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FEMS Microbiology Letters 195 (2001) 179^183
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Involvement of acetyl phosphate in the in vivo activation of the
response regulator ComA in Bacillus subtilis
Seong-Bin Kim
a;c
, Byung-Sik Shin a , Soo-Keun Choi a , Chi-Kyung Kim c ,
Seung-Hwan Park b; *
a
b
Laboratory of Microbial and Bioprecess Engineering, Korea Research Institute of Bioscience and Biotechnology (KRIBB), P.O. Box 115, Yusong,
Taejon 305-600, South Korea
Genome Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), P.O. Box 115, Yusong, Taejon 305-600, South Korea
c
Department of Microbiology, Chungbuk National University, Cheong-ju 361-763, South Korea
Received 21 September 2000; received in revised form 20 December 2000; accepted 31 December 2000
Abstract
Development of genetic competence in Bacillus subtilis is regulated by ComP^ComA, a two-component signal transduction system. The
response regulator ComA is primarily activated by ComP, a histidine kinase that mediates response to nutrient conditions and cell density,
and the activated ComA is required for transcription of the srf operon, which is essential for the development of genetic competence and
surfactin production. In this study we suggested that the ComA could also be activated by a small molecule phospho-donor, acetyl
phosphate. Examination of srfA-lacZ expression indicated that a significant amount of srfA expression still occurs in the comP mutant
during growth in a sporulation medium containing excess glucose. Analysis of a comP and pta mutant suggests that srfA activation seen in
the comP mutant is dependent on the expression of pta, which encodes phosphotransacetylase (Pta). As Pta is responsible for the catalysis
for conversion of acetyl coenzyme A to acetyl phosphate, we conclude that the expression of srfA seen in the comP mutant is mainly due to
the activation of ComA by acetyl phosphate. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science
B.V. All rights reserved.
Keywords : Two-component signal transduction; ComA; Acetyl phosphate ; srfA-lacZ ; Bacillus subtilis
1. Introduction
Bacillus subtilis has natural competence, generally de¢ned as a physiological state that permits a bacterial cell
to bind and take up high-molecular mass DNA [1]. Early
regulatory steps in competence development are activated
by two extracellular factors, ComX pheromone and competence stimulation factor (CSF), which accumulate in
culture media as cells grow to high density. These two
factors were known to deliver signals from outside by
two di¡erent sensing pathways and converge to activate
expression of srfA [2]. By signaling of ComX pheromone,
ComP histidine kinase autophosphorylates on a histidine
residue. In vivo, the phosphate from ComPVP is transferred to the ComA response regulator. Then, the phosphorylated ComAVP binds to and activates the transcrip-
* Corresponding author. Tel. : +82 (42) 860-4410;
Fax: +82 (42) 860-4594; E-mail: [email protected]
tion of srfA [3], and resultantly the competence regulatory
gene comS, which lies within and out-of-frame with the
second gene of the fourth amino acid-activating domain
of the srfA operon is also transcribed [4]. Interestingly,
however, Solomon et al. reported that comP mutants still
could activate the ComA response regulatory protein, and
this indicates that there must be an additional source of
phosphate for activation of ComA in the cell [2]. Despite
the drastic decrease of srfA transcription by the comP
mutation, the accumulation of L-galactosidase speci¢c activity from srfA-lacZ in a comP mutant remains V5% of
that in the wild-type. Recent studies on Escherichia coli
have shown that the major secondary source of phosphoryl groups is a small molecule phosphate donor, acetyl
phosphate [5^7]. Several response regulatory proteins,
such as CheY [5], PhoB [6], and OmpR [7], have been
shown to be phosphorylated in vitro when incubated
with acetyl phosphate. It was also reported that partially
puri¢ed ComA protein, prepared from E. coli cells overexpressing ComA, could be phosphorylated in vitro by
0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 0 0 8 - 8
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incubation with acetyl phosphate [8]. Genetic studies have
indicated that acetyl phosphate functions as the principle
phospho-donor in E. coli mutant strains that are de¢cient
in the cognate histidine kinase [9]. In B. subtilis, however,
there is no evidence that acetyl phosphate is a phosphodonor in the two-component signal transduction system in
vivo. In this study, we present evidence that acetyl phosphate is a phospho-donor that can activate the ComA
response regulator in B. subtilis, and that this activation
is dependent on the regulation of pta that encodes a phosphotransacetylase (Pta). Glucose and fructose, which can
activate pta, stimulated the activation of ComA, but pyruvate did not induce this activation.
2. Materials and methods
2.1. Bacterial strains and media
The bacterial strains used in this study are described in
Table 1. E. coli DH5K was used for plasmid construction.
All are derived from the parent strain JH642. Strains were
constructed by transformation with plasmid and chromosomal DNA. LB medium was routinely used for cultivating E. coli and B. subtilis. Difco sporulation medium
(DSM) was used for growth and maintenance of B. subtilis. Competent cells were obtained in Spizizens medium
as described by Albano et al. [10]. For carbon source,
pyruvate and fructose were added to sterile DSM to yield
1% ¢nal concentration.
2.2. L-Galactosidase assay
The inoculum for the L-galactosidase assay was prepared by growing cells on a DSM agar plate at 30³C overnight with appropriate antibiotics. The cells were then
harvested by washing the plate surface with 1.0 ml of
prewarmed DSM, and liquid medium was inoculated to
an initial absorbance level at 600 nm of about 0.02. Cultures were grown at 37³C and 300 rpm, and growth was
monitored by measuring A600 . The assays were performed
with toluenized cells, as described by Nicholson and Set-
low [11]. The speci¢c activity was expressed as Miller units
[12].
2.3. Plasmid and strain constructions
Plasmid pDG1728 contains a sequence homologous to
the amyE locus and carries a promoterless copy of lacZ
[13]. The srfA promoter region from 3291 to +140 relative
to the srfA transcription start site, with £anking EcoRI
and BamHI restriction sites, was cloned into the lacZ fusion vector, pDG1728, and the fusion was recombined
into the chromosome by double-crossover, selecting for
spectinomycin resistance. To construct a comP mutant, a
DNA fragment of about 2.2 kb containing the comP front
region was ampli¢ed by PCR and then cloned into
pGEM-7Zf (+) (Promega), giving pComP-F. Then, the
cat gene was ligated into ClaI and SpeI restriction sites
of pComP-F, giving pComP(F)-cm. Finally, the fragment
of comP back region was cloned by PCR. An approximately 1.6-kb PCR fragment restricted with ClaI and
EcoRI was cloned into pComP(F)-cm, giving prComP.
This plasmid was integrated into the chromosome by
means of double-crossover. To construct a comA mutant,
a DNA fragment of about 2.0 kb containing the comA
gene was ampli¢ed by PCR and then cloned into
pGEM-7Zf (+), giving pComA. A deletion^insertion
mutation of the comA gene was constructed by replacing
the 0.5-kb ClaI^SpeI fragment of pComA with a 1.1-kb
ClaI^SpeI fragment of the cat gene, cloned from pDG1661
by PCR. The resulting plasmid, prComA, was
integrated into the chromosome by means of a doublecrossover.
2.4. Site-directed mutagenesis of comA
To substitute an alanine (A) for the aspartic acid (D)
residue at position 55 on the comA sequence, an Altered
sites0 II in vitro Mutagenesis system (Promega) was used.
Mutagenesis was performed following the instructions of
the manufacturer. This mutation (D55A in the comA sequence) was con¢rmed by DNA sequencing. Mutations
were con¢rmed by Southern hybridization.
Table 1
Bacterial strains used in this study
Strain
Genotype
JH642
BSK9901
BSK9902
BSK9903
BSK9904
BSK9905
BSK9906
BSK9907
trpC2
trpC2
trpC2
trpC2
trpC2
trpC2
trpC2
trpC2
pheA1
pheA1
pheA1
pheA1
pheA1
pheA1
pheA1
pheA1
Source
amyE: :(srfA-lacZ spc)
vcomP: :cat amyE: :(srfA-lacZ spc)
vcomA: :cat amyE: :(srfA-lacZ spc)
vpta: :erm amyE: :(srfA-lacZ spc)
vccpA: :erm amyE : :(srfA-lacZ spc)
vcomP: :cat vpta: :erm amyE: :(srfA-lacZ spc)
vcomP: :cat vccpA: :erm amyE: :(srfA-lacZ spc)
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S.-B. Kim et al. / FEMS Microbiology Letters 195 (2001) 179^183
181
from srfA-lacZ grown in DSM-G did not decrease even
3 h after the end of the exponential phase (T3 ).
In order to analyze the regulation of srfA expression in
cells grown in medium containing a high concentration of
glucose, the e¡ect of the comP mutation on the expression
of srfA was examined. As expected, expression of srfAlacZ was greatly reduced in the comP mutant grown in
DSM (Fig. 1B), which was seen previously [4]. However,
the expression of srfA was largely restored in the comP
mutant by the addition of glucose to DSM (Fig. 1B).
These results suggest that the residual srfA expression in
the comP mutant seen in DSM-G is largely dependent on
the presence of glucose in the medium. The expression of
srfA in the comP mutant was increased much more in this
experiment than in the previous report of Solomon et al.
[4]. There was no detectable expression of srfA-lacZ from
a comA null mutant grown in either DSM or DSM-G
(Fig. 1B).
It has been reported that the unphosphorylated form of
ComA protein can bind to a DNA fragment containing
the srfA promoter in vitro, although the binding a¤nity is
enhanced when ComA is phosphorylated [8]. As the conserved aspartate at position 55 of ComA is suggested to be
the phosphorylation site of ComA [15], we constructed a
comA mutant with alanine in place of the conserved aspartate (comAD55A ) to determine whether unphosphorylated ComA could stimulate the expression of srfA. The
speci¢c activity of L-galactosidase from srfA-lacZ in the
comAD55A mutant was very low in both DSM and DSM-
Fig. 1. Expression of srfA-lacZ in wild-type (A) and a comP mutant (B)
in the presence and absence of glucose in DSM. Samples were taken for
determination of L-galactosidase speci¢c activity at 30-min or 1-h. intervals. (A) Expression of srfA in wild-type is constantly maintained in the
stationary phase of growth under the presence of glucose. b, L-galactosidase activity in DSM ; F, L-galactosidase activity in DSM-G. (B) Expression of srfA in comP mutant is activated in the presence of glucose.
R, L-galactosidase activity in DSM; 8, L-galactosidase activity in
, L-galactosidase activity of comA mutant in DSM; S, LDSM-G;
galactosidase activity of comA mutant in DSM-G. Arrows indicate the
onset of stationary phase (T0 ).
3. Results and discussion
3.1. E¡ect of glucose on the srfA expression
The expression of srfA, required for activation of
ComK, increases as cells enter the stationary phase, and
this activation of srfA transcription decreases at the lategrowth phase [14]. However, the addition of a high concentration of glucose to the culture medium resulted in
constitutive expression of srfA during the late-growth
phase (Fig. 1A). The L-galactosidase speci¢c activity
Fig. 2. Model for the role of acetyl phosphate in the regulation of srfA
expression and competence in B. subtilis. The expression of srfA through
these pathways is reached in the end of exponential phase. Upon entry
into stationary phase, CSF reaches 50^100 nM, concentrations that
stimulate sporulation and inhibit ComA-dependent gene expression. But,
the expression of srfA in the presence of glucose or fructose is maintained. Results presented in this report indicate that acetyl phosphate
may be involved in the expression of srfA in the stationary phase in
which much acetate is produced through acetyl phosphate by AckA and
unknown pathways.
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Table 2
E¡ect of carbon source on the expression of srfA-lacZ in various B. subtilis mutants
Medium
DSM
DSM-G
DSM-F
DSM-P
L-Galactosidase speci¢c activity-Miller units (T0 /T3 )
Genotype
BSK 9902 (comP)
BSK 9904 (pta)
BSK 9905 (ccpA)
BSK 9901 (wild-type)
BSK 9906 (comP+pta)
3.3/2.5
203/187
237/217
27/12
215/64
214/95
241/98
255/86
190/69
197/133
227/144
224/67
188/43
235/218
277/206
210/56
3.4/2.6
57/36
58/47
4.0/4.5
Values represent means of at least two independent experiments. T0 is the onset of the stationary phase and T3 is 3 h after T0 . Glucose, fructose and
pyruvate were added to ¢nal 1% in DSM medium.
G, similar to that in the comA null mutant (data not
shown), indicating that the unphosphorylated form of
ComA does not appear to have a role in the regulation
of srfA. All of these results indicate that the majority of
srfA expression during growth in DSM-G, in particular at
late-growth phase, does not depend on the presence of
ComP, and that there must be an additional source of
phosphate for activation of ComA in these cells.
3.2. E¡ect of pta regulation on the ComA activation
To examine if acetyl phosphate could be another phosphate donor in the activation of ComA, we analyzed the
e¡ect of pta regulation on the expression of srfA. In a
previous study, we showed that the expression of pta,
which encodes a Pta responsible for conversion of acetyl
coenzyme A to acetyl phosphate, is activated by the addition of excess glucose or fructose, and that this activation
is dependent on CcpA [16,17]. As the expression of pta
depends on the presence of glucose in the medium, it is
possible that expression of srfA observed in DSM-G may
be due to induction of pta expression by the presence of
excess glucose. As shown in Table 2, in the comP mutant,
the expression of srfA was greatly increased by the addition of glucose or fructose. In contrast, the expression of
srfA in the pta mutant was almost not e¡ected by the
presence of glucose or fructose. We also observed that
the pattern of srfA expression in the pta mutant was almost the same as that in the ccpA mutant. Because the
expression of pta requires CcpA [17,18], the expression of
srfA in the ccpA mutant should be similar to that in the
pta mutant. The dependence of srfA expression on the
presence of sugar also could be observed in the wildtype : the addition of glucose or fructose increased the
expression of srfA in the wild-type strain, especially during
the late-growth phase (T3 ). All of these results indicate
that the induction of srfA expression seen in the presence
of glucose or fructose is critically dependent on the regulation of the pta gene, suggesting possible involvement of
acetyl phosphate in the activation of ComA (Fig. 2).
It is interesting to note that the addition of pyruvate did
not greatly induce the expression of srfA in the comP mutant. Although pyruvate could be considered a good
source for production of acetyl phosphate, we observed
that the expression of the pta gene is not induced by the
addition of 1% pyruvate in DSM (data not shown). In B.
subtilis, CcpA-dependent catabolite repression requires the
seryl-phosphorylated form of both the Hpr and Crh proteins; and two glycolytic metabolites, fructose-1,6,-diphosphate (FDP) and glucose-6-phosphate (G6P), have been
proposed as e¡ectors of CcpA [19,20]. As the presence
of pyruvate did not e¡ect the expression of srfA, it is
possible that activation of pta by CcpA also requires either
FDP or G6P as a coactivator.
We also measured expression of srfA in the comP-pta
double mutant. Although the expression of srfA was signi¢cantly lower than that in either single mutant, there
was still detectable expression of srfA in the double mutant in the presence of glucose or fructose (Table 2). In B.
subtilis, acetate is mainly produced via the Ack^Pta pathway during growth with excess carbohydrate. However, a
signi¢cant amount of acetate is still produced in the pta
mutant, indicating that there may be another pathway
responsible for acetate production [18]. Thus it is possible
that the expression of srfA seen in the comP-pta double
mutant may be due to acetyl phosphate produced from
acetate by reverse reaction of AckA. As the Pta^AckA
pathway is the only route for production of acetyl phosphate, examination of a comP-pta-ackA triple mutant
should be done to prove this hypothesis. It is also possible
that residual expression in the comP-pta double mutant
may have resulted from activation of ComA by another
histidine kinase, although there is no available evidence in
B. subtilis to support this speculation.
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