Carbon catabolite protein A (CcpA) is a global regulator of carbon
metabolism in gram-positive bacteria, repressing transcription of genes
for the utilization of secondary carbon sources in the presence of a
readily metabolized carbon source and activating transcription of
genes, such as ackA and pta, that are required for carbon excretion. The promoter region of the Bacillus
subtilis ackA gene contains two catabolite responsive elements
(cre sites), of which only the site closest to the promoter
(cre2) binds CcpA to activate transcription. A region
immediately upstream of the cre2 site is also important for
transcriptional activation. The required elements in this region were
further defined by mutagenesis. CcpA binds to the ackA
promoter region in gel shift assays even in the presence of mutations
in the upstream element that block transcriptional activation,
indicating that this region has a function other than promoting binding
of CcpA.
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TEXT |
Carbon catabolite repression in
Bacillus subtilis and other gram-positive bacteria is
controlled by a mechanism different from that in gram-negative
bacteria. The key regulator is carbon catabolite protein A (CcpA),
which represses the transcription of various genes encoding proteins
involved with the utilization of secondary carbon sources
(12). CcpA also activates the transcription of genes
involved in carbon excretion. These genes include pta and
ackA, which function together to convert acetyl coenzyme A to acetate for excretion into the growth medium (11, 22,
26).
The CcpA protein is a member of the LacI-GaIR family of transcriptional
repressors (13). Members of this family contain an
amino-terminal helix-turn-helix DNA binding domain and carboxy-terminal regions involved with effector recognition and oligomerization (28). The activity of CcpA is controlled by HPr or the HPr
homologue Crh, both of which are phosphorylated by an ATP-dependent
kinase during growth in glucose (8, 9, 23, 24). Mutations
which block this signaling pathway cause loss of glucose repression of
many target genes and loss of transcriptional activation of ackA and pta (1, 2, 22, 27).
Activation of ackA expression during growth in glucose is
dependent on the cre2 CcpA binding site (centered at 
56.5
relative to the transcription start site) and sequences upstream of
cre2 (11, 27). The molecular mechanism of
transcriptional activation by CcpA is unknown, and the role of the
region upstream of the CcpA binding site has not been characterized.
Deletion analysis of the ackA upstream region.
Deletions of the region upstream of cre2 were generated by
PCR, and transcriptional fusions to lacZ were generated
using the plasmid pFG328 (11) and inserted in single copy
into the B. subtilis chromosome by recombination into an
SP
prophage. Expression of the ackA-lacZ fusions was
monitored during growth in TSS medium (5) containing 1%
Casamino Acids (Difco) in the presence or absence of glucose (1%).
Deletion constructs up to and including ACKBAM11, which
contains 28 bp of sequence upstream of cre2, exhibited normal induction of ackA-lacZ expression (Table
1). ACKBAM6, containing 23 bp
upstream of cre2, exhibited a small decrease in activation.
These results map the 5' end of the sequence elements required for
complete activation of the ackA-lacZ fusion in the presence
of glucose to between 23 and 28 bp upstream of cre2.
Random mutagenesis.
To identify the sequence elements required
for activation, the 33 bp upstream of cre2 were randomly
mutagenized by amplifying this region using an oligonucleotide
containing 6% non-wild-type bases (94% wild-type) at each position in
the region. This frequency of doping is predicted to create a pool of
oligonucleotides with 1 to 3 mutations per individual oligonucleotide
with a 
1% probability of the wild-type sequence (15).
The oligonucleotide included 13 bp of wild-type cre2
sequence at its 3' end to ensure efficient annealing of primers
containing mismatches at the 3' end of the target sequence. The
resulting pool of PCR fragments was inserted into the plasmid pFG328 to
generate ackA-lacZ transcriptional fusions, and the plasmids
were propagated as mixed pools and then introduced into the B. subtilis chromosome by recombination of the fusion into an SP
prophage. Isolates that retained normal activation of
ackA-lacZ transcription during growth on tryptose blood agar
base (Difco) containing
5-bromo-4-chloro-3-indolyl--D-galactopyranoside (40 µg/ml) and glucose (1%) were selected for further analysis, and the
DNA sequence of the ackA upstream region was determined. Of
the 34 candidates chosen, 17 were wild type, 2 had three mutations, 4 had two mutations, and 11 had one mutation. The results revealed two
sequence elements in which no mutations appeared, the first between
positions 82 and 77 (region I) and the second between positions
70 and 67 (region II) (Fig. 1A).
These elements were presumed to be important for the activation of
transcription.
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FIG. 1.
Sequence of the ackA upstream region.
Numbering is relative to the transcription start site. (A) Random
mutagenesis of the region between nucleotides 96 and 64. Up arrows
indicate mutations that allowed a significant degree of activation of
the ackA promoter in the presence of glucose. Down arrows
indicate mutations that did not allow significant activation. Two
triple mutations were obtained: A(95)C A(91)G G(86)T and G(94)T
A(91)G T(89)A. Four double mutations were obtained: A(76)T
A(72)T, A(95)T A(72)T, A(95)C C(74)A, and T(93)A C(74)T.
Eleven single mutations were obtained: A(65)T, T(66)G, C(71)T,
C(74)A, A(75)T, A(76)T, T(77)C, T(77)A, T(83)A, G(88)T,
and T(96)A. (B) Alignment of sequences upstream of the
ackA and pta cre sites. (C) Site-directed
mutagenesis. Mutations were generated by PCR and introduced restriction
sites at the positions shown. ACKKPN3-6 deleted a T residue
at position 67. ACKBCL1-2T is the same as ACKBCL2-3
with the introduction of an additional T residue between
positions 69 and 70.
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The importance of these elements is also supported by conservation
between the ackA and pta upstream regions (Fig.
1B), suggesting that transcriptional activation of ackA and
that of pta operate by similar mechanisms. Interestingly, 7 of the 12 differences between the ackA and pta
regions were found as single mutations in the random mutagenesis and
therefore do not drastically affect expression of ackA. Four
of the remaining five differences are at positions which were invariant
in these experiments but are surrounded by positions which did change.
The final difference between ackA and pta is
within region II [C(68)T], suggesting that T may be allowed at this
position or that there are context-specific effects.
-Galactosidase measurements for isolates with the mutations
T(96)A, T(83)A, T(77)A, C(74)A, and T(66)G,
each of which contained only a single mutation, are shown in Table
2. The activation ratio ranged from 2.5- to 3.6-fold, relative to 3.4-fold for the wild-type fusion, and the
expression level varied from half to twice that of the wild-type
fusion. The C(74)A mutant exhibited a basal level and
glucose-activated transcriptional level two-fold over the wild-type
level. This fusion was confirmed to be in single copy (data not shown).
To determine if this increase in expression was dependent on CcpA, the
mutant fusion was introduced into a ccpA mutant strain.
While no activation in the presence of glucose was observed, the basal
activity of the fusion was still increased over that of the wild-type
fusion, indicating that CcpA is required for activation but that some
other factor is leading to the increase in basal activity.
Site-directed mutagenesis of the region upstream of
cre2.
Site-directed mutagenesis was performed on the
sequence elements highlighted by the random mutagenesis (Fig. 1C). Each
mutation drastically affected activation in the presence of glucose,
confirming the importance of these sequence elements (Table 2). These
constructs were generated in the context of the entire ackA
upstream region, indicating that the presence of cre1 does
not obviate the requirement for these elements. The ACKBAM9-10
and ACKBCL2-3 mutant fusions were also introduced into
BR151MACcpA::Spc, and the residual activation was
found to require CcpA.
Binding of CcpA to the ackA upstream region.
The
sequence elements required for activation could function as a protein
binding site, assist in the binding of CcpA to the cre2 site, or confer a DNA conformation required for
activation of the ackA promoter. To test the hypothesis that
the region upstream of ackA cre2 is important for CcpA
binding, a gel mobility retardation assay was performed using
wild-type and mutant DNA fragments. CcpA was purified to homogeneity by
incorporating a six-histidine tag at the amino terminus followed by
passage through a nickel-nitrilotriacetic acid (Ni-NTA) column,
essentially as described for Bacillus megaterium CcpA
(1). The His-tagged CcpA bound efficiently to a 163-bp wild-type ackA cre2-containing DNA fragment (Fig.
2a). It was previously shown that
mutagenesis of cre2 conferred a loss of transcriptional
activation from the ackA promoter, most likely due to the
lower affinity of CcpA for the mutant cre2 site
(27). It was determined that CcpA can still bind in vitro
to the DNA fragment containing the mutant cre2, but with a
significant reduction in affinity (data not shown). Since the sequence
upstream of cre2 might affect CcpA binding to
cre2, binding of CcpA to the mutant DNA fragments was
tested. As represented by the site-directed mutant ACKKPN3-6,
CcpA was still able to bind the fragments in each
site-directed mutant (Fig. 2a and data not shown). In a competition assay (Fig. 2b), ACKSAL1-2 and ACKBAM9-10 DNA
fragments were able to sequester CcpA from the labeled wild-type DNA as
efficiently as did unlabeled wild-type DNA. It therefore appears that
the upstream elements are not required for CcpA binding.
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FIG. 2.
(a) Binding of CcpA-His to an ackA
cre2-containing DNA fragment. The ackA DNA (163 bp;
positions 139 to +24 relative to the transcription start site) was
generated by PCR, labeled using T4 polynucleotide kinase and
[ -32P]ATP, and purified with the Qiagen QIAquick PCR
purification kit. Mutant DNA was generated in parallel to the wild-type
fragment. The nonspecific DNA probe (169 bp) contained a portion of the
ccpA coding region. The intact ccpA coding region
was isolated by PCR and inserted into the plasmid pQE30 (Qiagen) to
generate the amino-terminal six-histidine tag, introduced into E. coli M15(pREP4) (Qiagen), and expressed upon addition of
isopropyl--D-thiogalactopyranoside to a final
concentration of 2 mM. The His-tagged protein was purified using
nickel-nitrilotriacetic acid spin columns (Qiagen), and imidazole (250 mM) was used to elute the protein. Probe DNA (1 ng) was incubated with
CcpA in a solution containing 10 mM Tris-HCl (pH 7.5), 1 mM
dithiothreitol, 1 mM EDTA, 50 mM KCl, 5% glycerol, 50 µg of bovine
serum albumin/ml, and 0.05% igepal CA-630 (Sigma) in a 20-µl volume
for 10 to 30 min at 37°C (18). Samples were mixed with
nondenaturing sample buffer (40% sucrose, 0.25% bromphenol blue) and
loaded directly onto a 5% polyacrylamide gel prepared in 6.7 mM
Tris-HCl (pH 8.0)-1 mM EDTA-2.5% glycerol. Gels were run at 200 V
for 1.5 h in a Protean II xi Cell (Bio-Rad) gel electrophoresis
unit, dried, and exposed to X-ray film (Kodak) for autoradiography.
Lanes 1 to 6, wild-type DNA (1 ng); lanes 7 to 12, ACKKPN3-6; lanes 13 and 14, CcpA9-10 (nonspecific sequence).
Lanes 1, 7, and 13, no protein; lanes 2 and 8, 1 ng of CcpA-His (0.66 nM); lanes 3 and 9, 3 ng of CcpA-His (2.0 nM); lanes 4, 10, and 14, 7 ng of CcpA-His (4.6 nM); lanes 5 and 11, 10 ng of CcpA-His (6.6 nM);
lanes 6 and 12, 20 ng of CcpA-His (13 nM). (b) Competition gel
retardation assay. Unlabeled wild-type and mutant DNA was tested for
the ability to sequester CcpA-His from the labeled wild-type probe (1 ng). The unlabeled DNA contained wild-type sequence (lanes 3 and 4),
ACKSAL1-2 sequence (lanes 5 and 6), or ACKBAM9-10
sequence (lanes 7 and 8). Lanes 3, 5, and 7, 10 ng of unlabeled
DNA; lanes 4, 6, and 8, 100 ng of unlabeled DNA; lanes 2 to 8, 50 ng of
CcpA (33 nM); lane 1, no protein; lane 2, no competing DNA.
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The requirement for these DNA elements for transcriptional activation
of ackA suggests that additional factors may be involved. It
is known that phosphorylated HPr or Crh is required for activation in
vivo (27); however, in other systems these proteins have been shown to affect the efficiency of binding of CcpA to
cre sites without directly contacting the DNA themselves
(6, 7). Another unknown protein may be binding to the
region upstream of the cre2 site. RNAP has been shown to
respond to two bound activators to initiate transcription
(14). For example, in an artificial promoter construct,
cI contacts the 
subunit of RNAP while cyclic AMP receptor
protein (CRP) contacts the carboxy-terminal domain of the 
subunit
(-CTD) of RNAP to stimulate transcriptional initiation
(16). In the activation of the divergent malEp
and malKp promoters, CRP effects a shift of the activator
MalT to its functional sites in phase with the 10 region of the
promoter to allow a favorable interaction with RNAP
(25). For the proP P2 promoter, CRP (at
121.5) and Fis (at 41) act together to stimulate transcription
initiation (19); each of these proteins individually makes
contact with the -CTD portion of RNAP. It is possible that some form
of coactivation is occurring at the ackA promoter, which
includes binding of CcpA to cre2 and binding of some other
factor to the region upstream of cre2. Alternatively, the
upstream region may interact directly with RNAP, perhaps functioning as
an UP element to contact the -CTD; if so, activation must be
dependent on CcpA binding, perhaps to reposition the upstream region to
permit contact with RNAP. Proteins such as integration host factor have
been shown to bend the DNA to allow optimal contact between the
subunits of RNAP and a DNA sequence or another protein binding upstream
of the promoter (21). UP elements, which like the
ackA upstream region contain A- and T-rich sequences, have been identified upstream of Escherichia coli and B. subtilis promoters and have been shown to interact with -CTD
(3). This interaction between the UP element and the subunit of RNAP may assist the binding of RNAP to the promoter and
further steps of transcription initiation (4). CcpA could
also bend the DNA to facilitate an interaction between RNAP and region
I and/or region II.
Further study will be required to determine the function of the
upstream sequence elements and the molecular mechanism of transcriptional activation. Since these elements are conserved in
pta, it is likely that activation is similar for the two
genes; this would provide coordinate expression consistent with
cotranscription of these genes in E. coli (17).
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