Forty millimolar fructose-1,6-bisphosphate FBP or glucose-6-phos-phate Glc6-P increases the affinity of HPrSerP to CcpA at least twofold, but have no effect on CrhP–CcpA binding.. In an a
Trang 1cre in carbon catabolite regulation of Bacillus subtilis
Gerald Seidel, Marco Diel, Norbert Fuchsbauer and Wolfgang Hillen
Lehrstuhl fu¨r Mikrobiologie, Institut fu¨r Mikrobiologie, Biochemie und Genetik der Friedrich-Alexander Universita¨t Erlangen-Nu¨rnberg, Germany
Carbon catabolite regulation (CCR) in Gram-positive
bacteria with low GC content is one of the most
ver-satile regulatory processes known in bacteria In
Bacillus subtilis, the central regulator of CCR, called
CcpA, represses or activates more than 300 genes
involved in carbon and nitrogen utilization [1–4] and is
active in the exponential but also in the stationary
growth phases [5–8] Therefore, the probably
multifac-eted regulatory mechanism of CcpA-mediated CCR is
of considerable interest CcpA is a member of the
LacI⁄ GalR family of bacterial regulators and binds to
catabolite responsive elements (cre) in dependence of
different effectors While members of the LacI⁄ GalR
family usually respond to low molecular weight
compounds, the main effectors for CcpA are the Ser46 phosphorylated histidine-containing protein (HPrSerP) and the Ser46 phosphorylated catabolite repression HPr (CrhP) [3] HPr can also be phosphorylated at histidine 15 acting as a phosphotransferase in the phosphoenolpyruvate:sugar phosphotransferase system (PTS) In contrast, Crh residue 15 is a glutamine, which cannot be phosphorylated by the PTS Mutation
of the respective genes, ptsH and crh, results in complete loss of CCR [9–13] HPr and Crh are phos-phorylated at Ser46 by the ATP-dependent HPr kinase⁄ phosphorylase (HPrK ⁄ P) in response to high glycolytic activity [9] There is increasing evidence that HPrSerP and CrhP can lead to different responses
Keywords
carbon catabolite regulation; CrhP; HPrSerP;
fluorescence spectroscopy; surface plasmon
resonance
Correspondence
W Hillen, Lehrstuhl fu¨r Mikrobiologie,
Institut fu¨r Mikrobiologie, Biochemie und
Genetik der Friedrich-Alexander Universita¨t
Erlangen-Nu¨rnberg, Staudtstr 5, 91058,
Erlangen, Germany
Fax: +49 9131 8528082
Tel: + 49 9131 8528081
E-mail: whillen@biologie.uni-erlangen.de
(Received 28 January 2005, revised 16
March 2005, accepted 23 March 2005)
doi:10.1111/j.1742-4658.2005.04682.x
The phosphoproteins HPrSerP and CrhP are the main effectors for CcpA-mediated carbon catabolite regulation (CCR) in Bacillus subtilis Complexes of CcpA with HPrSerP or CrhP regulate genes by binding to the catabolite responsive elements (cre) We present a quantitative analysis
of HPrSerP and CrhP interaction with CcpA by surface plasmon resonance (SPR) revealing small and similar equilibrium constants of 4.8 ± 0.4 lm for HPrSerP–CcpA and 19.1 ± 2.5 lm for CrhP–CcpA complex dissoci-ation Forty millimolar fructose-1,6-bisphosphate (FBP) or glucose-6-phos-phate (Glc6-P) increases the affinity of HPrSerP to CcpA at least twofold, but have no effect on CrhP–CcpA binding Saturation of binding of CcpA
to cre as studied by fluorescence and SPR is dependent on 50 lm of HPrSerP or > 200 lm CrhP The rate constants of HPrSerP–CcpA– cre complex formation are ka ¼ 3 ± 1 · 106m)1Æs)1 and kd¼ 2.0 ± 0.4· 10)3Æs)1, resulting in a KD of 0.6 ± 0.3 nm FBP and Glc6-P stimu-late CcpA–HPrSerP but not CcpA-CrhP binding to cre Maximal HPr-SerP-CcpA–cre complex formation in the presence of 10 mm FBP requires about 10-fold less HPrSerP These data suggest a specific role for FBP and Glc6-P in enhancing only HPrSerP-mediated CCR
Abbreviations
CcpA, catabolite control protein A; CCR, carbon catabolite regulation; cre, catabolite responsive elements; CrhP, catabolite repression HPr phosphorylated at serine 46; F-6-P, fructose-6-phosphate; FBP, fructose-1,6-bisphosphate; Glc1-P, glucose-1-phosphate; Glc6-P, glucose-6-phosphate; HPrK ⁄ P, HPr kinase ⁄ phosphorylase; HPrSerP, histidine containing protein phosphorylated at serine 46; PTS, phosphotransferase system; RU, response units; SPR, surface plasmon resonance.
Trang 2The ptsH1 mutant encoding HPr46A shows reduced
CCR at many genes because CrhP substitutes only
partially for HPrSerP crh mutants, on the other hand,
do not exhibit reduced CCR [14] The properties of
both effectors in CCR can depend on the growth
con-ditions: The B subtilis hut operon responds only to
HPrSerP in Luria–Bertani medium, but to HPrSerP
and CrhP in minimal medium [15] CrhP is the sole
effector for CCR of citM in minimal medium with
suc-cinate [16] These observations may be related to the
recently observed carbon source-dependent difference
in crh and ptsH expression: the PTS sugars mannitol,
fructose, sucrose and glucose lead to an increase of
ptsH expression, whereas succinate or citrate increase
crh expression [17] CCR of some promoters, e.g
ctaBCDEF and creup dependent regulation of the gnt
operon, is not affected by the ptsH1 mutation, but no
data regarding the participation of CrhP are available
[8,18] No difference is observed to HPr-kinase
cata-lysed in vitro phosphorylation of HPr and Crh [9] but
the stimulatory effect of HPrSerP on CcpA binding to
cre is stronger than that of CrhP [11,12] Structural
differences were revealed by NMR and X-ray
indi-cating dimerization of Crh, but not of HPr [19–21]
Low molecular mass effectors, which would be
typical inducers for members of the LacI⁄ GalR
fam-ily, are discussed controversially as effectors for
CcpA Fructose-1,6-bisphosphate (FBP) and
glucose-6-phosphate (Glc6-P) enhance HPrSerP binding to
CcpA [22], and FBP and NADP showed cooperative
stimulation of CcpA binding to amyO in the
pres-ence of HPrSerP [23] Glc6-P also stimulated CcpA
binding to cre in the absence of HPrSerP [18,24]
Taken together, there are many observations of
dif-ferential CcpA-mediated CCR, involving two
phos-phoproteins and several low molecular weight
effectors In an attempt to quantitatively describe the
binding of HPrSerP and CrhP to CcpA, the effects
of FBP and Glc6-P and their stimulation of cre
binding we used surface plasmon resonance (SPR)
and fluorescence to observe formation of these
com-plexes We describe a new role for FBP and Glc6-P
in CCR because they enhance HPrSerP-mediated
binding of CcpA to cre, but have no effect on
CcpA–CrhP–cre interaction
Results
HPrSerP and CrhP binding to CcpA
SPR analyses of the protein–protein interactions of
HPr, Crh and their serine phosphorylated forms with
CcpA from B subtilis have been carried out on
Bia-core CM5 chips, to which CcpA was covalently cou-pled in flowcell 2 TetR was used as control in flowcell 1 and showed no affinity for any of these proteins Increasing concentrations (from 10 to
100 lm) of HPr or Crh did not show any binding of either protein indicating their weak affinities for CcpA In contrast, HPrSerP or CrhP bind to CcpA under these conditions (Fig 1) A saturation response difference of 250–280 reponse units (RU; 1000 RU,
1 ng bound ligand) was obtained for concentra-tions above 100 lm when a chip with 2100 RU of immobilized CcpA was used The equilibrium con-stants of HPrSerP and CrhP binding to CcpA were determined by titration under steady-state conditions using 1700–2300 RU of coupled CcpA and a flow rate of 5 lLÆmin)1 (Supplementary material, Fig 1) Langmuir fits of the results revealed the rather small dissociation constants of 4.8 ± 0.4· 10)6m for HPr-SerP and 19.1 ± 2.5· 10)6m for CrhP We did not detect any indication for cooperativity in the fit (Fig 2) This result is in agreement with the hypo-thesis that only one form of the phosphoproteins and one interaction modus are involved in complex formation of HPrSerP or CrhP with CcpA Struc-tural analyses suggested that Crh may exist as a dimer at high concentrations [19–21], however, we detected only one band in 7.5% native PAGE indi-cating that our protein preparation contains only one form of CrhP (data not shown) Furthermore, native PAGE of phosphorylated HPrSerP or CrhP did not show any nonphosphorylated HPr or Crh (data not shown) In addition, the saturation response for CrhP is the same as that for HPrSerP bound to the same CcpA loaded chip Since the SPR signal corres-ponds directly to the bound mass, and as both
pro-Fig 1 Surface plasmon resonance analyses of effector binding to CcpA The figure shows the sensorgrams obtained from the inter-action analysis of CcpA with HPr, HPrSerP, Crh and CrhP Dilutions (10 l M and 100 l M ) of each protein were pumped at 5 lLÆmin)1 over a CM5 chip loaded with TetR (control) in flowcell 1 and CcpA
in flowcell 2 The left diagram shows sensorgrams from injections
of HPr or HPrSerP and the right diagram shows the respective sen-sorgrams for injections of Crh or CrhP The concentrations of each cofactor are shown.
Trang 3teins have almost equal molecular weights, this
strongly indicates binding of the same forms of
HPr-SerP and CrhP to CcpA In conclusion, we assume
that only the monomeric state of CrhP is present
under the conditions of this study
Effects of FBP and Glc6-P on HPrSerP- and CrhP–CcpA interaction
The effects of FBP and Glc6-P were determined by SPR at 1 lm of HPrSerP or 4 lm of CrhP so that about 20% of the immobilized CcpA is complexed The addition of FBP or Glc6-P at millimolar concen-trations led to increased complex formation of HPr-SerP (Fig 3) Titration with rising concentrations of
up to 40 mm of FBP or Glc6-P did not yield satura-tion (Fig 3B and D) The stimulasatura-tion of HPrSerP binding to CcpA by these two effectors is highly spe-cific because neither fructose-6-phosphate (F-6-P) nor glucose-1-phosphate (Glc1-P) showed any influence
on binding (Fig 3A and C) Titrations of CcpA with HPrSerP at 40 mm FBP or Glc6-P resulted in a
KD (40 mm FBP) of 1.7 ± 0.3· 10)6m and a KD (40 mm Glc6-P) of 2.2 ± 0.1· 10)6m, respectively (data not shown) Therefore, FBP stimulates CcpA– HPrSerP complex formation at least twofold The SPR increase at saturation is about the same for titrations with or without FBP or Glc6-P indicating that roughly the same mass binds to CcpA, ruling out a possible oligomerization of HPrSerP Addition
of only FBP or Glc6-P to the CcpA chip did not yield a signal (data not shown)
Fig 2 Equilibrium titration of CcpA with HPrSerP and CrhP The
figure shows a plot of the equilibrium responses from each
sensor-gram vs the corresponding HPrSerP (d) and CrhP (s)
concentra-tions Equilibrium constants were derived by the displayed
Langmuir fits.
Fig 3 Effects of low molecular mass coeffectors on HPrSerP and CrhP binding to CcpA Sensorgrams resulting from running 1 l M of HPr-SerP or 4 l M of CrhP over a CM5 chip with TetR in flowcell 1 and CcpA in flowcell 2 In addition, 5–40 m M FBP, F-6-P (A and B), Glc6-P or Glc1-P (C and D) were added (A) The left diagram shows sensorgrams from passages of 1 l M HPrSerP with or without 40 m M FBP or F-6-P The right diagram displays sensorgrams resulting from injections of 4 l M CrhP with or without 40 m M FBP or F-6-P (B) The left dia-gram shows a titration of CcpA with mixtures of 1 l M HPrSerP and increasing concentrations (5–40 m M ) of FBP The right diagram shows the analogous titration with 4 l M CrhP instead of HPrSerP (C) The left diagram shows sensorgrams from passages of 1 l M HPrSerP with or without 40 m M Glc6-P or Glc1-P The right diagram shows passages of 4 l M CrhP with or without 40 m M Glc6-P or Glc1-P (D) The left part shows sensorgrams from a titration of CcpA with mixtures of 1 l M HPrSerP and increasing concentrations (5–40 m M ) of Glc6-P The sensor-grams in the right diagram show the respective titration with 4 l M CrhP instead of HPrSerP Analytes and their concentrations are shown in the diagrams.
Trang 4CrhP binding to CcpA was not affected by FBP,
Glc6-P, F-6-P or Glc1-P (Fig 3) This result is
surpri-sing and suggests distinct functions of these
phospho-proteins Since neither nonphosphorylated HPr nor
Crh interacted with CcpA in the presence of FBP,
Glc6-P, F-6-P or Glc1-P in the concentration range
used in these experiments (data not shown), these low
molecular weight coeffectors specifically affect the
HPrSerP–CcpA complex
Stimulation of CcpA-cre complex formation
by HPrSerP, CrhP, FBP and Glc6-P
The interaction of CcpA with cre was analysed by
fluorescence and SPR A C-terminally His-tagged
CcpA-1W mutant carrying a single tryptophan residue
at the N terminus was used for the fluorescence
meas-urements The regulatory activity of this mutant was
determined in B subtilis WH440 DccpA carrying a
xynP¢::lacZ fusion, transformed with either pWH1533,
pWH1541 or pWH1542 expressing CcpA, His-tagged CcpA or His-tagged CcpA-1W, respectively (Table 1) The three strains expressed about the same b-galactosi-dase activities in dependence of the respective carbon sources (Table 2) We therefore conclude that CcpA-1W exhibits the same regulatory properties as the wild-type CcpA-1W was prepared to homogeneity and showed increased fluorescence emission upon addition
of cre DNA and HPrSerP (Fig 4) or CrhP (data not shown) No fluorescence change was observed when HPrSerP or CrhP was added without cre DNA, or in the presence of an oligonucleotide without cre (data not shown) Thus, the fluorescence change of CcpA-1W is indicative for cre binding No fluorescence change was observed with HPr instead of HPrSerP (Fig 4) Titration of a CcpA-1W⁄ cre DNA mixture with either HPrSerP (Supplementary material, Fig 2)
or CrhP (data not shown) led to increasing fluores-cence, indicating complex formation of CcpA with cre About 2.5-fold more CrhP than HPrSerP was needed
Table 1 Plasmids and strains used in this study.
Table 2 Effect of the ccpA deletion and in trans complementation of the xynP ¢::lacZ fusion with wildtype ccpA and the His-tagged ccpA mutants.
Strain and (relevant genotype)
b-Galactosidase activity in different media
Trang 5to obtain the same degree of CcpA binding to cre.
This result corresponds to the weaker binding of CrhP
to CcpA described above We were unable to
deter-mine the binding constant of the CcpA-1W–HPrSerP–
cre complex by fluorescence, since the required low
CcpA-1W concentration is below the detection limit
The influence of effectors on CcpA binding to cre
were also analysed by SPR About 1000 RU
biotinylat-ed 48-bp cre DNA were bound to a Biacore SA chip in
flowcell 2 and 48-bp nonspecific DNA in flowcell 1 and
titrated with 100 nm to 75 lm HPrSerP at 10 nm CcpA
or with 100 pm to 10 nm of CcpA at 25 lm HPrSerP
The results indicated that at least 10 lm of HPrSerP
and nanomolar concentrations of CcpA would have
to be used for quantifications We did not observe a
steady-state response within a feasible time (data not
shown), and concentrations above 5 lm of HPrSerP
led to nonspecific interactions with the Biacore SA
chip Nonspecific interaction of HPrSerP or CrhP did
not occur with the Biacore CM5 chip We have used a
new method to couple aminomodified DNA to that
chip and measured CcpA–cre binding, HPrSerP
stimu-lation of CcpA–cre binding and their reaction rates
Initial experiments confirmed that CcpA binds weakly
to cre (data not shown) as published previously [25]
Stimulation of xylAcre binding of nanomolar
concen-trations of CcpA occurs only at micromolar
concentra-tions of HPrSerP or CrhP but not with HPr or Crh
Thus, xylAcre was titrated with HPrSerP or CrhP at a
fixed concentration of 10 nm of CcpA (Fig 5A and B)
The results demonstrate that 50 lm HPrSerP leads to
complete saturation of cre, while the same concentra-tion of CrhP yields only partial saturaconcentra-tion, resembling its weaker affinity for CcpA (Fig 2)
The effects of FBP and Glc6-P on cre binding were also analysed by fluorescence and SPR Fluorescence was observed in mixtures containing 0.075 lm of CcpA-1W, 0.225 lm cre DNA and 0.3 lm HPrSerP or 0.75lMCrhP These conditions yielded about 30% of the maximal fluorescence change, indicating partial formation of the CcpA–HPrSerP–cre complex Titra-tion with FBP (Fig 6A) or Glc6-P (Supplemental Fig 3A) yielded an increased fluorescence until satura-tion was reached at 2 mm FBP and 10 mm Glc6-P, repectively This experiment showed the same fluores-cence intensity obtained in the titrations with HPrSerP
Fig 4 Fluorescence analysis of effector stimulated binding of CcpA
to cre Fluorescence emission spectra of 0.15 l M CcpA-1 W with
and without HPr or HPrSerP in the presence and absence of cre
are shown as indicated in the figure Black line, 0.15 l M CcpA-1 W;
dark green line, 0.15 l M CcpA-1 W with 1.5 l M HPr; red line,
0.15 l M CcpA-1 W with 1.5 l M HPrSerP; light green line, 0.15 l M
CcpA-1 W with 1.5 l M HPr and 0.225 l M xylAcre; blue line,
0.15 l M CcpA-1 W with 1.5 l M HPrSerP and 0.225 l M xylAcre.
Fig 5 HPrSerP and CrhP concentration dependence of the CcpA– xylAcre association rate (A) Titration of cre with HPrSerP at 10 n M
of CcpA The HPrSerP concentration for each sensorgram is shown The baseline responses were found for 10 n M of CcpA with
or without 50 l M of HPr (B) Titration of cre with CrhP in the pres-ence of 10 n M CcpA The baseline response was found for 10 n M
of CcpA with 50 l M of Crh The CrhP concentration for each sen-sorgram is shown.
Trang 6(Supplemental Fig 2) We conclude that FBP or
Glc6-P stimulates binding of HGlc6-PrSerGlc6-P to CcpA thereby
increasing HPrSerP-CcpA-cre complex formation,
which is monitored by flourescence In contrast,
titra-tions with F-6-P or Glc1-P did not result in any
change of fluorescence Replacing HPrSerP by CrhP
in these titrations did not yield any stimulation of
complex formation by FBP (Fig 6B) or Glc6-P
(Supplemental Fig 3B), either, whereas the subsequent
increase of the CrhP concentration resulted in
com-plete complex formation To verify this result by SPR,
experiments using mixtures of 10 nm CcpA and 1 lm HPrSerP or 5 lm CrhP yielding partial CcpA-HPrSerP
or CcpA-CrhP complex formation with cre on a CM5 chip were titrated with FBP Fig 6C shows the sensor-grams of both titrations Ten millimolar FBP resulted
in complete HPrSerP–CcpA–cre complex formation In contrast, the binding of CrhP–CcpA to cre is not affec-ted by up to 20 mm FBP We conclude again that FBP and Glc6-P stimulate the HPrSerP-dependent binding of CcpA to cre, but have no effect on CrhP-dependent binding
Fig 6 Fluorescence and SPR analysis of
FBP effects on HPrSerP and CrhP mediated
cre binding by CcpA (A) Plot of I ⁄ I 0 (I 0 :
CcpA-1 W fluorescence intensity only) vs.
the effector concentrations for titrations of
0.075 l M CcpA-1 W and 0.225 l M cre (n),
or of 0.075 l M CcpA, 0.225 l M cre and
0.3 l M HPrSerP (m) with FBP, and the
titra-tion of 0.075 l M CcpA-1 W, 0.3 l M HPrSerP
and 0.225 l M cre with F-6-P (d) (B)
Fluor-escence titration of 0.075 l M CcpA-1 W,
0.75 l M CrhP and 0.225 l M cre with FBP.
At 12 m M of FBP the CrhP concentration
was raised to 3.75 l M and 5.75 l M Points
after addition of cre and corepressor are
marked by arrows and labels (C)
Sensor-grams showing the influence of FBP
con-centrations of 2–20 m M added to 10 n M
CcpA, xylAcre and 1 l M HPrSerP (left
dia-gram) or 5 l M of CrhP instead of HPrSerP
(right diagram) The baseline sensorgram
results from the analysis of a mixture of
10 n M CcpA and 10 m M FBP The FBP
con-centrations are shown at the right side of
each sensorgram.
Trang 7Association and dissociation kinetics of the
CcpA–HPrSerP–xylAcre complex
Dissociation of the CcpA–HPrSerP–cre complex is very
fast when buffer is injected A much slower dissociation
was observed when HPrSerP was included in that
buf-fer (Fig 7A) Variation of the HPrSerP concentration
yielded a constant dissociation rate above 10 lm (data
not shown) Since the maximal rate of
CcpA–HPr-SerP–cre association occurs at 50 lm of HPrSerP (see
Fig 5A) we have used this concentration to avoid bulk
effects during the experiment, which are due to
nonspe-cific signal changes caused by differences between
sam-ple composition and the running buffer We assume
that all CcpA is complexed with HPrSerP under these
conditions Therefore, the association and dissociation
rate constants of the HPrSerP–CcpA–cre complex were
determined with 50 lm HPrSerP in all buffers and
increasing concentrations from 1 to 30 nm of CcpA
We fitted the sensorgrams according to the 1 : 1
Lang-muir binding model implemented in the biaevaluation
3.1 software, assuming association and dissociation of
the CcpA–HPrSerP complex from cre under these
conditions The respective sensorgrams and fits for the
rate constants are shown in Fig 7B, yielding a ka of
3 ± 1· 106m)1Æs)1 and a kd of 2.0 ± 0.4· 10)3s)1
resulting in an apparent KDof 6 ± 3 · 10)10 m at an
average deviation v2ass.¼ 3–4 and v2
diss.¼ 1 The sen-sorgrams from a titration of cre with increasing
con-centrations of CcpA in the presence of 5 lm HPrSerP
and 10 mm FBP are shown in Fig 7C The same fitting
as above assuming association or dissociation of a
CcpA–HPrSerP–FBP complex from cre yields the
con-stants ka¼ 2.2 ± 0.5 · 106m)1Æs)1 and kd¼ 2.7 ±
0.8· 10)3s)1 resulting in an apparent KD of 1.2 ±
0.4· 10)9m at vass2¼ 2–3 and v2
diss:¼ 1 These con-stants are very similar to the ones obtained without
FBP at 50 lm HPrSerP suggesting that FBP decreases
the amount of HPrSerP necessary for complete binding
of CcpA to cre
Discussion
Many qualitative and some quantitative studies of
var-ious effector molecules affecting CcpA–cre interaction
have led to a general mechanism of action for CCR in
B subtilis[11,12,23,25,26] However, the current model
does not explain all results, e.g it is not clear how
sim-ilar PTS sugars such as glucose, fructose or mannitol
lead to quite different extents of CCR, and how carbon
sources like glucitol or succinate lead to ptsH- or
crh-dependent CCR [9,11,16,27,28] The different roles of
HPr and Crh in CcpA-mediated CCR are particularly
Fig 7 Kinetic analysis of CcpA-HPrSerP binding to xylAcre by SPR (A) Sensorgrams of mixtures containing 10 n M CcpA and 5 l M HPr-SerP are shown In the red sensorgram the dissociation is observed in running buffer and in the blue sensorgram the dissoci-ation is observed first in running buffer with and then without 5 l M
HPrSerP as indicated (B) The rate constants were obtained from titrations of xylAcre with mixtures of 1–30 n M B subtilis CcpA and
50 l M HPrSerP (running buffer with 50 l M HPrSerP) or (C) 1–30 n M
B subtilis CcpA, 5 l M HPrSerP and 10 m M FBP (running buffer with 5 l M HPrSerP and 10 m M FBP) The concentrations of the CcpA–HPrSerP are assumed to be the same as those of CcpA and are depicted in the respective colour The fits of the association phases are drawn as black lines.
Trang 8mysterious, since their phosphorylation by HPrK⁄ P is
similarly effective [9,29] CrhP may be specifically active
in CCR brought about by nonsugar compounds as
des-cribed for citM [16] The approximately fourfold
stron-ger affinity of HPrSerP for CcpA compared to CrhP
found here may contribute to the weaker stimulation of
CrhP for CcpA binding to xylAcre, glpFKcre, ptacre
and xynPcre [11,12,30], but it seems likely that other
factors also contribute to differential regulation For
example, the KDof the B subtilis HPrSerP–CcpA
com-plex of 5 lm is almost identical to that determined
for the respective Lactobacillus casei proteins (4 lm),
but despite the fact that B subtilis- and B
megateri-um-derived HPrSerP showed the same fivefold lower
KD for binding to L casei CcpA, only the B subtilis
but not the B megaterium ccpA mutant can be
com-plemented by L casei ccpA [26] This indicates that
CcpA–cre complex formation may be influenced by
more factors than the CcpA–HPrSerP affinity
Stimulation of HPrSerP–CcpA complex formation by
FBP and Glc6-P has been observed qualitatively before
[22] Footprinting has indicated that HPrSerP and CrhP
mediated CcpA–cre complex formation are stimulated
by FBP [11,12] The data presented here establish for the
first time distinct mechanisms for these two effectors as
only HPrSerP binding to CcpA responds to the
coeffec-tors FBP and Glc6-P Consequently, only the HPrSerP–
CcpA–xylAcre interaction is stimulated by FBP and
Glc6-P, but not CrhP–CcpA–xylAcre complex
forma-tion The stimulatory concentrations of approximately
10 mm FBP or Glc6-P are within the range of
physiolo-gical variance of these compounds [31,32] FBP or
Glc6-P reduce the concentration of HGlc6-PrSerGlc6-P necessary for
complete occupation of CcpA, and, in turn, 10 mm FBP
leads to an approximately tenfold reduction of the
amount of HPrSerP necessary for complete occupation
of cre by CcpA–HPrSerP Thus, in the presence of these
mediators at least 40-fold more CrhP compared to
HPr-SerP would be necessary to mediate full repression
These properties could explain the ptsH-specific CCR in
the presence of glucitol [11] because this non-PTS sugar
is converted to FBP [33] Furthermore, the stimulatory
effect of Glc6-P could explain the stronger CCR exerted
by glucose as compared to other PTS sugars [9,27,28]
Crh-mediated CCR occurs in the presence of succinate
and glutamate [16] Since this is a physiological situation
with low intracellular concentrations of Glc6-P and
FBP, there may be yet unknown effectors for CcpA
The equilibrium constants of HPrSerP and CrhP
binding to CcpA from B subtilis are quite low, but
they are very well adjusted to the cellular
concentra-tions of 1 lm of CcpA and 0.1–2 mm of HPrSerP, as
found in Bacilli and Streptococci in the presence of
glucose [34,35] The low affinity of HPrSerP to CcpA makes the in vitro analysis of the coupled binding to cre difficult This explains the unusually high concen-trations of CcpA that had to be used to detect DNA binding in all previous studies, except for binding to amyO [23] and rocGcre [13] We have previously deter-mined a low apparent equilibrium constant of KD¼
200 nm for the CcpA–HPrSerP–cre complex from
B megaterium by EMSA and SPR [25], because we assumed a KD of at least 500 nm for the CcpA–SerP complex and consequently used not enough HPr-SerP to obtain saturation of CcpA These conditions also masked the effects of FBP and Glc6-P The KDof the CcpA–HPrSerP complex and the titrations of cre with HPrSerP at a constant CcpA concentration deter-mined here show that at least 50 lm of HPrSerP is required to assure complete complex formation of HPrSerP with CcpA, a prerequisite for quantification
of the CcpA–HPrSerP–cre interaction
The rate and equilibrium constants determined here agree well with those determined for other members of the LacI⁄ GalR family of bacterial regulators, like PurR
in the presence of guanine (ka¼ 1.5 ± 2 · 107m)1Æs)1;
kd¼ 1.2 ± 0.2 · 10)3s)1; KD¼ 0.8 ± 1 · 10)10m) [36] and LacI (ka ¼ 2 · 106m)1Æs)1; kd¼ 3.5 · 10)4s)1;
KD¼ 2 · 10)10m) [37] However, there may be two dif-ferent types of CcpA–cre interactions CcpA binding to the cre sites at the xylA, xynP [11], pta [12], glpFK [30]
or gnt [38] promoters is very weak or not detectable without cofactors, whereas binding to amyO [23] or rocGcre [13] is strong HPrSerP at 0.68 lm stimulated CcpA binding to amyO only 10-fold and 2 mm FBP with 0.68 lm HPrSerP stimulated it 300-fold, whereas CcpA–xylAcre binding is stimulated at least 1000-fold
in the presence of 50 lm HPrSerP [23] Thus, different cre sequences found in many genes or operons may respond in a differential manner to FBP- or Glc6-P-mediated stimulation
Experimental procedures
Plasmid construction and bacterial strains Strains and plasmids used in this study are listed
in Table 1 For in frame deletion of ccpA in B subtilis two DNA fragments were amplified from chromosomal DNA from B subtilis 168, where primer pairs dccpA1 (5¢-ATA ATAATAGAGCTCGCTGTGCCGATTTTGAAACAAG-3¢) and dccpA2 (5¢-TATTATTATAGCGGCCGCAATATT GCTCATCCTAAAACC-3¢) yielded fragment 1 and dccpA3 (5¢-ATAATAATAGCGGCCGCTGAAGCACTGCAGCAT CTGATG-3¢) with dccpA4 (5¢-TATTATTATGGTACCT TTTCGGTGCCGTTCCTCC-3¢) yielded fragment 2
Trang 9Frag-ment 1 comprises the sequence from 500 bp upstream to
13 bp downstream of ccpA translational start with SacI
and NotI restriction sites at the 3¢- and 5¢-termini,
respect-ively Fragment 2 includes the ccpA sequence from
base-pair 684–438 bp of ytxD downstream from ccpA with NotI
and KpnI restriction sites at the 3¢- or 5¢- ends,
respect-ively Plasmid pWH618 was constructed by cloning these
fragments into pBluescriptII SK + via the restriction sites
SacI and NotI for fragment 1 and NotI and KpnI for
frag-ment 2 The product carries a ccpA fragfrag-ment lacking bases
13–684 (corresponding to residues Thr5–Leu228) The NotI
restriction site was positioned between bases 13 and 684
resulting in a linker with three alanines replacing CcpA
residues 6–227 Strain WH440 was generated by
cotrans-formation of B subtilis 168 with the plasmid pWH618 and
chromosomal DNA from B subtilis QB7144 (xynP¢::lacZ)
Transformants were selected for the presence of the cat
resistance gene linked to the xynP¢::lacZ fusion from
QB7144 on CSK medium supplemented with 1% glucose,
0.2% xylose and 80 mgÆmL)1X-Gal and 5 mgÆmL)1
chlor-amphenicol Blue stained colonies showing deregulation of
xynP¢::lacZ were picked for verification of the deletion
of ccpA by Western blotting For complementation of
WH440 ccpA was amplified with the primer pair ccpAmut1
(5¢-ATAATATCTAGAACCAAGTATACGTTTTCATC-3¢)
and ccpAstd2 (5¢-TATTATTATGGATCCTTTTCTTA
TGACTTGGTTT-3¢) This fragment contains the ccpA
promoter 290 bp upstream from the start codon [39] This
fragment was cloned into the shuttle vector pHT304 [40]
via the restriction sites XbaI and BamHI resulting in
pWH1533 For construction of the vector pWH1541 ccpA
was amplified by ccpAmut1 and ccpAnot (5¢-TATTAT
TATGCGGCCGCTGACTTGGTTGACTTTCTA-3¢) using
pWH1533 as template The His-tag encoding
seq-uence was amplified by primers hisnot (5¢-ATAA
TAGCGGCCGCGGGCGGTCATCACCATCACCATCAC
TA-3¢) and hisbam (5¢-TATTATTATGGATCCTTAGC
TTCCTTAGCTCCTGA-3¢) from vector pQE17 After
restriction of the ccpA fragment with XbaI and NotI and
the His-tag encoding fragment with NotI and BamHI, both
were cloned in a three-armed ligation into pHT304 via the
restriction sites and XbaI and BamHI For construction of
pWH1542 ccpA was mutagenized via two-step
mutagene-sis using primers ccpAmut1, hisbam and ccpA +1W
(5¢-CGTAATATTGCTCCACATCCTAAAACC-3¢) The
resulting fragment encoding C-terminally His-tagged ccpA
carrying an additional tryptophan residue at the N terminus
was cloned into pHT304 via XbaI and BamHI For
over-expression of HPr and Crh from B subtilis or HPr from
B megaterium either ptsH genes or crh were cloned into
pET3c via NdeI and BamHI resulting in pWH466,
pWH467 and pWH1576 For overexpression Escherichia
coli FT1 [41] was transformed with the latter plasmids
For overexpression ccpA from B subtilis was subcloned
from pWH1533 into pWH1520 resulting in pWH1537
By analogy ccpA-1Whis was subcloned from pWH1542 yielding pWH1544 B megaterium WH419 overexpressed either proteins after transformation with pWH1537 or pWH1544
b-Galactosidase assays
Cells for b-galactosidase assays were grown overnight at
37C in CSK minimal medium From overnight cultures the same medium and CSK supplemented with 0.2% xylose or with 1% glucose, 0.2% xylose were inocculated to D600¼ 0.1 and grown at 37C until a D600value of 0.4 was reached One-hundred microlitres bacterial culture were diluted with
900 lL Z-buffer (60 mm Na2HPO4, 40 mm NaH2PO4,
10 mm KCl, 1 mm MgSO4, 50 mm b-mercaptoethanol,
pH 7) After lysis with lysozyme and Triton-X-100 b-galac-tosidase activity was determined as described earlier [28]
Preparation of proteins CcpA from B subtilis was expressed in B megaterium WH419⁄ pWH1537 and C-terminally His-tagged CcpA-1 W
in B megaterium WH419⁄ pWH1544 (Table 1) as described [24] For purification the cells were disrupted by
ultrasonifi-cation, centrifuged for 45 min at 48 400 g at 4C and incu-bated with 5 lgÆmL)1 RNaseA and 10 lgÆmL)1 DNaseI (Sigma, Munich, Germany) Wild-type CcpA was purified
by subsequent cation exchange chromatography on POROS
20 HS (Perseptive Biosystems, Framingham, MA, USA), desalting (Pharmacia Biotech, Freiburg, Baden Wuerttem-berg, Germany), anionic exchange chromatography on Fractogel EMD TMAE (Merck, Darmstadt, Hesse, Ger-many) and gelfiltration on Superdex G75 (Pharmacia Bio-tech) C-terminally His-tagged CcpA-1 W was purified using Ni-affinity chromatography on POROS 20 MC (Perseptive Biosystems) Further purification was achieved
by gelfiltration on Superdex G75 (Pharmacia Biotech) HPr from B megaterium was overproduced in E coli FT1⁄ pWH1576, HPr from B subtilis in E coli FT1 ⁄ pWH466 and Crh in E coli FT1⁄ pWH467 The crude lysates have been incubated with 5 lgÆmL)1 RNaseA and
10 lgÆmL)1DNaseI (Sigma), then prepurified by heat dena-turation for 20 min at 70C and 65 C, respectively After centrifugation from the precipitated proteins, HPr or Crh could be extracted from the supernatant Phosphorylation
of either protein was performed in the prepurified crude lysate using a HPr kinase extract as described [25] Purifica-tion of HPr, HPrSerP, Crh or CrhP was achieved by anion exchange chromatography on DEAE Sephacel (Pharmacia Biotech) and subsequent gelfiltration on Superdex G75 (Pharmacia Biotech) The activities of both phosphorylated proteins was assumed to be 100%, as there is no obvious method for activity determination However, we assume that the potentially active fractions are the same for both,
Trang 10similarly heat stable proteins Therefore, the ratio of
con-stants should be reliable
Determination of protein concentration
Protein concentrations were measured using a Bio-Rad
(Munich, Germany) Bradford dye binding assay BSA was
used as a standard The concentration of purified CcpA-1
W protein was confirmed by UV spectroscopy at 280 nm
using an extinction coefficient of e280nm¼ 21300 m)1Æcm)1
Preparation of cre DNA
Forty-eight nucleotide synthetic oligonucleotides
contain-ing xylAcre (forward: 5¢-CTAATAAAATTAATCATTTT
GAAAGCGCAAACAAAGTTTTATACGAAG-3¢;
back-ward: 5¢-CTTCGTATAAAACTTTGTTTGCGCTTTCAAA
ATGATTAATTTTATTAG-3¢) and 26-nt oligonucleotides
containing xylAcre (forward: 5¢-AATCATTTTGAAAGC
GCAAACAAAGT-3¢; backward: 5¢-ACTTTGTTTGCG
CTTTCAAAATGATT-3¢) or a nonspecific DNA sequence
(5¢-AATCATTTATGGCATAGGCAACAAGT-3¢;
back-ward: 5¢-ACTTGTTGCCTATGCCATAAATGATT-3¢)
were hybridized and used for analyses without further
puri-fication Both forward 26-nt oligonucleotides carry a C6
aminolinker at the 5¢-end All oligonucleotides were
pur-chased with or without modification at MWG Biotech
(Ebersberg, Germany) The concentration of the hybridized
DNA was determined using an extinction coefficient of
e¼ 1186 · 10)6m)1Æcm)1 as determined from the
nucleo-tide composition
SPR measurements
SPR measurements with CcpA, HPrSerP or CrhP each from
B subtilis or xylAcre, were analysed using a BIAcoreX
instrument operated at 25C (BIAcoreX, Uppsala, Sweden)
For the analysis of protein–protein interactions CcpA was
immobilized by amine coupling on the carboxylated dextran
matrix of a CM5 sensorchip (Biacore AB) in flowcell Fowcell
1 contained TetR from E coli and was used as a reference
For immobilization on the activated chip matrix (injection of
35 lL of a mixture containing 50 mm N-hydroxysuccinimide
and 200 mm
N-ethyl-N¢-(3-dimethylaminopropyl)carbodi-imide hydrochloride in desalted, sterile water) the proteins
were injected at 500 nm concentrations in 10 mm
sodiumace-tate, pH 5 After coupling of the proteins the residual
activa-ted carboxyl groups were deactivaactiva-ted by injection of 1 m
ethanolamine hydrochloride⁄ NaOH, pH 8.5 Both proteins,
CcpA and TetR-B⁄ D, were adjusted to equal immobilization
levels of 1700–2100 RU on different sensorchips During
immobilization and interaction analyses HBS⁄ EP buffer
(0.01 m Hepes pH 7.4, 0.15 m NaCl, 3 mm EDTA, 0.005%
polysorbate) purchased from Biacore was used as a running
buffer at a flowrate of 5 lLÆmin)1 For the interaction analy-ses, the injected analyte volume was adjusted to the amount needed for a constant response difference indicating the equi-librium of interaction of CcpA with HPrSerP or CrhP The concentration of the complex is measured directly as the steady state response [R(eq)] in SPR As the analyte is constantly replenished during sample injection, the concen-tration of free analyte is equal to the bulk analyte concentration The equilibrium constants were determined
by Langmuir fits of plots from the steady state response vs the analyte concentrations Evaluation was done using the Langmuir equation for 1 : 1 ligand binding of the program sigmaplotTM
8.0 (SPSS Inc., Chicago, IL, USA) Each equi-librium constant and deviations were determined from three different titrations For interaction analyses of CcpA with xylAcrewe immobilized amino-modified 26-meric DNA (see preparation of cre DNA) containing the xylAcre or a non-specific DNA sequence on the surface of Biacore CM5 chips
We used a new method for coupling of amino-modified DNA to Biacore CM5 chips This method uses cetyltrimeth-ylammoniumbromide (CTAB) micelles as carriers to immo-bilize DNA on the carboxymethylated dextran matrix (H Sjo¨bom, Biacore AB, Uppsala, Sweden, personal com-munication) We coupled hybridized nonspecific DNA in flowcell 1 and xylAcre containing DNA in flowcell 2 by injec-tion of mixtures containing 5 lm of amino-modified DNA, 0.6 mm CTAB in 10 mm Hepes at a pH of 7.4 over a CM5 chip that was activated as described above During coupling
we used HBS-N (10 mm Hepes, 150 mm NaCl) as a running buffer at a flow rate of 5 lLÆmin)1 After deactivation of residual activated carboxyl groups as described above
280 RU DNA remained stably attached to the chip, but only 30–60 RU were functional as calculated from the maximum response of CcpA-HPrSerP binding to xylAcre For all CcpA–cre interaction analyses HBS-EP buffer pur-chased from Biacore AB was used as a running buffer The mass transport limitation was tested by alteration of flow rates A flow rate of 40 lLÆmin)1was suitable for all experi-ments to minimize mass transport To regenerate the chip surface the dissociation of the CcpA–HPrSer46P complex was stopped by injection of 80 lL HBS-EP buffer at 40 lLÆ min)1after each injection Fits showed that concentrations
> 30 nm CcpA or CcpA–HPrSerP complex, which saturate the cre coupled to the chip, result in biphasic sensorgrams
We analysed only sensorgrams from 1 nm to 30 nm CcpA in the presence of HPrSerP or HPrSerP and FBP The titrations for the kinetic measurements have been carried out twice for each protein complex, CcpA–HPrSerP or CcpA–HPrSerP– FBP FBP (Fluka) F-6-P, Glc6-P or Glc1-P (Sigma) were diluted immediately before each experiment in HBS-EP buf-fer to 100 mm stock solutions and if necessary adjusted to
pH 7.4 In order to prevent bulk effects the HBS-EP running buffer was adjusted to the concentration of these compounds and then supplied with HPrSerP if required