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Journal of Bacteriology, December 1999, p. 7634-7638, Vol. 181, No. 24
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Mutations in Catabolite Control Protein CcpA
Showing Glucose-Independent Regulation in Bacillus
megaterium
Elke
Küster-Schöck,1,
Andrea
Wagner,1
Uwe
Völker,2 and
Wolfgang
Hillen1,*
Lehrstuhl für Mikrobiologie,
Friedrich-Alexander-Universität Erlangen-Nürnberg, 91058 Erlangen,1 and Laboratorium für
Mikrobiologie, Philipps-Universität Marburg and
Max-Planck-Institut für Terrestrische Mikrobiologie,
Marburg,2 Federal Republic of Germany
Received 26 April 1999/Accepted 1 October 1999
 |
ABSTRACT |
We identified five single amino acid exchanges in CcpA that lead to
permanent repression of the xylose utilization genes in the absence of
glucose. Other proteins from the CcpA regulon also show
glucose-independent regulation in the mutants. The mutant CcpA proteins
bind to the DNA target catabolite responsive elements without the
corepressor HPr-Ser-P.
| |
TEXT |
Catabolite control protein CcpA is
the central regulator of carbon catabolite repression (CCR) in
Bacillus megaterium, Bacillus subtilis, and other
gram-positive bacteria of low G+C content (5, 11, 12, 21, 22,
25). Genes and operons coding for the utilization of less
favorable carbon sources, such as xylose, are regulated by CcpA on the
level of transcription in the presence of rapidly metabolizable sugars
like glucose or fructose (12). The mechanism of CCR is
distinct from the one described for Escherichia coli
(reviewed in reference 27). CcpA can either repress
or activate transcription. Activation was shown in two cases,
ackA in B. subtilis (31) and the
las operon of Lactococcus lactis (22).
Repression by CcpA was demonstrated for multiple genes and operons in
B. subtilis, B. megaterium, and other
gram-positive bacteria (10).
CcpA binds to DNA target sites termed catabolite responsive elements
(CRE). Repression depends on the presence of HPr-Ser-P or Crh-Ser-P;
the former is a component of the phosphoenolpyruvate:sugar phosphotransferase system whose phosphorylation state reflects glycolytic activity (30). The requirement for a corepressor for CcpA was confirmed by DNA footprinting studies involving CRE sequences from the xyl and gnt operons. In
addition to HPr-Ser-P, glucose-6-phosphate also triggered CRE binding
by CcpA in both systems in in vitro assays (7, 9, 24).
Similar experiments with the xynB CRE showed that Crh-Ser-P
can substitute for HPr-Ser-P as a corepressor, and both Crh-Ser-P and
HPr-Ser-P can trigger CcpA-regulated CCR of the lev operon
in vivo (8, 23). In contrast to these results, in vivo CCR
of amyE is independent of phosphorylated HPr (14,
33), and even though CcpA-CRE interaction was strengthened by a
combination of HPr-Ser-P and fructose-1,6-bisphosphate or NADP, it did
not improve repression in in vitro transcription (15).
Accordingly, CcpA is thought to receive signals from HPr-Ser-P or
Crh-Ser-P and possibly from other effectors.
A direct interaction of CcpA with HPr-Ser-P has been demonstrated
(3, 13), and a putative HPr-Ser-P binding site on CcpA was
recently identified (17). CcpA is a member of the LacI-GalR family of bacterial repressors, and sequence similarities, limited proteolysis, and mutational data suggest a common three-dimensional fold for CcpA, LacI, and PurR (13, 16, 17, 34). On the other
hand, HPr-Ser-P does not bind in the effector binding cleft where
isopropyl-
-D-thiogalactopyranoside (IPTG) binds to LacI and hypoxanthine binds to PurR (see Fig. 4). It is therefore
interesting to collect further evidence about the activation of CcpA
for CRE binding.
CcpA mutagenesis and screen for glucose-independent
repression.
We have conducted a screen for CcpA variants which
repress the xylose utilization genes of B. megaterium in the
absence of a repressing carbon source. A plasmid library of mutated
ccpA alleles was generated by in vitro mutagenesis with
nitrite treatment, as described previously (18). It was
transformed into B. megaterium WH353 [lac 
ccpA
gdh2
(xylA-spoVG-lacZ) xylR],
which carries an in-frame ccpA deletion and a
xylA-lacZ fusion as a probe for catabolite repression
activity. An additional chromosomal deletion in xylR ensured
that repression of xylA-lacZ transcription is only caused by
the plasmid-encoded CcpA. We screened the transformants on M9 minimal
medium containing succinate, which is neutral in CCR, as a carbon
source and
5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal)
to identify CcpA mutants which permanently repress xyl expression, thus displaying the repression phenotype
ccpAk.
Screening about 12,000 colonies yielded 58 white or light blue colonies
which were colony purified. The plasmids were isolated, passaged
through E. coli, and retransformed into B. megaterium WH353. If the original phenotype was retained we
quantified repression by determining -galactosidase activities. A
total of 28 of the original clones were regarded as glucose independent
in CCR (ccpAk phenotype), since they repressed
xylA-lacZ expression in succinate to 50% or less of
wild-type expression in the absence of glucose. Total cell extracts
from the mutants were analyzed by immunoblotting with anti-CcpA
antiserum as described previously (16, 19). All of the
mutant proteins were present at levels similar to that of wild-type
CcpA expressed from the same vector (data not shown).
Sequence analysis revealed that 13 of the 28
ccpAk alleles carried distinct mutations and
that most of them caused multiple
amino acid substitutions. They were
separated by subcloning utilizing
unique restriction sites in
ccpA. The subclones were rescreened
as described above. From
the original screen and the subcloning
we obtained the following five
CcpA mutants, each with a single
amino acid substitution, which exert
permanent, glucose-independent
repression: CcpA
kE77L,
CcpA
kI227V, CcpA
kD275G, CcpA
kM282,
and CcpA
kT306I.
None of the mutants represses
xyl expression to the level
obtained by the wild type with glucose, and they all show increased
repression upon addition of glucose (Fig.
1). Thus, the mutants
are only partially
independent of glucose. We have observed severe
growth deficiencies in
all of the
ccpAk strains (data not shown). A
more complete, permanent regulation
phenotype might not show up in our
screen if the phenotype is
linked to poor growth.
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FIG. 1.
-Galactosidase activities expressed from
xylA-lacZ in B. megaterium WH353 regulated by
different CcpA mutants. Cells were grown in M9 medium with 0.5%
succinate or with 0.5% succinate plus 0.2% glucose. The dotted line
marks 50% of the wild-type (wt) expression with succinate. The amino
acid substitutions in CcpA are shown under the respective columns.
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|
2D gel electrophoresis of protein extracts from wild-type and
ccpAk mutant cells.
To analyze the effects
of the ccpAk mutations on the entire CcpA
regulon we used two-dimensional (2D) gel electrophoresis. Total soluble
protein extracts were prepared from the B. megaterium ccpA
deletion mutant WH353 carrying the empty vector pWH1509K (26) or derivatives of pWH2051 carrying the genes for
wild-type CcpA or one of the five single amino acid CcpAk
mutants. Protein extract (100 mg) was then subjected to 2D protein electrophoresis as described by Völker et al. (32),
and the protein profiles of the different strains were compared after silver staining. A typical gel is shown in Fig.
2 (left panel).
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FIG. 2.
CcpA-mediated changes in the protein synthesis profile
of B. megaterium. The panel on the left shows a silver
nitrate-stained 2D gel loaded with 100 µg of crude protein extract
from B. megaterium WH353(pWH2051) expressing wild-type (wt)
CcpA which had been grown on glucose. The small panels on the right
display enlarged regions from this gel and the corresponding regions
from gels prepared with extracts of the same strain grown on succinate
and of CcpAkE77L grown on succinate. Arrowheads indicate
proteins showing the expected CCR or carbon catabolite activation
pattern, and glucose-independent regulation in CcpAkE77L.
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If a strain expressing wild-type CcpA is grown in glucose, spots for
proteins whose expression is CCR dependent should be
absent or reduced
in intensity compared to those of the same strain
grown in succinate,
i.e., without the repressing carbon source.
A comparison with a strain
carrying only the vector revealed high
expression of those proteins in
the presence of glucose (data
not shown), demonstrating that the
regulation was mediated by
CcpA. Using this approach, we recognized 39 spots showing the
pattern for CcpA-dependent repression. Examples are
shown in Fig.
2 (small panels, top row). Furthermore, we identified
four proteins
showing CcpA-dependent activation of expression (Fig.
2,
small
panels, bottom row). These spots were almost undetectable in the
absence of glucose or if the cells were lacking CcpA. These numbers
of
proteins do not reflect the total size of the CcpA regulon
because the
expression of many proteins needs to be specifically
induced, and we
only looked at soluble proteins in the absence
of inducers. The limited
detectability of intensity differences
in silver-stained gels further
reduces the number of regulated
proteins that are
recognized.
CcpA-mediated regulation should be independent of glucose in the
CcpA
k mutants. Spot intensities of cells of
CcpA
k mutants grown in succinate resemble those found with
cells of
the wild type grown in glucose. Examples obtained with
CcpA
kE77L are shown in Fig.
2 (rightmost small panels). The
CcpA
kE77L strain showed the expected pattern for 37 of 39 repressed
proteins and for 3 of 4 activated proteins identified in the
wild
type. Similar results were obtained with the strains with other
single amino acid mutations, as follows: for CcpA
kI227V, 35 of 39 and 4 of 4; for CcpA
kD275G, 31 of 39 and 4 of 4; for
CcpA
kM282V, 30 of 39 and 4 of 4; and for
CcpA
kT306I, 35 of 39 and 3 of 4 (results are the numbers of
repressed
and activated proteins, respectively). In summary, more than
75%
of the 43 proteins regulated in a glucose-dependent fashion in
the
wild type were regulated in the absence of glucose in each
of the
CcpA
k strains. Therefore, permanent, glucose-independent
repression
by the five CcpA variants is not limited to
xylA.
Purification of mutant proteins and PAGE DNA retardation
assays.
We tested DNA binding of the mutant proteins with and
without the corepressor HPr in vitro. For this, the five
ccpAk alleles leading to single amino acid
substitutions were cloned into overexpression vectors, and the proteins
were overproduced in B. megaterium and purified by column
chromatography (data not shown) as has been described for the wild-type
protein (9). The apparently homogeneous preparations were
then used for DNA retardation experiments. To obtain the corepressor
HPr-Ser-P, the gene for HPr from B. megaterium was cloned
into the same overexpression system, and overproduced in B. megaterium (33a). Purification was carried out
essentially as described previously for HPr from Staphylococcus
aureus (1). The protein was subsequently phosphorylated at Ser46 with partially purified HPr kinase from B. megaterium; the protocol was taken from Deutscher and Saier, Jr.
(4), with minor adaptations. The preparation contained more
than 90% HPr-Ser-P as estimated by nondenaturating polyacrylamide gel
electrophoresis (PAGE) (data not shown).
For the PAGE DNA retardation assays, a synthetic double-stranded
oligonucleotide containing CRE (26mer, as described in reference
13) was mixed with purified CcpA and HPr-Ser-P at
concentrations
of 5 µM (DNA), 10 µM (CcpA), and 10 µM (HPr-Ser-P)
in a buffer
containing 100 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 10%
glycerol.
The mixture was incubated at 37°C for 10 min prior to the
loading
of 5 µl of it on a nondenaturing 5% polyacrylamide minigel
(9
by 6 cm). The gel was run in 100 mM Tris-HCl (pH 7.5)-1 mM EDTA
at
110 V for 45 min and stained with ethidium
bromide.
Under the conditions employed, the wild-type CcpA protein could only
form a complex with the DNA fragment if the corepressor
HPr-Ser-P was
present, as shown in Fig.
3. This effect
is specific
as it cannot be induced by the addition of unphosphorylated
HPr
(data not shown). Figure
3 shows that the same fragment is
complexed
by all five CcpA
k proteins in the absence of the
corepressor. The addition of HPr-Ser-P
does not increase the amount of
complex formed (only shown for
CcpA
kE77L). Thus, all
CcpA
k proteins exhibit HPr-Ser-P-independent DNA binding in
vitro.
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FIG. 3.
Retardation of CRE by wild-type (wt) and mutant CcpA.
Purified CcpA and HPr-Ser-P were combined with a 26mer oligonucleotide
containing CRE as indicated above the lanes. Numbers indicate the
position of the amino acid substitution in each CcpA mutant. Samples
were incubated at 37°C for 10 min prior to loading and run on a
non-denaturing 5% polyacrylamide gel. DNA was visualized with ethidium
bromide.
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Position of the mutations in the three-dimensional structure.
It was surprising that the five amino acid substitutions causing the
ccpAk phenotype are located at distant positions
on the protein chain. To further assess their location we took
advantage of the putative common three-dimensional fold of CcpA and the
LacI-GalR family of bacterial regulators (13, 16). Sequence
comparisons of CcpA proteins to other family members showed that they
constitute a distinct subgroup among LacI-GalR regulators
(17), but overall structural similarities should be
sufficient to evaluate amino acid location. The model of CcpA shown in
Fig. 4 is based on the three-dimensional
structure of PurR (29) (Protein Data Bank entry 1PNR). The
positions mutated in the CcpAk proteins are indicated. The
most striking common feature is their location in the protein core and
not in the DNA binding heads. They highlight different regions of the
protein with possible functional importance: the dimerization surface,
the effector binding cleft, and the corepressor binding site.
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FIG. 4.
Ribbon representation of a model of CcpA based on the
PurR structure. One monomer is dark grey, and the other one is white; a
stick model of the complexed DNA is shown in black, and the functional
domains are named underneath. Circles in the darker monomer mark
positions mutated in CcpAk variants, and the respective
amino acid substitutions are shown above the model. The side chains of
the original amino acids at these positions are depicted as
ball-and-stick models in both monomers.
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Two of the mutations, those in CcpA
kI227V and
CcpA
kM282V, affect residues whose analogs in LacI (Phe226
and Tyr282) and PurR
(Tyr227 and Tyr282) play a role in the
dimerization of the C-terminal
subdomains of the protein core (
6,
29). The structure and
relative positions of the C-terminal
subdomains do not change
significantly upon switching between the DNA
binding and nonbinding
conformations. They form the support for a hinge
movement of the
two N-terminal subdomains which opens and closes the
effector
binding clefts (
20,
28). It is therefore not
obvious how a
mutation in this region could lead to permanent
repression by
CcpA. The mutations introduce no drastic changes in the
chemical
properties of the affected residues in terms of size or
hydrophobicity.
These small changes might be the reason that
dimerization is still
possible, and their effector-independent
DNA-binding implies a
function of the C-terminal CcpA subdomain more
pronounced than
those recognized for LacI and
PurR.
The E77L and D275G substitutions are close to the effector binding
cleft (Fig.
4) which is involved in ligand recognition
and binding in
PurR and LacI and undergoes structural changes
in switching between the
DNA binding and nonbinding states (
20,
28). The side chain
of Glu77 faces the effector binding cleft.
It is flanked by other
residues making direct contact with the
effectors such as the analogous
residues to Asp275 of CcpA which
is mutated in CcpA
kD275G.
Asp is well conserved at this position among the members
of the
LacI-GalR family and a general role in ligand binding is
assumed
(
17,
34). The occurrence of
ccpAk
mutations in the effector binding cleft indicates that
low-molecular-weight
effectors such as glucose-6-phosphate and NADP
(
9,
15,
24)
could bind in that region, but a direct
interaction of these compounds
with CcpA and their physiological
function in this regulation
remain to be
proven.
Thr306, which is changed to Ile in CcpA
kT306I, is in a
solvent, exposed position, neighboring the proposed binding surface
for
HPr-Ser-P (
17). Amino acid exchanges in that surface result
in a loss of CCR in vivo and no or reduced interaction with HPr-Ser-P
(
17). In contrast, CcpA
kT306I leads to permanent
repression and binding of DNA. The amino
acid at position 306 is
conserved among CcpA-like proteins but
not among other members of the
LacI-GalR family (
17). Only one
protein of the CcpA
subfamily, RegA from
Clostridium acetobutylicum,
carries an
Ile at this position, as does CcpA
kT306I. Interestingly,
RegA complements a
B. subtilis ccpA mutant
to constitutive
repression of
amyE (
2). There is no explanation
why a change of the hydrophilic Thr to the hydrophobic Ile at
this
surface position renders CcpA regulation glucose independent,
but
CcpA
kT306I gives further evidence for the functional
importance of
this
region.
In summary, the five amino acid substitutions conferring permanent,
glucose-independent regulation by CcpA indicate that mutations
in three
different regions of the protein can alter DNA binding.
Mutations in
the corepressor binding cleft are consistent with
the assumption that
CcpA may be triggered by low-molecular-weight
effectors. The binding
site for HPr-Ser-P has no equivalent in
related repressors, but
CcpA-specific conservation and the gain-of-function
mutation
characterized here emphasize its special role for CcpA-mediated
regulation.
| |
ACKNOWLEDGMENTS |
We thank Kerstin Mahr for help with some experiments, Sabine
Pöhlmann for a gift of purified HPr-Ser-P protein, Alexandra Kraus and Richard Brennan for fruitful discussions, and Alexandra Schütz for expert technical assistance with 2D protein electrophoresis.
This study was supported by the EU Biotech Program, the Deutsche
Forschungsgemeinschaft through SFB 473, and the Fonds der Chemischen Industrie.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Lehrstuhl
für Mikrobiologie, Friedrich-Alexander-Universität
Erlangen-Nürnberg, Staudtstr. 5, 91058 Erlangen, Federal Republic
of Germany. Phone: 49-9131-8528081. Fax: 49-9131-8528082. E-mail:
whillen{at}biologie.uni-erlangen.de.
Present address: Department of Biology, Massachusetts Institute of
Technology, Cambridge, Massachusetts.
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Journal of Bacteriology, December 1999, p. 7634-7638, Vol. 181, No. 24
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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