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Journal of Bacteriology, July 1999, p. 4125-4128, Vol. 181, No. 13
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Mutations in Catabolite Control Protein CcpA
Separating Growth Effects from Catabolite Repression
Elke
Küster,
Tanja
Hilbich,
Michael K.
Dahl, and
Wolfgang
Hillen*
Lehrstuhl für Mikrobiologie, Institut
für Mikrobiologie, Biochemie und Genetik der
Friedrich-Alexander-Universität Erlangen-Nürnberg,
Erlangen, Federal Republic of Germany
Received 1 February 1999/Accepted 20 April 1999
 |
ABSTRACT |
Carbon catabolite repression in Bacillus megaterium is
mediated by the transcriptional regulator CcpA. A chromosomal deletion of ccpA eliminates catabolite repression and reduces the
growth rate on glucose. We describe four single-amino-acid mutations in
CcpA which separate the growth effect from catabolite repression, suggesting distinct regulatory pathways for these phenotypes.
| |
TEXT |
Bacillus megaterium
exerts carbon catabolite repression (CCR) on transcription of the
xyl operon to preferentially use energetically favorable
carbon sources (29). As in Bacillus subtilis, two proteins play a central role in CCR, namely, the catabolite control protein CcpA and HPr of the phosphoenolpyruvate-sugar
phosphotransferase system (6, 7, 9, 11). Upon
phosphorylation of HPr at residue Ser46 by the metabolite-activated
ATP-dependent kinase PtsK (27), it binds to CcpA
(3), leading to recognition of DNA sequences called
catabolite responsive elements (cre) (6, 7, 13).
These are found in the xyl operon and in front of many
catabolic genes and operons (10), several of which have been
shown to be subject to CCR (reviewed in reference
8). Immunological evidence suggests that CcpA-like
proteins are common to many gram-positive bacteria (18), and
recent results have demonstrated that CcpA homologues are involved in
CCR in Staphylococcus xylosus, Lactobacillus
casei, Lactobacillus pentosus, Lactococcus lactis, and Listeria monocytogenes (1, 4, 21, 22,
25). Thus, CcpA-mediated CCR turns out to be a widespread
mechanism among gram-positive bacteria. In addition to the loss of CCR, a deletion of ccpA in B. megaterium, B. subtilis, and Lactococcus lactis also leads to a
markedly decreased growth rate on minimal medium with various carbon
sources (11, 22, 33).
There may be different pathways by which the catabolic signal is
transmitted to CcpA-dependent target sites. CcpA binds without a
cofactor to cre in front of amyE of B. subtilis (13), and in vitro repression is enhanced by
combinations of HPr(Ser-P) and fructose-1,6-bisphosphate or NADP
(14), whereas binding to cre sequences present in
other operons is triggered by glucose-6-phosphate or HPr(Ser-P) alone
(7, 24). Thus, CcpA may discriminate between different
signals and respond with various outputs. We therefore asked whether
the two major phenotypes, namely, CCR and the influence on growth rate,
can be separated by mutations of ccpA.
Random mutagenesis of ccpA.
We randomly mutagenized the
ccpA gene and set up an in vivo screen for novel or altered
functions. The ccpA gene, including its putative promoter,
was cloned into M13mp18 (34), and single-stranded DNA of the
resulting phage mWH560 was treated with nitrite and amplified by
error-prone PCR, as described previously (26). The resulting
fragments were cloned into pWH2051 to give a pool of randomly mutated
ccpA genes. The 5' flanking region of ccpA in
pWH2051 is shorter than in the otherwise isogenic plasmid pWH2005, which has been used for regulatory studies of CcpA (11, 16). However, CCR mediated by pWH2005 or pWH2051 carrying the wild-type gene
is identical (data not shown).
Screening for ccpA alleles that differentially affect
CCR and growth.
We screened the mutant pool for ccpA
alleles that allow a chromosomal ccpA deletion mutant to
grow normally on glucose as the sole carbon source without restoring
CCR of the xyl operon (ccpAc,
defective in CCR only). We also looked for strains deficient in growth
but active in CCR (ccpAg, deficient in growth
only). For this purpose, we constructed B. megaterium WH353
[lac 
ccpA gdh2
(xylA1-spoVG-lacZ)
xylR] carrying a xylA-lacZ fusion and
chromosomal deletions in ccpA and xylR to observe
only the effects exerted by plasmid-encoded ccpA. The
bacteria were transformed with the mutant pool of ccpA by
protoplast transformation (28). After regeneration, the
cells were plated on M9 minimal medium containing glucose (0.2%) as a
regulatory carbon source, succinate (0.5%) as a carbon source which is
not effective in CCR, and X-Gal
(5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside) (80 mg/liter). We screened for large blue colonies
(ccpAc) and small white colonies
(ccpAg). Plasmid DNA was isolated, passaged
through Escherichia coli, and again transformed into
B. megaterium WH353 or WH419 [lac ccpA gdh2(xylA3-spoVG-lacZ)] (kindly provided by A. Wagner, Erlangen, Germany), and the phenotypes were reconfirmed on
plates. Since B. megaterium WH419 carries a functional
xylR gene, all experiments with this strain were carried out
in the presence of 0.5% xylose in the growth medium. Results obtained
with WH419 and WH353 were identical. The screen yielded six
ccpAc alleles and one
ccpAg mutant from about 2,500 primary transformants.
Characterization of ccpA mutations affecting catabolite
repression only (ccpAc).
Sequencing of the
ccpA alleles revealed that all ccpAc
mutants carried multiple mutations, as listed in Table
1. We separated the mutations by
combining ccpAc alleles with the wild type at
the unique AflII site, followed by screening as described
above. The subclones were named for the parental mutation, with the
addition of "n" or "c", indicating an N- or C-terminal mutation
with respect to the restriction site. The superscript "c" was
retained for those alleles that confer the ccpAc
phenotype. The relative growth rates for the original mutants and all
subclones were estimated from colony sizes on plates and are noted in
Table 1. The ccpAc mutants were
indistinguishable in colony size from those containing wild-type
ccpA.
To quantify CCR exerted by
ccpA alleles,
-galactosidase
activity in M9 medium with succinate or glucose and succinate as
sole
carbon sources was recorded. As expected from their blue
phenotype on
plates, the original
ccpAc mutants show reduced
CCR compared to wild-type
ccpA, even though
the remaining
repression efficiency is higher than that of the
ccpA
mutant (Table
1). The mutants were considered CCR negative
if they
exerted 25% or less wild-type repression. The subclones
of two
multiply mutated
ccpAc alleles
(
ccpAc9 and
ccpAc12) did not show reduced CCR
upon separation of the original mutations
(Table
1). Three
ccpA alleles with single mutations
(
ccpAc10n,
ccpAc19n, and
ccpAc20n) conferred wild-type-like
growth along with reduced CCR (Table
1). To ensure that effects were
not caused by different levels
of CcpA expression, we performed Western
blot analyses with extracts
of total cell protein, as described
previously (
16). All of
the mutant
ccpA alleles
expressed CcpA to levels similar to the
wild type (data not
shown).
For those
ccpAc mutants caused by a
single-amino-acid exchange, the growth phenotype was quantified on M9
minimal medium with
glucose (0.5%). Growth on plates was scored by
determining the
number of CFU per colony (Fig.
1). The values reflect the colony
sizes
given in Table
1. The growth rates in liquid medium (Fig.
1) are
consistent with these observations for mutants CcpA
c10n and
CcpA
c19n, which grow at the same rate as the wild type.
CcpA
c20n exhibits slower growth than the deletion mutant in
liquid
culture, which is in contrast to its behavior on solid medium.
The activity of CcpA in CCR is dependent on the agitation of liquid
cultures, with activities at low levels of agitation being comparable
to those observed on solid media (
2). The growth differences
in CcpA
c20n could be due to a similar effect, which may
imply that growth
regulation by CcpA
c20n is sensitive to
environmental conditions.
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FIG. 1.
Growth of selected mutants on solid and liquid minimal
media with 0.5% glucose as the sole source of carbon. To score growth
on solid medium, cells were streaked on plates and incubated at 30°C
for 20 h. Single colonies were excised with the surrounding agar
and resuspended in liquid medium. The number of CFU was determined by
plating aliquots from serial dilutions (hatched bars). Growth rates in
liquid medium were determined at 32°C with vigorous agitation (open
bars).
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|
To interpret possible defects caused by the mutations, we made use of
the common three-dimensional fold of LacI and PurR,
which probably
holds for the entire LacI/GalR family of bacterial
regulators,
including CcpA (
32), and is supported by limited
proteolysis
(
12) and mutational data (
16,
17). Their
functional
implications are discussed in the light of
structure-function
relationships in LacI (
5,
20) and PurR
(
30).
The three singular
ccpAc mutations are located
in the N-terminal DNA-binding domain of CcpA, as depicted in Fig.
2. The Thr4-to-Ser
exchange in
CcpA
c10n aligns with Thr3 of PurR, where it is located
N-terminally
to the positioning
-helix 1 of the helix-turn-helix
motif (Fig.
2) and does not contact DNA (
30). Mutational
analysis of LacI
revealed that the equivalent Thr5 residue could be
replaced only
by Ser, yielding a partially active repressor
(
15). Our observation
of diminished repression of
xylA-lacZ by CcpA
c10n resembles that result.
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FIG. 2.
Sequence alignment of the N termini of B. megaterium (Bme) and B. subtilis (Bsu) CcpA and the
purine and lactose repressors of E. coli (Eco). Identical
amino acids are shaded. Numbered boxes below the sequences denote
-helices, as determined for PurR. The positions of amino acid
exchanges of mutants isolated in this study are given above the
sequences.
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|
Arg47 is mutated to His in CcpA
c19n. The equivalent
residues Ser46 of PurR and Ile48 of LacI are involved in anchoring the
DNA-binding domain to the core of the protein (
30,
31). Any
mutation at this position is likely to affect the positioning
of the
headpieces relative to the protein core and thus their
alignment on the
operator.
The
ccpAc20n mutation, Asn49 to Ser, is located
next to the hinge helix which connects the DNA-binding domain to the
core of
the protein and contacts DNA. Asn residues are frequently found
at this position in the LacI/GalR family (
32). PurR contains
a Ser which contacts the DNA at a phosphate residue (
30).
Thus,
the Asn49-to-Ser exchange in CcpA may affect DNA binding. In
contrast
to this, the corresponding residue in LacI, Asn50, is involved
in interdomain contacts between the headpiece and the core (
20,
31). The LacI Asn50-to-Ser mutant is fully active, while other
residues at position 50 yield inactive mutants (
15). Thus,
the
underlying reason for the reduced CCR may be altered DNA binding,
but this conclusion is not unambiguously deducible from these
data.
Taken together, all three
ccpAc mutations are at
positions likely to directly or indirectly affect DNA binding. This
relates
the loss of CCR to impaired recognition of
cre. The
fact that
loss of CCR is not accompanied by a growth defect may be
explained
in two ways. Either DNA sequences recognized for growth
regulation
are different from the
cre mediating CCR in
xyl or from the
cre consensus sequence in general
or the interaction with possibly
unknown cofactors leads to a titration
effect that is lacking
in the
ccpA mutant but not in the
ccpAc mutants.
Characterization of a ccpA mutant affecting growth only
(ccpAg).
The only CcpAg
mutation, CcpAg14, limited growth to small colonies on all
media tested, but not as small as those of WH353 (ccpA). Therefore, the growth phenotype of
ccpAg14 is designated "+/
" in
Table 1. Quantification of its growth behavior on solid and liquid
media led to values similar to those of the deletion mutant. The
-galactosidase activity of the xylA-lacZ fusion shows
that the ccpAg14 mutant is even more
efficient in repression than is the wild type (Table 1).
CcpAg14 is expressed to a level similar to that of the
wild-type protein, as determined by immunoblotting (data not shown).
The
ccpAg14 mutation leads to a
single-amino-acid exchange, Ala17 to Thr. This residue is located in
the recognition helix
(Fig.
2). The analogous Thr16 of PurR is involved
in protein-DNA
interaction and contacts two bases directly
(
30). The equivalent
Gln18 of LacI also determines operator
recognition (
19,
20).
One would therefore assume that the
ccpAg14 mutation leads to an altered
DNA-binding specificity of CcpA.
However, because catabolite repression
of
xylA is even somewhat
more effective with
ccpAg14 than in the wild type,
cre of
xylA must be efficiently recognized.
Thus,
the growth rate reduction may not involve DNA binding, although
the
properties of this mutant would also be consistent with speculation
that a different
cis-acting sequence mediates growth rate
control.
We have established for the first time that growth rate control and CCR
are separable functions of CcpA. The underlying molecular
basis could
be interaction with different signal molecules, differences
in signal
transfer from the effector binding site to the headpiece,
or
recognition of different
cis-acting sequences. The four
single
mutants are affected in amino acids involved in DNA recognition
or interdomain contacts relevant for recognition of DNA. Although
altered cofactor binding cannot be rigorously ruled out, binding
of
CcpA to as-yet-unidentified DNA sites is likely to be part
of its
function in growth
regulation.
| |
ACKNOWLEDGMENTS |
We thank Holger Ludwig, Bianka Wolf, and Michael Stock for help
with some of the experiments and Jörg Stülke and Alexandra Kraus for fruitful discussions.
This study was supported by a personal grant of the
Friedrich-Ebert-Stiftung to E.K. and by the EU Biotech Programme, the Fonds der chemischen Industrie, and the Deutsche Forschungsgemeinschaft through SFB 473.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Lehrstuhl
für Mikrobiologie, Institut für Mikrobiologie, Biochemie
und Genetik der 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.
| |
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Journal of Bacteriology, July 1999, p. 4125-4128, Vol. 181, No. 13
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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