Modulation of Monomer Conformation of the BglG Transcriptional Antiterminator from Escherichia coli

Strains.The following E. coli K-12 strains were used: BL21(DE3) [hsdS gal (λcI857 ind1 Sam7 nin5) lacUV5-T7 gene1], obtained from Novagen, and SG13009, obtained from QIAGEN, were used for the expression of His-tagged proteins; MC1061 [hsdR, mcrB, araD, 139Δ(araABC-leu) 7679 ΔlacX74 galU galK rpsL thi] was used for the expression of proteins tagged with maltose-binding protein (MBP); SU202, which harbors a chromosomal copy of the lacZ gene under the control of a hybrid LexA operator (op408/op+) (11), was used to study heterodimerization; K38 (HfrC trpR thi λ⁺) was used as a host for the preparation of membrane fractions.

Media.Luria-Bertani (LB) medium, M63 salts minimal medium, and M9 minimal medium were prepared essentially as described by Miller (22). Ampicillin (200 μg/ml), kanamycin (30 μg/ml), and chloramphenicol (25 μg/ml) were included in the medium for growing strains containing plasmids that confer resistance to either one of these antibiotics.

Plasmids.All plasmids used in this study and the proteins they encode are listed in Table 1. Plasmid pLLZ-F encodes BglF expressed from the P_LtetO-1 promoter. A 1,938-bp fragment, encoding BglF and containing the Acc65I site at one end and the MluI site at the other, was generated by PCR by using pT7OAC-F (1) as a template. This fragment was ligated to the 4,211-bp Acc65I-MluI fragment of pZS*tetp/oFhf. pZS*tetp/oFhf, a derivative of pZS*2tet0-1/Tev⁺tet, was obtained from E. Bibi (18). More details on plasmid constructions are available from the authors upon request.

Chemicals.[γ-³²P]ATP (6,000 Ci/mmol) was obtained from NEN. Phosphoenolpyruvate (PEP), pyruvic acid, pyruvate kinase, ATP, isopropyl β-d-thiogalactopyranoside, DNase I, RNase A, N,N′-(p-phenylene)dimaleimide (p-PDM), and endonuclease (benzonase) were obtained from Sigma. 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride was obtained from Calbiochem. Amylose resin, maltose, and monoclonal anti-MBP antibodies were obtained from New England Biolabs. Peroxidase-conjugated AffiniPure goat anti-mouse was obtained from Jackson ImmunoResearch Laboratories Inc. Anhydrotetracycline hydrochloride was obtained from Acros.

Protein purification.MBP-tagged BglG, PRD1, or PRD2 was expressed in MC1061 and purified as described previously for MBP-BglG (12). His-tagged enzyme I (EI), HPr, and IIB^bgl were expressed in BL21(DE3). His-tagged IIA^bgl was expressed in SG13009. His-tagged proteins were purified as described previously (7), except that the extracts were incubated for only 1 h with the Ni-nitrilotriacetic acid resin. 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride was used during the purification procedure instead of phenylmethylsulfonyl fluoride.

Far-Western analysis.Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Gels were subjected to far-Western analysis as described previously (23) or stained with Coomassie blue. No denaturing agents were used throughout the entire procedure performed to study the interaction of the BglG compact and noncompact forms with IIB^bgl by far-Western analysis.

Two-hybrid analysis.For a quantitative assay of heterodimerization, the SU202 strain, which harbors a chromosomal copy of the lacZ gene under the control of a hybrid LexA operator (op408/op+), was cotransformed with the derivatives of pDP804 and pMS604 (11). Cells were grown in a minimal medium containing 0.4% succinate as the carbon source and 0.5 mM isopropyl β-d-thiogalactopyranoside. Assays for β-galactosidase activity were carried out as described by Miller (22).

Preparation of membrane fractions.Membrane fractions enriched for BglF were prepared as described previously (1). BglF was expressed from its gene cloned under the T7 promoter in pT7OAC-F. The expression of T7 RNA polymerase from plasmid pGP1-2, which is compatible with the pT7 plasmid, was induced thermally. The E. coli K38 strain was used as a host.

In vitro phosphorylation and dephosphorylation.In vitro (de)phosphorylation was carried out essentially as described before (8). Briefly, to study phosphorylation of BglG, [³²P]PEP was prepared and separated from [³²P]ATP. IIB^bgl (2 μg) was labeled by incubation at 30°C in a mixture containing EI (0.2 μg), HPr (1.5 μg), IIA^bgl (2 μg), [³²P]PEP (10 μM), and PLB buffer (50 mM Na₂HPO₄ [pH 7.4], 0.5 mM MgCl₂, 1 mM NaF) lacking dithiothreitol, in a final volume of 10 μl. After incubation for 10 min, MBP-BglG (5 μg) was added, and the reaction mixtures were further incubated in PLB buffer for 10 min at 30°C. To study dephosphorylation of P-BglG by β-glucosides, the reaction was dialyzed against PLB buffer lacking dithiothreitol to separate ³²P-BglG from residual [³²P]PEP. Subsequently, 0.1% salicin and BglF-containing membranes (400 μg) were added to the dialyzed ³²P-BglG (80 μg), and incubation was continued at 30°C. Aliquots were removed at various times. Reactions were terminated by the addition of electrophoresis sample buffer containing or lacking β-mercaptoethanol. Equal amounts of samples were subjected to SDS-PAGE.

In vivo cross-linking of MBP-BglG in the presence of BglF and arbutin.MC1061 cells, harboring plasmids pANSMG and pLLZ-F, were grown with aeration at 37°C in M9 minimal medium containing 0.4% glycerol as a carbon source. When indicated, BglF expression from pLLZ-F was induced by the addition of anhydrotetracycline hydrochloride to a final concentration of 100 ng/ml. When specified, 0.5% arbutin (hydroxyphenyl-β-d-glucopyranoside) was added to the medium. When cells reached an optical density at 600 nm of 0.6, in vivo cross-linking with p-PDM was performed as described before (12). Cells were subsequently handled and analyzed as previously described (21), except that when cells were grown in the presence of arbutin, 0.5% arbutin was maintained in all solutions throughout the procedure.

SDS-PAGE, Western blot analysis, and autoradiography.Proteins were incubated with electrophoresis sample buffer containing 62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 0.01% bromophenol blue, and, only when indicated, 5% β-mercaptoethanol. Electrophoresis of proteins was carried out by SDS-PAGE (8 or 10% polyacrylamide). Samples were fractionated next to SeeBlue Plus2 prestained marker (Invitrogen). After electrophoresis, gels were either stained with Coomassie blue, blotted onto a nitrocellulose filter for Western blot analysis as described before (8), or dried and exposed to Kodak X-OMAT X-ray film at −70°C.

The active site-containing domain of BglF binds to the BglG compact form in vitro.In a recent publication from our laboratory it was shown that the transcriptional antiterminator BglG can fold into a compact conformation in which the PRD1 and PRD2 domains are in close proximity (12). In this conformation, two cysteines, one in each PRD, are brought to a distance that enables them to form of a disulfide bond as a result of air oxidation upon cell rupture. Consequently, a fraction of the BglG protein can be detected as a faster migrating form when analyzed in the absence of reducing agents. Another recent publication from our laboratory demonstrated that BglG interacts with the BglF membrane sensor and with its active site-containing domain, IIB^bgl (17). One tool used for studying this interaction was the far-Western technique. We showed that BglG, which was analyzed by the standard SDS-PAGE procedure, i.e., in the presence of β-mercaptoethanol, and blotted onto a membrane, reacted with IIB^bgl. Hence, we established that the active site-containing domain of BglF binds to the noncompact form of BglG, the only BglG form that was detected under these conditions. To examine whether BglF can bind also to the compact form of BglG, we used the following modification of the far-Western technique: we separated the two forms of BglG on a nonreducing gel, blotted both forms onto a filter, and tested the ability of IIB^bgl to bind to them. Hence, purified MBP-BglG (BglG fused to MPB) was analyzed on an SDS-polyacrylamide gel with no reducing agents and blotted onto a nitrocellulose filter. The membrane was incubated with His-tagged IIB^bgl (the IIB^bgl domain fused to six histidines) and then with antibodies against the His tag (Fig. 1A, lane 3). Both BglG forms, the slower migrating noncompact form and the faster migrating compact form, reacted with IIB^bgl. The lower intensity observed with the compact form can be attributed, at least in part, to its relative low amount (Fig. 1, lane 1, Coomassie blue stain). BglG analyzed in the presence of β-mercaptoethanol served as a control (Fig. 1, lanes 2 and 4). When MBP alone was probed with His-IIB^bgl, no binding was observed (reference 17 and data not shown), ruling out the possibility that the MBP moiety mediates the interaction. These results demonstrate that the active site-containing domain of BglF interacts with both forms of BglG. These interactions are direct and do not require additional proteins, at least in vitro. Because the far-Western technique is not sensitive enough to give a quantitative estimate for the interaction, we cannot draw any conclusion concerning the relative affinity of IIB^bgl for the two BglG forms. Hence, we cannot rule out the possibility that IIB^bgl interacts preferentially with one of the BglG forms.

• Open in new tab
• Download powerpoint

FIG. 1.

The two forms of BglG, the compact and the noncompact, interact with the active site-containing domain of BglF in vitro. (A) Purified MBP-BglG was analyzed by SDS-PAGE in the absence or presence of β-mercaptoethanol (βME) and stained with Coomassie blue. (B) As for panel A, but the proteins were blotted onto a nitrocellulose membrane and probed with His-tagged IIB^bgl and then with anti-His antibodies. The use of denaturing agents was avoided throughout the entire procedure. Arrowheads indicate the positions of the compact and noncompact forms of MBP-BglG.

The compact BglG form is detected in the phosphorylated fraction of BglG and is dephosphorylated by BglF.It has previously been shown that the phosphorylated forms of intact BglF and its active site-containing domain, IIB^bgl, can phosphorylate BglG in vitro (1, 7). In contrast, either the entire BglF protein or its truncated IIBC^bgl derivative (IIB^bgl fused to the membrane domain IIC^bgl) but not IIB^bgl alone is required for BglG-P dephosphorylation in the presence of β-glucosides (7). At this time, we asked whether the compact form of BglG is detected after in vitro phosphorylation. Because MBP-BglG and BglF are similar in size and comigrate in SDS-PAGE, the detection of ³²P-BglG after incubation with intact ³²P-BglF is not possible. Therefore, we used IIB^bgl, the active site-containing domain of BglF, to follow phosphorylation of the compact form of BglG. To this end, MBP-BglG was added to ³²P-IIB^bgl, which was prelabeled in a reaction mixture containing [³²P]PEP and purified His-tagged EI, HPr, IIA^bgl, and IIB^bgl. After further incubation, the products were subjected to SDS-PAGE analysis in the absence or presence of β-mercaptoethanol (Fig. 2A, lanes 1 and 2, respectively). Both forms of BglG, the compact and the noncompact, were detected as phosphorylated proteins by this analysis (Fig. 2A, lane 1). However, we cannot rule out the possibility that the compact form was obtained due to oxidation of the noncompact form subsequent to the phosphorylation. Although we cannot proclaim that IIB^bgl directly phosphorylates the compact form of BglG, this result demonstrates that the compact BglG form can exist as a phosphorylated protein.

• Open in new tab
• Download powerpoint

FIG. 2.

Reversible phosphorylation of the compact and noncompact forms of BglG in vitro. (A) Phosphorylation of BglG was carried out by incubating purified MBP-BglG with ³²P-IIB^bgl for 10 min. IIB^bgl was prelabeled by incubating purified EI, HPr IIA^bgl, and IIB^bgl, all His-tagged, with [³²P]PEP for 10 min. (B) Dephosphorylation of BglG was obtained by incubating ³²P-MBP-BglG, phosphorylated as described in panel A and then separated from the residual [³²P]PEP by dialysis, with membranes containing BglF and 0.1% salicin. Aliquots were removed at the indicated time points. Samples were analyzed by SDS-PAGE in the absence or presence of β-mercaptoethanol (βME) followed by autoradiography. Arrowheads indicate the position of the compact form of MBP-BglG on the gels.

Next, we tested the ability of the BglG compact form to be dephosphorylated by BglF in the presence of β-glucosides. The results shown in Fig. 2B demonstrate that the two ³²P-BglG forms, the compact and the noncompact, are dephosphorylated in a time-dependent manner when incubated with BglF-enriched membranes and the β-glucoside salicin (Fig. 2B, lanes 2 to 4). Therefore, the two forms of BglG are both recognized and dephosphorylated by BglF in the presence of β-glucosides.

Binding of BglG to BglF is mediated by its PRD2 domain and is hampered by PRD1.To characterize the interaction of BglG with BglF, we aimed at determining the domain in BglG which mediates the interaction. To this end, we studied the interaction of the separated PRDs of BglG with BglF or with its active site-containing domain, IIB^bgl, both in vivo and in vitro, and compared the results to those obtained with the entire BglG protein.

First, we tested the interaction of purified BglG or the separated PRDs with purified IIB^bgl by the far-Western technique. To this end, similar amounts of BglG, PRD1, and PRD2 fused to MBP (MBP-BglG, MBP-PRD1, and MBP-PRD2, respectively) were subjected to SDS-PAGE (Fig. 3, Coomassie stain). The proteins were then blotted onto a nitrocellulose filter and incubated with His-tagged IIB^bgl and antibodies against the His tag (Fig. 3, far-Western). Α strong signal was detected for the interaction of either intact BglG or its PRD2 domain with IIB^bgl (Fig. 3, lanes 4 and 6, respectively). A relatively weak signal was detected for the interaction of the PRD1 domain with IIB^bgl (Fig. 3, lane 5). When MBP alone was probed with His-IIB^bgl, no binding was observed (data not shown), ruling out the possibility that the interaction with IIB^bgl is mediated by the MBP moiety. Hence, PRD2 recognizes and interacts with IIB^bgl much better than PRD1, suggesting that the PRD2 domain is responsible for the interaction of BglG with IIB^bgl.

• Open in new tab
• Download powerpoint

FIG. 3.

Far-Western analysis of the interaction between the PRDs of BglG and the active site-containing domain of BglF. (A) Purified BglG, PRD1, and PRD2, each fused to MBP, were analyzed by SDS-PAGE and stained with Coomassie blue. (B) As for panel A, but the proteins were blotted onto a nitrocellulose membrane, probed with His-tagged IIB^bgl, and then with anti-His antibodies.

To study the interaction of the PRDs with BglF or with IIB^bgl in vivo, we used the bacterial LexA-based two-hybrid system (11). In this system, the proteins of interest are fused either to the DNA-binding domain of the wild-type LexA repressor (LexADBD) or to an altered-specificity LexADBD and introduced into a strain that harbors a chromosomal copy of lacZ under the control of a LexA hybrid operator (SU202). Transcriptional repression is achieved upon coexpression of both hybrid proteins, provided that they bind to each other. We fused BglF, IIB^bgl, BglG and the two PRDs to LexADBD, wild type or mutant, and calculated the transcriptional repression obtained when different combinations of these chimeras are coexpressed in SU202. The results are presented in Table 2. The leucine zipper domains of Fos and Jun, fused to the wild-type and mutant LexADBD, respectively, served as positive controls, and the two LexADBDs served as a negative control. As shown before (17), coexpression of either IIB^bgl or BglF with the entire BglG protein fused to the LexADBDs resulted in formation of stable heterodimers that functioned as repressors. The lower repression obtained with BglF is attributed to the constraint caused by anchoring the BglF-LexADBD chimera to the membrane. Replacing BglG with the PRD1 domain either reduced or almost eliminated formation of heterodimers with IIB^bgl or BglF, respectively. In contrast, not only did PRD2 bind to IIB^bgl and to BglF, as indicated by the high transcriptional repression obtained when the respective combinations of chimeras were coexpressed, but these interactions were stronger than those obtained when the entire BglG was coexpressed with IIB^bgl or BglF. Hence, in agreement with the results obtained with the far-Western technique, these results imply that the interaction of BglG with BglF or IIB^bgl is mediated by the PRD2 domain of BglG. Moreover, the results obtained with the LexA-based two-hybrid system suggest that the presence of PRD1 in the entire BglG protein interferes with or reduces the interaction of PRD2 with BglF or IIB^bgl.

Formation of the BglG compact monomer is modulated in vivo by BglF and the β-glucoside inducer.We have recently shown that the compact conformation of BglG, which is present mainly or solely in the monomeric fraction, can be detected in vivo by using cell-penetrating reagents that cross-link C102 in PRD1 to C243 in PRD2 (12). To examine whether folding of the BglG monomer is affected by BglF and β-glucosides, which regulate BglG phosphorylation, dimerization, and activity, we examined whether BglF and the stimulating sugar have an effect on the relative level of the compact form in the cell. To this end, cells expressing MBP-BglG and expressing or not expressing BglF were grown in the presence or absence of the β-glucoside arbutin. The cells were incubated with p-PDM, a homobifunctional dimaleimide cross-linker, collected by centrifugation, washed, resuspended in sample buffer, and analyzed by SDS-PAGE and Western blot analysis. The amount of the compact form observed after this procedure is lower than the genuine level in the cell (see Discussion). Hence, we refer here to the relative level of the compact form. As can be seen in Fig. 4, the level of the cross-linked product, i.e., the compact form of BglG, which was very low in cells not expressing BglF, increased in cells expressing BglF (Fig. 4, compare lanes 1 and 2). The relative amount of the compact BglG form in cells expressing BglF decreased when arbutin was added to the growth medium (Fig. 4, compare lanes 2 and 3). To correct for potential loading errors, we compared the amounts of the compact and noncompact forms by comparing the intensities of the upper and lower bands, respectively, in the different lanes. The relative values are given in the legend to Fig. 4. These results, which were confirmed in several independent experiments, imply that the formation of the compact conformation of BglG, in which the PRD1 and PRD2 are in close proximity, is enhanced by BglF, provided that β-glucosides are not present in the growth medium. The presence of β-glucosides in the growth medium leads to a reduction in the relative amount of the compact form in a BglF-dependent manner, as β-glucosides added to cells not expressing BglF did not affect the level of the compact BglG form (data not shown). Therefore, BglF and β-glucosides affect the relative amount of the compact BglG form in the cell in opposite ways: the presence of BglF increases it, whereas the addition of β-glucosides decreases it.

• Open in new tab
• Download powerpoint

FIG. 4.

Modulation of compact BglG monomer formation by BglF and β-glucosides in vivo. Cells expressing MBP-BglG (lane 1) or both MBP-BglG and BglF (lanes 2 to 3) were grown in minimal medium, lacking (lanes 1 to 2) or containing (lane 3) 0.5% arbutin, to an optical density at 600 nm of 0.6. Pelleted cells were washed and resuspended in phosphate-buffered saline, pH 7.0, with arbutin added only to cells grown with arbutin. p-PDM, a cross-linker that penetrates cells and cross-links cysteines in the BglG compact monomer, was added to a final concentration of 1 mM. Following incubation at 30°C for 30 min, samples were collected by centrifugation, washed, resuspended in sample buffer, and analyzed by SDS-PAGE and Western blot analysis. The two forms of BglG were detected with anti-MBP antibodies. The relative intensities of the upper bands in the three lanes, compared by a densitometer, are 1.00:0.98:1.00. The relative intensities of the lower bands are 1.00:3.03:1.10. The arrowhead indicates the position of the compact form of MBP-BglG.