LevR, which controls the expression of the lev operon of Bacillus subtilis, is a regulatory protein containing an N-terminal domain similar to the NifA/NtrC transcriptional activator family and a C-terminal domain similar to the regulatory part of bacterial anti-terminators, such as BglG and LicT. Here, we demonstrate that the activity of LevR is regulated by two phosphoenolpyruvate (PEP)-dependent phosphorylation reactions catalysed by the phosphotransferase system (PTS), a transport system for sugars, polyols and other sugar derivatives. The two general components of the PTS, enzyme I and HPr, and the two soluble, sugar-specific proteins of the lev-PTS, LevD and LevE, form a signal transduction chain allowing the PEP-dependent phosphorylation of LevR, presumably at His-869. This phosphorylation seems to inhibit LevR activity and probably regulates the induction of the lev operon. Mutants in which His-869 of LevR has been replaced with a non-phosphorylatable alanine residue exhibited constitutive expression from the lev promoter, as do levD or levE mutants. In contrast, PEP-dependent phosphorylation of LevR in the presence of only the general components of the PTS, enzyme I and HPr, regulates LevR activity positively. This phosphorylation most probably occurs at His-585. Mutants in which His-585 has been replaced with an alanine had lost stimulation of LevR activity and PEP-dependent phosphorylation by enzyme I and HPr. This second phosphorylation of LevR at His-585 is presumed to play a role in carbon catabolite repression.
The phosphoenolpyruvate–carbohydrate phosphotransferase system (PTS) is responsible for the uptake and concomitant phosphorylation of a number of sugars in both Gram-negative and Gram-positive bacteria (for a review, see Postma et al. (1993). The phosphorylation cascade from phosphoenolpyruvate (PEP) to the sugar involves enzyme I (EI), a histidine-containing phosphocarrier protein (HPr), and a sugar-specific multidomain enzyme II (EII). The EII is either a single polypeptide with several domains or several different polypeptides (Reizer and Saier, 1997). The PTS is involved in several regulatory mechanisms, such as chemotaxis, transcriptional regulation and carbon catabolite repression (Saier, 1989; Postma et al., 1993). The glucose-specific EIIA is central to carbon catabolite repression in Escherichia coli, whereas HPr plays this role in Bacillus subtilis. The HPr protein of Gram-positive bacteria can be phosphorylated at two different sites: (i) the catalytic histidine-15 is phosphorylated by EI in the presence of PEP necessary for sugar transport; and (ii) the regulatory seryl residue 46 is phosphorylated by an ATP-dependent protein kinase whose activity is stimulated by glycolytic intermediates such as fructose-1,6-bisphosphate (for a review, see Reizer et al., 1993).
Positive and negative controls are mediated via PEP-dependent phosphorylation. Activation of the transcriptional regulators requires phosphorylation by EI and HPr, whereas phosphorylation via EI, HPr and the corresponding sugar-specific EII leads to inhibition of the regulator. The EIIBgl (BglF) phosphorylates the antiterminator BglG reversibly, thereby mediating induction by β-glucosides (Amster-Choder et al., 1989; Schnetz and Rak, 1990). Biochemical evidence for the phosphorylation of SacT, SacY, LevR and LicT by HPr has also been obtained (Arnaud et al., 1996; Deutscher et al., 1997; Stülke et al., 1995; Tortosa et al., 1997). The regulation of SacY activity is different, as the phosphorylation of SacY by HPr at His-99 probably has a negative regulatory role (Tortosa et al., 1997).
The levanase operon of B. subtilis (levDEFG sacC ) encodes a fructose-specific PTS (lev-PTS) and the extracellular levanase, which hydrolyses fructose polymers and sucrose (Martin et al., 1987; Martin-Verstraete et al., 1990). Together with EI and HPr, LevD and LevE have been shown to form a phosphorylation chain. LevD is phosphorylated by HPr, and P-LevD transfers the phosphoryl group to LevE (Charrier et al., 1997). The expression of this operon is induced by fructose and repressed by glucose (Martin-Verstraete et al., 1995). Fructose induction depends on the NifA-type activator LevR (Débarbouilléet al., 1991), which is negatively regulated by the fructose-specific enzymes IIA/IIB of the PTS encoded by levD and levE and positively controlled by HPr (Martin-Verstraete et al., 1990; Stülke et al., 1995; Charrier et al., 1997). Phosphorylation of LevR by EI and HPr has been demonstrated previously (Stülke et al., 1995). In the LevR protein, the regulatory part of the protein appears to have evolved by duplication and can be divided into domains B and C, which are similar to each other. Domains B and C of LevR contain the duplicated P1 and P2 domains found in all the antiterminators (Tortosa et al., 1997). Domain B is necessary for the stimulation by HPr, and domain C was thought to be the target for negative regulation by the lev-PTS (Stülke et al., 1995).
To understand the complex mechanisms of the regulatory interactions between PTS components and the LevR protein, we synthesized domains B and C of LevR separately and demonstrated their phosphorylation. Using genetic and biochemical approaches, we identified the phosphorylation sites of LevR. Phosphorylation at His-585 in domain B requires only EI and HPr. In contrast, His-869 in domain C is only phosphorylated if the fructose-specific PTS components LevD and LevE are also present.
PEP and the general PTS components phosphorylate domain B of LevR
Genetic data indicate that LevR activity is controlled by the PTS via two mechanisms. Positive control is mediated by EI and HPr, whereas negative control also requires two components of the lev-PTS (Stülke et al., 1995). LevR was phosphorylated in vitro by PEP, EI and HPr. On the basis of data obtained with truncated LevR proteins, it has been suggested previously that the regulatory domain B (amino acids 411–689) is necessary for HPr-mediated stimulation of LevR activity, whereas domain C (amino acids 785–938) of LevR (Fig. 1) was predicted to be the target for lev-PTS-mediated induction by fructose (Stülke et al., 1995). To confirm the role of domain B in HPr-dependent regulation of LevR, a set of truncated LevR polypeptides was tested for phosphorylation in vitro by labelled PEP, EI and HPr (Fig. 2). LevR798 and LevR723 containing part or none of domain C were still phosphorylated by EI and HPr (Fig. 2, lanes 3 and 4). LevR407, which contains neither domain B nor domain C, was not phosphorylated (Fig. 2, lane 6). This confirms the genetic data obtained by Stülke et al. (1995) and indicates that domain B is the target of HPr-dependent regulation. However, PEP-dependent phosphorylation of domain C by EI, HPr, LevD and LevE remained to be demonstrated.
EI, HPr, LevD and LevE are required for the phosphorylation of domain C of LevR
To demonstrate that EI and HPr phosphorylate domain B and to investigate the role of domain C in PTS-dependent regulation, we synthesized the domains separately. Plasmids pRL56 and pRL78 encoding GST fused to domain B or domain C, respectively, were constructed. The corresponding polypeptides were purified as described in Experimental procedures.
The purified domain B of LevR was phosphorylated in the presence of [32P]-PEP, EI and HPr. This phosphorylation was not affected by the presence of LevD or LevE (data not shown). Purified GST–domain C was not phosphorylated by [32P]-PEP in the presence of EI and HPr (Fig. 3, lane 2) or EI, HPr and LevD (Fig. 3, lane 3). The presence of all the four PTS proteins, EI, HPr, LevD and LevE, was required for phosphorylation of the GST–domain C hybrid (Fig. 3, lane 4). These four PTS proteins have previously been shown to form a phosphorylation chain in vitro (Charrier et al., 1997). Thus, domain B is phosphorylated by EI and HPr, whereas phosphorylation of domain C requires the presence of LevD and LevE.
Mutation of His-869 of LevR leads to the loss of lev-PTS-catalysed phosphorylation
The phosphate bonds in phosphorylated LevR were rapidly hydrolysed under acidic conditions but not under alkaline conditions (pH higher than 10), suggesting that the phosphoryl groups in this protein are bound to a histidine or a cysteine forming either a phosphoamidate or thiophosphate bond (Mattoo et al., 1984; Postma et al., 1993). As the conserved regulatory domain of the PTS-regulated proteins, including the BglG family of antiterminators and LevR, does not contain a conserved cysteine, the amino acid phosphorylated by the PTS in LevR is probably a histidine. Domain C of LevR contains four histidines. However, only His-869 is conserved among domain C and members of the SacT/BglG family of antiterminators (Fig. 1). Conserved histidine is also phosphorylated in SacY, LicT and BglG (Chen et al., 1997; Deutscher et al., 1997; Tortosa et al., 1997).
To test whether His-869 of LevR is phosphorylated, a mutation was introduced into the GST–domain C hybrid protein, replacing His-869 with a non-phosphorylatable alanine residue. The protein was purified and tested for phosphorylation. Unlike the wild-type GST–domain C hybrid, the corresponding H869A modified protein was not phosphorylated by EI, HPr, LevD and LevE (Fig. 3, lanes 4 and 7), suggesting that His-869 is the site of negative regulation by the lev-PTS.
Mutation of His-869 of LevR leads to constitutive expression of the lev operon
To test the involvement of His-869 in the regulation of LevR activity, the levR allele containing the H869A mutation was integrated into the amyE locus of strain QB5182, which contains a deletion of levR and a sacC′–lacZ transcriptional fusion. Expression of the lacZ fusion in the levR H869A mutant was compared with that in the isogenic levR+ and levR8 (encoding LevR truncated at position 798) strains as controls. The different levR alleles were also introduced into isogenic strains carrying either a ΔptsGHI or a levE7 mutation. Both negative and positive control by the PTS was abolished in the ΔptsGHI strain, whereas only negative regulation by the lev-PTS was absent in the levE7 mutant containing a nonsense mutation in levE (Martin-Verstraete et al., 1990). β-Galactosidase activities of the levR mutants were assayed after growth in CSK medium in the presence or in the absence of 0.2% fructose (Table 1). Like the levR8 mutation, the levR H869A mutation led to high constitutive expression of the sacC′–lacZ fusion. The activity of the LevR H869A polypeptide was not altered significantly in a levE7 mutant (Table 1). These results confirm that histidine-869 of LevR is the site of negative regulation by the lev-PTS. Nevertheless, LevR H869A and LevR798 were positively regulated by HPr: fivefold and 300-fold lower β-galactosidase activities, respectively, were measured in a ΔptsGHI background. In these mutants, LevR contains the presumed site of HPr-catalysed phosphorylation in domain B, as both proteins were phosphorylated by EI and HPr (Fig. 2, lane 3; data not shown).
Table 1. . Regulation of transcription of the levanase operon by the PTS in the presence of the levR gene containing point mutations. a. The modified levR alleles were introduced at the amyE locus of strains containing a levR gene disruption, a sacC′–lacZ transcriptional fusion in a wild-type ΔptsGHI and levE7 background. The corresponding strains are listed in Table 2.b. The β-galactosidase activities were measured in extracts prepared from exponentially growing cells (A600 0.7–1).
Replacement of His-506 affects LevR activity
His-506 and His-567, both located in domain B of LevR, are conserved in the regulatory domain of the antiterminators (Fig. 1) (Tortosa et al., 1997). Mutations replacing His-506 or His-567 with an alanine were, therefore, introduced in the levR gene. EI and HPr phosphorylated the LevR H506A and LevR H567A polypeptides (data not shown), suggesting that neither His-506 nor His-567 is the phosphorylation site in domain B. The levR H506A allele was also integrated into the amyE locus of a ΔlevR, a ΔlevRΔptsGHI and a ΔlevR levE7 strain, all carrying a sacC′–lacZ transcriptional fusion. The phenotype of the levR H506A mutant is complex. LevR H506A activity, measured after growth in CSK, was 13-fold higher than that observed for wild-type LevR and was induced only threefold by the presence of 0.2% fructose (Table 1). In a levE7 background, β-galactosidase activity only reached the level obtained after induction by fructose in the levR H506A mutant. Although these results suggest that His-506 is involved in the regulation of LevR, the basal activity of LevR H506A, 13-fold higher than in the wild-type LevR, argued against His-506 being the site of positive control by HPr.
We studied further the role of His-506 in the regulation of LevR activity. A double mutant, levR H506A H869A, was constructed and integrated at the amyE locus of ΔlevR, ΔlevRΔptsGHI and ΔlevR levE7 mutants. Expression of the sacC′–lacZ transcriptional fusion was measured in the resulting strains tested after growth in CSK medium, in the presence and in the absence of 0.2% fructose (Table 1). The levR H506A H869A mutant exhibited constitutive expression of the lev operon and remained threefold positively regulated by HPr (Table 1). LevR proteins truncated at position 798 or 723 and containing the H506A mutation exhibited a 65- and 18-fold lower activity in the Δpts strain than in the wild-type strain, respectively (data not shown), indicating the existence of a site in domain B other than His-506 for positive regulation by HPr.
His-585 of LevR is the site of HPr-dependent phosphorylation
To identify the site of regulation by HPr, we compared the sequence of LevR and CelR, a putative regulatory protein of cellobiose catabolism in Bacillus stearothermophilus exhibiting the strongest similarity to LevR (Lai and Ingram, 1993). CelR contains a His at the position corresponding to His-585 in LevR, unlike the BglG family of antiterminators. To test whether histidine-585 is phosphorylated by HPr, levR H585A and levR H585A H869A mutants were constructed as described in Experimental procedures.
PEP-dependent phosphorylation of partially purified LevR H585A and LevR H585A H869A was tested in vitro (Fig. 4). In these two polypeptides, His-585 is replaced by an alanyl residue. Neither was phosphorylated by EI and HPr (Fig. 4, lane 3; data not shown), suggesting that His-585 is the site of HPr-catalysed phosphorylation in LevR.
We also tested whether the addition of LevD and LevE to EI and HPr would allow phosphorylation of LevR H585A and LevR H585A H869A. EI, HPr and LevD are not sufficient to phosphorylate these proteins, but the addition of LevE restored the phosphorylation of LevR H585A (Fig. 4, lanes 6–8). However, EI, HPr, LevD and LevE failed to phosphorylate LevR H585A H869A (Fig. 4, lane 5), indicating that His-869 is most probably the site of phosphorylation by the lev-PTS. The levR H585A allele was also integrated into the amyE site of the ΔlevR, ΔlevRΔptsGHI and ΔlevR levE7 strains, and the effect of the levR H585A mutation on the expression of the sacC′–lacZ transcriptional fusion was studied (Table 1). Being negatively regulated by the lev-PTS, but presumably missing the positive effect of EI and HPr, LevR H585A exhibited low basal activity. The activity was sevenfold and threefold higher in the Δpts and levE7 background, respectively, probably because of the loss of the negative effect of the lev-PTS on LevR H585A activity.
Direct phosphorylation of LevR by P-LevE
Although P-LevD and P-LevE are necessary for the phosphorylation of LevR at His-869, there is no evidence that the phosphoryl group is not transferred from P-HPr to His-869. P-LevD and P-LevE may only modulate HPr-dependent phosphorylation of LevR at His-869. To assess the role of LevD and LevE, P-LevE was separated from the proteins necessary for its phosphorylation as follows. EI, HPr and LevD, each carrying a polyhistidine tag, were used to phosphorylate LevE in the presence of [32P]-PEP, and P-LevE was then separated from the other proteins by binding them to Ni–NTA agarose (Fig. 5B, lanes 1 and 2). P-LevE was used in vitro to phosphorylate LevR+, LevR H869A, LevR H585A, LevR H506A H585A and LevR H506A H585A H869A. All LevR proteins containing His-869 were phosphorylated by P-LevE (Fig. 5A, lanes 3–5), whereas LevR H869A and LevR H506A H585A H869A were not (Fig. 5A, lane 6 and Fig. 5B, lane 3). These results confirm that His-869 is necessary for the phosphorylation of domain C. The absence of phosphorylation of domain B in LevR H869A by P-LevE rules out a substantial contamination of the P-LevE preparation with HPr. Phosphorylation of the LevR polypeptide presented in Fig. 5 must, therefore, be caused by direct transfer of the phosphate group from P-LevE to His-869 of LevR.
Repressor-controlled gene expression is only one of several mechanisms used by bacteria to induce catabolic genes. Other induction mechanisms described in bacteria include transcriptional activators and antiterminators (Rutberg, 1997). The two latter mechanisms were found to be associated mainly with the induction of operons encoding sugar-specific components of the PTS. The activity of such transcriptional activators and antiterminators is often regulated by PEP-dependent phosphorylation catalysed by proteins of the PTS (Deutscher et al., 1997). The antiterminator BglG is phosphorylated by the corresponding sugar-specific PTS component EIIBgl at a single histidyl residue (Chen et al., 1997), whereas several histidyl residues in LicT and SacY are phosphorylated by HPr (Deutscher et al., 1997; Tortosa et al., 1997).
We studied the transcriptional activator LevR, which controls the expression of a fructose-specific PTS and contains regulatory domains similar to those present in the antiterminators. We found that it was phosphorylated twice, once by HPr and once by the sugar-specific component LevE. Phosphorylation of LevR in domain C requires the presence of PEP, EI, HPr, LevD and LevE and substantially reduced LevR activity. Mutants defective in levD or levE (Martin-Verstraete et al., 1990) or expressing LevR, in which the presumed phosphorylation site His-869 had been replaced with an alanine, exhibit constitutive expression from the lev promoter. In all these mutants, LevR is not phosphorylated in domain C. Although we could not completely exclude that modification at His-869 prevents the phosphorylation of another histidyl residue of domain C, both in vivo and in vitro data give strong evidence that His-869 is the site of phosphorylation by the lev-PTS. Phosphorylation of His-869 therefore seems to regulate induction of the lev operon in response to the availability of a substrate of the lev-PTS (Fig. 6). In the absence of a substrate of the lev-PTS, fructose or mannose (Martin-Verstraete et al., 1990; 1996), the sugar-specific components LevD and LevE, although produced in low amounts, are assumed to be present mainly in the phosphorylated form. This allows phosphorylation and inactivation of LevR by P-LevE, and the lev operon will be poorly expressed. In contrast, in the presence of fructose, P-LevE is assumed to transfer its phosphate group mainly to the incoming sugar, leading to dephosphorylation and activation of LevR. Interestingly, like the IIB domain of EIIBgl (Chen et al., 1997), P-LevE seems to be able to transfer its phosphate group either to a histidyl residue of a regulatory protein or to a hydroxyl group of a sugar. To demonstrate unequivocally that P-LevE is the phosphate donor for the phosphorylation of LevR and to exclude the possibility that one of the other PTS proteins present in the phosphorylation assay can phosphorylate domain C of LevR when P-LevE is present, we separated P-LevE from the other three PTS proteins necessary for its phosphorylation (Charrier et al., 1997). The results obtained with purified P-LevE and several LevR variants confirmed that P-LevE is the phosphate donor for phosphorylation of the domain C of LevR at His-869.
Phosphate transfer between proteins of the PTS is usually considered to be reversible (Postma et al., 1993). If this was the case for the lev-PTS, dephosphorylation of the PTS components caused by any rapidly metabolizable PTS substrate would lead to dephosphorylation of LevR and, consequently, to induction of the lev operon. Interestingly, it has been observed that, although P-His-HPr can phosphorylate LevD rapidly, the phosphate group transfer from P-LevD to HPr is very slow (Charrier et al., 1997). This unidirectional phosphoryl group transfer, possibly resulting from steric hindrance of the interaction of P-LevD with HPr, could allow LevD, LevE and LevR to remain phosphorylated even when a rapidly metabolizable PTS substrate, such as glucose or mannitol, is present in the growth medium.
The regulatory domain of LevR seems to have evolved by gene duplication, as the two domains (B and C) exhibit extended similarity. Interestingly, we could not demonstrate that His-506 in LevR (corresponding to His-869 in domain B) was phosphorylated by the PTS, although mutations replacing His-506 with an alanine affected LevR activity. These effects could be caused by structural changes accompanying the mutation at His-506. However, we cannot exclude the possibility that His-506 is phosphorylated by intramolecular transfer of a phosphate group from either His-585 or His-869. The regulatory domain of LevR is also similar to the regulatory domains of antiterminators (Tortosa et al., 1997). Anti-terminators usually contain two histidyl residues corresponding to His-869, one located in subdomain P1 and one in subdomain P2. In the case of LicT and SacY, these two histidyl residues, His-100 (His-99 for SacY) and His-207, are phosphorylated by P-His-HPr (Deutscher et al., 1997; Tortosa et al., 1997). As HPr and LevE interact with the same PTS protein, LevD, it is possible that the active sites of these two proteins have a similar secondary structure that allows them to interact with the same conserved phosphorylation sites in transcriptional activators and antiterminators.
PEP-dependent phosphorylation of domain B of LevR requires the presence of EI and HPr, but not of LevD and LevE. The absence of phosphorylation of LevR H585A by EI and HPr strongly suggests that this histidyl residue is the site of phosphorylation by HPr-His-P. According to genetic data, phosphorylation in domain B stimulates LevR activity. Mutants defective in EI or HPr or expressing LevR, in which His-585 has been replaced with an alanine, exhibited LevR activity that is lower than that in levD or levE mutants (Stülke et al., 1995). PEP-dependent phosphorylation in domain B is probably a mechanism for catabolite repression of the lev operon. In the presence of rapidly metabolizable PTS sugars, such as glucose or mannitol, LevR has to compete with the corresponding EIIs for the phosphate group on P-His-HPr, which will be transferred mainly via the sugar-specific PTS components to the incoming sugar. As a consequence, domain B of LevR will not be phosphorylated and, hence, will be present in a less active form, even when fructose is present in the growth medium. Genetic data have been used to propose a very similar carbon catabolite repression mechanism for the bgl operon of B. subtilis (Krüger et al., 1996). The activity of LicT, the antiterminator controlling the expression of the B. subtilis bgl operon, is completely dependent on a functional PTS. LicT is phosphorylated by PEP, EI and HPr at histidyl residues (Deutscher et al., 1997), and this phosphorylation reaction has been suggested to be used as a carbon catabolite repression mechanism for the bgl operon. Interestingly, alignment of the regulatory domains of LevR and the antiterminators indicates that His-585 corresponds to any of the histidyl residues phosphorylated in the antiterminators (Tortosa et al., 1997). However, there is a histidine corresponding to His-585 in the transcriptional regulator CelR that is believed to control expression of the B. stearothermophilus cel operon (Lai and Ingram, 1993).
In summary, we have demonstrated that the transcriptional activator LevR is phosphorylated by two PEP-dependent phosphorylation reactions, according to the model presented in Fig. 6. His-585 has been identified as the probable phosphorylation site for EI and HPr, whereas phosphorylation by LevE-P most probably occurs at His-869. Phosphorylation of domain B by P-His-HPr stimulates LevR activity and is probably involved in a CcpA-independent carbon catabolite repression mechanism. Phosphorylation at His-869 by P-LevE substantially reduces LevR activity and plays a role in the induction of the B. subtilis lev operon. Further detailed studies will be necessary to elucidate the effects of the two PEP-dependent PTS-catalysed phosphorylation reactions on the activation of transcription of the lev promoter by LevR.
Bacterial strains and culture media
Bacterial strains used in this work are listed in Table 2. E. coli TGI [K-12 Δ(lac pro ) supE thi hsd5/F′traD36 proA+B+lacIqlacZΔM15] was used for cloning experiments, E. coli CJ236 (dut ung thi relA/pJC105 [CmR]) for oligonucleotide-directed mutagenesis and E. coli BL21 [ompT hsdSB (mb−rb−) gal dcm] for overproduction of proteins. E. coli was grown in LB broth and B. subtilis in SP medium and C minimal medium. CSK is C medium supplemented with potassium succinate (6 g l−1) and potassium glutamate (8 g l−1) (Martin-Verstraete et al., 1990). LB and SP plates were prepared by the addition of 17 g l−1 Bacto agar (Difco).
Standard procedures were used to transform E. coli (Sambrook et al., 1989), and transformants were selected on LB plates supplemented with ampicillin (100 μg ml−1). B. subtilis was transformed as described previously with plasmid or chromosomal DNA (Kunst and Rapoport, 1995), and transformants were selected on SP plates containing chloramphenicol (Cm; 5 μg ml−1), kanamycin (Km; 5 μg ml−1), erythromycin (Em; 1 μg ml−1) plus lincomycin (Lin; 10 μg ml−1), spectinomycin (Spc; 60 μg ml−1) or tetracycline (Tc; 15 μg ml−1).
Amylase activity of B. subtilis was detected after growth on tryptose blood agar base (TBAB; Difco) supplemented with 10 g l−1 hydrolysed starch (Connaught). Starch degradation was detected by sublimating iodine onto the plates.
lacZ expression in liquid medium was studied as follows. Cells were grown in CSK medium supplemented with 2 g l−1 fructose. Cells were harvested at an optical density at 600 nm (OD600) of 0.6–0.8 for cultures grown in CSK and an OD600 of 1 for cultures grown in CSK with sugar. β-Galactosidase activities were determined according to the method of Miller (1972) with cell extracts obtained by lysozyme treatment. One unit of β-galactosidase activity is defined as the amount of enzyme that produces 1 nmol of o-nitrophenol per min at 28°C. The specific activities are expressed as mean values derived from three to five independent experiments.
Standard procedures were used to extract plasmids from E. coli (Sambrook et al., 1989). Restriction enzymes, phage T4 DNA polymerase, phage T4 DNA ligase and T4 polynucleotide kinase were used, as recommended by the manufacturers. DNA fragments were purified from agarose gel with a Prep-A-Gene kit (Bio-Rad).
To perform site-directed mutagenesis, the Muta-Gene M13 in vitro mutagenesis kit (Bio-Rad) was used, based on the method described by Kunkel et al. (1987). M13mp18C1 phage DNA (Martin-Verstraete et al., 1994), containing a 4.8 kb PstI–SacI fragment of the levR gene, was used to introduce point mutations into the codons of histidine residues conserved in the BglG/SacT family of regulators (Fig. 1) and of His-585. Double and triple mutants in the levR gene were obtained by the same method using derivatives of the M13mp18C1 carrying one or two mutations in levR as the template for the mutagenesis. The presence of the mutations was confirmed by DNA sequencing by the dideoxy chain termination method (Sanger et al., 1977).
Vector pAC5 (Martin-Verstraete et al., 1992) carries the pC194 chloramphenicol resistance gene cat and a lacZ gene between two fragments of the B. subtilis amyE gene. This vector was used to introduce single copies of the different alleles of the levR gene into the chromosome of B. subtilis. The following constructions were performed: M13 replicative forms corresponding to phages carrying the different levR alleles were extracted and digested with SacI and BamHI. The SacI–BamHI fragments containing the levR alleles were ligated into pAC5 leading to a partial deletion of lacZ. The pAC5 derivatives are listed in Table 3.
Table 3. . pAC5 and PBQ200 derivatives containing different levR alleles.
The levR genes containing various point mutations were integrated at the amyE locus of the B. subtilis chromosome of strain QB5182 (trpC2ΔlevR ::aphA3 sacC′–lacZ+erm) or QB5075 (trpC2 levE7ΔlevR ::aphA3 sacC′–lacZ+erm) (see Table 2).
The levR alleles were also inserted into pBQ200 containing the degQ36 promoter (Martin-Verstraete et al., 1994). pAC5 derivatives containing different levR alleles were digested with Bst BI, made blunt using the Klenow fragment of DNA polymerase I and digested with BamHI. The Bst BI–BamHI fragments were ligated between the SmaI and BamHI sites of pBQ200 to give the plasmids listed in Table 3.
The domains B and C of LevR were fused to the GST protein as follows: the part of the levR gene corresponding to amino acids 407–723 (domain B) or 723–938 (domain C) was amplified by polymerase chain reaction (PCR) using oligonucleotides creating BamHI and EcoRI restriction sites. The BamHI–EcoRI fragments were inserted into the expression vector pGexKT (Hakes and Dixon, 1992). The resulting plasmids, pRL56 and pRL78, encode domains B and C, respectively, of LevR fused to the GST protein. Plasmid pRL80 encodes a GST fusion to domain C carrying the H869A mutation.
For the partial purification of LevR, the pBQ200 derivative plasmids carrying the various levR alleles were introduced into strain QB5277 (Table 2; Stülke et al., 1995) containing both deletions of the levR gene and the genes encoding the general and lev-specific components of the PTS. The resulting strains overproduce LevR during stationary phase after growth in LB medium. The various modified LevR polypeptides were partially purified from crude extracts by ammonium sulphate precipitation as described previously (Martin-Verstraete et al., 1994). The modified LevR polypeptides made up about 5% of the total proteins.
To purify GST–domain B, GST–domain C and GST–domain C H869A, plasmids pRL56, pRL78 and pRL80 were introduced into E. coli strain BL21 (Novagen) containing the plasmid pREP4 groESL and, thus, overproducing the bacterial chaperones GroES and GroEL (Amrein et al., 1995). Transformants were grown at 37°C in LB medium until the culture reached an OD600 of 1. The fusion genes present in pGexKT were induced by the addition of 0.15 mM IPTG and incubation for a further 3 h at room temperature.
The cells were pelleted by centrifugation, resuspended in TPBS buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4) and disrupted by sonication. Proteins were purified as described by Hakes and Dixon (1992). Fractions containing GST–domain B or GST–domain C were pooled. Protein concentrations were determined spectrophotometrically (Bio-Rad protein assay). Protein solutions were stored at −20°C.
To prepare P-LevE without a His-tag, the same phosphorylation conditions were used as for LevE(His)6 (Charrier et al., 1997). After phosphorylation of LevE, the phosphorylation mixture was treated three times with Ni–NTA resin to separate phosphorylated LevE from EI(His)6, HPr(His)6 and LevD(His)6.
In vitro phosphorylation of LevR
[32P]-PEP was prepared from [γ-32P]-ATP as described previously (Roossien et al., 1983). [32P]-PEP was separated from [γ-32P]-ATP by ion exchange chromatography (Mattoo and Waygood, 1983). Partially purified LevR (30 μg) or purified GST–domain polypeptides (20 μg) were phosphorylated by incubation in a 25 μl reaction mixture containing EI (0.2 μg), HPr (0.1 μg), MgCl2 (10 mM), Tris-HCl (50 mM, pH 7.4) and 1 μM [32P]-PEP (0.5 μCi) for 20 min at 37°C. For the phosphorylation of LevR or GST–domain C by LevE, LevD(His)6 (0.5 μg) and LevE(His)6 (5 μg) were added to this reaction mixture. The optimal conditions of phosphorylation of LevD and LevE by EI, HPr and PEP have been studied extensively (Charrier et al., 1997). The reaction was stopped by adding electrophoresis sample buffer containing 0.1% SDS. Electrophoresis of proteins was carried out on SDS–polyacrylamide gels. Gels were dried and exposed for 2–16 h to autoradiography (Kodak Biomax).
†The two first authors contributed equally to this work
We are grateful to W. Hengsterberg for providing us with enzyme I from S. carnosus, to M. Débarbouillé and A. Klier for helpful discussions and to J. Bignon for excellent technical assistance. C. Dugast is acknowledged for typing the manuscript, A. Bosch, C. van Herrewege and Alex Edelman for their assistance in preparing this manuscript. This research was supported by grants from the Centre National de la Recherche Scientifique, The Pasteur Institute, The University Paris 7 and by the European Community Biotech Program BIO4-CT96-0380.