JOURNAL OF BACTERIOLOGY, Aug. 1967, p. 434-440Copyright @ 1967 American Society for Microbiology

Vol. 94, No. 2Printed in U.S.A.

Metabolic Requirements for Microcycle Sporogenesisof Bacillus megaterium'P. K. HOLMES AND HILLEL S. LEVINSON

Pioneerinig Researchl Division, U.S. Army Natick Laboratories, Natick, Massachusetts 01760

Received for publication 25 May 1967

Spores of Bacillus megaterium QM B1551 germinated, elongated, and resporu-lated (microcycle sporogenesis) in simple chemically defined media which permittedno cell division. The second-stage spores thus produced were heat-stable and re-quired heat activation for germination. The orignal amount of spore deoxyribo-nucleic acid tripled before completion of the cycle. Acetate and a small amount ofa tricarboxylic acid cycle intermediate were the minimal organic metabolic require-ments for microcycle sporogenesis. During this cycle. germinated cells oxidizedacetate only after a delay, whether or not glucose was initially present. Spores thatwere germinated in the absence of a carbon source first oxidized an endogenoussubstrate, and then developed the ability to oxidize acetate.

By diluting a complex medium shortly afterspores had germinated, Vinter and Slepecky (14)demonstrated that the first cell formed on germi-nation of a Bacillus cereus or of a B. megateriumspore was capable of resporulating without inter-vening cell division (microcycle sporogenesis).Microcycle sporogenesis may offer an optimallyuncomplicated and synchronous system for thestudy of a morphogenetic cycle-the cycle fromthe spore stage through the vegetative state tothe spore stage. The present work was done todiscover the minimal nutritional requirements forthis cycle in B. megaterium, to compare germi-nation properties of spores produced in micro-cycle sporogenesis with those produced in com-plex media, and to determine whether the primarycell (the cell resulting from the germination of aspore) was initially repressed for acetate oxida-tion.

MATERIALS AND METHODS

Spores of B. megaterium QM B1551 were grownon a medium containing 0.5% Liver Fraction B(Wilson Laboratories, Chicago, Ill.) buffered at pH6.5 with 10 mm potassium phosphate, and wereharvested, washed, and lyophilized as previouslydescribed (9). Unless otherwise noted, spores formicrocycle cultures were suspended at 30 mg/ml inwater and heat-activated at 60 C for 10 min, whengerminated with glucose, and 30 min, when germi-nated with iodide. Of the heated spore suspension,0.1 ml was added to 2.9 ml of microcycle medium in

1 Some of the data in this paper were presented atthe 66th Annual Meeting, American Society forMicrobiology, Los Angeles, Calif. 1-5 May, 1966.

50-ml Erlenmeyer flasks to give a final spore concen-tration of 1.0 mg/ml. Cultures were incubated at 30 Con a shaker reciprocating 100 times/min with a 3-inch(7.6-cm) stroke.The fraction of spores completing microcycle was

determined by microscopic counts of sporangia instained culture smears, or by a test for heat resistance(70 C for 10 min) involving the usual plating proce-dure. Cell counts were made in a Petroff-Hausserchamber.

Glucose was measured as total reducing sugar withdinitrosalicylate (12). Deoxyribonucleic acid (DNA)was extracted and measured by the diphenylaminemethod of Burton (3). Two perchloric acid extrac-tions at 70 C removed the maximal amount of DNAfrom both spores and vegetative cells; salmon DNAwas the standard. Dipicolinic acid (DPA) was esti-mated by the method of Janssen et al. (8). Oxygenuptake was determined by conventional Warburgtechniques; 3 mg of spores in a total volume of 3 mlwas used in each flask.

Cell extracts for enzyme assays were prepared byshaking 50 mg of spores or an equal number of pri-mary cells together with 4.5 g of glass beads (No. 13Ballotini, ca. 0.1 mm diameter) and 5 ml of 0.1 Mtris(hydroxymetkyl)-aminomethane (Tris) buffer (pH7.2) for 40 sec in a Nossal cell disintegrator (McDon-ald Engineering Co., Cleveland, Ohio) at 4 C. Thistreatment ruptured more than 99% of the spores andcells. The homogenate was centrifuged at 10.000 X gfor 20 min; the supernatant extract was used in theassays. Protein nitrogen was determined. bv thenesslerization technique of Miller and Miller (10),as the trichloroacetic acid precipitable fraction ofextracts.

Per cent germination was estimated by micro-scopic counts of cells staining with 0.5% methylene

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B. MEGATERIUM MICROCYCLE SPOROGENESIS

blue. The optical density of cultures was determinedat 560 m,t.

Aconitase activity was measured spectrophoto-metrically (1).

Actinomycin D was generously provided by MerckSharp and Dohme Research Laboratories, WestPoint, Pa.

RESULTSDescription of the cycle. A high percentage of

microcycle sporogenesis occurred in a chemicallydefined medium containing 10 mm glucose, 3 mMNH4Cl, 6 mm K2HPO4, and 1.5 mm K2SO4(initial pH 7.6). Heat-activated spores (1 mg/ml)were 96% germinated after 15 min of incubationin this glucose-ammonia medium. With germi-nation, the optical density of the culture and theheat resistance of the cells decreased (Fig. 1).The optical density increased as the primary cellsemerged and enlarged. Another increase in op-tical density beginning at about 10 hr was corre-lated with the appearance of second-stage sporesand heat resistance. The DPA content of thecells increased after the 9th hr when nonrefractileforespores became visible (Fig. 2). The DNAcontent of the cells began to increase between 1.0and 1.5 hr, doubled by 4.0 hr, and tripled by theend of the microcycle (Fig. 2). A tripling ofDNAduring sporulation of B. cereus has also beendescribed by Young and Fitz-James (15). Therewas no cell division in this medium; the totalnumber of cells remained constant throughoutthe 18 to 20 hr of incubation. The cells that didnot form second-stage spores neither divided norlysed.

In the experiment represented by Fig. 1, about75% of the originally germinating spores formedsecond-stage spores; in other experiments, as highas 99% sporulation was recorded. At 18 hr, theprimary cell, now a mature sporangium, con-tained a second-stage spore and was still partiallyencased in the coat of the initial spore (Fig. 3).After 48 hr of incubation, the sporangia hadlysed, leaving free second-stage spores.

Germination properties of second-stage spores.Washed, but not lyophilized, second-stage spores,free from sporangia, were tested for germinationin potassium phosphate-buffered (50 mm, pH 7.6)glucose or L-alanine. Like the lyophilized initialspores, the second-stage spores required heat ac-tivation for rapid germination in glucose (TableI). However, neither heated nor unheated second-stage spores responded to L-alanine.

Second-stage spores which were not heat-ac-tivated produced only about 25% as manycolonies on Nutrient Agar (Difco) as did heat-activated second-stage spores (data not shown).

Heat-activated second-stage spores germinated

7.0'-IDLE

x ~~~~~z 75cpH6w0p 2E ~~~~~~~0

5.0 and:Lu ~~~~~~~Spores. Z54GluoseI eat 25 0 CD

A' rsistant

0 24 68 01 12 14 1618CULTURE AGE. HRS

FIG. 1. Events occurring during microcycle sporo-genesis of Bacillus megalerium QM B1551 in glucose-ammonia medium.

a)

0

'-,

zC

13.0O

6.5

8)

7t.a)

60 ,50 0

40 -

30 A20C-10

0 2 4 6 8 10 12 14 16 18 20 22

AGE OF PRIMARY CELLS, HRS.

FiG. 2. Concentrations of deoxyribonucleic acid(DNA) and of dipicolinic acid (DPA) extracted fromprimary cells of Bacillus megaterium QM B1551undergoing microcycle sporogenesis in glucose-ammo-nia medium.

and elongated, but were unable to completemicrocycle sporogenesis in the glucose-ammoniamedium which supported microcycle sporo-genesis of initial spores.

Nutritional requirements for microcycle sporo-genesis. (i) Metals. Initial spores contained asufficient endogenous supply of divalent metals tosupport the formation of second-stage spores inthe glucose-ammonia medium. Material releasedinto the medium by the initial spores duringearly stages of germination was not required forsubsequent sporulation. The sporulation cyclecould be interrupted after 15 min of incubation(when 96% of the initial spores had germinated),and the germinated spores could be washed ex-

tensively in fresh glucose-ammonia medium, with-out altering the progress of the cycle.

(ii) Phosphate. No second-stage spores wereformed in the absence of phosphate. In glucose-ammonia medium, buffered at pH 7.5 with 6 mMTris or sodium cacodylate replacing phosphate,

0

DNA

0

DPA

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HOLMES AND LEVINSON

spore coatWA

primary cell

\second stagespore

lam

FIG. 3. An 18-hr primary cell of Bacillus megaterium QM B1551 containing a second-stage spore. Shadowedwith Cr at angle of 100. The primary cell has emerged from the ruptured spore coat of the initial spore, and asmaller, mature, second-stage spore lies within it. Siemens Elmiskop JA electron microscope, 80 kv, 50-pt objectiveaperture.

glucose was oxidized very slowly, and at 24 hr thecells were swollen but not elongated [cf. Hyatt andLevinson (7) ]. Normal formation of second-stage spores was supported by phosphate at con-centrations from 5 to 50 mm in glucose-ammoniamedium, or in glucose-ammonia medium con-taining 6 mM Tris.

(iii) Sulfate. No second-stage spores wereformed in the absence of sulfate. Sulfate appearedto be necessary for the rapid oxidation of glucose[cf. Hyatt and Levinson (6)] and the rapid utiliza-tion of the acids produced; in sulfate-deficientglucose-ammonia medium, the pH of the culturefell and rose slowly, but visible forespores werenot produced by 48 hr.

(iv) Carbon and nitrogen sources. The concen-trations of the carbon and nitrogen sources in theglucose-ammonia medium described above (10,umoles of glucose and 3 ,umoles of NH4Cl per mgof spores) allowed maximal second-stage sporula-tion. Increasing or decreasing the concentrationof either compound separately impaired or de-layed completion of the cycle (Table 2). Simul-

TABLE 1. Germinationi of initial and second-stagespores of Bacillus megaterium QM B1551S

Gernination

Germinant Initial spores Second-stagespores

25C 60C 25C 60C

%o % % %oPhosphate buffer...... 0 0 1 3Glucose, 10 mm....... 4 84 2 63L-Alanine, 100 mm.... 11 57 4 2

a Aqueous suspensions of washed spores weretreated for 10 min at 25 or 60 C before addition ofgerminant and incubation for 30 min at 30 C.

taneous increase in the concentration of bothcompounds resulted in cell division and in de-creased sporulation of primary cells.

Glucose in the glucose-ammonia medium wasoxidized during the first 4 to 5 hr of the sporula-tion cycle, the acids produced lowering the pH to

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B. MEGATERIUM MICROCYCLE SPOROGENESIS

TABLE 2. Effect ofglucose and ammonia concenitra-tions on the development of primary cells of

Bacillus megaterium QM BI55Ja

Concn

Glucose

m M

25252525

10lOb

1010

555

5

NH4CI

mM

5

3

21

5

3b

2I

5

3

21

Forespores, Refractilenonre- second-stagefractile spores

063151

0

0

6650

6323

40

00

0

0

81410

6233

Celldivision

420

0

0

710

0

0

0

0

0

0

a Cultures were examined after 24 hr of incuba-tion at 30 C. Data are percentages of all germi-nated cells.

b Represents concentrations used in the glucose-ammonia medium.

a minimum at 3 hr. The pH then rose as the acidswere oxidized (Fig. 1).

Glucose, per se, was not required for micro-cycle sporogenesis. Ionically germinated (10 mMKI) initial spores formed second-stage spores ina medium containing, as carbon sources, 40 mMsodium acetate plus 1 mm concentrations of anyof the following: glucose, ribose, citrate, isoci-trate, glutamate, succinate, fumarate, or oxalace-tate. Acetate alone did not support second-stagespore formation. Compounds which did not com-plement acetate for second-stage spore formationincluded ethyl alcohol, lactate, pyruvate, glycerol,dihydroxyacetone, a-ketoglutarate, malate,ribose-5-phosphate, 6-phosphogluconate, glu-cose-6-phosphate, L-methionine, L-cysteine, L-

lysine, L-isoleucine, L-proline, DL-threonine, L-

aspartate, and L-asparagine. An acetate-succinatemedium containing 40 mm sodium acetate, 2 mMsodium succinate, 6 mm K2HPO4, and 10 mm KI(as germinant) supported 70 to 90% second-stagespore formation, and was used to study the devel-opment of the ability to oxidize acetate. In con-

trast to the situation with the glucose-ammoniamedium, ammonia was not required in theacetate-succinate medium for the production ofsecond-stage spores. No spores were produced inthe presence of 2 mm succinate as sole carbonsource.Development of the ability to oxidize acetate.

Acetate oxidation via the tricarboxylic acid cycleby primary cells appeared to be a prerequisite formicrocycle sporogenesis, as it is for sporulation inother Bacillus species (5, 13). The competence tooxidize acetate apparently did not persist throughthe spore stage to the primary cell; primary cellsdeveloped this competence after germination.

Spores germinated ionically (10 mm KI) inacetate-succinate medium showed no acid-de-pendent oxygen uptake until 2 hr after germi-nation (Fig. 4). The appearance of acid-depen-dent oxygen uptake was prevented by inhibitorsof protein synthesis (chloramphenicol, 15 ,ug/ml;8-azaguanine, 50 ,ug/ml; or actimomycin D, 50,ug/ml). It is inferred that enzymes necessary foracetate oxidation were synthesized after germina-tion. One enzyme associated with acetate oxida-tion through the tricarboxylic acid cycle, aconi-tase, appeared only in the older primary cells(Table 3), a development which, in its relation tosporulation, is similar to that in B. cereus and B.subtilis (5, 13).

Primary cells needed no exogenous carbonsource to develop competence to oxidize acids.lonically germinated spores allowed to respireendogenously were able to oxidize, without delay,acids presented to them after 3 hr of incubation

w AC- SUCC, 0-time

!a 300- AC+ SUCC, 3 hr-J

D0

. 200- . AC + SUCC+ (1), 3hr

ENDOGENOUS

/0*; ,...---.--.AC + SUCG + (I),O- time

O 23 4 567

INCUBATION TIME, HRS

FIG. 4. Oxygeni uptake by primary cells under vari-ous conditions. Initial spores of Bacillus megateriumQM B1551 germinated with 10 mvi K!, 6 mM K2HPO4,and 1.5 mmv K2SO4 (endogenous). Acetate (AC), 40mM, and succinate (S UCC), 2 m.mr, added with orwithout inhibitor (I) either at zero-time or after 3 hrof incubation at 30 C (arrow). Inhibitors (chloram-phenicol, 15 ,g/ml, or actinomycin D, 50 ,g/ml) pro-duced the same results whether added at the same timeas, or 15 min prior to, the addition of the acids. Oxygenuptake on acetate (no succiniate) was only slightlyabove endogenous (not shown). Sporulation occurredonly in the cultures represenited by the top two curves(AC + SUCC, 0-time; and AC + SUCC, 3 hr).

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HOLMES AND LEVINSON

(Fig. 4). By 3 hr, these endogenously respiringprimary cells contained aconitase (Table 3).

Fluoroacetate, an inhibitor of acetate oxida-tion, decreased second-stage spore formation inglucose-ammonia medium (Table 4); this in-hibition was relieved with citrate or succinate.Maximal fluoroacetate inhibition of sporulationoccurred when the inhibitor was present beforethe pH had risen; fluoracetate added after 6 hrhad less effect (Table 4).The acid-oxidizing ability of spores incubated

in the glucose-ammonia medium (as judgedfrom the rise in pH) appeared as the glucoseconcentration was decreasing (Fig. 1). Adding

TABLE 3. Aconitase activity in extracts of sporesand primary cells of Bacillus megaterium

QM BISSJ5

Aconitase activity per 108 cells

Age of cellsGlucose-ammonia Carbon -free

medium medium

hr unit unit

0 <0.02 <0.020.5 <0.023.0 2.59 1.674.0 2.88

a Prior to extraction, cells were incubated forthe stated times in either glucose-ammonia me-dium or in carbon-free medium (10 mM KI, asgerminant, substituted for glucose in glucose-ammonia medium). Cuvettes contained 10 umolesof sodium DL-iSocitrate, 100 ,moles of Tris buffer(pH 7.2), 0.05 ml of cell extract, in a volume of3 ml. One unit represents a change in opticaldensity at 240 nm of 0.001 per min.

TABLE 4. Inhibition of microcycle sporulation byfluoroacetate added at various times to spores

and primary cells of Bacillus megateriumQM B1551 in glucose-ammonia medium

Microcycle sporulation" (20 hr) withaddition to culture of

Time of additionFluoroacetate FluoroacetateFluooacmat (20 mm) and(20 mM) succinate (40 mm)

hr S %0 9 851 11 852 20 803 20 784 19 755 22 816 55 83

a With no additions to the culture, 75% of thecells completed microcycle sporulation.

extra glucose to the medium at any time before9 hr of incubation resulted in renewed pro-

duction of acid (Fig. 5) with complete suppressionof second-stage sporulation. These additions ofglucose did not promote cell division unless themedium was heavily buffered (50 mM P04',pH 7.5). Addition of glucose after 9 hr inhibitedfurther appearance of forespores.

Inhibition of sporulation by shift-up and shift-down. Increasing the available nutrients pre-

vented microcycle sporogenesis. It has been seen

that additional glucose suppressed sporulation,without promoting cell division. The additionof complex media, such as Nutrient Broth (Difco)or Arret-Kirshbaum (2) broth, at various timesto primary cells in glucose-ammonia mediumcompletely suppressed sporulation and promotedcell division as late as 6 hr, well after the oxi-dation of acids had begun. After 6 hr in glucose-ammonia medium, as cells became "committed"to sporulation, they became decreasingly capableof colony formation on solid complex media(Fig. 6). Primary cells were also less capable offorming colonies on the complex solid mediafor a period between 2 and 6 hr (Fig. 6), but theycould still divide. For instance, 4-hr cells ex-

amined in liquid culture divided once after ad-dition of complex medium and then either ceaseddivision or lysed. Vinter and Slepecky (14) havereported a similar sensitivity to shift-down,rather than to shift-up, in primary cells of B.cereus.

In the glucose-ammonia medium a temporaryshift-down was achieved (for the period from

8

-7

pH6

5

0 2 4 6 8 10 12

CULTURE AGE, HRSFIG. 5. Changes in pH of microcycle cultures of

Bacillus megaterium QM B1551 in glucose-ammoniamedium supplemented at intervals with 10,moles ofglucose per ml of culture. The upper curve representspH in the unsupplemented culture; arrows indicateaddition ofglucose to separate cultures.

I I I I I I

It Il

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B. MEGATERIUM MICROCYCLE SPOROGENESIS

Cf)w

z

0

0

z

Li

C-)

0~

5

4

3

2

-.

* 0

- o 006 ~~~~~0

8i S 00 .0

0 ~~0.* 0

00

00

S 00

0 ! 80 2 3 4 5 6 7 8 9

AGE OF PRIMARY CELLS, HRS.

FIG. 6. Colony-forming ability of Bacillus mega-terium QM B1551 primary cells, taken from glucose-ammonia medium at intervals and plated on NutrientAgar (a) or Arret-Kirshbaum agar (0).

0.25 to 1.25 hr) either by chilling the culture to2 C, or by incubation with 2 mm rather than 10mm glucose. When the temperature of the chilledculture was restored to 30 C, the cells elongatedand formed division septa, but did not sporulate.Similarly, when the normal glucose concentrationwas returned to the glucose-deprived culture,the cells elongated but failed to sporulate.

DISCUSSION

The endogenous reserves of B. megateriumQM B1551 were oxidized upon germination(Fig. 4), but did not support either cell divisionor sporulation. The minimal organic require-ments for microcycle sporogenesis, acetate anda tricarboxylic acid cycle intermediate, wereinadequate for continued growth. In this respect,primary cells resembled the late vegetative stagegranulated cells of B. cereus (11), which sporu-lated, but did not multiply, in a medium con-

taining acetate, citrate, and glutamate. Thegranulated cells, however, contained a functionaltricarboxylic acid cycle; the newly germinatedprimary cells described here apparently did not.A functioning tricarboxylic acid cycle was

probably a prerequisite for microcycle sporu-lation. Fluoroacetate inhibited sporulation onlywhen added before the acids were oxidized, andinhibition was relieved with tricarboxylic acidcycle components. Although extracts of sporesof B. megaterium contain appreciable amounts ofisocitric dehydrogenase, fumarase, and malicdehydrogenase (Holmes and Levinson, unpub-lished data), at least one of the enzymes involvedin the tricarboxylic acid cycle, aconitase, wasabsent during germination, but was present

during acid oxidation. Since ionically germinatedcells oxidized acetate rapidly only in the presenceof small amounts of tricarboxylic acid cycleintermediates, we infer that glucose, in the glu-cose-ammonia recycling medium, may serve tosupply the newly germinated cell with primingamounts of 4-carbon compounds for the opera-tion of the tricarboxylic acid cycle.

Acetate oxidation and sporulation are ap-parently repressed in newly germinated primarycells as well as in log-phase vegetative cells. Inthe case of spores germinated in the absence of acarbon source, the repression and derepressionof acetate oxidation were apparently under thecontrol of endogenous metabolism. Whetherendogenous repression played a role when glu-cose was present has not been determined.The inhibitory effects of chilling and of tem-

porary glucose deprivation on microcycle sporu-lation may be related by a common influenceon the initial rate of cell development; bothtreatments probably lowered the rate of glucoseturnover. Yet glucose turnover was not a re-quirement for sporulation, as cells which germi-nated in the absence of a carbon source latersporulated successfully in acetate and succinate.The presence of glucose during early stages ofrecycle may have committed the cell to a phaseof development which must be completed beforesporulation can begin, and which, if temporarilyinterrupted by a premature lowering of the glu-cose concentration, proceeds at a decreased rate.

Grelet (4) has shown that B. megaterium isderepressed for sporulation when starved forany one of several compounds, including glucose,phosphate, sulfate, or nitrate, and has concludedthat "sporogenesis is the response of a geneticallyapt bacterium to an increase of the generationtime" (cf. R.S. Hanson, Bacteriol. Proc., p. 96,1966). Under microcycle conditions, the genera-tion time is extended indefinitely; in the absenceof sufficient nutrient for cell division, nuclearreplication proceeds and the primary cell sporu-lates.

ACKNOWLEDGMENT

We are grateful to John H. Freer, Department ofMicrobiology, New York University School of Medi-cine, New York, N. Y., for the electron micrograph.

LITERATURE CITED1. ANFINSEN, C. B. 1955. Aconitase from pig heart

muscle, p. 695-698. In S. P. Colowick and N.0. Kaplan [ed.], Methods in enzymology, vol.1. Academic Press, Inc., New York.

2. ARRET, B., AND A. KIRSHBAUM. 1959. A rapiddisc assay method for detecting penicillin inmilk. J. Milk Food Technol. 22:329-331.

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3. BURTON, K. 1956. A study of the conditions andmechanism of the diphenylamine reaction forthe colorimetric estimation of deoxyribonu-cleic acid. Biochem. J. 62:315-323.

4. GRELET, N. 1957. Growth limitation and sporula-tion. J. Appl. Bacteriol. 20:315-324.

5. HANSON, R. S., V. R. SRINIVASAN, AND H. 0.HALVORSON. 1963. Biochemistry of sporula-tion. I. Metabolism of acetate by vegetativeand sporulating cells. J. Bacteriol. 85:451-460.

6. HYATT, M. T., AND H. S. LEVINSON. 1957. Sulfurrequirement for postgerminative developmentof Bacillus megaterium spores. J. Bacteriol.74:87-93.

7. HYATT, M. T., AND H. S. LEVINSON. 1959. Util-ization of phosphates in the postgerminativedevelopment of spores of Bacillus megaterium.J. Bacteriol. 77:487-496.

8. JANSSEN, F. W., A. J. LUND, AND L. E. ANDER-SEN. 1958. Colorimetric assay for dipicolinicacid in bacterial spores. Science 127:26-27.

9. LEVINSON, H. S., AND M. T. HYATT. 1964. Effect ofsporulation medium on heat resistance, chem-ical composition, and germination of Bacillusmegaterium spores. J. Bacteriol. 87:876-886.

10. MILLER, G. L., AND E. E. MILLER. 1958. Deter-mination of nitrogen in biological materials.Anal. Chem. 20:481-488.

11. SRINIVASAN, V. R. 1965. Intracellular regulationof sporulation of bacteria, p. 64-74. In L. L.Campbell and H. 0. Halvorson [ed.], SporesIII. American Society for Microbiology, AnnArbor.

12. SUMNER, J. B., AND G. F. SOMERS. 1944. Labora-tory experiments in biochemistry. AcademicPress, Inc., New York.

13. SZULMAJSTER, J., AND R. S. HANSON. 1965.Physiological control of sporulation in Bacillussubtilis, p. 162-173. In L. L. Campbell and H.0. Halvorson [ed.], Spores III. AmericanSociety for Microbiology, Ann Arbor.

14. VINTER, V., AND R. A. SLEPECKY. 1965. Directtransition of out-growing bacterial sporesto new sporangia without intermediate cell di-vision. J. Bacteriol. 90:803-807.

15. YOUNG, I. E., AND P. C. FITZ-JAMES. 1959.Chemical and morphological studies of bac-terial spore formation. I. The formation ofspores in Bacillus cereus. J. Biophys. Biochem.Cytol. 6:467-482.

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