inducción de esporulación por decoyinina

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COMMUNICATION J. Biochem. 91, 1089-1092 (1982)

Induction of Bacillus subtilis Sporulation by Decoyinine and

the Concomitant Disappearance of ppGpp in Vegetative Cells

Kenji IKEHARA, Mayuko OKAMOTO, and Kin-ichi SUGAEDepartment of Chemistry, Faculty of Science, Nara Women's University,Kita-uoya-nishi-machi, Nara, Nara 630

Received for publication , December 14, 1981

Sporulation of Bacillus subtilis, growing exponentially in the presence of rapidlymetabolizable nutrients, was induced by addition of decoyinine (an antibiotic inhibitor of GMP synthesis), and intracellular amounts of ppGpp were determined after2 m formic acid extraction by polyethyleneimine (PEI)-cellulose thin-layer chromatography. Consequently, it was found that the ppGpp in vegetative cells abruptlydisappeared after the addition of decoyinine. This indicates that the disappearanceof ppGpp is closely correlated to the initiation of B. subtilis sporulation.

Sporulation of Bacillus subtilis, which is inducedby exposing cells to a limited supply of an essentialnutrient (carbon, nitrogen, or phosphorus source),has been used as a simple model system for studieson cellular differentiation or development (1-4).However, the search for the factor which regulatesthe initiation of sporulation has had only limitedsuccess until now. Recently, Freese and colleagueshave reported that sporulation can be induced inthe presence of excess nutrients under conditions

causing partial deprivation of GTP and GDP, andthat under all sporulation conditions, the guaninenucleotides decrease (5-9). From this, they sup-posed that GTP or GDP may be a factor for thecontrol of sporulation initiation. On the otherhand, we have proposed a novel hypothesis thatppGpp detected in B. subtilis vegetative cells is afactor for the regulation, based on the findingsthat ppGpp is present at a high level in the vegetative cells growing in nutrients-rich media (10),and that the nucleotide disappears from the cells

upon deprivation of the carbon source followedby the sporulation (11). But, these phenomena

might also be explained by the view that the

ppGpp in the vegetative cells plays a role not in

the initiation of sporulation but in the regulation

of metabolic pathways upon energy starvation,

such as catabolite repression. To determine which

of the above two possible explanations is valid and

to elucidate the relationship between the partial

decrease of GTP (and GDP) and the disappearance

of ppGpp, sporulation was induced in the presence

of excess carbon, nitrogen and phosphorus sources

by partial inhibition of GMP synthesis caused byaddition of decoyinine (6), and intracellular

amounts of ppGpp were measured after extraction

with 2 m formic acid.

B. subtilis 60015 (trp-, met-) was grown in

mS7 medium at 37•Ž with reciprocal shaking.

The mS7 medium was prepared by a slight modi

fication of S7 medium (6), and contained 100 mm

potassium morpholino propane sulfonic acid (pH7.1), 20 mm K-glutamate, I % glucose, 10 mm

(NH4)2SO4, 1 mm phosphate buffer (pH 7.1), 1 mm

MgCl2, 0.7 mm CaCl,, 50 ƒÊm MnClz, 5 ƒÊm ZnCl2,5 ƒÊm FeCls, 50 ƒÊg/ml L-tryptophan, and 20 ƒÊg/ml

Vol. 91, No. 3, 1982 1089



1090 COMMUNICATION

L-methionine. The growth was followed by tur

bidometry at 660 nm in a Hitachi model 100-10

spectrophotometer.

When an antibiotic inhibitor of GMP synthe

sis, decoyinine, was added to the exponentially

growing culture (Assn=0.5) to a final concentration

of 0.4 mg/ml, B. subtilis sporulation was initiated

efficiently as described by Mitani et al. (6), in spite

of the presence of excess nutrients. The decoy

inine, which was provided by Dr. Y. Fujita of

Hamamatsu Univ., was a gift from Dr. G.B.

Whitfield (The Upjohn Co., Kalamazoo, Mich.,

U.S.A). Under the sporulation conditions, re

fractile prespores were observed under a phase

contrast microscope at a ratio of about 40% to

total cells at 8 h after the addition of decoyinine,whereas none were detected at all in the control

culture without the antibiotic. This was also

confirmed by measurements of spore titer as shown

in Table ‡T. The total viable cell number was

measured by plating on tryptose agar plates and

the spore number was determined by heating the

diluted sample for 20 min at 75•Ž and then plating

on the plates. The sporulation frequency mea

sured at 20 h after addition of the antibiotic (T25)

was 0.38 but was only 1.7 x 10-5 without the com

pound.Intracellular phosphorylated compounds were

extracted from the continuously 3=P-labeled cells

grown in mS7 medium containing 20 ƒÊCi/ml of

H332PO, in the presence or absence of decoyinine,

as shown in Fig. 1. Conditions for extraction of

nucleotides with 2 m formic acid (pH 1.5), chro

matographic separation of them on PEI-cellulose

thin-layer plates (Polygram CEL300, Machery

TABLE ‡T. Spore formation induced by addition ofdecoyinine.

a Decoyinine was added to the culture to a final concen

tration of 0.4 mg/ml and each cell number was measuredat 20 h after the addition (T20). b Each cell number wasmeasured at T20without decoyinine.

Fig. 1. Growth curves of B. subtilis 60015 (circles) andrelative change of ppGpp (triangles) in cells grown inmS7 medium in the presence (open symbols) or absence(closed symbols) of decoyinine. Turbidity at 660 nmwas measured spectrophotometrically, and the ppGppcontent is plotted as the relative quantity to the nucleotide in cells just before the addition of decoyinine (T0).Procedures for the quantification have been describedpreviously (11). For the points indicated by upward-pointing arrows, cells were harvested and phosphorylated compounds were extracted with 2 M formic acid.The downward-pointing arrow indicates the time atwhich decoyinine was added to the exponentially grow-ing cells in mS7 medium.

Fig. 2. Autoradiogram of 2 M formic acid extracts of

B. subtilis cells grown in mS7 medium containing 20

ƒÊ Ci/ml of H332PO4, in the presence of decoyinine.

Extracts of equal amounts of cells were developed on

PEI-cellulose thin-layer plates with 1.5 M potassium

phosphate buffer (pH 3.4). Cells were harvested andsamples were extracted with 2 M formic acid at the times

indicated in Fig. I.

J. Biochem.



DISAPPEARANCE OF ppGpp FOLLOWING DECOYININE ADDITION 1091

TABLE ‡U. Nucleotide concentrations in B. subtilis cells

during sporulation induced by addition of decoyinine.

a The time after addition of decoyinine.

Nagel) and autoradiography have been previouslydescribed in detail (10). Figure 2 shows an auto-radiogram of 32P-labeled compounds which wereextracted from the cells grown in the presence ofdecoyinine and separated by one-dimensionalchromatography with 1.5 M potassium phosphatebuffer (pH 3.4). It can be seen in the figure thatspots of ppGpp and a material numbered 2 ( spot2 compound) detected in the extracts from thevegetative cells abruptly disappeared or decreasedafter the addition of the antibiotic. This wasconfirmed by quantitative measurements of the

compounds (Table II). For the measurements,regions on one-dimensional chromatograms corresponding to the radioactive areas detected on thefilms were cut out and counted in a Packard modelTRI-CARB 300C liquid scintillation system. 3.10x 104 cpm of 32P-radioactivity on the chromato

gram corresponded to 1 nmol of phosphate underthe conditions. Table II also shows that a usualguanine nucleotide, GTP, decreases partially afterthe addition of decoyinine, as previously describedby Lopez et al. (8), while ATP increases. On the

other hand, as was expected, the ppGpp from cellsin the control culture without decoyinine wasessentially constant even at T, (Table II). Figure3 shows a two-dimensional autoradiogram ofnucleotides extracted from B. subti/is vegetativecells. As a matter of course, the spot referred toas ppGpp in Fig. 3 certainly comigrated with theauthentic ppGpp on a separate two-dimensionalPEI-cellulose plate (solvent system 1; (10)).

Here, we wish to emphasize that the approachused in this work is much more useful for studying

the role of the ppGpp in vegetative cells, becausethe synthesis of catabolic enzymes (inositol de-

Fig. 3. Two-dimensional autoradiogram of 2 M formic

acid extracts of B. subtilis vegetative cells just before

addition of decoyinine (T0). The plate was developed

with solvent system I (first; 3.3 M ammonium formate,

0.68 M boric acid (pH 7.0), and second; 1.5 M potassium

phosphate buffer (pH 3.4) (10)).

hydrogenase, acetoin dehydrogenase, and sorbitol

dehydrogenase) remains repressed after the sporu

lation is induced by the addition of decoyinine,

as reported by Lopez et al. (12). Therefore, the

results in this paper support our hypothesis that

the disappearance of ppGpp is closely correlated

with the initiation of sporulation, not with the

regulation of metabolic pathways upon energysource deprivation.

Freese et al. have previously reported from

their extensive experiments that the decrease of

GTP (and GDP) concentration alone suffices to

initiate B. subtilis sporulation and GTP (and GDP)

may always play a decisive role in the initiation

(13, 14). However, we would again insist that

the ppGpp in B. subtilis vegetative cells may be

an effector, which regulates or represses the initi

ation of sporulation, based on the following facts;

(i) The ppGpp possesses unusual structural features. (úA) The hyperphosphorylated nucleotide is

present at sites from which it is hardly extractable

under mild conditions. We could confirm that

the ppGpp was not extracted by addition of formic

acid to a final concentration of 0.1 N to the grow-

ing culture, whereas it was easily extracted from

the same culture by addition of the compound to

a final concentration of 2 N (Ikehara et al., unpub

lished data). (úB) Certainly, the concentration of

GTP decreases after the addition of decoyinine

(Table ‡U, 8), but the decrease of ppGpp is muchmore abrupt and more extensive than that of

Vol. 91, No. 3, 1982



1092 COMMUNICATION

GTP (Table ‡U, 11). These facts may indicate

that ppGpp is more favorable as a regulation

factor than a usual nucleotide, GTP or GDP.

From this, it can be reasonably inferred that the

deprivation of the carbon source causes the partial

decrease of the intracellular concentration of GTP

and/or GDP which is followed by the disappear

ance of ppGpp, and that the disappearance of

ppGpp induces initiation of spore formation.

As seen in Fig. 2 and Table ‡U, the spot 2

compound also disappeared or decreased after the

addition of the antibiotic. This compound may

be related to B. subtilis sporulation, and, moreover,

it may be a unique nucleotide, judging from the

position of the spot after two-dimensional thin-

layer chromatography (Fig. 3). Experiments on

the structure and function of the spot 2 material

are now in progress in our laboratory.

We thank Dr. Yasutaro Fujita (Hamamatsu University

Medical School) for kindly supplying the antibiotic,

decoyinine, and for helpful discussion. We also thank

Professor Michio Kurata (Institute for Chemical Re-

search, Kyoto University) for allowing us to use a

microdensitometer (Rigaku-Denki model MP-3) for

quantification of the ppGpp detected on the films afterautoradiography.

REFERENCES

1. Piggot, P.J. & Coote, J. (1976) Bacteriol. Rev. 40,

908-962

2. Freese, E. (1976) Spore Res. 1, 1-323. Doi, R.H. (1977) Annu. Rev. Genet. 11, 29-484. Sonenshein, AL. & Campbell, K.M. (1978) in

Spore VII (Cambliss, G. & Vary, J.C., eds.) pp. 179-192, American Society for Microbiology, Wash

ington, D.C.5. Freese, E., Heinze, J., Mitani, T., & Freese, E.B.(1978) in Spore VII (Cambliss, G. & Vary, J.C., eds.)pp. 277-285, American Society for Microbiology,Washington, D.C.

6. Mitani, T., Heinze, J., & Freese, E. (1977) Biocheni.Biophys. Res. Cononun. 77, 1118-1125

7. Heinze, J., Mitani, T., Rich, K.E., & Freese, E.(1978) Biochim. Biophvs. Acta 521, 16-26

8. Lopez, J.M., Marks, C.L., & Freese, E. (1979)Biochim. Biophvs. Acta 587, 238-252

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1119 112510. Ikehar-, K., Ando, H., Takada, Y., & Sugae, K.(1981) J. Biochem. 89, 511-516

11. Ikehara, K., Maeda, K., Makino, S., & Sugae, K.(1981) J. Biochem. 89, 517-521

12. Lopez, J.M., Uratani-Wong, B., & Freese, E. (1980)J. Bacteriol. 141, 1447-1449

13. Lopez, J.M., Dromerick, A., & Freese, E. (1981)J. Bacteriol. 146, 605-613

14. Freese, E. (1981) in Sporulation and Germination(Levinson, H.S., Sonenshein, A.L., & Tipper, D.J.,eds.) pp. 1-12, American Society for Microbiology,

Washington, D.C.

J. Biochem.

inducción de esporulación por decoyinina

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