Analysis of the Interaction between the Global Regulator Mlc and EIIBGlc of the Glucose-specific Phosphotransferase System in Escherichia coli*

Sabine SeitzDagger , Sung-Jae LeeDagger , Carole Pennetier§, Winfried BoosDagger , and Jacqueline Plumbridge§

From the Dagger  Department of Biology, University of Konstanz, D-78457 Konstanz, Germany and the § Institut de Biologie Physico-Chimique (UPR9073), 13, rue Pierre et Marie Curie, 75005 Paris, France

Received for publication, November 26, 2002, and in revised form, January 14, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mlc is a global regulator acting as a transcriptional repressor for several genes and operons of Escherichia coli encoding sugar-metabolizing enzymes and uptake systems. The repressing activity of Mlc is inactivated by binding to the dephosphorylated form of EIICBGlc (PtsG), which is formed during the transport of glucose. Here, we demonstrate that EIIBGlc, the cytoplasmic domain of PtsG, alone is sufficient to inactivate Mlc but only when EIIBGlc is attached to the membrane by a protein anchor, which can be unrelated to PtsG. Several EIIBGlc mutants, which were altered in and around the phosphorylation site (Cys-421) of EIIBGlc, were tested for their ability to bind Mlc and to affect transcriptional repression by Mlc. The exchange of Cys-421 with serine or aspartate still allowed binding to Mlc, and in addition, derepression became constitutive, i.e. independent of phosphoenolpyruvate-dependent phosphotransferase system (PTS) phosphorylation. Mutations were made in the surface-exposed residues in the vicinity of Cys-421 and identified Arg-424 as essential for binding to Mlc. Binding of Mlc to the EIIBGlc constructs in membrane preparations paralleled their ability to derepress Mlc-dependent transcription in vivo. These observations demonstrate that it is not the charge change at Cys-421, produced by PTS phosphorylation, that allows Mlc binding but rather the structural change in the environment surrounding Cys-421 that the phosphorylation provokes. Native Mlc exists as a tetramer. Deleting 18 amino acids from the C-terminal removes a putative amphipathic helix and results in dimeric Mlc that is no longer able to repress.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Bacteriological Methods-- Most of the strains used are derivatives of JM101 carrying a ptsG-lacZ fusion on a lambda -lysogen (2). JM-G2 carries the mlc:tet mutation, and JM-96 is Delta ptsG:cat, ptsM8, which eliminates glucose uptake by the PTS. JM-G77 is Delta ptsG:cat and Delta ptsHIcrr:kan, which eliminates all PTS phosphorylation. These strains have been described previously (2, 3). IT1168 (ptsG:kan) (19) was used for membrane preparations. beta -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-Delta 410-419-PstI, C-terminal (5'-AACTGCAGTTAACCGTTATACATCGCGTCTTTTACC-3') or the primer mlc-Delta 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(Delta 5-390) was described previously (8). PtsG(Delta 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).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -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(Delta 9) still strongly repressed the expression of ptsG-lacZ, Mlc(Delta 18) was unable to do so.


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Fig. 1.   Protein molecular size (MS) determination of Mlc and the Delta 9 and Delta 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, MlcDelta 9, 173 kDa; filled circles, MlcDelta 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, MlcDelta 9 and MlcDelta 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 beta -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.

Simultaneously Mlc(Delta 18) lost its ability to be bound by PtsG present in everted membrane vesicles in vitro, whereas Mlc(Delta 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, MlcDelta 9, and MlcDelta 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.

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(Delta 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 Delta ptsHIcrr, Delta ptsG strain (JM-G77) in vivo is indicated.

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 lambda -lysogen on the chromosome of the Delta ptsHIcrr, Delta ptsG strain (JM-G77). This strain produces chromosomal levels of Mlc. In a ptsG+, Delta 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 (Delta ptsHIcrr, Delta 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 Delta ptsHIcrr, Delta 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 (lambda RS/ptsG-lacZ, Delta ptsG, Delta ptsHIcrr) and JM-G96 (lambda RS/ptsG-lacZ, Delta 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. beta -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.

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 Delta ptsHIcrr, Delta 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 (Delta ptsG, Delta 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.

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 Delta ptsHIcrr, Delta 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+Delta 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 Delta 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+Delta 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 Delta ptsG strain, exhibit essentially wild type character in the ptsHIcrr+Delta ptsG. The other mutants that were fully or partially active in the Delta 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 (Delta 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+ Delta 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.

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 Delta 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.

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.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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(Delta 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 Delta 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 Delta 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 beta -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 alpha -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 beta 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.

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 beta 1 and the short loop to beta 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 Delta ptsG, Delta 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).

    ACKNOWLEDGEMENTS

We thank Joachim Diez for preparing the three-dimensional presentation of Fig. 9; Boris Görke for advice on use of the Gp8 linker; Saul Roseman and Mitchell Lewis for pointing out the putative alpha -helices in the C terminus of Mlc and NagC; Bernard Erni for the gift of plasmids; and Hiroji Aiba for comments on the manuscript.

    FOOTNOTES

* This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie (to W. B.) and from the CNRS and the Université Paris 7 (to UPR9073).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 33-1-58-41-51-52; Fax: 33-1-58-41-50-20; E-mail: plumbridge@ibpc.fr.

Published, JBC Papers in Press, January 14, 2003, DOI 10.1074/jbc.M212066200

    ABBREVIATIONS

The abbreviations used are: PTS, PEP-dependent phosphotransferase system; PEP, phosphoenolpyruvate; HPr, histidine-containing protein of the PTS; EIICBGlc, membrane-bound subunit of the glucose-specific PTS transporter encoded by ptsG; EIICGlc, integral membrane domain of EIICBGlc; EIIBGlc, cytoplasmic domain of EIICBGlc; EIIAGlc, soluble protein of the glucose-specific PTS transporter encoded by crr; Gp8-IIB, hybrid protein between Gp8 of M13 and EIIBGlc; TM, transmembrane helice; MOPS, 4-morpholinepropanesulfonic acid; IPTG, isopropyl-beta -D-thiogalactopyranoside.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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