From the
In the Gram-positive bacterium, Lactococcus lactis,
nonmetabolizable cytoplasmic sugar phosphates, accumulated by the
phosphoenolpyruvate:sugar phosphotransferase system, are rapidly
dephosphorylated and expelled from the cell upon addition of glucose
(inducer expulsion). Our recent studies have established that a
metabolite-activated, ATP-dependent protein kinase that phosphorylates
serine-46 in HPr of the phosphoenolpyruvate:sugar phosphotransferase
system activates a sugar phosphate phosphatase, thus initiating the
inducer expulsion process. A membrane-associated, HPr(Ser(P))-dependent
phosphatase has been identified, solubilized from the membrane,
separated from other cellular phosphatases, and purified to near
homogeneity. It exhibits a low subunit molecular mass (10 kDa) and
behaves on gel filtration columns like a monomeric enzyme. It has broad
substrate specificity, optimal activity between pH 7.0 and 8.0, is
dependent on a divalent cation for activity, and is not inhibited by
fluoride. It is stimulated more than 10-fold by HPr(Ser(P)) or a mutant
derivative of HPr, S46D HPr, in which the regulatory serine is changed
to aspartate, which bears a permanently negative charge as does
phosphate. Stimulation is due both to an increase in the maximal
velocity (V
The phenomenon of ``inducer expulsion'' is well
established in Gram-negative bacteria such as Escherichia coli (Haguenauer and Képès, 1971; Winkler, 1971) and in
Gram-positive bacteria such as Streptococcus pyogenes and Lactococcus lactis (Reizer and Panos, 1980; Thompson and
Saier, 1981). Although the mechanism is still not understood in E.
coli, recent work with Gram-positive bacteria has implicated the
proteins of the phosphoenolpyruvate:sugar phosphotransferase system
(PTS).
Recent work employing vesicles of L. lactis has provided
direct, compelling evidence for the involvement of HPr(Ser(P)) in (a) the inhibition of sugar uptake via the PTS, (b)
the activation of a cytoplasmic sugar phosphate phosphatase, and (c) the consequent phenomena of inducer exclusion and
expulsion (Saier, 1985; Ye et al., 1994a, 1994b). Activation
of the sugar phosphate phosphatase by the HPr(Ser(P)) mutant analogue,
S46D HPr (Reizer et al., 1989, 1992; Wittekind et
al., 1989, 1990, 1992), has recently been demonstrated in
permeabilized vesicles of L. lactis (Ye et al.,
1994a, 1994b). Evidence for the involvement of HPr(Ser(P)) and S46D HPr
in the regulation of non-PTS carbohydrate permeases in Lactobacillus brevis (Ye et al., 1994c, 1994d; Ye and
Saier, 1995a, 1995b) and in catabolite repression in Bacillus
subtilis (Deutscher et al., 1994, 1995; Hueck et
al., 1994)
Previously published work did not lead to identification of the
sugar phosphate phosphatase that is activated by HPr(Ser(P)) and S46D
HPr. A sugar phosphate phosphatase was purified and characterized from L. lactis (Thompson and Chassy, 1983), but its involvement in
inducer expulsion was not demonstrated. We therefore set out to
identify the enzyme that is the target of HPr(Ser(P)) action. We were
able to show that the phosphatase of Thompson and Chassy (here termed
phosphatase I or Pase I) is not activated by S46D HPr. By contrast, a
membrane-associated sugar phosphate phosphatase (here termed
phosphatase II or Pase II) was found to be activated by S46D HPr. This
enzyme could be solubilized from the membranes with the use of 8 M urea and 4% butanol and purified to near homogeneity. Its unusual
properties and its functional interactions with HPr and various mutant
derivatives of HPr are herein described.
[
The
membranes were resuspended in HEDM buffer and treated with 8 M urea and 4% 1-butanol at 0 °C for 30 min as previously
described (Kundig and Roseman, 1971; Saier et al., 1977)
before centrifugation at 200,000
The dialyzed, urea-butanol-treated
supernatant (40 ml) was transferred (at a flow rate of 0.5 ml
min
The
membranes were treated with 8 M urea and 4% butanol
essentially as previously described (Kundig and Roseman, 1971; Saier et al., 1977) to release Pase II. After centrifugation to
remove the membranes, and after removal of the butanol and urea by
dialysis, the enzyme was purified by ion exchange chromatography and
gel filtration as described under ``Experimental
Procedures.'' An estimate of the -fold purification was not
possible because of the presence of both Pase I and Pase II and
possibly other sugar phosphate phosphatases. However, an approximately
50-fold increase in specific activity was observed during purification
of the activity released from the membranes by butanol-urea extraction.
Fig. 1presents the elution profiles of activity from the
DEAE-Sephacel I column (Fig. 1A) and the Sephadex
G-50-50 gel filtration column (Fig. 1B). Pase I
eluted from the ion exchange column at a salt concentration of about
0.2 M while Pase II eluted at about 0.33 M NaCl. The
two activity peaks were clearly separated. While Pase I activity was
not detectably stimulated by S46D HPr, Pase II activity was stimulated
5-10-fold ().
Gel filtration (Fig. 1B) of the concentrated Pase II off of the
DEAE-Sephacel column yielded a single major peak of activity, which
eluted shortly after cytochrome c (M
Inducer expulsion in Gram-positive bacteria possessing a PTS
is mediated by a metabolite-activated, ATP-dependent protein kinase. A
cytoplasmic sugar phosphate phosphatase has been implicated in the
process (See Introduction). Prior to this report, nothing was known
about the enzyme that catalyzed this reaction. Using S46D HPr
stimulation of sugar phosphate phosphatase activity as an assay, we
have purified a phosphatase (termed Pase II) from L. lactis extracts that exhibits unusual properties as follows. 1) It has an
exceptionally small catalytic subunit, possessing a molecular mass of
less than 10,000 Da, based on both gel filtration of the native enzyme
and SDS-gel electrophoresis of the denatured enzyme. A small size had
been suggested early in our studies by the observation that the enzyme
passed through dialysis tubing at a rate comparable to that of
HPr.
The results summarized in this paper serve
to characterize the phosphatase that is most probably responsible for
the inducer expulsion phenomenon in L. lactis and possibly in
other Gram-positive bacteria as well. Its small size suggests that it
will prove ideal material for structural studies employing both
multidimensional NMR and x-ray crystallography. Since high resolution
three-dimensional structures of HPr are already available (Wittekind et al., 1989, 1990, 1992; Herzberg et al., 1992), the
Pase II-HPr(Ser(P)) interaction should be molecularly definable (Chen et al., 1993).
Assay conditions were as reported under
``Experimental Procedures'' with mannitol-1-P (20
mM) as the substrate. Values of three experiments were
averaged and are reported ± S.D.
Phosphatase II (15
µl of 0.2 mg/ml of the purified enzyme) was incubated in an assay
solution containing 20 mM Mg
Assays were performed as indicated under
``Experimental Procedures.'' 20 mM mannitol-1-P was
present in all assays as the substrate. Values of three experiments
were averaged and are reported ± S.D.
We thank Mary Beth Hiller and Lisa Preble for
assistance in the preparation of this manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
) and a decrease in the
Michaelis-Menten kinetic constant (K
) for
sugar phosphate. The enzyme exhibits a K
for S46D HPr of 15 µM. Although the enzyme is
thermally stable, activation by HPr(Ser(P)) is heat sensitive.
(
)In these organisms, HPr is
phosphorylated both by phosphoenolpyruvate and enzyme I of the PTS on
histidyl residue 15 and by ATP and a metabolite-activated protein
kinase on seryl residue 46 (Deutscher and Saier, 1983; Deutscher et
al., 1986; Reizer et al., 1989). Kinetic analyses showed
that inducer expulsion is a two step process: intracellular sugar
phosphate is first hydrolyzed to free sugar and inorganic phosphate in
the cytoplasm, and the free sugar is then expelled into the external
medium (Reizer et al., 1983; Reizer and Saier, 1983; Sutrina et al., 1988). In early studies, HPr(Ser) phosphorylation and
inducer expulsion appeared to be related since conditions that gave
rise to inducer expulsion also promoted phosphorylation of HPr on
serine (Thompson and Saier, 1981; Reizer et al., 1992).
(
)has recently been presented.
Assay Conditions
Sugar phosphate
phosphatase activity was routinely determined by a modification of the
method described by Parvin and Smith(1969). Briefly, 10-25 µl
of enzyme was added to a medium containing 50 mM MES (pH 7.0),
20 mM MgSO, and various concentrations (usually 10
or 20 mM) of one of several sugar phosphates. Assay solutions
(50 µl, final volume) were incubated at 40 °C for 10-30
min. Isobutanol (1 ml) and 4.25% (w/v) ammonium molybdate in 0.43 M sulfuric acid (0.3 ml) were then added, and phosphate was
extracted into the organic phase by vigorous mixing for 20 s. When the
organic phase had cleared, 1 ml was removed and mixed by immediately
vortexing with 1 µl of 4% (w/v) SnCl
in concentrated
HCl before measurement of the optical density at 760 nm.
C]TMG-6-phosphate was prepared by using
starved cells of L. lactis as described by Thompson and
Saier(1981). [
C]TMG-6-phosphate phosphatase
activity was measured as follows. 1.5 mM
[
C]TMG-6-phosphate (specific activity, 0.5
mCi/mmol, purified as previously described (Ye et al., 1994a,
1994b)) was added to the same assay solution described above lacking a
sugar phosphate substrate. The assay solution was then incubated for up
to 2 h before measuring the proportion of free
[
C]TMG by ion exchange chromotography (5
0.5 cm, column size) as previously outlined (Kundig and Roseman, 1971).
PTS enzyme I and HPr were assayed by complementation of mutant crude
extracts as previously described (Mitchell et al., 1993).
Protein concentration was determined by the procedure of Lowry et
al.(1951).
Organism and Growth Conditions
L.
lactis ML3 (12 liters for a typical enzyme preparation) was grown
at 37 °C for 16 h in LB medium containing 25 mM galactose
as previously described (Ye et al., 1994a, 1994b). A final
yield of about 45 g (wet weight) of cells was usually obtained.
Purification of Sugar Phosphate Pase
II
All purification procedures were conducted at 0-4
°C. The washed cell pellet (about 45 g) was resuspended to a volume
of 60 ml in HEPES buffer (pH 7.3) containing 1 mM EDTA, 1
mM dithiothreitol, and 5 mM MnSO (HEDM
buffer), as well as 0.1 mM phenylmethylsulfonyl fluoride. The
cells were disrupted by three successive passages through a French
pressure cell (11,000 pounds per inch
(76 megapascal)).
Cell debris was removed by centrifugation at 10,000
g for 20 min, and the membranes were collected by
ultracentrifugation at 200,000
g for 90 min.
g for 2 h. The
supernatant was collected and dialyzed overnight against 11 liters of
20 mM Tris-HCl, pH 7.0.
) onto a DEAE-Sephacel anion-exchange column (3
20 cm) previously equilibrated with HEDM buffer. The proteins
were eluted with a gradient of 0.1-0.4 M NaCl in HEDM
buffer (see Fig. 1A).
Figure 1:
Elution
profiles of phosphatase I and phosphatase II from DEAE-Sephacel ion
exchange (A) and of phosphatase II from Sephadex G-50 (B). Conditions were as outlined under ``Experimental
Procedures.'' In A, elution was effected with a linear
salt gradient of 0.1-0.4 M NaCl as indicated by the dashedline. Phosphatase II eluted at an estimated
salt concentration of 0.33 M. In B, catalase (M = 220,000) eluted at the portion
indicated by arrow1, and cytochrome c (M
= 12,000) eluted at the portion
indicated by arrow2.
Fractions from the
DEAE-Sephacel column that contained Pase II activity were pooled and
concentrated to 3 ml by lyophilization. After addition of 0.5 ml of
glycerol, the enzyme was loaded onto a Sephadex G-50-50 column
(1.2 32 cm) preequilibrated and eluted with HEDM buffer.
Fractions containing sugar phosphate phosphatase activity were
combined, analyzed for purity, and used for characterization of enzymic
properties (see Fig. 1B). Although Pase II was of about
the same size as HPr, it exhibited no detectable HPr activity in
complementation assays (Mitchell et al., 1993). Reciprocally,
all HPr and mutant HPr derivative preparations tested for activation of
Pase II lacked detectable phosphatase activity. The Pase II preparation
purified through gel filtration was used in all analyses reported
except for those presented in .
Gel Electrophoretic
Analysis
SDS-electrophoresis was performed with a
PhastSystem Separation and Control Unit (Pharmacia, Uppsala, Sweden). 5
µl (from 0.1 to 5 µg of protein) of each sample was mixed with
SDS sample buffer (60 mM Tris-HCl, pH 7.0, 2% SDS, 10%
glycerol, 0.025% bromphenol blue) and boiled at 100 °C for 2 min
before loading on a PhastGel (Gradient 8-25) for separation by
SDS-polyacrylamide gel electrophoresis. The protein bands were
visualized employing staining with Coomassie Blue R-250 by the
procedure of Diezel et al.(1972).
Preincubation of Pase II at Various
Temperatures
Aliquots of Pase II, purified through the gel
filtration column, were incubated for 10 min in tubes submerged in a
water bath at various temperatures ranging from 40 to 100 °C. They
were then chilled to 0 °C in preparation for assay of the
mannitol-1-P (20 mM) hydrolysis rate with or without 50
µM S46D HPr employing the standard assay conditions
described above.
Reagents
Radiolabeled
[C]TMG (58 mCi/mmol) was from DuPont-NEN.
Bio-safe II scintillation mixture was purchased from Research Products
International Corp. (Mt. Prospect, IL). Analytical gradient
anion-exchange resin AG1-X2 was obtained from Bio-Rad. Sephadex
G-50-50 was from Sigma, and DEAE-Sephacel anion-exchange resin
was obtained from Pharmacia Biotech Inc. PhastGels (Gradient
8-25) for SDS-polyacrylamide gel electrophoresis separations were
from Pharmacia Biotech AB (Uppsala, Sweden). Nonradioactive compounds,
phosphorylated sugars, and other reagents were obtained from Sigma.
Solubilization and Purification of Pase
II
Crude extracts of L. lactis exhibited low
degrees of stimulation of mannitol-1-P and
[C]TMG-phosphate phosphatase activities upon
addition of S46D HPr (). When the membrane fraction was
separated from the soluble protein fraction by high speed
centrifugation (200,000
g for 2 h), the resuspended
membranes exhibited activity that was enhanced by addition of S46D HPr
to a greater extent than was observed for the crude extract. The
soluble protein fraction exhibited more activity than did the
membranes, but this activity exhibited almost no stimulation by S46D
HPr (). As noted below, the soluble fraction was enriched
for Pase I while the membranes were enriched for Pase II. Membranes
were therefore used as the preferred source of Pase II.
12,000, see Fig. 1B). Its size was estimated at
9,000-10,000 Da. Gel electrophoresis revealed a single band that
ran just ahead of HPr using the Coomassie Blue protein stain (data not
shown). Pase II gave the following N-terminal sequence: APLKGRF. Only a
single predominant amino acid (>80%) was present at each position.
The amounts of these amino acids, relative to each other, were about
the same, suggesting that they derived from a single protein. This
enzyme was used for all remaining experiments reported below.
Substrate Specificity of Pase II
presents data revealing the substrate
specificity of the enzyme and its -fold stimulation by S46D HPr with
each of the potential substrates tested. Almost all sugar phosphates
tested were hydrolyzed, although to varying degrees, and the -fold
stimulation by S46D HPr varied between 5- and 10-fold. In no case did
S46A HPr stimulate the activity of Pase II. Fructose 1,6-diphosphate
and p-nitrophenyl phosphate were poor substrates, and the low
activity observed with these substrates was not appreciably stimulated
by S46D HPr.
Dependence of Pase II Activity on Divalent
Cations
Using mannitol-1-P as the substrate, Pase II
activity was found to be dependent on divalent cations. Regardless of
concentration, Mg appeared to be most effective.
Mg
stimulated half-maximally at a concentration of
about 1 mM, and high concentrations were not inhibitory (data
not shown). The stimulatory effect of Mg
was
independent of S46D HPr. Other divalent cations (Mn
,
Co
, Fe
, and Zn
)
stimulated to lesser degrees with decreasing efficacies in the order
mentioned. Fluoride, which inhibited Pase I almost quantitatively under
our standard assay condition (see also Thompson and Chassy, 1983), had
no effect on the activity of Pase II when present in 2-fold excess over
the Mg
concentration.
pH Dependence of Pase II
The pH curves
with and without S46D HPr are shown in Fig. 2. In both cases, the
pH optimum was about 7 to 8. Stimulation by S46D HPr was maximal within
this pH range.
Figure 2:
pH activity curve for purified phosphatase
II. Buffers used at 50 mM were as follows: pH 3-5,
sodium acetate; pH 6-8, MES buffer adjusted to the appropriate pH
with HCl; pH 9-10, sodium borate. Other conditions were as
outlined under ``Experimental Procedures'' with 20 mM mannitol-1-P as the substrate.
Kinetic Characteristics of S46D HPr-stimulated Pase
II Activity
In the absence of S46D HPr, Pase II exhibited
low affinity for its sugar substrates. For example, the apparent K for mannitol-1-P was 50 mM.
When 35 µM S46D HPr was added, the K
decreased to about 7 mM, and the V
increased about 50% (Fig. 3). No apparent cooperativity
(sigmoidicity of the vversusS curve) was
observed either in the presence or the absence of S46D HPr.
Figure 3:
Lineweaver-Burk, double reciprocal plot of
the mannitol-1-P hydrolysis rate as a function of the sugar phosphate
concentration. Conditions other than substrate concentration were as
outlined under ``Experimental Procedures'' with 50 mM MES buffer, pH 7.0. Phosphatase II, purified through gel
filtration, was used in the assay. The calculated K and V values at 40 °C were as follows: without
S46D HPr, 50 mM and 2.0 µmol/mg protein/min, respectively,
and with S46D HPr at 35 µM, 8 mM and 3.8
µmol/mg protein/min, respectively.
The
dependence of the activity on S46D HPr is shown in Fig. 4. Under
the assay conditions used, 50 µM S46D HPr gave maximal
activity with half-maximal activation occurring at about 13
µM. The curve was hyperbolic, consistent with a lack of
cooperativity for substrate binding. In agreement with these results,
chemical cross-linking experiments with disuccinimidyl tartarate
conducted as described by Belogrudov et al.(1995) revealed
that Pase II alone or S46D alone each gave only one band on
SDS-polyacrylamide gel electrophoresis at 9-10 kDa, but when
these two proteins were mixed prior to the addition of disuccinimidyl
tartarate, an additional band of about 18-20 kDa was observed. A
1:1 stoichiometry of binding is therefore suggested.
Figure 4:
Activation of phosphatase II as a function
of S46D HPr concentration with 20 mM mannitol-1-P as the
substrate in the presence of 50 mM MES buffer, pH 7.0, and 20
mM MgCl under standard assay conditions. The K for S46D HPr was calculated to be 13
µM.
Stimulatory Effects of Various HPr Derivatives on
Pase II Activity
I presents the effects of
HPr, HPr(Ser(P)), and various mutant derivatives of HPr on the activity
of the purified Pase II. When used at a concentration of 15
µM, HPr(Ser(P)) was substantially more stimulatory than
was S46D HPr. When used at near saturating concentrations (50
µM), S46D HPr and the double mutant, H15E,S46D HPr, were
equally stimulatory. All other derivatives of HPr tested were
essentially without effect (I).
Thermal Stability of Pase II and Thermal Inactivation
of the S46D Stimulatory Effect
Fig. 5shows the
effects of a short preincubation of Pase II at temperatures ranging
from 40 to 100 °C on the activities of the purified enzyme. Basal
Pase II activity using mannitol-1-P as substrate was unaffected by
thermal exposure, even at a preincubation temperature of 100 °C. By
contact, the stimulatory effect of S46D HPr was gradually lost to
increasing degrees when the enzyme was exposed to temperatures ranging
from 50 to 100 °C. The enzyme preincubated at 100 °C exhibited
little stimulation. The results suggest that while the active site of
the enzyme is heat stable, the S46D HPr interaction site is less
stable.
Figure 5:
Effects of preincubation at various
temperatures on Pase II activity with and without S46D HPr. The
purified enzyme was heated 10 min at the temperature indicated, chilled
at 0 °C, and assayed with or without S46D HPr at a concentration of
50 µM. The assays were performed at 40 °C with 20
mM mannitol-1-P as substrate as outlined under
``Experimental Procedures.''
(
)2) While Pase II elutes from DEAE Sephacel
ion exchange columns at a high (0.33 M) NaCl concentration,
Pase I elutes at a much lower salt concentration (0.2 M),
suggesting that Pase II may be a much more acidic protein than is Pase
I. 3) The activity of Pase II, like that of Pase I, is stimulated by
various divalent metal ions, with Mg
being preferred.
However, unlike most phosphatases, including Pase I (Thompson and
Chassy, 1983), fluoride is apparently not inhibitory under the assay
conditions used. 4) In contrast to Pase I, which has a low pH optimum
(Thompson and Chassy, 1983), Pase II exhibits a high pH optimum. 5)
Pase II seems to be predominantly peripherally associated with the
particulate fraction while Pase I is predominantly soluble in crude
extracts. The former enzyme can be quantitatively solubilized by
treatment with 8 M urea in the presence of 4% butanol
according to the procedure of Kundig and Roseman(1971). This fact
suggests that the enzyme is not an integral constituent of the
membrane. 6) In contrast to Pase I, which is not affected by HPr and
its derivatives, Pase II is specifically activated by HPr(Ser(P)), S46D
HPr, and H15E,S46D HPr. Non-phosphorylated wild-type HPr and other
mutant derivatives of HPr did not activate Pase II. The binding
stoichiometry is apparently 1:1. These facts suggest that Pase II
directly binds HPr(Ser(P)) to initiate inducer expulsion in lactococci.
7) Physiological concentrations of S46D HPr both lower the K
for sugar phosphate and increase the V
for sugar phosphate hydrolysis, thereby
accounting for stimulation of the activity of Pase II. It is
interesting to note that while Pase II is activated by H15E,S46D HPr as
well as S46D HPr, only the latter HPr derivative could bind to the CcpA
protein, a mediator of catabolite repression in B.
subtilis(8) .
Finally, while Pase II activity
appears to be heat stable, its activation by S46D HPr is heat labile.
This last observation may suggest that the interaction of Pase II with
S46D HPr is dependent on a conformation of the enzyme that is not
required for catalysis.
Table: Stimulation of phosphatase activity in a
crude extract of L. lactis and in various partially purified fractions
by S46D HPr
Table: Substrate specificity of S46D HPr-stimulated
sugar phosphate phosphatase II from L. Lactis
and 50 mM MES buffer (pH 7.0) for 15 min at 40 °C as detailed under
``Experimental Procedures.'' The substrate concentration was
20 mM. When TMG-6-phosphate was the substrate, 0.2 mM [
C]TMG-6-phosphate (specific activity, 0.5
mCi/mmol) was present in the assay solution. HPr derivatives were used
at a concentration of 100 µM. Values of three experiments
were averaged and are reported ± S.D.
Table: Stimulation of purified phosphatase II
by various HPr derivatives
C]TMG,
-D-[methyl-
C]thiogalactopyranoside;
MES, 2-(N-morpholino)ethanesulfonic acid.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.