COMMUNICATION

Induction
the

of Bacillus

Concomitant

subtilis

Sporulation

Disappearance

Kenji IKEHARA,

Mayuko

by

of ppGpp

OKAMOTO

for publication

, December

Decoyinine

91,

1089-1092

(1982)

and

in Vegetative

Cells

, and Kin-ichi SUGAE

Department
of Chemistry,
Faculty of Science , Nara
Kita-uoya-nishi-machi,
Nara , Nara 630
Received

. Biochem.

Women's

University,

14, 1981

Sporulation
of Bacillus subtilis , growing exponentially
in the presence of rapidly
metabolizable
nutrients , was induced by addition of decoyinine (an antibiotic inhib
itor of GMP synthesis) , and intracellular amounts of ppGpp were determined after
2 m formic acid extraction
by polyethyleneimine
(PEI)-cellulose
thin -layer chro
matography.
Consequently,
it was found that the ppGpp in vegetative cells abruptly
disappeared after the addition of decoyinine.
This indicates that the disappearance
of ppGpp is closely correlated to the initiation of B . subtilis sporulation.

Sporulation
of Bacillus subtilis, which is induced
by exposing cells to a limited supply of an essential
nutrient (carbon, nitrogen , or phosphorus
source),
has been used as a simple model system for studies
on cellular differentiation
or development
(1-4).
However, the search for the factor which regulates
the initiation of sporulation
has had only limited
success until now. Recently, Freese and colleagues
have reported that sporulation
can be induced in

might

also

ppGpp

in

the

initiation

of

metabolic

such
of

the

to

elucidate
of

ppGpp detected in B. subtilis vegetative cells is a

factor for the regulation,
based on the findings
that ppGpp is present at a high level in the vege

The

tative cells growing in nutrients-rich

media (10),
and that the nucleotide disappears from the cells
upon deprivation
of the carbon source followed
by the sporulation
(11). But, these phenomena

7.1),

of

(and

induced

60015
at

S7

FeCls,

which

is valid
the

and
partial

disappearance
in

the

presence
sources

synthesis

caused

and

intracellular

measured

(trp-,

after

was

met-)

with

by

extraction

was

prepared
(6),

by

and

phosphate

50 g/ml

a slight

acid

I %

glucose,

buffer

(pH

L-tryptophan,

MnClz,

modi
100

sulfonic

50 m

in

shaking.

contained

propane

CaCl,,

grown

reciprocal

K-glutamate,
1 mm
mm

starvation,

determine

the

(6),

in

regulation

phosphorus

GMP

37

medium

mm

0.7

and

were

morpholino
20

and

was

the
not

acid.

medium
of

the

between

decoyinine

subtilis

mS7

in
energy

GDP)

that

a role

explanations

of

ppGpp

view

To

nitrogen

medium

(NH4)2SO4,

1089

relationship

inhibition

potassium

5 m

the

2 m formic

fication

MgCl2,

possible

of

B.
mS7

upon

two

carbon,

addition

with

but

repression.

GTP

partial

the
plays

sporulation

sporulation

amounts

by
cells

pathways

above

of excess

posed that GTP or GDP may be a factor for the

control of sporulation
initiation.
On the other
hand, we have proposed a novel hypothesis that

3, 1982

of

of ppGpp,

by

explained
vegetative

as catabolite

decrease

the presence of excess nutrients under conditions

causing partial deprivation of GTP and GDP, and
that under all sporulation
conditions, the guanine
nucleotides decrease (5-9).
From this, they sup-

Vol. 91, No.

be
the

10 mm
7.1),

5 m
and

mm
(pH

1 mm
ZnCl2,

20 g/ml

1090

COMMUNICATION

L-methionine.

The

bidometry

growth

at

660

nm

an

antibiotic

was

in

followed

Hitachi

by

model

tur

100-10

spectrophotometer.
When
sis,

decoyinine,

growing
of

culture

0.4

of

B.

as

the

by

of

excess

which

was

U.S.A).
fractile

(The

Upjohn

Under

the

prespores

contrast
cells

at

whereas

none

culture

without

confirmed
in

The
by

the

number

diluted
on

sample

the

sured

plates.
at

was

for

20

0.38

20

but

was

40%

in

the

This

control

was

titer

cell

as

also
shown

number

was

plates

determined

by

heating

75

to

decoyinine,

agar

addition
only

phase

of

spore

and

sporulation

after

re
a

tryptose

at

of
G.B.

Mich.,

about

all

viable

min

The

of

at

on

Fujita
Dr.

under

addition

total

spite

decoy

conditions,

of

was

Y.

from

antibiotic.

plating

in

Kalamazoo,

detected
the

Table T.

Dr.

gift

ratio

measurements

measured
spore

a
the

initiated

(6),
The

observed

at

were

by

by
a
Co.,

after

al.

nutrients.

were

8 h

et

sporulation

microscope

total

concentration
was

Mitani

was

synthe

exponentially

a final

provided

Univ.,

Whitfield

to

GMP

the

sporulation

described

Hamamatsu

of

to

subtilis

presence

inine,

added

(Assn=0.5)

mg/ml,

efficiently

inhibitor

was

then

and
the
plating

frequency
of

the

1.7 x 10-5

mea

antibiotic

without

(T25)
the

com

pound.
Intracellular
extracted
grown
H332PO,
as

shown

nucleotides
matographic

phosphorylated

from
in

the

compounds

continuously

medium

the

presence

or

1.

Conditions

2 m

formic

in
in

Fig.
with

containing

separation

thin-layer

plates

TABLE T.

Spore

of

(Polygram

formation

were

3=P-labeled

mS7

of
for
(pH
on

1.5),

of
chro

PEI-cellulose

CEL300,

induced

of

decoyinine,
extraction

acid
them

cells

20 Ci/ml

absence

Fig. 1. Growth curves of B. subtilis 60015 (circles) and

relative change of ppGpp (triangles) in cells grown in
mS7 medium in the presence (open symbols) or absence
(closed symbols) of decoyinine. Turbidity at 660 nm
was measured spectrophotometrically,
and the ppGpp
content is plotted as the relative quantity to the nucleo
tide in cells just before the addition of decoyinine (T0).
Procedures for the quantification have been described
previously (11). For the points indicated by upwardpointing arrows, cells were harvested and phospho
rylated compounds were extracted with 2 M formic acid.
The downward-pointing
arrow indicates the time at
which decoyinine was added to the exponentially growing cells in mS7 medium.

Machery

by

addition

of

decoyinine.

Fig.
B.

2.

Autoradiogram

subtilis

cells

Ci/ml
Extracts

a Decoyinine was added

tration of 0.4 mg/ml and
at 20 h after the addition
measured at T20without

to the culture to a final concen

each cell number was measured
(T20). b Each cell number was
decoyinine.

of

H332PO4,

of

equal

PEI-cellulose

indicated

in

buffer
were

in

in

(pH

extracted
Fig.

2 M

formic

mS7
the

amounts

cells

plates
3.4).
with

acid

medium

of
were

with
Cells

2 M formic

extracts

of

containing

presence
of

thin-layer

phosphate
samples

of
grown

20

decoyinine.
developed

1.5
were

harvested
acid

on

potassium
and

at

the

times

J.

Biochem.

I.

DISAPPEARANCE
TABLE U.
during

OF

Nucleotide
sporulation

FOLLOWING

concentrations
induced

a The time after

ppGpp

addition

by

in B.

addition

of decoyinine

of

subtilis

DECOYININE

ADDITION

cells

decoyinine.

.
Fig.

3.

acid

extracts

Two-dimensional

Nagel) and autoradiography

have been previously
described in detail (10). Figure 2 shows an autoradiogram
of 32P-labeled
compounds
which were
extracted from the cells grown in the presence of
decoyinine
and separated
by one-dimensional
chromatography
with 1.5 M potassium
phosphate
buffer (pH 3.4). It can be seen in the figure that
spots of ppGpp and a material numbered 2 ("spot
2" compound)
detected in the extracts from the
vegetative cells abruptly disappeared
or decreased
after the addition
of the antibiotic.
This was
confirmed
by quantitative
measurements
of the

addition

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

regulation

gram corresponded
to 1 nmol of phosphate under
the conditions.
Table II also shows that a usual

may

always

(13,

14).

guanine nucleotide, GTP, decreases partially after

the addition of decoyinine, as previously described
by Lopez et al. (8), while ATP increases.
On the
other hand, as was expected, the ppGpp from cells
in the control
culture without
decoyinine
was
essentially constant even at T, (Table II). Figure
3 shows a two-dimensional
autoradiogram
of
nucleotides
extracted from B. subti/is vegetative
cells. As a matter of course, the spot referred to
as ppGpp in Fig. 3 certainly comigrated with the
authentic ppGpp on a separate two-dimensional
PEI-cellulose plate (solvent system 1; (10)).
Here, we wish to emphasize that the approach
used in this work is much more useful for studying
the role of the ppGpp in vegetative cells, because
the synthesis of catabolic enzymes (inositol de-

the

ppGpp

an

effector,

Vol.

91, No.

3, 1982

1091

of

of

with

solvent

0.68

M boric

I (first;
and

buffer

(pH

3.4)

(10)).

induced

the

the

source

extensive

GTP

(and

initiate

al.

sporu

decoyinine,

Therefore,

our
is

the

hypothesis
closely

sporulation,

that

correlated
not

pathways

have

with

upon

GDP)

the

energy

play

B.

and

decisive
we

role

sporulation,

based

ppGpp
(A) The

hyperphosphorylated

present

at

from

mild
ppGpp

acid

to

culture,

the

same

a final
lished
GTP

not

a final

ing

which

conditions.
was

it

by

(Table U,

but
and

the

the

the

be
initi

facts;
fea

nucleotide
is hardly
could

confirm

addition

of

0.1

N to

the

of

extensive

that

of formic
the

grow-

extracted
the

addition

is

extractable

by

of

decrease

more

that

may

following

easily

Certainly,

after

initiation
insist

structural

of 2 N (Ikehara

(B)

8),
abrupt

was

addition

concentration

decreases

the

We

concentration

culture

data).

it

extracted

whereas

the

to

GDP)

cells

unusual

tures.

under

in

represses

on

possesses

sites

(and

again

or

of

suffices

vegetative

regulates

from

decrease

GTP

would

subtilis

which

of

the
alone

sporulation

in

reported

that

concentration

However,

The

previously

experiments

B. subtilis

ation

more

sorbitol

the

of

(12).

ppGpp

of

metabolic

et

their

the

and

deprivation.
Freese

(i)

of

initiation
of

al.

formate,

after

support

before

developed

1.5 M potassium

addition

et

paper

disappearance

with

the

Lopez

this

just

was

second;

repressed

by

by

in

plate

dehydrogenase,

remains

reported

cells

3.3 M ammonium

7.0),

acetoin

is

The

(pH

dehydrogenase)

results

(T0).

system

of 2 M formic

vegetative

acid

hydrogenase,

as

subtilis

decoyinine

phosphate

lation

autoradiogram

B.

from

compound
et

al.,

to
unpub

concentration

of

of

decoyinine

ppGpp

is

than

that

much
of

1092

COMMUNICATION

GTP

(Table U,

that
factor

is

than

From

of

and/or

the

GDP

ance

of

ppGpp

in

Fig.

also

addition

of

related
may

be

of

and

a
of

structure

after

are

now

We

thank

in

Dr.

Medical

for

and

Professor

Michio
Kyoto

in

Yasutaro

School)

decoyinine,

search,

function

progress

for

our

Fujita

University)

and,

of the

ppGpp

the
may

moreover,

judging

the

from

the
thin-

Experiments
"spot

2"

(Hamamatsu
supplying

on
material

(Institute
for

We
for

allowing

detected

University
the

discussion.

(Rigaku-Denki

quantification

2"

after

laboratory.

kindly

Kurata

"spot

compound

3).
of

helpful

microdensitometer

the

decreased

of

two-dimensional

(Fig.

and

formation.

nucleotide,

spot

chromatography

the

disappearance

sporulation,

unique
the

GTP

disappear

the

This

the

partial
of

the

spore

or

antibiotic.

B. subtilis

the

Table U,

disappeared

the

to

position

that

by

GDP.
that

concentration

initiation

seen

or

causes

followed

and

regulation

inferred

source

is

ppGpp,

As

layer

carbon

indicate

GTP

reasonably

intracellular

induces

may
as

nucleotide,

be

which

compound

be

can

facts

favorable

usual

it

of the

decrease

These

more

this,

deprivation

it

11).

ppGpp

thank

Chemical
us

model
on

antibiotic,
also

to

Reuse

MP-3)
the

films

a
for

after

autoradiography.

2. Freese, E. (1976) Spore Res. 1, 1-32

3. Doi, R.H. (1977) Annu. Rev. Genet. 11, 29-48
4. Sonenshein, AL. & Campbell, K.M. (1978) in
Spore VII (Cambliss, G. & Vary, J.C., eds.) pp. 179192, 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
9. Vasantha, N. & Freese, E. (1980) J. Bacteriol. 144,
1119 1125
10. 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.

REFERENCES
1.

Piggot,

P.J.

&

Coote,

J. (1976)

Bacteriol.

Rev.

40,

908-962

J. Biochem.