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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
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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.
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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
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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.
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