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INTRODUCTION |
Mlc is a global transcriptional regulator (repressor) in
Escherichia coli serving several genes and operons encoding
a variety of sugar-metabolizing enzymes and transport systems
(1-7). The major target for Mlc regulation is the
phosphotransferase
(PTS)1-dependent
transport of glucose. The expression of the genes for both of the PTS
enzyme II systems capable of transporting glucose, the glucose-specific
transporter (EIICBGlc = PtsG) and the less specific,
so-called mannose transporter (EIIABCDMan = PtsM, ManXYZ)
as well as the genes for the general components of the PTS, enzyme I,
and HPr, are all controlled by Mlc. In addition to these PTS proteins,
MalT, the central transcriptional activator of the maltose regulon, is
subject to transcriptional control by Mlc. The maltose regulon consists
of 10 genes encoding enzymes and an ABC transporter involved in the
uptake and the metabolism of maltose and maltodextrins, which produce
intracellular glucose and glucose-1-P.
The particular feature that makes Mlc an attractive subject of study is
its mode of regulation. Unlike normal prokaryotic transcriptional
regulators, no low molecular weight molecule, which could act as an
inducer to inactivate Mlc and prevent its binding to DNA, has been
identified. The induction of Mlc-controlled genes is dependent upon the
activity of the PTS, and it is the binding (sequestration) of Mlc to
PtsG, when PtsG is actively transporting glucose, that inactivates Mlc
(8-10). An analogous system, in which the activity of the signal
transduction protein GlnK, is regulated by sequestration by the
ammonium transporter, AmtB, has been recently described (11). Other
examples of membrane association affecting the activity of
transcription regulators are known (12, 13), suggesting that this might
be a general method used by bacteria to achieve cellular compartmentalization.
Binding of Mlc to PtsG was shown to be dependent upon the
phosphorylation state of Cys-421 in the EIIBGlc domain of
PtsG. Mlc interacts with the non-phosphorylated Cys-421 form of PtsG,
and phosphorylation by the PTS inhibits this binding. Deletions in PtsG
that remove parts of the integral membrane EIIC domain but retain one
or more transmembrane helices (TM) still allow binding of Mlc to
membranes carrying these deleted PtsG in vitro and
derepression of Mlc-controlled genes in vivo (8). The
largest deletion studied within EIICGlc, which was still
able to regulate Mlc, removed TMs numbered 1-8 (14) but retained a
putative ninth helix (15) and the interdomain "linker" sequence
(16). This linker is conserved in several members of the glucose
subfamily of enzyme IIs (16). Deletions that removed all of the
EIICGlc domain and produced a soluble EIIBGlc
protein were no longer able to regulate Mlc-controlled genes (8).
However, Nam et al. (9) showed, by surface plasmon resonance, that the soluble EIIBGlc domain did interact
with Mlc. This raised the question of whether the role of the ninth
helix and linker sequence is just to permit the binding of the soluble
EIIBGlc domain to the membrane or whether these sequences
are also part of the recognition site for Mlc. The work described here
was undertaken to investigate this question and to try to identify the
regions of EIIBGlc that are in contact with Mlc.
The solution structure of EIIBGlc is known from NMR studies
(17). The phosphorylation site, Cys-421, is located on a convex surface
and is part of a polar patch surrounded by a ring of hydrophobic residues. The amino acid residues showing a significant amide shift
upon phosphorylation of Cys-421 were located in this patch (18). As the
binding of Mlc to PtsG is sensitive to the phosphorylation state of
Cys-421, we reasoned that the best candidates to interact with Mlc
would be amino acids in this region. To identify contact residues,
alanine mutations were constructed in these amino acids, and their
effect on Mlc binding was investigated in vivo and in vitro.
Mlc is a tetramer with the DNA binding domain, a helix-turn-helix
motif, at the N-terminal. The part of the molecule responsible for
interaction with PtsG and tetramerization is within the C-terminal region. As a first approach to define the regions of Mlc important for
the contact with PtsG, C-terminal deletions were made in Mlc. These
showed that a tetrameric form of Mlc was necessary to bind to
EIIBGlc and to regulate Mlc-controlled genes in
vivo.
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EXPERIMENTAL PROCEDURES |
Bacteriological Methods--
Most of the strains used are
derivatives of JM101 carrying a ptsG-lacZ fusion on a
-lysogen (2). JM-G2 carries the mlc:tet mutation, and
JM-96 is
ptsG:cat, ptsM8, which eliminates
glucose uptake by the PTS. JM-G77 is
ptsG:cat and
ptsHIcrr:kan, which eliminates all PTS phosphorylation.
These strains have been described previously (2, 3). IT1168
(ptsG:kan) (19) was used for membrane preparations.
-galactosidase activities were measured as described by Miller
(20).
Construction of Two C-terminal Deleted Mlc Proteins--
To
construct plasmids encoding Mlc with the 9 and 18 C-terminal amino
acids deleted, two PCR fragments were amplified using the primer
BamH1-mlc, N-terminal
(5'-CGGATCCGATGGTTGCTGAAAACCAGCC-3') and the primer
mlc-
410-419-PstI, C-terminal
(5'-AACTGCAGTTAACCGTTATACATCGCGTCTTTTACC-3') or the primer
mlc-
402-419-PstI, C-terminal
(5'-AACTGCAGTTATGCAGCGCCTGCCATCGTGC-3') using pSL104
(8) (encoding wild type Mlc with N-terminal His6 tag) as
template. The two PCR fragments were digested with BamHI and
PstI (restriction sites in the oligonucleotides are
underlined) and ligated into pGDR11, a pQE31 derivative harboring the
lacIq gene (8). The resulting plasmids,
pMlc410 and pMlc401, encode Mlc proteins with 9 and 18 amino acids
deleted from the C terminus and an N-terminal His6 tag.
Construction of PtsG Deletions and Gp8-IIB Hybrid
Proteins--
The pTZ19R-derived plasmid expressing PtsG(
5-390)
was described previously (8). PtsG(
5-353) was made using an
oligonucleotide, Glc17Nsi
(5'-CCAATGCATTTCTGTGGCTGTTCCCGATCGTC-3') homologous to DNA
corresponding to amino acids 354-360 with a 5' extension carrying an
NsiI site (underlined) in phase with the NsiI
site covering amino acids 4-5 of ptsG. A PCR fragment was
amplified between Glc17Nsi and an oligonucleotide specific to the
lac insert of pTZ19R vector DNA (Lac22). This fragment was
digested with NsiI and EcoRI and inserted between
the same sites of pTZ(PtsG) to replace the full-size ptsG
gene. To construct the Gp8-IIB hybrid proteins, two PCR fragments, one
corresponding to Gp8 and the other corresponding to the IIB domain,
were independently amplified and used to replace the wild type
ptsG of pTZ(PtsG). Glc17Bgl (5'-TACAGATCTCTGTGGCTGTTCCCGATCGTC-3') corresponds
to amino acids 354-360 of PtsG, and Glc15Bgl
(5'-ACTAGATCTGAAGATGCAAAAGCGACA-3') corresponds to
amino acids 391-396, and both have a 5' extension including a
BglII site (underlined). DNA fragments corresponding to the EIIBGlc domain were amplified with these
oligonucleotides and the primer specific to the pTZ19R vector DNA
(Lac22) and the fragments digested with BglII and
EcoRI. The gene for Gp8 of M13 was amplified with oligonucleotides gp8Nsi
(5'-AGAATGCATTGAAAAAGTCTTTAGTCCTCAAAGCC-3') and
gp8Bgl (5'-ACTAGATCTGCTTGCTTTCGAGGTGAATTTC-3'), and
restriction sites are underlined. gp8Nsi is homologous to amino
acids 2-9 of Gp8 with a 5' extension replacing the Met codon and
carrying an NsiI site in phase with the NsiI site
of ptsG. The gp8-Bgl oligonucleotide is homologous to the
C-terminal amino acids 73-67 of Gp8 with a 5' extension replacing the
TGA stop codon and including a BglII site in phase with the
BglII site of oligonucleotides Glc15Bgl and Glc17Bgl. The
amplified fragment was digested with NsiI and
BglII and inserted together with either of the
BglII-EcoRI IIB domain fragments into the
pTZ(PtsG) NsiI-EcoRI plasmid backbone. Gp8 plus
the Glc17Bgl fragment gave pTZ(Gp8-TM9-IIB), whereas the Glc15Bgl
fragment gave pTZ(Gp8-IIB). The pTZ(Gp8) control plasmid was made by
replacing the BglII-EcoRV fragment of
pTZ(Gp8-IIB) with oligonucleotides incorporating tandem stop
codons in phase with Gp8 between BglII and EcoRV
restriction sites. Pwo was used for PCR amplifications and the
structure of the resulting plasmids verified by sequencing (by MWG
Biotec AG).
Mutagenesis--
Oligonucleotide mutagenesis was carried out by
the Kunkel method as described by Sambrook et al. (21) on
the single strands synthesized from pTZ19R(Gp8-IIB). The plasmid
expressing Gp8-IIB with the C421S mutation was made as described
above for the wild type construct using pMaG-C421S as template (a gift
from B. Erni).
Membrane Preparation and Binding Interaction with
Mlc--
IT1168 and JM-G77 transformed with different PtsG constructs
were used for membrane preparations. Protocol 1, as described in Lee
et al. (8), was followed for measuring binding of Mlc. Antibodies against EIIBGlc were obtained from a rabbit
immunized with purified EIIBGlc with a C-terminal
His6 tag obtained from cells transformed with pJBH (22) and
against Mlc using Mlc-His6 purified from cells transformed
with pSL104. In vitro phosphorylation to label PTS proteins
in total extracts was performed as described previously (8).
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RESULTS |
The Role of the C Terminus of Mlc--
Mlc, when analyzed by
SDS-PAGE, exhibits an apparent molecular weight of 46,000 (9). When
analyzed by molecular sieve chromatography (Fig.
1) under non-denaturing conditions, it
eluted from the column as a protein of 182,000 daltons, indicative of a
tetrameric protein. Nam et al. (9) have similarly
estimated a native molecular weight of 172,000. Mlc and NagC (a
homologous transcriptional repressor that controls genes for
N-acetylglucosamine utilization (23)) share high sequence
identity throughout their whole length. In both NagC and Mlc, the
C-terminal sequence is predicted to form an
-helix that shows
significant signs of an amphipathic character (Fig.
2). Therefore, it seemed likely that this
helix might be involved in the tetramerization of Mlc, analogous to the
coiled coil of the E. coli Lac repressor (24). We made two constructs lacking the last 9 or 18 C-terminal amino acids of Mlc. All
three proteins (the two C-terminal deletions and the wild type Mlc)
contain a His6 tag at their N termini for easy purification. Fig. 1 shows the results of molecular sieve
chromatography with the three proteins. The variant lacking half of the
putative amphipathic helix was still able to form a tetramer, whereas
the variant lacking all of the helix eluted as a dimer. The in
vivo effect of these proteins on the Mlc-dependent
ptsG-lacZ gene fusion is shown in Fig.
3. Although wild type and
Mlc(
9) still strongly repressed the expression of
ptsG-lacZ, Mlc(
18) was unable to do so.

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Fig. 1.
Protein molecular size
(MS) determination of Mlc and the
9 and 18 forms by gel
filtration. Proteins of the calibration kit with molecular sizes
in the range 25-232, shown as open diamonds, were used to
calibrate the Superdex 200 HR10/30 column (Amersham Biosciences).
Kav values were determined according to the
manufacturer's instructions. The Kav values for
the different Mlc forms were calculated, and the corresponding
molecular sizes were determined. Filled squares, wild type
Mlc, 183 kDa; filled triangles, Mlc 9, 173 kDa;
filled circles, Mlc 18, 95 kDa.
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Fig. 2.
Helical wheel representation of the last 18 amino acids of Mlc. Charged amino acids are indicated with
minus signs or plus signs. The hydrophobic amino
acids are predominately on one side (right as drawn),
whereas the charged and polar amino acids are on the other side.
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Fig. 3.
Effect of Mlc, Mlc 9
and Mlc 18 on expression of
ptsG-lacZ. JM-G2 carrying the
ptsG-lacZ fusion and the mlc:tet mutation was
transformed with plasmids pSL104, encoding full-size Mlc with an
N-terminal His6 tag; pMlc410 and pMlc401, which encode Mlc
proteins with 9 and 18 amino acids deleted from the C terminus; and
pGDR11, the vector plasmid. They were grown in minimal casamino acids
medium, and -galactosidase activities were measured after growth
with 50 µM IPTG. Activities are given as the percentage
of the expression in the vector control. 100% = 0.207 µmol/min/mg of
protein (~230 Miller units). wt, wild type; aa,
amino acid.
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Simultaneously Mlc(
18) lost its ability to be bound by PtsG present
in everted membrane vesicles in vitro, whereas Mlc(
9) was
still able to bind PtsG, as did the wild type (Fig.
4). Thus, tetramerization of Mlc appears
to be necessary for repression at Mlc-dependent operons as
well as for binding of Mlc by PtsG.

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Fig. 4.
Binding of Mlc,
Mlc 9, and Mlc 18 to
membranes carrying PtsG. Mlc binding to everted vesicles
containing overproduced PtsG(EIICBGlc) derived from pTSG11
(22) in IT1168. Membranes were incubated with
Mlc-His6 (2 µg) or its deleted variants and separated by
centrifugation. Equivalent amounts (10% of total) of pellet
(P) and supernatant (S) were separated by
SDS-PAGE. std, 50 kDa prestained marker; purified
Mlc, 0.2 µg of Mlc was detected by Western blotting with
anti-Mlc antibodies; wt, wild type; aa, amino
acid.
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Attachment of the EIIBGlc Domain to the Membrane Is a
Prerequisite for Regulation by Mlc--
We followed the example of
Görke and Rak (25) and used Gp8, the major coat protein of
bacteriophage M13, as a simple membrane anchor to replace the integral
membrane EIICGlc domain of PtsG. Gp8 is synthesized as a
73-amino-acid long precursor that is processed during membrane
insertion to the mature 50-amino-acid long form. Mature Gp8 consists of
20 amino acids in the periplasm, 20 amino acids spanning the plasma
membrane, and a C-terminal tail of 10 amino acids in the cytoplasm. Two
constructs were made. In the first (Gp8-TM9-IIB), the whole of
Gp8 was inserted between amino acids 5 and 354 of PtsG, whereas in the
shorter construct (Gp8-IIB), Gp8 was located between amino acids 5 and
391 of PtsG (Fig. 5). The longer hybrid
protein includes TM9 and the linker between the two domains. We
verified that the deletion of amino acids 5-353 from PtsG produces a
membrane-bound protein capable of regulating the activity of Mlc (Fig.
5). On the other hand, deleting between amino acids 5 and 390 produces
a soluble protein, PtsG(
5-390), which cannot regulate Mlc activity
in vivo (8).

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Fig. 5.
Structure of PtsG, deleted derivatives and
hybrid constructs with Gp8. The IIC domain of PtsG carries nine
TMs, represented as small boxes. It is joined to the soluble
IIB domain by a conserved linker sequence. Deletions were made within
the IIC domain that removed all nine or the first eight of the TMs or
replaced them with the heterologous membrane anchor, Gp8, shown as a
shaded box. The ability of these constructs to induce
Mlc-repressed genes in the ptsHIcrr, ptsG
strain (JM-G77) in vivo is indicated.
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The ability of the deleted PtsG derivatives and Gp8-IIB hybrids to bind
Mlc in vivo and in vitro was measured. A
convenient in vivo test is the ability of plasmid-encoded
Gp8-IIB constructs to derepress the ptsG-lacZ fusion carried
by a
-lysogen on the chromosome of the
ptsHIcrr,
ptsG strain (JM-G77). This strain produces chromosomal
levels of Mlc. In a ptsG+,
ptsHIcrr strain, which is missing the common central
components of the PTS (EI, HPr, and EIIAGlc), there is no
PTS phosphorylation so that PtsG is permanently unphosphorylated,
i.e. in the form that will bind Mlc. This produces derepressed expression of Mlc-regulated genes, even in the absence of
glucose transport. If, in addition, ptsG is deleted
(
ptsHIcrr,
ptsG), there is no PtsG, Mlc
stays bound to its DNA targets, and the expression of the fusion stays
at the fully repressed, basal level. Introducing PtsG or membrane-bound
deletions of PtsG on plasmids into this strain produces high level
expression of the ptsG-lacZ fusion (8). Both Gp8-IIB
constructs, expressed from IPTG-inducible plasmids, in the
ptsHIcrr,
ptsG strain were active in
derepression of ptsG-lacZ, whereas the Gp8 domain by itself
was inactive (Table I, Fig. 5).
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Table I
Effect of mutations in Gp8-IIB on the regulation by M1c
JM-G77 ( RS/ptsG-lacZ, ptsG,
ptsHIcrr) and JM-G96 ( RS/ptsG-lacZ,
ptsG, ptsM8) were transformed with the series of plasmid
derivatives of pTZ19R expressing Gp8-IIB and the mutant versions. The
bacteria were grown in MOPS cas amino acids (0.5%) media with the
carbon source indicated, 0.5mg/ml ampicillin, and 0.1 mM
IPTG. 1 mM cAMP was included for JM-G77. -galactosidase
activities (Miller units) are given for cultures growing in late
exponential phase and are the mean of three or more independent
cultures (except for those with basal level expression). A. In the
absence of EI, HPr, and EIIAGlc, all PTS proteins are
dephosphorylated, binding of Mlc to Gp8-IIB should be maximal, and the
ptsG-lacZ expression should be high. B. In the
pts+ strain growing on glycerol, all PTS proteins
are phosphorylated so that binding of Gp8-IIB to M1c should be minimal
and ptsG-lacZ expression is basal. C. Growth on the PTS
sugar G1cNAc produces dephosphorylation of the specific EIINag
transporter and partial dephosphorylation of other EIIs by cross
dephosphorylation. D. Extracts from bacteria grown in phosphorylating
conditions (B) were labelled in vitro with
[32P]ATP and analysed by SDS-PAGE (Fig. 7). The table
indicates whether the Gp8-IIB protein was labeled by such treatment.
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We confirmed that the Gp8-IIB construct was capable of binding Mlc to
the membrane fraction in vitro by centrifugation as described previously (8). Fig.
6A shows the ability of
Gp8-IIB, contained in isolated everted membrane vesicles made in the
ptsHIcrr,
ptsG strain, to bind Mlc, whereas
Gp8 alone is unable to bind Mlc. Western blotting with
anti-EIIBGlc antibodies showed that the amounts of
overproduced PtsG and Gp8-IIB in the membrane preparations were
comparable (Fig. 6B). If the amino acids present between
positions 353 and 390 of PtsG were essential for the interaction with
Mlc, then we would expect Gp8-EIIBGlc to be inactive.
However, since it regulates ptsG-lacZ expression in
vivo and binds Mlc in vitro, we can conclude that only
the soluble IIB domain of PtsG, downstream of amino acid 390, is
necessary for binding of Mlc. Thus, it demonstrates that TM9 and the
linker region of PtsG are dispensable for Mlc binding but that the
membrane attachment, be it via EIICGlc or via Gp8, is
not.

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Fig. 6.
Binding of Mlc to membranes carrying PtsG,
wild type Gp8-IIB, or mutated Gp8-IIB. As shown in A,
everted membrane vesicles were prepared from strain JM-G77
( ptsG, ptsHIcrr) expressing PtsG, Gp8-IIB
or the mutated versions grown in minimal glycerol media and induced for
3 h with 50 µM IPTG. Aliquots of membranes
corresponding to ~2 mg of total cellular protein were incubated with
Mlc-His6 (1 µg) and separated by centrifugation. Any
residual supernatant (S) was removed by washing the pellet
(P) with 100 µl of buffer. Equivalent amounts of pellet
and supernatant were analyzed as described in the legend for Fig. 4.
std, prestained protein molecular size markers. As shown in
B, the same amount of each pellet fraction was analyzed by
SDS-PAGE and Western blotting with antibodies to
EIIBGlc.
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The Local Environment around the Phosphorylation Site in
EIIBGlc Is Important for Mlc Binding--
To clarify the
interaction between the IIB domain of PtsG and Mlc, a series of
mutations was made in the vicinity of Cys-421 on the Gp8-IIB construct,
and the effect of the mutations on the binding of Mlc and subsequent
inactivation of Mlc as repressor was studied. Gp8-IIB and its
variants were expressed from plasmids in strains that were either
capable of PTS-dependent phosphorylation or lacked the PTS enzymes.
The various mutations were first tested in the
ptsHIcrr,
ptsG strain (JM-G77), in which there is no PTS
phosphorylation. The binding of EIIBGlc to Mlc is expected
to be maximal, and thus, derepression of Mlc-dependent genes will be at their highest level. Table I lists the mutations made
and shows the results. The change of the phosphorylatable Cys-421 to
Ser or Asp still allowed derepression. The change of Cys to the
negatively charged Asp could have mimicked the condition of PtsG
phosphorylation and therefore could have prevented Mlc-binding. Since
the opposite result was obtained, it seems unlikely that the presence
of the negative charge on Cys-421 is mediating Mlc release. This
implies that the change in charge at Cys-421 is not the direct signal
for Mlc interaction, and indeed, it shows that the Cys residue itself
is not essential for binding Mlc. As the signal to bind Mlc is
generated by the dephosphorylation of Cys-421, it seemed likely that
residues surrounding Cys-421, which are affected by its phosphorylation
(18), are important for the contact with Mlc. Thus, we changed the
surface-exposed residues surrounding Cys-421 to Ala.
In contrast to the changes at Cys-421, the change of the nearby Arg-424
to Ala produces a protein that is completely incapable of
derepressing Mlc-regulated genes (and therefore incapable of binding to
Mlc, see below). The ptsG-lacZ fusion remains fully repressed (Table I). Changes to Ala at positions Asp-419, Ile-422, Arg-426, and Ile-458 were still capable of fully derepressing Mlc,
whereas Ala substitutions at positions Thr-423 and Gln-456 showed
reduced derepression. The dramatic effect of the R424A mutation
prompted us to test other substitutions at this position, in particular
changes to histidine and lysine, to try to conserve the positive charge
of arginine. Both were also inactive in derepression of
ptsG-lacZ.
The series of Gp8-IIB mutants was also tested under conditions of PTS
phosphorylation (in strain JM-G96, which is
ptsHIcrr+
ptsG). During growth on
glycerol (i.e. in the absence of glucose), the central PTS
proteins phosphorylate Cys-421, and expression of Mlc-regulated genes
is low. This is the case for the wild type Gp8-IIB construct and for
all the mutants except the two Cys-421 mutations, which produce high
ptsG-lacZ activities in this strain (Table I). Changes at
Cys-421 were expected to produce proteins showing the same activity in
both pts+ and
pts backgrounds. Yet
the Cys-421 mutants behave somewhat differently under phosphorylating
and non-phosphorylating conditions (see "Discussion").
To determine whether the mutant Gp8-IIB constructs were still sensitive
to regulation by PTS phosphorylation, we grew the ptsHIcrr+
ptsG strain on
N-acetylglucosamine. We demonstrated previously that growth
on other PTS sugars, such as N-acetylglucosamine, provides
conditions where the phosphate group on Cys-421 of EIIBGlc
is continuously removed by cross-dephosphorylation between the different EIIs of the PTS so that Mlc-regulated genes are derepressed (3, 8). This allows the distinction between an inactive Gp8-IIB (one
that is defective in binding Mlc) and a protein that shows wild type
behavior (phosphorylation-dependent Mlc-binding) (Table I).
The exchange of Arg-424 to Ala, His, or Lys produces proteins that do
not derepress ptsG-lacZ under any conditions (Table I);
thus, we conclude that mutations at Arg-424 lead to proteins that are
genuinely impaired in binding Mlc. D419A and I422A, which were fully
active in the ptsHIcrr
ptsG strain, exhibit essentially wild type character in the
ptsHIcrr+
ptsG. The other mutants
that were fully or partially active in the
ptsHIcrr
strain are more or less defective under dephosphorylating conditions,
hinting at problems in Cys-421 dephosphorylation. In particular, the
R426A variant showed somewhat reduced activity under these conditions.
The Ability To Be Phosphorylated Does Not Always Correlate with the
Ability to Regulate Mlc--
In another set of experiments, we tested
the ability of the different Gp8-IIB variants to be phosphorylated
in vitro by the general components of the PTS. Total
sonicated extracts of the pts+ strain (JM-G96)
containing the plasmid-encoded Gp8-IIB constructs were treated with
32[P]ATP (which is converted in the extracts to PEP), and
the labeled proteins were separated by SDS-PAGE and visualized by
phosphorimaging (Fig. 7). In this
analysis, phosphorylated Gp8-IIB can easily be recognized as a discrete
band that is absent in the Gp8 control lane. The exchange of Arg-424
with Ala or His, but not with Lys, destroys the ability of Cys-421 to
be phosphorylated in vitro, although all three variants fail
to derepress the ptsG-lacZ fusion. The other interesting
case is the exchange of Arg-426 with Ala. Here the apparent
phosphorylation seen in Fig. 7 is very low, although in vivo
under phosphorylating conditions (pts+), there
is complete repression, and in the absence of any phosphorylation (
ptsHIcrr), there is full derepression (Table I). Note,
however, that derepression during growth of the
pts+ strain on N-acetylglucosamine
was lower. All other variants were phosphorylated, as was the wild
type, including those variants that were partially defective in
derepressing Mlc (T423A, Q456A) and even the double mutant Q456A/I458A,
which was inactive in derepressing Mlc (Table I).

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Fig. 7.
Phosphorylation of Gp8-IIB mutants in
vitro. Bacteria (JM-G96, pts+
ptsG) carrying the plasmids expressing the Gp8-IIB
mutants were grown in MOPS media with 0.4% glycerol, 0.5% casamino
acids, 0.5 mg/ml ampicillin, and 0.1 mM IPTG until late
exponential. The cells were harvested, washed, and broken by
sonication. Aliquots (about 50 µg) were incubated with 5 µCi of
[32P]ATP in 10 mM Hepes, pH 8.0, 5 mM MgCl2, 0.2 mM ATP for 10 min at
30 °C, mixed with SDS sample buffer, warmed to 52 °C, and then
immediately analyzed on 15% SDS-PAGE. The proteins were
electrophoretically blotted to a nitrocellulose membrane, and labeled
proteins were identified by phosphorimaging. The position of the
Gp8-IIB protein (17 kDa) is indicated. wt, wild
type.
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Binding of Mlc to the Gp8-IIB Variants in Membranes Parallels Their
Ability to Derepress Mlc-controlled Genes--
In the last set of
experiments, we tested the ability of Mlc to bind to some Gp8-IIB
variant strains contained in everted membrane vesicles. To
compare these strains under the conditions of PTS phosphorylation used
in the in vivo tests, we used two different strains. The
first strain (JM-G77) lacks both PtsG and the general PTS components.
In this strain, Gp8-IIB is never phosphorylated, and binding of Mlc to
membranes should be maximal. All the mutants tested except R424A,
R424K, and R424H did bind Mlc to the membranes from this strain like
the wild type (Fig. 6). R424H exhibited reproducibly residual binding
of Mlc that was not reflected in the repression test. This residual
binding, in contrast to the binding of the other Gp8-IIB hybrids, was
partly washed off by resuspending the pellet in buffer and
recentrifuging, and the nature of this binding is not understood at
present. Thus, with the possible exception of R424H, the ability of
membranes containing the Gp8-IIB mutants to bind Mlc in
vitro was closely correlated with their ability to regulate
ptsG expression in vivo.
The second strain used (IT1168) is also ptsG but contains
all the general PTS components and was grown under conditions allowing the phosphorylation of Gp8-IIB (Fig. 8).
During the preparation of PtsG-containing-membranes, normally there is
a loss of the relatively labile phosphate from Cys-421 of PtsG (and
from His residues of other PTS proteins). This means that membranes
containing PtsG isolated from the pts+ strain
become partially dephosphorylated and are able to bind Mlc. In fact,
they require incubation with PEP in the presence of the soluble PTS
proteins immediately before isolation of the membranes to phosphorylate
PtsG and decrease binding of Mlc (8). For the Cys-421 and R424A
mutants, the pattern of Mlc binding is identical in the two strains.
For R426A, binding of Mlc in the pts+ strain was
reduced as compared with the
ptsHIcrr strain. This could
mean that the spontaneous dephosphorylation of this mutant is
decreased.

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Fig. 8.
Binding of Mlc to membranes of a
pts+ strain containing PtsG and wild type
or mutated versions of Gp8-IIB. As shown in A, everted
membrane vesicles derived from strain IT1168(ptsG:kan) with
plasmids expressing PtsG, Gp8-IIB, and variants were incubated with
Mlc-His6 (1 µg) and analyzed as described in the legend
for Fig. 6. P, pellet; S, supernatant;
std, prestained protein molecular size markers. As shown in
B, the pellet fractions were analyzed as described in the
legend for Fig. 6.
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The amount of overproduced Gp8-IIB variants in the membrane
preparations was checked using antibodies against the soluble EIIBGlc in Western blots (Figs. 6B and
8B) and was roughly comparable in all preparations. All the
hybrid proteins were stable, and no apparent signs of degraded
proteins were detected.
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DISCUSSION |
Soluble EIIBGlc Is Sufficient to Interact with Mlc when
Attached to Membranes via a Heterologous Membrane Anchor--
We
showed previously that deleted versions of PtsG that retained the
EIIBGlc domain, the linker between the EIICGlc
and EIIBGlc domains, and the ninth potential TM helix of
EIICGlc were capable of binding Mlc to membranes and
to derepress Mlc-regulated genes. An excess of soluble
EIIBGlc domain (C-terminal His6 version) was
rather inefficient at displacing Mlc from the PtsG-containing membranes
(8). This suggested that binding of Mlc was associated with the
EIIBGlc domain but that some other part of PtsG, in
addition to the soluble EIIBGlc domain, for instance the
linker region between EIICGlc and EIIBGlc, was
necessary to bind Mlc to membranes. Nam et al. (9) showed a
phosphorylation-dependent interaction between the soluble
EIIBGlc domain and Mlc by surface plasmon resonance.
However, it remained unclear whether or not the affinity of this
interaction is the same as with the EIIBGlc domain, when it
is attached to EIICGlc in wild type PtsG. This raised the
question of whether the role of TM9 and the linker was just to anchor
the EIIBGlc domain to the membrane and produce a physical
separation of Mlc from its DNA targets or whether it was part of a
genuine binding site. Here we show that indeed the role of TM9 is to
provide a membrane anchor and that it can be replaced by the
membrane-spanning Gp8 protein from M13, entirely unrelated to PtsG.
There are several considerations to be discussed in respect to this
finding. If the PtsG-Mlc interaction is solely via the EIIBGlc domain, why does not soluble EIIBGlc
elicit derepression by binding Mlc in solution? As the soluble versions
of EIIBGlc are completely inactive in derepressing
Mlc-repressed genes, this implies that the complex between Mlc and
soluble EIIBGlc is still able to bind to the target DNA. In
this case, in vivo derepression of Mlc must be just a
consequence of physically sequestering Mlc from the DNA. However, as
Görke and Rak (25) have elegantly shown, by using Gp8 to anchor
the BglG transcriptional antiterminator to the membrane, actively
transcribing DNA can, in principle, interact with membrane-bound
transcriptional regulators. This would suggest that a simple
sequestration model is not sufficient and that the quality of the
interaction between Mlc and EIIBGlc is different in the
membrane environment. Alternatively, the conformational change in
membrane-associated Mlc, which provokes the switch to the non-DNA
binding form, could be mediated by another, as yet unidentified,
component in the membrane. Such a component would be unable to affect
the interaction of Mlc with soluble EIIBGlc.
Another aspect that should be considered is the stoichiometry of the
interaction between PtsG and Mlc, but for which we have no precise
information. PtsG is believed to be dimeric in the membrane (26),
whereas soluble EIIBGlc is presumably monomeric. We ignore
the quaternary structure adopted by Gp8-IIB in the membrane. An altered
multimeric structure of Gp8-IIB as compared with PtsG could affect
either the stoichiometry or the affinity of its binding to Mlc as
compared with PtsG. Mlc is a tetramer, and as we have shown, the
Mlc(
18) form, which does not form a tetramer, cannot bind to PtsG.
It is perhaps relevant that higher concentrations of IPTG (100 µM) are necessary to achieve full derepression of the
ptsG-lacZ fusion with Gp8-IIB rather than 10 µM, which is sufficient with full-size PtsG. This could mean that the Gp8-IIB construct has a lower affinity for Mlc than for
PtsG. In addition, we found that the binding capacity of the Gp8-IIB-containing membranes for Mlc appears to be lower than that of
PtsG-containing membranes. Although in our standard assay, PtsG-containing membranes could remove 2 µg of Mlc from the
supernatant, Gp8-IIB could only bind 1 µg.
Cys-421 Phosphorylation and Binding of Mlc--
Three groups
(8-10) showed previously that binding of Mlc to PtsG depended upon the
phosphorylation state of Cys-421 of PtsG in membranes. Mutations of
this critical Cys to Ser or Asp, which we hoped would mimic the
unphosphorylated and phosphorylated states respectively of
EIIBGlc, both produce derepressed expression of
Mlc-regulated genes. Mutations of serine to aspartate (in HPr
from Bacillus subtilis (27)) and of histidine to aspartate
(in LicT (28)) have been described that produce proteins with the
characteristics of the Ser(P) and His(P) phosphorylated forms of the
proteins. However, for Gp8-IIB, both C421S and C421D mutants behave as
activated forms of the EIIBGlc protein, binding Mlc
constitutively. Moreover, the Asp form produces full activity, showing
that the negative charge of aspartate is not sufficient to antagonize
Mlc binding and arguing that the charge change, produced by
phosphorylation of EIIBGlc, is neither causative nor
sufficient to account for the changed interaction with Mlc. In
addition, these findings show that the sulfur of Cys-421 is not
recognized by Mlc since the Cys-421 mutants are still capable of
binding Mlc. The C421S mutant was less active in the
pts+ strain than in
ptsHIcrr. This
was surprising; we had expected the Cys-421 mutations to be insensitive
to the PTS phosphorylation status of the bacteria. Possibly, the C421S
mutation is interacting with EIIAGlc, which is present in
the pts+ strain but absent in
ptsHIcrr.
Although Cys-421 is not essential for Mlc binding, Mlc binding responds
to the phosphorylation state of Cys-421, and so we turned our attention
to the amino acids whose NMR spectra are affected by Cys-421
phosphorylation (18). Cys-421 is located at the C terminus of a
-strand on the solvent-exposed surface and is surrounded by
hydrophilic residues (Fig. 9). To
identify amino acids that might contact Mlc, we changed these residues to alanine. At residues Asp-419, Ile-422, Arg-426, and Ile-458, the
alanine substitutions were still capable of full derepression of the
fusion, and therefore capable of binding of Mlc; at Thr-423 and
Gln-456, alanine replacements had a decreased ability to derepress, whereas mutations at Arg-424 were inactive and fully repressed (Table I
and Fig. 9). The conservative replacements of Arg-424 with Lys or His
were also inactive in displacing Mlc in vivo.

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Fig. 9.
Location of the mutations
on the IIB structure. A stereo representation of the
EIIBGlc structure (Protein Data Bank number 1IBA) is shown.
The -carbon ribbon of the molecule is shown in blue, and
the Van der Waals' surface is represented as a gray
surface. Cys-421, highlighted in magenta, occurs at the
end of the 1 strand in the solution structure of EIIBGlc
(17, 18). The molecule is orientated so that the protruding convex
surface, with the hydrophilic residues surrounding Cys-421, is facing
toward the viewer. The residues changed in this study are
colored. Mutations at Arg-424, shown in red, are
inactive in derepressing Mlc-regulated genes. Mutations to alanine at
the amino acids shown in green were capable of full
derepression of Mlc-controlled genes, and alanine substitutions at
amino acids shown in yellow were partially defective.
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Lanz and Erni (16) had previously studied R424K and R426K mutations on
full-size PtsG. Both proteins behaved similarly in their tests: they
were inactive for glucose transport, and they were capable of being
phosphorylated by EIIAGlc but unable to transfer the
phosphate to glucose. The behavior of the Gp8-IIB constructs carrying
Arg-424 and Arg-426 replacements in respect to Mlc binding is, however,
quite different. The R426A substitution still allowed Mlc binding, but
all the Arg-424 replacements were inactive. The Ala replacements at
positions 424 and 426 were introduced onto the full-size PtsG, and as
in the case of the Lys changes studied by Lanz and Erni (16), both are
inactive for glucose transport. Consistent with the results for the
Gp8-IIB constructs, full-size PtsG carrying the R424A mutation was
incapable of derepressing ptsG-lacZ, whereas PtsG-R426A was
still active for Mlc regulation.
The R424A replacement on Gp8-IIB was not labeled with
[32P]phosphate, whereas the R426A replacement was only
weakly labeled, although it was fully active in derepressing
ptsG-lacZ in vivo. A possible explanation of this weak
labeling is that the alanine substitution has reduced the spontaneous
dephosphorylation of EIIBGlc in vitro without
eliminating the PTS-catalyzed phosphorylation in vivo.
Therefore, R426A, isolated from the pts+ strain,
might already be phosphorylated by unlabeled phosphate. During
preparation of the extracts, it does not loose the phosphate and
therefore can not be effectively labeled by 32P in
vitro.
The observation that Gp8-IIB-R424K is labeled with
32P in vitro agrees with the Lanz and Erni (16)
finding that PtsG-R424K was phosphorylated by EIIAGlc-P.
The fact that it is inactive for depressing ptsG-lacZ shows that an arginine at position 424 is essential for Mlc binding per
se, i.e. irrespective of whether the alterations at
this position affect Cys-421 phosphorylation.
The residues Thr-423 and Gln-456, whose replacement with Ala reduces
the interaction of Gp8-IIB with Mlc, are found on one side of Arg-424,
that which is distal from Cys-421 (Fig. 9). The effect of these
mutations could be indirect and alter the conformation of Arg-424,
which we propose is a major recognition determinant for Mlc. Although
the I458A mutation is still capable of fully derepressing Mlc, the
double Gln-456/Ile-458 mutation becomes completely inactive, which
could be due to global structural perturbations. The NMR spectra
changes showed significant sharpening of the amide lines of Ile-422,
Thr-423, and Arg-424 upon phosphorylation, indicating a stiffening of
the three-dimensional structure (18), and this conformational change
could be the signal detected by Mlc. As shown in the stereo
representation of EIIBGlc, the three amino acids, Cys-421,
Ile-422, and Arg-424, are at the apex of a prominent structure formed
by the end of
1 and the short loop to
2 (17, 18) (Fig. 9), which
should provide a contact surface with Mlc.
Aside from the dominant effect of Arg-424 on Mlc binding, the
interaction between Mlc and PtsG must be more complex. The amino acid
sequence around Cys-421 is conserved in other members of the glucose
family of EIIs (16). For example, the sequence between amino acids
Asp-419 and Arg-426 (DACITRLR) around the phosphorylated Cys is found
in both the NagE protein (EIICBANag) of E. coli
and PtsG (EIICBAGlc) from B. subtilis.
Plasmid-encoded NagE or PtsG of B. subtilis did
not elicit any derepression of ptsG-lacZ in the
ptsG,
ptsHIcrr strain. This
demonstrates that something in addition to the immediate protein
environment of Cys-421/Arg-424 contributes to Mlc recognition.
Mutations in the EIICGlc domain have been characterized
that change the sugar specificity of PtsG transport (29, 30) and simultaneously affect regulation by producing partially derepressed expression of Mlc-regulated genes under non-inducing conditions (31-33). Although these mutations affect Mlc-binding, they cannot be
in amino acids essential for interacting with Mlc since the entire
EIICGlc region (amino acids 5-390) can be deleted without
abolishing Mlc binding. The existence of such mutations implies a tight
coupling between sugar transport through EIICGlc, the
phosphorylation by EIIBGlc, and the conformation around
Arg-424 and Cys-421. A conformational coupling between the IIC and IIB
domains has been detected previously in the mannitol enzyme IICBA
transporter (34).