-.-J-Jw

I32t

TIME IN HOURS

FIG. 3. Growth and sporulation patterns of Bacil-lus megaterium (A) in molybdenum-supplementedpurified medium at 50 Ag/ml and (B) in copper-supplemented purified medium at 0.06 Ag/ml. Percent cell types at 0, 14, 18, 24, 28, 88, 48, and 62 hr:(1) per cent vegetative cells; (2) per cent sporangia;(8) per cent spores. Total growth indicated by opticaldensity (OD).

TABLE 1. Concentration of cupric sulfaterequired for the sporulation of

Bacillus megaterium

Per cent cell types at 66 hr

Sporangia

0002004010

No growth

Spore

52

4870999086985

No growth

occasional sporulation in the purified mediumgrown in glass flasks. Various salts of the sus-pected metals were added to the purified mediumat concentrations not effecting growth. Theseincluded 100lg/ml of regular (chemically pureor reagent, unavailable as spec-pure) Na2SiO3.5H20; AlCJ8; LiCl; BaCl2; H3BO4; and spec-pure CoSO4 7H20; 50 Ag/ml of spec-pure (NH4)86Mo7024 4H20; 1 ,ug/ml of regular NiCl2CrO3; and 0.1 ,ug/ml of spec-pure CuSO4.5H20.Of all the salts tested, only spec-pure (NH4)6.Mo7024*4H20 and CuSO4 5H20 had any effecton sporulation during the 66-hr culture cycle.The molybdenum salt at a concentration of 50pug/ml and the copper salt at 0.1 pug/mi inducedcomplete sporulation. Also, each of the fourspec-pure medium trace salts was increased to 100pg/ml separately and together in a mixture. Theindividual salts did not activate sporulation,and only a small response was evident with thefour-salt mixture, eliminating the possibilitythat the purified salts were not being added insufficient quantities to bring about sporulation.Growth and sporulation patterns of B. mega-

terium in the purified medium supplementedwith (NH)6Mo7024-4H20 (50 pg/ml) andCUSO4 *5H20 (0.05 pAg/ml) are illustrated inFig. 3.

Addition of (NH4)6Mo7024 4H20 to thepurified medium reduced growth by 20% overthat observed in the unpurified, but was onlyslightly less than that obtained in the purifiedform (Fig. 3A). The peak of growth was reachedat 28 hr with a concoinitant sporangia maximum;however, although spores were noted, sporangiapersisted longer than in the unpurified medium,indicating some difficulty in the liberation of thespores from the sporangia. The addition ofCUSO4 *55H20 to the purified medium restoredthe normal growth and sporulation patterns(Fig. 3B). Growth reached its peak around 28 hrwith a closely associated sporangium peak;completion of sporulation was noted by 38 hr.

Table 1 shows the culture response of B.megaterium to various concentrations of CUSO4.5H20 when grown in the purified sucrose-mineral salts medium. The addition of 0.005pug/ml was without effect on sporulation, whereas0.01 and 0.025 pAg/ml resulted in 48 and 70%spores, respectively; a level of 0.05 pug/ml in-duced complete sporulation (99%). From 0.05pAg/ml to 1.0 pg/ml concentrations, sporulationwas complete, but only after a progressively

CUS045H20

pg/mt00.0050.010.0250.05t0.100.501.01.52.0

Vegeta-tive

959852281

10102

85

* Lag phase: - indicates less than hour given;+ indicates greater than hour given.

t CuSO4.5H20, 2.0 X 10-7 M (0.0127 ,ug/ml ofcupric ions).

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lengthened lag period, probably due to inhibi-tion of spore germination. At 2.0 ,ug/ml, com-plete toxicity was observed. Therefore, 0.05,ug/ml (2.0 X 10-7 M CuSO4-5H20; 0.0127,ug/ml of cupric ions) was taken as the optimalconcentration for growth and sporulation, andthis amount was utilized in all subsequent ex-periments.

Reagent-grade Cu (NO3)2 -3H20, Cu(C2H302)2,and CuCl2 2H20 were also tested for sporulationinduction, and all satisfied this requirement atthe same cupric ion concentration. The effectsof cuprous ions were not surveyed, because thecuprous compounds were too insoluble for ex-perimental use, and were easily oxidizable tocupric ions under present conditions.CUS04 *5H20 was added to the purified sucrose-

mineral salts medium at various stages of growth(Table 2). Whenever cupric sulfate was addedto the medium and sterilized with other mediumcomponents, complete sporulation occurred. But,when added separately to the medium aftersterilization, little or no sporulation occurred. Asimilar response was observed when added atthe various stages of the growth cycle. Only the6-hr addition responded with partial sporula-tion. These results suggested a copper bindingwith the medium constituents during steriliza-tion. The approximate time for the copper needfor sporulation has not been resolved.DPA analysis was carried out on spores of B.

megaterium produced in unpurified and copper-supplemented purified medium. The DPA con-tent of the spores from the unpurified sucrose-mineral salts medium was 78.4 ,g/mg (dryweight), whereas spores produced in the purifiedcopper-supplemented medium contained 57.3,ug/mg (dry weight).Both B. cereus var. albolactis ATCC 7004 and

B. cereus var. mycoides also showed a copperrequirement for sporulation (Table 3). Albolactisand mycoides grew well, and sporulated in theunpurified sucrose-mineral medium. The my-coides variety displayed some difficulty inliberating its spores by 66 hr. After purificationof the medium, sporulation was lost in bothinstances. Supplementation of the medium withCuSO4.5H20 induced sporulation in both organ-isms; this response resembled that observed inthe unpurified medium, including the sporan-gium-spore liberating difficulty of B. cereus var.mycoides. Molybdenum addition induced only apartial response in B. cereus var. albolactis and

TABLE 2. Time-interval additions of cupric sulfateto the purified sucrose-mineral salts medium

CuSO4.5H20*

hr

0(BS)t0(AS) t

369141821242838

Per cent cell types at 66 hr

Vegetative Sporangia Spore

177883796879493959692

01110901000000

99122

544367548

Growth phase

LagLagLagEarly-logMid-logLate-logEnd-logDeclineDecline

* 0.05 ,Ag/mi of CuS04 5H20.t BS: before sterilization; AS: after steriliza-

tion.

TABLE 3. Sporulating response of Bacillus cereusvar. albolactis ATCC 7004 and B. cereus var.

mycoides in the unpurified, purified,supplemented, and purified-supple-

mented sucrose-mineralsalts medium

B. cereus var. B. cereus var.albolactis mycoides

MediumVeg- Spar- Spore Veg- Spor- Sporta- angia reta- angia retive aga tiveana

Unpurified ........ 2* 0 98 18 42 40Purified ............ 97 0 3 80 0 20Purified + Cut...... 17 0 83 8 43 49Purified +Mol ... 56 4 40 84 9 7Purified + Cut, Mot. 6 0 94 4 0 96

* Figures indicate per cent types at 66 hr.t 0.05 jAg/mI of CuSO4*5H20.t 50 jig/ml of (NH4)6MO7024*4H20.

only a small response in B. cereus var. mycoides.A simultaneous addition of copper and molybde-num salt stimulated complete sporulation inboth organisms by 66 hr.The effects of the omission of FeSO4 -7H20,

ZnSO4 .7H20, MnSO4, or CaCl2 from the un-unpurified and purified sucrose-mineral salts(supplemented with copper, molybdenum, orcopper-molybdenum) media are given in Table 4.

Deletion of either the iron or zinc salt from theunpurified medium showed no reduction in

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TABLE 4. Effect of the deletion of Fe, Zn, Mn, andCa on sporulation in a Cu-, Mo-, or Cu-

and Mo-supplemented medium

Per cent spores at 66 hr

Salt deleted Purified plusUnpurified

Cu Mo Cu, Mo

FeSO4 *7H20.... 98 20 92 95ZnSO4*7H20 ..O 94 24 88 97MnSO4 ......... 18 20 13 37CaCl2 .......... 98* 87t 90t

* 40% nonrefractile.t 65% nonrefractile.t 58% nonrefractile.

sporulation of B. megaterium; however, a manga-nese deficiency resulted in loss of sporulation.Omission of CaC12 resulted in complete sporuila-tion, but 40% of the spores formed were nonre-

fractile. Deletion of either iron or zinc from thepurified copper-supplemented medium resultedin sporulation reduction to 20 and 24%, respec-tively. The omission of manganese and calciumresponded similarly to that observed in theunpurified medium; the manganese exclusionreduced sporulation to 20%; the calcium deletiondid not interfere with sporulation, but 65% ofthe spores were nonrefractile. The exclusion ofiron and zinc from purified molybdenum-sup-plemented sucrose-mineral salts medium didnot display the same effect as that observed inthe copper-supplemented form. In this instance,complete sporulation still occurred after ironor zinc salt deletion; however, manganesedeficiency still resulted in the loss of sporulation.This suggested that the molybdenum substitutedfor iron or zinc but not for manganese. In thecase of copper-molybdenum supplemented puri-fied medium, the response was similar to themolybdenum-supplemented conditions. Com-plete sporulation was observed with iron or zincdeficiencies, and the absence of manganesereduced the spore crop to 37%.With the elimination of the iron salt from the

purified copper-supplemented medium, sporula-tion was reduced to 26%; the addition of 0.1and 1.0 ,ug/ml elevated the spore yield to 30 and58%, respectively, whereas 2.5 yg/ml or 9.0 X10-6 M induced complete sporulation. Thislatter concentration contained 0.5 ,ug/ml offerrous ions, which was designated as the metal

ion concentration essential for complete sporula-tion under these conditions.The iron salt was added to the culture at

various time intervals during the various growthand sporulation stages to determine the phasethat the metal cation must be present for maxi-mal sporulating activity. Its addition at 0 and 6hr (lag phase of culture) did not decrease sporula-tion, but at 12 hr (lag early log phase) sporeformation was reduced to 34%. Salt additionsfrom 15 through 38 hr, i.e., from mid-logarithmicgrowth through the decline stages, were ineffec-tive for sporulation induction. This suggestedthat the ferrous ion must be present prior to orat the time of spore germination, early outgrowth,and probably the first few cell divisions.The omission of zinc from the copper-supple-

mented medium resulted in only 27 % spores. Anaddition of 0.1, 1.0, and 2.5 ,4g/ml to the mediumsupported only 19, 37, and 30% spores, respec-tively; however, the addition of 5.0 ,g/ml or1.7 X 10-6 M ZnSO4 *7H20 induced completesporulation. This concentration contained 1.1,ug/ml of zinc ions, which was denoted as theminimal salt and metal ion concentration essentialfor sporulation under the specified conditions.

Addition of zinc at any time between 0 and 28hr induced greater than 80% spores; however,if added at 38 hr, sporulation was reduced to 22%,suggesting that zinc was not required untilgrowth had ceased.The omission of manganese from the copper-

supplemented medium resulted in only 20%sporulation. An addition of 0.001 and 0.01,ug/ml resulted in 29 and 36% sporulation,respectively; an increase to 0.1 ag/mgor 6.65 X10-7 M stimulated complete sporulation. Thisconcentration contained 0.0365 ,g/ml of manga-nous ions, and was designated as the minimalsalt and ion content vital for sporulation underthe prescribed conditions.

Additions of manganese at 0, 6, and 12 hrresulted in greater than 90% spores; but, whenadded at 15 or 18 hr (mid or late log phase),sporulation was reduced to 75%. Addition at 21through 38 hr or at the end of growth resultedin loss of sporulating activity.

Omission of calcium from the copper-supple-mented medium did not interfere with sporula-tion, but 71% of these spores were nonrefractile.Addition of 0.01, 0.1, or 1.0 ,ug/mg decreasedthe nonrefractile spores to 36, 27, and 22%,respectively; however, addition of 2.5 ,ug/ml

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or 2.25 X 10-5 M CaCl2 reduced them to 13%.This concentration contained 0.90 ,ug/ml ofcalciulm ions, and was considered as the minimalsalt content necessary for the production ofrefractile spores.

Addition of calcium between 0 and 28 hr, i.e.,any time between inoculation and the appear-ance of sporangia, was followed by formation ofnormal refractile spores. The addition of calciumat 38 hr was no longer effective, since sporulationwas complete and about 55% of these were non-refractile.

DISCUSSIONPurification of a sucrose-mineral salts medium

disclosed a previously undescribed copper andmolybdenum requirement for the sporulation ofB. megaterium. Copper had not been implicatedas a requirement for bacterial sporulation;however, its presence had been demonstrated invegetative cells and spores by spectrochemicalanalysis (Curran, Brunstetter, and Myers, 1943),colorimetric means (Slepeckv and Foster, 1959),diphenylcarbazone staining (Troger, 1959), andelectroparanmagnetic resonance studies (Windleand Sacks, 1963). Powell and Strange (1956)presented data indicating a strong chelationbetween dipicolinic acid and copper.

Various biological roles have been associatedwith copper in animal, plant, and bacteria]systems (McElroy and Glass, 1950; Underwood,1959; Stiles, 1961), and among these the require-ment for copper in various enzymatic reactionsas a cofactor or a metal ion activator (Dixon andWebb, 1958; Weinberg, 1962). It is feasible thatcopper might activate a specific enzyme at aparticular stage of cell development for sporula-tion to occur.Of much interest is the work of Mulder (1939,

1953), who showed the necessity of copper forthe growth and sporulation of various fungi.Aspergillis niger grew in the absence of copperbut would not sporulate; however, with theaddition of sufficient copper, normal mycelialdevelopment was evident with the formationof normal spores. Copper may have a similarbiochemical function in fungal and bacterialsporulation.

Although molybdenum was stimulatory, itstotal effect was not as distinct as that demon-strated with copper. Because the concentrationof ammonium molybdate was 1,000-fold higherthan that of the copper salt, it would appear

that a sufficient concentration of cupric ionsmight be introduced as a contiguous trace con-taminant; however, the spectrographic analysisreport of the molybdenum salt indicated thatthe copper present was below the stimulatingthreshold.As with copper, molybdenum has not been

implicated previously with bacterial sporulation;however, various other biological roles have beenknown for molybdenum in plants, animals, andmicroorganisms (Underwood, 1959; Stiles, 1961).Molybdenum-enzyme associations have beenshown for nitrate reductase (Nason, 1962) andin symbiotic and nonsymbiotic nitrogen fixation(Nicholas, 1963; Nason and McElroy, 1963).Iron and zinc requirements were established

for the sporulation of B. megaterium in the puri-fied medium; this was in direct contrast to resultswith the unpurified medium where no loss ofsporulation was recognized upon deletion. Thissuggests that a sufficient quantity of these metalswas introduced into the unpurified medium astrace contaminants of the chemically pure orreagent-grade chemicals.The essentiality of iron and zinc for sporula-

tion has been considered by others, but theresults are vague since the media were composedof chemically pure or reagent-grade chemicals.Iron was considered by Brewer et a]. (1946),who showed that ferrous or ferric ions were notessential for sporulation of B. anthracis in achemically defined medium, but its presenceraised the total spore yield; by Ward (1947),who noted that iron increased total sporulationof B. coagulans var. thermoacidurans grown onproteose peptone agar; and by Grelet (1952a,b) who observed that an iron deficiency did notaffect sporulation of B. megaterium in a glucose-mineral salts medium.

In a glucose-mineral salts medium, otherstudies have demonstrated the presence of ironin vegetative cells and spores by various means(Curran et a]., 1943; Powell and Strange, 1956;Beskid and Lundgren, 1961; Lundgren andCooney, 1962).That iron was shown to be necessary at the

early stages of growth agrees with mediumanalysis studies of Beskid and Lundgren (1961)and cell and spore analyses by Lundgren andCooney (1962) showing that iron was utilizedbv B. cereus during early growth and returned tothe medium by the end of sporulation. This isin contrast to the observations of Powell and

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Strange (1956), who reported an increase in theiron content in cells of B. cereus and B. subtiiduring sporulation.A requirement for zinc had not been established

clearly. It was reported for B. anthracis (Breweret al., 1946) and B. megaterium (Grelet, 1952a,b) that zinc omission had little effect on sperula-tion; however, Ward (1947) reported that zincincreased the spore yield of the thermophileB. coagulant var. thermoacidurans, whereasLundgren and Beskid (1960) observed that zincwas essential for the sporulation of B. cereus.Species or media differences might account forthese discrepancies, but it is more than likelythat trace-metal contamination is the probableexplanation. A zinc requirement could not bedemonstrated in the present study unless thehighly purified medium was used.The necessity for zinc just prior to maximal

growth is in accord with Lundgren and Cooney(1962), who demonstrated with B. cereus grownat 37 C that zinc uptake occurred during sporula-tion.

Since iron and zinc ions were substituted bymolybdenum ions, it is possible that molybdenummay be the essential ion, but substitutable byeither iron or zinc.Manganese is the most firmly established metal

requirement for sporulation (Charney, Fisher,and Hegarty, 1951; Grelet, 1952a, b; Curranand Evans, 1954; Amaha, Ordal, and Touba,1956; and Powell, 1957) and can be demonstratedreadily in most unpurified media. The require-ment was more specific than iron or zinc sincemolybdenum did not substitute for it. Manga-nese was found to be required somewhere betweenmid or late logarthmic growth, agreeing withWeinberg (1955).

In both the unpurified and purified forms ofthe medium utilized in this study, calcium wasshown to be a requisite for the formation ofrefractile spores, and was shown to be necessaryat the time of sporangium formation. This is inclose agreement with observations of Vinter(1956), who demonstrated that the calciumcontent of cells increased with the formation ofthe spores within the sporangium. As the sporesmatured, they progressively accumulated calciumuntil they contained several times more calciumthan their respective vegetative cells. In additionto being indispensable for the formation ofnormal refractile spores, calcium is known to be

required for thermoresistance and, in this capac-ity, it is associated closely with DPA. Thiseffect of calcium on spore thermoresistance wasobserved by Curran et al. (1943) in 12 differentsporeformers; by Grelet (1952a, b), by Vinter(1956), and Slepecky and Foster (1959) in B.megaterium; by Amaha and Ordal (1957) in B.coaulans; and by Sugiyama (1951) in Clostridiumbotulinum. Black, Hashimoto, and Gerhardt(1960) demonstrated that, in endotrophic sporula-tion, calcium had to be added to the water-sus-pended vegetative cells for the formation ofnormal heat-stable spores containing the normalDPA content. Halvorson and Howitt (1961)and Cooney and Lundgren (1962), using Ca45,showed that calcium uptake was closely asso-ciated with sporulation and DPA synthesis, andboth were present in approximately an equimolarratio.Many other possible roles may be ascribed to

metal ions in bacterial sporulations, for example,involvement in various membrane effects (Lacey,1961); however, much more work, preferablywith the use of extremely sensitive techniques,must be performed.

ACKNOWLDGMENT

This investigation was supported by grantNSF G-23576 from the National Science Founda-tion.

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