Journal qf Generul Microbiology (1989), 135, 1335-1 345. Printed in Great Britain
1335
Dissection of the Expression Signals of the spoUA Gene of Bacillus subtilis:
Glucose Represses Sporulation-specific Expression
By S H I N Y A Y A M A S H I T A , ' ? F U J I 0 K A W A M U R A , '
HIROFUMI YOSHIKAWA,' H I D E 0 TAKAHASHI,'*
YASUO KOBAYASHI* AND H I U G A SAITOII
Institute of'Applied Microbiology, University of Tokyo, Bunkyo-Ku, Tokyo 113, Japan
Department of' Applied Biochemistry, Hiroshima University, Fukuyama 720, Japan
(Received 12 January 1989 ;accepted 30 January 1989)
The expression of the spo0A-lac2 fusion gene was partially repressed in the presence of an
excess of glucose. Expression was restored either by the mutation sigA47 (crsA47)or by addition
of decoyinine, an inhibitor of GMP synthetase, to the medium. By constructing a lac2 fusion
with a smaller fragment of the spo0A gene, we observed a /I-galactosidase profile in which
expression was completely repressed by an excess of glucose. This expression was restored by the
addition of decoyinine. These results indicate that the expression of the spo0A gene is regulated
by at least two different mechanisms, one sensitive to glucose, the other not. Furthermore, the
glucose-sensitive regulation was shown to reside at the transcriptional level. It is likely that the
reduced expression of the spo0A gene in the presence of glucose at an early stage of sporulation
causes the repression of sporulation.
INTRODUCTION
Sporulation in Bacillus subtilis is repressed at a very early stage by the presence of an excess of
glucose (Freese et al., 1970; Schaeffer et al., 1965; Takahashi & MacKenzie, 1982). A number of
mutants that overcome this repressive effect of glucose were isolated by Takahashi (1979). These
mutations were located at six distinct loci, crsA to crsF, on the chromosome (Sun & Takahashi,
1982, 1984). Recently, it was shown that three crsA mutations, crsAl, crsA4 and crsA47, were
identical two-base changes within the sigA (previously called rpoD; see Losick et al., 1986)
coding sequence encoding the major 043factor of RNA polymerase (Kawamura et al., 1985),
and that they could suppress various sporulation defects (Kawamura et al., 1985; Leung et al.,
1985). Therefore, it was suggested that the 043factor was involved in catabolite repression by
glucose, and that it interacted either directly or indirectly with sp00 gene products during the
initiation of sporulation.
Freese and his co-workers found that under most sporulation conditions there was a
corresponding decrease in the intracellular levels of G D P and GTP (Freese, 1981 ; Freese et al.,
1970; Lopez et a/., 1979; Losick et al., 1986). They demonstrated the importance of guanine
nucleotide deprivation for the initiation of sporulation by treating cells with decoyinine, which
is an inhibitor of GMP synthetase. A partially growth-inhibitory concentration of this drug
induced sporulation even in the presence of an excess of glucose (Lopez et al., 1979, 1980).
The spo0 gene products are candidates for the sensors that measure the concentration of
nutrients in the environment. In recent years, several laboratories have isolated and studied the
spo0 genes in an attempt to understand the regulation of their expression and the function of
t Present address : Central Research Laboratory, Nippon Suisan Kaisha, Ltd, Hachioji-shi, Tokyo 192, Japan.
$, Present address : School of Medicine, Teikyo University, Hachioji-shi, Tokyo 192, Japan.
0001-51 19
0 1989 SGM
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1336
S . YAMASHITA A N D OTHERS
their gene products (Bouvier et al., 1984; Ferrari et al., 1985a; Kudoh et al., 1985; Dubnau et al.,
1988; Trach et al., 1985; Yoshikawa et al., 1986). The results of lac2 fusion analyses have
suggested that the expression of spoOB and spoOH genes is not affected by addition of an excess of
glucose (Smith et al., 1985; Dubnau et al., 1988).
We reported previously that the expression of a spo0A-lac2 fusion was stimulated at the end
of vegetative growth in Spo+ cells and that this stimulation was greatly reduced in various sp00
mutants (spoOA, spoOB, spoOE, SPOOFand spoOH) (Yamashita et al., 1986). It has been suggested
that spoOA, a mutation which exerts highly pleiotropic effects, plays the most important role in
the initiation of sporulation (Ferrari et al., 1985b; Piggot & Coote, 1976). To determine how
spoOA gene expression is regulated during vegetative growth and during the initial stage of
sporulation, we have studied the effect of glucose and decoyinine on spoOA gene expression by
using spoOA-lac2 fusions.
METHODS
Bacteria. The Bacillus subtilis strains used in this study were UOT-1285 [trpCZ lys-I aprAA3 nprEI8 nprRZ] and
UOT-1296 [trpC2 lys-I aprAd3 nprEZ8 nprR2 sigA47 (crsA47)I. These strains were derived from JH642 (Bacillus
Genetic Stock Center 1 A96), and the mutations aprAA3 nprE18 nrpR2 were transferred from DB-104 (Kawamura
& Doi, 1984). Detailed construction of these strains will be published elsewhere.
DNA sequencing analysis. DNA sequencing by the dideoxy chain termination method of Sanger et al. (1977) was
performed as described by Messing (1983) with the sequencing kit obtained from Amersham.
Enzymes and chemicals. Restriction endonucleases, DNA polymerase I large fragment, Ba131 nuclease and T4
DNA ligase were purchased from Takara Shuzo Co. and Boeringer Mannheim-Yamanouchi. 5-Bromo-4-chloro-3indolyl P-D-galactopyranoside (X-Gal) and o-nitrophenyl P-D-galactopyranoside (ONPG) were obtained from
Sigma. Decoyinine was kindly supplied by Ajinomoto Co.
Medium. The composition of 2 x SG medium (L.eighton & Doi, 1971) is similar to Schaeffer's sporulation
medium but contains twice the concentration of Nutrient Broth (Difco) and 0.1 % or 2.0% (w/v) glucose. SBM
(spore basal medium) was described by Takahashi (1979).
Assayfbr sporulation. B. subtilis cells were grown in 2 x SG medium containing 0.1 % or 2.0% glucose at 37 "C
with shaking. After removal of a sample for the assay of P-galactosidase activity, the remaining culture was
incubated for 24 h, and then sporulation frequency was measured by heating for 10min at 80°C followed by
plating. Viable cells were also determined after 24 h incubation.
P-Gafactosidase actioity assay. B. subtilis cells lysogenized with #XM phages harbouring spo0A-lac2 fusion
genes were grown at 37 "C in 2 x SG medium containing 0-1% or 2.0% glucose. At 30 min or 60 min intervals
during the subsequent growth, 0.1 or 0.2 ml of the culture was withdrawn for the assay. P-Galactosidase activity
was assayed according to the method of Miller (1972) with the modification described by Wang & Doi (1984). One
unit of P-galactosidase activity is defined as the amount of enzyme which produced 1 nmol o-nitrophenol min-I at
28 "C, pH 7.0. The activity was calculated according to the following equation:
P-galactosidase unit = (1000 x A420)/(tx V x OD,(,,)
where t represents the time of the enzyme reaction in min, V is the volume of culture used in the assay in ml, and
the ODbboreflects the cell density just before assay.
RNA analysis. RNA was purified from B. subtilis UOT-1285 cells harvested at stage t2 of a culture incubated in
2 x SG medium containing 0.1% or 2.0% glucose, iis described by Gilman & Chamberlin (1983). The RNA
samples were spotted onto a nylon filter and probed with spo0A-specific DNA (1.4 kb of s p d A ClaI-Sac1
fragment electrophoretically purified from 4CA1; Yamashita et al., 1986). The 32P-labelled DNA probe
containing the spoOA gene was prepared by nick-translation using a nick-translation kit (Bio-Rad).
Structure ojrecombinant phages 4 C A Z I , 4 C A Z 6 and 4 C A Z 7 . All the constructed fusions are illustrated in Fig.
1. The construction of 4CAZl was described previously (Yamashita et al., 1986). Another recombinant phage,
4CAZ6, was constructed in a similar manner to that described previously (Yamashita et al., 1986)after treatment
of 4CAl with Ba131 nuclease. The resultant phage @CAZ6contained 230 bp of B. subtilis DNA preceding the
gene junction with the 9th codon of spo0A fused to the 9th codon of lac& and the upstream region of this fusion was
shortened by 41 bp as shown in Figs 1 and 2.
In order to ascertain that the spo0A-lacZ fusions were not transcribed from the vector sequence by read-through
transcription but from the spoOA promoter, we constructed a spo0A-lacZ fusion (4CAZ7) lacking the spoOA
promoter region by using the HpaI site (26 bp downstream from the transcription initiation site) as shown in Figs I
and 2.
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.----’,
Glucose repression of spoOA gene expression
193
$CAZl
1337
lacZ
+
Met
146
P
+ 26
$CAZ7
-’-
dCAZ6
+I
+ 146
+17
Fig. 1. Structure of 4CAZ1, 4CAZ6 and $CAZ7 carrying the spoOA-lacZ fusions. Three different
lengths of spoOA 5’-upstream regions containing N-terminal coding regions were fused to the facZ gene
and the fusions were then inserted into the unique BurnHI site (Ba) of 4 C M . The transcription starts at
nucleotide 1 (Ferrari et a f . ,19856).The D N A sequences at the junction sites were confirmed by D N A
sequence analysis.
+
$CAZ 1
r
-35
-10
4CAZ6
~ATCCCTC~~A~~TCTCAGAATACATACGG~~~~~~ATACAAAAGAACATTTTTCGACAAATTCACGTTTCCTT
-193
-150
GTTTGTCAAATT rCATTTTTACTCCAAAAACAGA~AAAAACATAGAATAACAAAGATATGCCACTAATATTGGTG
-100
- 35
- 50
4CAZ7
v
- 10
e
ATTATCATTT~TTAGAGCGTATATAGCGG~TCTCGAATCT~AACATGTAGCAAGGGTCAATCCTGTTAACTA
+1
4CAZ6
Hpa I
1
Met G l u LYS lie LYS V a l C Y S V a l A l a A s p A s p Asn A r g Glu
CATlTGCGGAGCAAGAAAC CTC GAG AAA A l l AAA CTT TGT GTT CCT GAT GAT AAT CCA GAG
4CAZ 1 7
1
~
Leu V a l Set- Leu Leu Ser Glu T y r l i e G l u G l y Cln Glu A s p Met Glu V a l l i e G l y
CTC GTA ACC CTG T T A ACT GAA T A T ATA CAA GGA CAC CAA CAC ATG CAA GTG ATC GGC
Fig. 2. D N A sequence of the spoOA 5’-upstream region. The deduced amino acid sequence is given
above the base sequence for the nontranscribed strand. The transcription initiation site is shown by a
filled arrow at nucleotide
1. The putative - 35 and - 10 regions of the spoOA promoter are
underlined, and putative - 35 and - 10 regions of the secondary spoOA promoter (see Discussion) are
dashed-underlined. The nucleotide junction sites of the spoOA sequence with phage vector $ C M and
with the fucZ gene in $CAZl, 6 and 7 are indicated by arrows.
+
RESULTS
Eflects of glucose on the expresson of spo0A-lac2 fusion and on sporulation
To examine the effects of glucose on both the expression of spo0A-lac2 and the sporulation
frequency, 4CAZ1 was introduced into the chromosome of a wild-type (UOT-1285) and of a
sigA47 mutant which was able to sporulate in the presence of high concentrations of glucose
(Takahashi, 1979). The /I-galactosidase activities (Fig. 3) and the sporulation frequencies (Table
1) of the lysogens were determined as described in Methods. The cells were grown in 2 x SG
medium because it gave fairly constant values of repressed sporulation frequency under glucoserich (2.0%) conditions as compared with Schaeffer’s sporulation medium. In the case of the
wild-type strain, not only the expression of the spo0A-lac2 fusion during an early stage of
sporulation but also the sporulation frequency was significantly reduced by the addition of 2.0%
glucose, while with the sigA47 mutant, both behaviours were little affected by glucose. Fig. 3 also
shows that the level of spoOA-directed P-galactosidase activity in the sigA47 mutant was much
higher than that in the wild-type during the vegetative and early stationary phases regardless of
the presence or the absence of glucose. The wild-type strain harbouring 4CAZ7, in which the
spoOA promoter had been removed, showed very low enzyme activity throughout growth
(Fig. 3).
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1338
S . YAMASHITA A N D OTHERS
1000
h
.-
C
c
W
x
.->
C
.-Y
cd
2 500
(d
2
B
C
cd
d
$
Qi
0
- 1 0
1
2
Time (h)
3
Fig. 3. Effect of glucose on the expression of spo0A-lucZ fusions 6CAZ1 and 6CAZ7. P-Galactosidase
activity (see Methods for definition of units) was measured for the following strains carrying 4CAZI :
UOT-1285 (wild-type) in the presence of 0.1 % glucose (0)or 2.0% glucose ( 0 )UOT-1296
;
(sigA47) in
the presence of 0.1% glucose ( 0 )or 2.0% glucose (a);and for UOT-1285 carrying 4CAZ7 in the
presence of 0.1 % glucose
0 indicates to, the end of exponential growth, unless otherwise stated.
a).
Table 1. Sporulation frequency of strains carrying 6 C A Z I
Cells were inoculated into 2 x SG medium containing the indicated concentration of glucose and
incubated at 37 "C with shaking. After 24 h, viable cells and spores were determined as described in
Methods.
Strain
UOT-1285 (wild-type)
UOT-1296 (sigA47)
Glucose
(%I
0.1
2-0
0.1
2.0
Viable cells ml-I
(V )
1.5 x
2.1 x
1.6 x
2.4 x
109
109
109
109
Spores ml-l
(S)
1.0 x
7.8 x
7.2 x
1.6 x
109
106
10'
10'
SlV
(%I
66
0.3
45
7
To clarify whether the effect of glucose repression on spoOA gene was at the transcriptional
level, mRNA dot-blot analysis was carried out. A typical result is shown in Fig. 4. It is evident
that spoOA transcription was repressed by addition of glucose during an early stage of
sporulation.
Eflects of decoyinine on the expression of spo0A-lacZ fusion and on sporulation
We examined the effects of decoyinine on both the expression of spo0A-lac2 fusion and the
sporulation frequency in the presence of 2 4 % glucose. First we used the synthetic medium
(SBM), since sporulation in this medium is repressed by glucose more drastically than in a rich
medium such as 2 x SG (see Tables 1 and 2a; Takahashi, 1979). Wild-type (UOT-1285) cells
harbouring 6CAZl were grown in SBM with 2.0%glucose and at OD,,, 0.5,2.5 mM-decoyinhe
was added to the medium, Samples were withdrawn for assay of P-galactosidase activity (Fig, 5)
and the remaining cultures were further incubated for 24 h to determine sporulation frequencies
(Table 2 a). Addition of decoyinine caused significant restoration of spo0A-lac2 expression in
the glucose-repressedcondition, although the level of the enzyme activity was lower than that in
the sigA47 mutant without decoyinine (Fig. 5). The addition of decoyinine also restored the
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1339
Glucose repression of spoOA gene expression
1
2
Fig. 4. Dot-blot analysis of B. subtilis RNA for spoOA sequence. Decreasing amounts of RNA (40.0,
20.0, 10.0, 5.0 and 2.5 pg per spot) from UOT-1285 cells harvested 2 h after the end of exponential
growth ( r J were spotted onto nylon filter and hybridized with 32P-labelledspd)A-specific DNA. Lane
1, in the presence of 2.0% glucose; lane 2, in the presence of 0.1 % glucose.
3
0
1
2
3
Time (h)
4
-
0
-
1
0
1
2
Time (h)
3
Fig. 6
Fig. 5
Fig. 5. Effect of sigA47 mutation or decoyinine on the expression of spo0A-lucZ fusion in SBM.
P-Galactosidase activity of strains carrying 4CAZ1 and grown in SBM containing 2.0%glucose was
measured for the following conditions: UOT-1285 (wild-type) in the absence (0)or presence of 2.5 mMdecoyinine (a);UOT-1296 (sigA47) in the absence of decoyinine (B).The arrow indicates the time
decoyinine was added (time 0).
Fig. 6. Effect of decoyinine on the expression of spoOA-lac2 fusion in the presence of glucose.
j-Galactosidase activity was measured for strain UOT-1285 (wild-type) harbouring 4CAZ1, cultured
in 2 x SG medium under the following conditions: in the presence of 0.1%glucose (0)or 2.0%glucose
(a);in the presence of 2.0%glucose with 2.5 mM-decoyinine added to the culture at to as indicated by
the arrow
(m).
sporulation frequency that was repressed by glucose, in agreement with the results of Mitani et
al. (1977).
Since it is difficult to study the expression of the spoOA gene in the transient stage from the
vegetative to the sporulation phase in SBM, 2 x SG medium was used to study the effect of
decoyinine on spoOA expression. The profiles of Q-galactosidase activity and the sporulation
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1340
S. YAMASHITA A N D O T H E R S
Table 2. Eflect of decoyinine on the sporulation frequency of strains carrying 4 C A Z 1
Strains were grown in the SBM (a) or 2 x SG medium (6) containing 2.0% glucose in the presence or
absence of 2.5 mhl-decoyinine and viable cells and spores were determined as described in Methods.
Dec., decoyinine.
Strain
(a)
UOT-1285
UOT-1296
(b) UOT-1285
Medium
SBM
SBM Dec.
SBM
2 x SG
2 x SG Dec.
+
+
Viable cells ml-l
( v>
Spores ml-l
(S)
SlV (%>
4.8 x 10'
1.3 x lo8
1.6 x 10'
2.2 x 109
1.7 x 109
2.0 x 103
1.5 x 107
1.7 x 107
6.5 x lo6
3.4 x 108
0.0004
12
11
0.3
20
frequencies are shown in Fig. 6 and Table 2(b),respectively. The restoration of sporulation by
decoyinine, to 20% of the non-repressed sporulation frequency from 0.3 % under the repressed
condition, could be seen also in 2 x SG medium. The stimulation of spo0A-lac2 expression
could be observed also in this medium by addition of decoyinine at the end of vegetative growth
(to).The results strongly suggest that the restoration of spo0A-lacZ activity and sporulation are
related phenomena. This idea is also supported by the results obtained by using the sigA47
mutant. This mutant is able to sporulate even under glucose-repressed conditions (Tables 1 and
2a), and spo0A-lacZ expression in this mutant was high even in the presence of an excess of
glucose, as shown in Figs 3 and 5.
Eject of glucose on the expression of spo0A-lacZ fusion in the wild-type strain harbouring
4 CAZ6
In order to determine the repression-sensitive region of the spoOA gene, we made another
recombinant phage, 4CAZ6, which contained the lac2 fusion with the spoOA gene having 27 bp
(9 codons) of the N-terminal region in addition to the 202 bp 5'-flanking region (Fig. 1). Fig. 7
shows the P-galactosidase activity profiles in strain UOT- 1285 carrying 4CAZ6. To our
surprise, the expression of the spo0A-driken P-galactosidase activity of 4CAZ6 during
vegetative growth was substantially diminished. Furthermore, it was of great interest that by
addition of 2.0% glucose, the induction of spo0A-lacZ was completely blocked. On the other
hand, in the case of strain UOT-1285 harbouring 4CAZ1, spo0A-lacZ expression was neither
affected during vegetative growth nor completely repressed at an early stage of sporulation in
the presence of an excess of glucose. These results suggested that the spoOA 5'-upsteam region
comprising 41 bp, between - 152 and - 193 bp, is not required for the stimulation of spoOA gene
expression sensitive to glucose at an early stage of sporulation but is essential for spoOA gene
expression during vegetative growth.
The enzyme activity directed by 4CAZ6 did not show a fall in activity after t 2 (Fig. 7 ) . It has
recently been reported that the repression of spoOA expression would be caused by the increased
amount of the spoOF gene product after t 2 (Yamashita et al., 1986; Chibazakura et al., 1988). It is
thus most likely that the deleted region in 4CAZ6 is required for efficient repression through the
increased amount of spoOF gene products.
In order to study further the effect of glucose on spoOA expression as well as sporulation we
used strain UOT- 1285 harbouring 4CAZ6, since 4CAZ6-directed expression of spo0A-lacZ
was completely repressed by an excess of glucose. Glucose was added to the culture at various
stages of growth, to a final concentration of 2.0%. The sporulation frequencies and the
P-galactosidase activity profiles are shown in Table 3 and in Fig. 8, respectively. Although
spo0A-lac2 expression was repressed by addition of 2.0% glucose until t l .5, neither spo0A-lac2
expression nor sporulation frequency was repressed by the addition of 2.0% glucose after t 1 . 5 .
The reason why sporulation was heavily repressed when glucose was added at
and tl.o
(Tables 1, 26 and 3) is not understood at present.
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Glucose repression of spoOA gene expression
-
1
0
1 2
Time (h)
3
4
I
1
0
1
Fig. 7
1
1
2
3
Time (h)
1
1
4
Fig. 8
Fig. 7. Effect of glucose on the expression of spo0A-lac2 fusion in strain UOT-1285 (wild-type)
harbouring 4CAZ6, in which lac2 is fused with the spo0A deletion (see Figs 1 and 2). P-Galactosidase
activity was measured for cells grown in the presence of 0.1%glucose (0)
or 2.0% glucose (m).
Fig. 8. Effect of the time of addition of 2.0 glucose on the expression of spoOA-lacZ fusion in strain
UOT- 1285 (wild-type) harbouring 4CAZ6. P-Galactosidase activity was measured for cells grown in
the presence of 0.1 %glucose alone (m), or 2,0%glucose added at t o . 5 (O), (a),t1.5
tz.o
t2.5
a),u),
(0).
Table 3. Eflect of the time of addition ofglucose on the sporulation frequency of UOT-1285
carrying 4 C A Z 6
Cells were grown in 2 x SG containing 0.1 % glucose at 37 "C with shaking, and 2.0%glucose was added
at the indicated times. After 24 h, viable cells and spores were determined as described in Methods.
Time of
glucose addition
Viable cells ml-1
(V )
i . ~ xi
34 x
24 x
3.6 x
24x
1.6 x
109
lo9
109
lo9
109
lo9
Spores ml-l
(S)
4.0 x
6-6 x
2.4 x
2.4 x
1.9 x
1.0 x
10'
lo3
104
109
109
109
SlV
(%I
29
0*0002
0.0001
67
79
63
Efect of decoyinine on the expression of spo0A-lacZ in the wild-type strain harbouring 4 C A Z 6
Next, we studied the effect of decoyinine on spo0A-lacZ expression and sporulation under the
glucose-repressed condition by using 4CAZ6. Strain UOT-1285 harbouring 4CAZ6 was grown
in the presence of 2.0% glucose, and 2.5 mhl-decoyinine was added into the medium at various
periods of growth. The sporulation frequencies are listed in Table 4, and the P-galactosidase
activity profiles are shown in Fig. 9. In the absence of decoyinine, P-galactosidase expression
was completely repressed in cultures containing 2.0% glucose, while the induction of the enzyme
activity was restored by addition of decoyinine to the cultures prior to the end of exponential
growth (to).However, this restoration effect was not observed when decoyinine was added after
to.5. It should be noted that enzyme induction occurred only at a definite stage around t l
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S . YAMASHITA A N D OTHERS
1ooc
h
m
U
.-
c
W
h
.->
Y
."
U
5oa
?i
2
Y
cb
B
d
@i
0
Time (h)
Fig. 9. Effect of the time of addition of decoyinine on the expression of spo0A-lucZ fusion in strain
UOT-1285 carrying 4CAZ6. P-Galactosidase activity was measured for cells grown in the presence of
0.1 % glucose alone (0),or in the presence of 2.0% glucose with 2-5 mM-decoyinine added at t-l.5 (O),
t-o.s (O), t-0.25 (A),
fo
to.j (O), and no addition of decoyinine (m).
u),
Table 4. Eflect of the time of addition of decoyinine on the sporulation frequency of UOT-1285
carrying 4 C A Z 6
Cells were grown in 2.0% glucose at 37 "C with shaking and received 2.5 mM-decoyinine at the
indicated times. Viable cells and spores were determined as described in Methods.
Time of
decoyinine addition
Viable cells ml-1
(V)
8.0 x
2.8 x
2.3 x
1.5 x
lo8
109
109
109
1.6 x 109
1.8 x 109
Spores ml-l
(S)
2.1 x 105
1.1 x 109
1.2 x 109
5.7 x 108
1.8 x lo8
1.0 x 108
SlV
(%I
0-03
39
52
38
11
6
regardless of the time of decoyinine addition. The glucose-repressed sporulation was restored to
near the non-repressed level when decoyinine was added no later than to. Thus the derepression
of sporulation seems to occur by the same mechanism as that which restores the spo0A-laddriven P-galactosidase activity.
DISC! USS I 0 N
As an approach to investigate how the spoOA gene of B . subtilis is regulated, we constructed
translational fusions between the N-terminal portion of spoOA protein and the lac2 protein of E.
coli, and inserted a single copy of these gene fusions into the B. subtilis chromosome using the
temperate phage 4 C M (Seki et al., 1986).
The expression of spo0A-lac2 in 4CAZl was stimulated at about the cessation of vegetative
growth; however, in the presence of an excess of glucose this stimulation of the spo0A-lac2
fusion did not occur. This indicates that at least part of the regulatory apparatus of the spoOA
gene is under the control of the glucose repression. Assay of spoOA mRNA at t2 indicates that the
reduced expression of spoOA-lac2 is due to the reduced synthesis of spoOA mRNA.
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Glucose repression of spoOA gene expression
1343
We found that the 5’-upstream region of the spoOA gene, from position - 152 to - 193, which
is upstream from the transcription initiation site reported by Ferrari et al. (1985b), was
responsible for the spoOA gene expression during vegetative growth. After deletion of this region
from the spo0A-lac2 fusion, the j-galactosidase assay showed a characteristic expression profile
which appeared after t , but was completely repressed in glucose-rich conditions. On the other
hand, the expression of spo0A in vegetative growth is not affected by an excess of glucose. These
results suggest that the expression of spoOA gene is subjected to at least two different regulatory
signals: one functions in vegetative growth through early stationary phase and is resistant to
glucose; the other is induced at stationaxy phase and is sensitive to glucose.
The sequence of the putative spoOA promoter whose - 35 and - 10 regions are underlined in
Fig. 2 has been reported by Ferrari et al. (1985b). Our results obtained with 4CAZ6 suggest that
the sequence between - 152 and - 193 may contain another promoter which is utilized during
vegetative growth. As indicated by the dashed underlining in Fig. 2, there is a putative promoter
sequence that closely resembles the promoter consensus sequence recognized by the major
vegetative sigma factor c~~~ (Moran et al., 1982). The - 35 (TTCACT) and - 10 (TAAAAT)
regions of this second spoOA promoter are identical to the consensus sequence for c~~~ promoter
( - 35 ‘TTGACA’, - 10 ‘TATAAT’) in 4 of 6 and 5 of 6 positions, respectively. However, since
4CAZ6 also has a downstream deletion relative to 4CAZ1, we cannot exclude the possibility
that the N-terminal coding region, +77 to 146 bp downstream from the transcription start,
may be required for the spoOA gene exlpression during vegetative growth.
It has recently been shown by S1 nuclease mapping of in vivo transcripts that spoOF is
regulated from dual promoters: one is insensitive to glucose and transcribed at low level only
during vegetative growth, while the other is sensitive to glucose and transcribed actively during
the sporulation phase (Lewandoski et a / . , 1986). It has been also shown that induction of both
spoOA and spoOF expression during sporiilation phase is required for spoOA, spoOB, spoOE, spoOF
and spoOH gene products (Yamashita et d.,
1986). However, it is not yet clear whether these spoO
gene products are directly required for the induction of spoOA and SPOOF.Although the
expression pattern of spoOA and SPOOFis similar, it is not known whether the mechanism
regulating the differential transcription from their promoters is shared.
The effect of glucose and decoyinine on spo0A-lac2 expression and sporulation was examined
in detail by using 4CAZ6, in which ~ p ~ l 0 A - 1expression
~~2
occurs only during the sporulation
initiation stage and is completely repressed by glucose. spo0A-lac2 expression and sporulation
were both repressed when glucose was added by tl.o, but not after t , . 5 , suggesting that enough
spoOA product has already been synthesized by t , to allow sporulation. The results from
experiments with decoyinine strongly suggest that the reduction of intracellular levels of GDP
and GTP caused by treatment with decoyinine results in relief from glucose repression of the
spoOA gene. It was necessary to add the decoyinine before to to restore the expression of spoOAlac2 fusion from glucose repression. Furthermore, the induction of expression of the spo0A-lac2
fusion always occurred at the same stage ( t , )in all restoration patterns. These results suggest that
there is a critical stage in the cellular ]physiological state; once the cells are past this specific
stage, spoOA expression cannot be restored by addition of decoyinine to the glucose-rich
medium. Thus it is likely that guanine nucleotide starvation has occurred before the specific
stage for the restoration of the expression of spo0A-lad.
Although the mechanism may be difkrent from the case of decoyinine, a sigA47 mutation also
activates the expression of spo0A-lac2 in glucose-rich conditions. This mutation not only
restores the sporulation frequency in glucose-rich conditions (Table 1) but also suppresses other
sporulation-deficient mutations in spoOB, spoOE and spoOF genes (Kawamura et al., 1985; Leung
et al., 1985). Moreover, our previous observations (Yamashita et al., 1986) and other
experiments using a sof-l mutant (Kawamura & Saito, 1983; Sharrock et al., 1984; Hoch et al.,
1985) suggest that the expression of spoOA gene is absolutely necessary for sporulation and that
other spoO genes, i.e. spoOB, spoOE and SPOOF,are required only to stimulate the spoOA gene. In
fact we have recently found that when present in multiple copies, spoO.4 could restore the ability
of these spo0 mutants to sporulate (unpublished results). These results suggest that the repression
of sporulation by glucose is due to the reduced expression of the spoOA gene, and also that the
+
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1344
S . YAMASHITA A N D OTHERS
elevated expression of spoOA can suppress both glucose repression of sporulation and some sp00
mutations, such as spoOB, spoOE and SPOOF.
It has been shown that the products of the spoOA and SPOOFgenes are homologous to a class of
protein that may have similar roles in diverse systems (Trach et al., 1985; Yoshikawa et al.,
1986; Ikeuchi et al., 1986). Many of the members of this gene family code for positive regulatory
elements and are considered to be components of information-transducing pathways, in that
each provides a mechanism by which bacteria can obtain some signals from their environment
and carry out appropriate responses. Thus spoOA and spoOF are candidates among the sp00 genes
for the sensor(s) that recognize(s) the cell’s nutritional status and transmit(s) this information for
the proper expression of genes.
We have shown here that the induction of spoOA expression at the initial stage of sporulation
is completely repressed by glucose. We have also shown that the induction of spoOA expression
occurs at an earlier stage than that of spoOF (Yamashita et al., 1986), and found that SPOOF
expression during the sporulation phase in a temperature-sensitive spoOA mutant was inhibited
immediately after the culture was transferred to high temperature (unpublished results),
indicating that the spoOA gene product acts directly as a positive regulator of SPOOFexpression
during an early stage of sporulation.
It is thus of great interest to clarify the mechanism by which spoOA expression is induced at the
end of exponential growth under nutritional deprivation and to study how glucose inhibits this
induction.
We are very grateful to Ajinomoto Co., for kindly supplying decoyinine. This research was supported by a
Research Grants for Life Science from the Institute of Physical and Chemical Research (Riken) and by a Grantin-Aid for Encouragement of Young Scientists to H.Y. from the Ministry of Education, Science and Culture of
Japan.
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