JOURNAL OF BACTERIOLOGY, Aug. 2009, p. 5205–5215 0021-9193/09/$08.00⫹0 doi:10.1128/JB.00526-09 Copyright © 2009, American Society for Microbiology. All Rights Reserved. Vol. 191, No. 16 Specific Interactions between Four Molybdenum-Binding Proteins Contribute to Mo-Dependent Gene Regulation in Rhodobacter capsulatus䌤 Jessica Wiethaus,1 Alexandra Müller,1 Meina Neumann,2 Sandra Neumann,1 Silke Leimkühler,2 Franz Narberhaus,1 and Bernd Masepohl1* Lehrstuhl für Biologie der Mikroorganismen, Fakultät für Biologie und Biotechnologie, Ruhr-Universität Bochum, 44780 Bochum, Germany,1 and Molekulare Enzymologie, Institut für Biochemie und Biologie, Universität Potsdam, 14469 Potsdam, Germany2 Received 20 April 2009/Accepted 28 May 2009 solely consist of either a mono-MOP or a di-MOP domain. Molbindins have been identified in many bacteria but not in E. coli. In addition to ModE (di-MOP domain), a C-terminal MOP domain exists in ModC (mono-MOP domain). The phototrophic purple bacterium Rhodobacter capsulatus codes for proteins containing either the FeMoco (molybdenum nitrogenase) or the Moco (xanthine dehydrogenase and dimethyl sulfoxide reductase). To regulate the internal Mo concentration, R. capsulatus synthesizes two ModE-like proteins, MopA and MopB. These regulators can replace each other in Mo-dependent repression of several genes, including modABC (31, 32). In the presence of Mo, MopA (but not MopB) activates transcription of the mop gene, coding for a mono-MOP molbindin. While mopA expression is repressed by Mo, the mopB gene is transcribed independent of Mo availability. As a consequence, the MopA/MopB ratio is thought to change in response to the intracellular Mo level. In this study we examined the oligomerization and Mobinding properties of R. capsulatus MopA and MopB. Furthermore, we investigated the interaction profiles of MopA and MopB with other MOP domain proteins, namely, the molbindin protein Mop and the high-affinity molybdate transporter ATPase ModC (Fig. 1). Our results show that MopA and MopB form homodimers independent of Mo availability. The interaction interface involves both the N-terminal HTH and the C-terminal di-MOP domains. Both MopA and MopB bind four molybdate oxyanions per dimer with high affinity. Furthermore, MopA interacts with ModC, while MopB interacts with Mop. The Mop hexamer itself is stabilized by binding of six molybdate oxyanions. Besides homodimers, MopA and Molybdenum is an essential trace element due to its role as a cofactor of numerous enzymes (13). Molybdenum nitrogenase has a unique iron-molybdenum cofactor (FeMoco), whereas all other molybdoenzymes, including nitrate reductase, xanthine dehydrogenase, and dimethyl sulfoxide reductase, contain a molybdopterin cofactor (Moco). Nitrogenase, which catalyzes the reduction of atmospheric dinitrogen (N2) to ammonia, is synthesized exclusively by diazotrophic bacteria and archaea. In contrast, Moco-containing enzymes have been identified in bacteria, archaea, plants, and animals. Many bacteria actively take up the oxyanion molybdate (MoO42⫺) by a high-affinity ABC-type transport system (27). This system is composed of the periplasmic substrate-binding protein ModA, the integral membrane protein ModB, and the cytoplasmic ATPase ModC. In Escherichia coli, expression of the modABC operon is repressed by the Mo-dependent regulator protein ModE (8). ModE functions as a homodimer with two distinct domains, an N-terminal DNA-binding helix-turnhelix (HTH) domain and a C-terminal Mo-binding domain (10). The latter consists of two so-called MOP domains per monomer. MOP domains of about 70 amino acids are responsible for cytoplasmic Mo binding and are found in three classes of proteins with distinct functions (23). Molbindins, which are thought to be involved in Mo homeostasis and Mo storage, * Corresponding author. Mailing address: Ruhr-Universität Bochum, Fakultät für Biologie und Biotechnologie, Lehrstuhl für Biologie der Mikroorganismen, 44780 Bochum, Germany. Phone: 49 (0) 234 32 25632. Fax: 49 (0) 234 32 14620. E-mail: bernd.masepohl@rub.de. 䌤 Published ahead of print on 5 June 2009. 5205 Downloaded from jb.asm.org at North Carolina State University Libraries on October 5, 2009 The phototrophic purple bacterium Rhodobacter capsulatus encodes two transcriptional regulators, MopA and MopB, with partially overlapping and specific functions in molybdate-dependent gene regulation. Both MopA and MopB consist of an N-terminal DNA-binding helix-turn-helix domain and a C-terminal molybdatebinding di-MOP domain. They formed homodimers as apo-proteins and in the molybdate-bound state as shown by yeast two-hybrid (Y2H) studies, glutaraldehyde cross-linking, gel filtration chromatography, and copurification experiments. Y2H studies suggested that both the DNA-binding and the molybdate-binding domains contribute to dimer formation. Analysis of molybdate binding to MopA and MopB revealed a binding stoichiometry of four molybdate oxyanions per homodimer. Specific interaction partners of MopA and MopB were the molybdate transporter ATPase ModC and the molbindin-like Mop protein, respectively. Like other molbindins, the R. capsulatus Mop protein formed hexamers, which were stabilized by binding of six molybdate oxyanions per hexamer. Heteromer formation of MopA and MopB was shown by Y2H studies and copurification experiments. Reporter gene activity of a strictly MopA-dependent mop-lacZ fusion in mutant strains defective for either mopA, mopB, or both suggested that MopB negatively modulates expression of the mop promoter. We propose that depletion of the active MopA homodimer pool by formation of MopA-MopB heteromers might represent a fine-tuning mechanism controlling mop gene expression. 5206 WIETHAUS ET AL. MopB form heteromers. In contrast to MopA homodimers, MopA-MopB heteromers are not capable of activating the mop promoter. Thus, heteromer formation might serve as a mechanism to control mop expression in response to Mo availability. MATERIALS AND METHODS Strains, plasmids, and growth conditions. The microbial strains and plasmids used in this study are listed in Table 1. Media, growth conditions, and antibiotic concentrations were as previously described (14, 24). Construction of a transcriptional mop-lacZ fusion plasmid. A DNA fragment carrying the R. capsulatus mop promoter region was PCR amplified (Table 2) and cloned into the SmaI site of pBluescript KS. Subsequently, the BamHI-HindIII fragment carrying the mop promoter was cloned into lacZ reporter plasmid pML5, leading to hybrid plasmid pLPRUB12. Finally, pLPRUB12 was introduced into R. capsulatus wild-type, mopA, mopB, and ⌬(mopA-mopB) mutant strains as described earlier (14, 17). -Galactosidase assays. R. capsulatus strains carrying mop-lacZ reporter plasmid pLPRUB12 were grown in Mo-free AK-NL minimal medium containing 20 mM ammonium. When required, Na2MoO4 was added in the range from 10 nM up to 100 M. Following growth to late exponential phase, -galactosidase activities were determined by the sodium dodecyl sulfate-chloroform method (20). Yeast two-hybrid (Y2H) studies. The R. capsulatus genes mop and modC were PCR amplified using appropriate oligonucleotides designed for amplification of full-length genes flanked by MunI restriction sites (Table 2). Cloning of MunI fragments into the EcoRI sites of the Escherichia coli-yeast shuttle vectors pEG202 (lexA-DBD) and pJG4-5 (B42-AD) generated in-frame fusions with either the DNA-binding domain (DBD) or the activation domain (AD) (Table 1). In addition, XhoI-EcoRI fragments containing either mopA or mopB from plasmids pAB4II and pAB5II were cloned into pEG202 and pJG4-5. DNA fragments coding for either the N-terminal HTH domain (mopAHTH) or the C-terminal di-MOP domain (mopAdi-MOP) of MopA were PCR amplified with primers flanked by EcoRI and XhoI restriction sites. EcoRI-XhoI fragments containing either mopAHTH or mopAdi-MOP were cloned into pEG202 and pJG4-5. The resulting DBD and AD fusion plasmids were cotransformed into yeast strain EGY48 (pSH18-34) containing a lacZ reporter gene controlled by the LexA operator by the polyethylene glycol-lithium acetate method (5). -Ga- lactosidase activities of yeast reporter strains were determined by the sodium dodecyl sulfate-chloroform method (24). Construction of mopA, mopB, mop, and modC expression plasmids. Construction of hybrid plasmids pJW32 (mopAhis) and pJW33 (mopBhis) has been described earlier (32). ApoI fragments with either mopAhis or mopBhis from plasmids pJW32 and pJW33 were cloned into the EcoRI site of plasmid pSUP401, which carries a kanamycin resistance gene for selection. The mop and modC coding regions were PCR amplified with primers encompassing recognition sites either for NdeI and XhoI (mop) or for NdeI and BamHI (modC) (Table 2). Subsequently, the NdeI-XhoI mop fragment was cloned into expression vector pET22b(⫹) to create a mophis fusion, whereas the NdeI-BamHI fragment containing modC was cloned into expression vector pET19 to create a hismodC fusion. In addition, the mopA, mopB, and mop coding regions were PCR amplified with primers carrying recognition sites for EcoRI and SalI. Subsequently, the EcoRI-SalI fragments were cloned into expression vector pASK-IBA3 to create mopAstrep, mopBstrep, and mopstrep fusions. Overexpression and purification of His-tagged proteins. Plasmids for overexpression of His-tagged proteins were transformed into E. coli strain BL21(DE3). Overexpression and purification of recombinant proteins was carried out as described previously (32). Copurification of His- and Strep-tagged proteins. Plasmids for overexpression of His- and Strep-tagged proteins were cotransformed into E. coli strain BL21(DE3). For overexpression of the recombinant proteins, 200 ml of selective LB medium was inoculated with 2 ml overnight culture of BL21(DE3) carrying the respective hybrid plasmids and cultivated at 37°C to an optical density at 580 nm of 0.7 before protein expression was induced by addition of anhydrotetracycline (for Strep-tagged proteins) and IPTG (isopropyl--D-thiogalactopyranoside) (for His-tagged proteins). After further incubation for 2.5 h, cells were harvested by centrifugation and resuspended in 20 ml of lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole). After disruption in a French pressure cell at 2000 lb/in2, the lysate was centrifuged at 22,548 ⫻ g for 30 min. When required, the supernatant was adjusted to 100 M Na2MoO4. Crude extracts were loaded onto Ni-nitrilotriacetic acid (NTA) agarose columns. After washing with buffer (50 mM NaH2PO4, 300 mM NaCl) with increasing imidazole concentrations (10 to 30 mM imidazole), His-tagged proteins were eluted by raising the imidazole concentration to 250 mM. When required, washing and elution buffers were adjusted to 10 M Na2MoO4. Aliquots of crude extracts and elution fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting using either the Penta-His horseradish peroxidase (HRP) conjugate (Qiagen, Hilden, Germany) or the Strep-Tactin HRP conjugate (IBA, Göttingen, Germany). Cross-linking experiments. His-tagged proteins were incubated with either 0.01% (MopAHis and MopBHis) or 0.05% (MopHis) glutaraldehyde in a total volume of 15 l at room temperature. When needed, reaction mixtures were adjusted to 10 mM Na2MoO4. Reactions were stopped after different time intervals with 2.5 l 1 M Tris (pH 8) before samples were analyzed by SDSPAGE and Western blotting. Gel filtration chromatography. Purified MopAHis, MopBHis, and MopHis proteins in elution buffer (100 mM NaCl, 50 mM NaH2PO4, 200 mM imidazole at pH 8) were loaded on a Superdex 75 HR 10/30 gel filtration column (Amersham Biosciences, Freiburg, Germany) preequilibrated with 100 mM NaCl, 50 mM NaH2PO4 at pH 8. Separation was performed at 4°C at a flow rate of 0.3 ml/min. The following standards were used to calibrate the column: albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), and RNase A (13.7 kDa). Dissociation constants of molybdate to MopA, MopB, Mop, and ModC. KD values for molybdate were determined by ultrafiltration using Microcon concentrators (molecular weight cutoff, 10,000; Millipore, Schwalbach, Germany) as described earlier (21). MopA, MopB, Mop, or ModC at 4 M was incubated with 0 to 16 M sodium molybdate and centrifuged at 14,000 ⫻ g for 5 min. As a control, molybdate was used in the absence of protein. Flowthrough fractions were incubated overnight in a 1:1 mixture with 65% nitric acid (Suprapur; Merck, Darmstadt, Germany) at 100°C and filled to a 10-fold volume with water prior to molybdenum analysis. Molybdenum contents were determined using a PerkinElmer Optima 2100DV inductively coupled plasma optical emission spectrometer. As a reference, the multielement standard XVI (Merck, Darmstadt, Germany) was used. Bovine serum albumin and R. capsulatus MoeA and MogA (22) were used for control experiments. RESULTS AND DISCUSSION Interaction profile of MOP domain proteins. R. capsulatus MopA and MopB consist of an N-terminal DNA-binding HTH Downloaded from jb.asm.org at North Carolina State University Libraries on October 5, 2009 FIG. 1. Interaction map of R. capsulatus MOP domain proteins. MOP domains in ModC, MopA, and MopB are shown as gray boxes. The Mop protein consists exclusively of a mono-MOP domain. Interactions were determined by Y2H studies, cross-linking experiments (CL), gel filtration chromatography (GF), and copurification experiments (CP). Homo- and heteromer formation is shown by black arrows, and oligomeric states (dimer or hexamer) are indicated. J. BACTERIOL. VOL. 191, 2009 GENE REGULATION BY MOLYBDENUM 5207 TABLE 1. Microbial strains and plasmids Species and strain or plasmid Relevant characteristicsa Source or reference Host for plasmid amplification Host for expression of recombinant proteins 11 Novagen, Darmstadt, Germany R. capsulatus B10S R423AI R423BI R423CI Spontaneous Smr mutant of R. capsulatus B10 mopA::关Gm⬎兴 insertion mutant of B10S mopB::关Gm⬎兴 insertion mutant of B10S ⌬(mopA-mopB)::关Gm兴 deletion mutant of B10S 14 15 15 15 S. cerevisiae EGY48 URA3 TRP1 HIS3 6op-LEU2 3 Plasmids pAB4II pAB5II pASK-IBA3 pAW2 pBluescript KS pEG202 pET19 pET22b(⫹) pJG4-5 pJW26 pJW32 pJW33 pJW50 pJW52 pJW69 pJW70 pJW80 pJW81 pJW82 pJW83 pJW84 pJW88 pJW89 pJW90 pJW95 pLPRUB12 pML5 pSH18-34 pSN2 pSN3 pSN7 pSN8 pSUP401 pUC18 derivative carrying mopA pUC18 derivative carrying mopB High-copy Strep tag expression vector; Ap pASK-IBA3 derivative carrying mopBstrep High-copy-number vector; Ap lexA-DBD HIS3; Ap High-copy His tag expression vector; Ap High-copy His tag expression vector; Ap PGAL1-B42-AD TRP1; Ap pASK-IBA3 derivative carrying mopAstrep pET22b(⫹) derivative carrying mopAhis pET22b(⫹) derivative carrying mopBhis pSUP401 derivative carrying mopAhis pSUP401 derivative carrying mopBhis pEG202 derivative containing DBD-modC pJG4-5 derivative containing AD-modC pEG202 derivative containing DBD-mop pEG202 derivative containing DBD-mopA pJG4-5 derivative containing AD-mopA pEG202 derivative containing DBD-mopB pJG4-5 derivative containing AD-mopB pJG4-5 derivative containing AD-mop pET22b(⫹) derivative carrying mophis pASK-IBA3 derivative carrying mopstrep pET19 derivative carrying hismodC pML5 derivative carrying mop-lacZ transcriptional fusion Mobilizable lacZ fusion broad-host-range vector; Tc URA3 8op-lacZ; Ap pJG4-5 derivative containing AD-mopAHTH pEG202 derivative containing DBD-mopAHTH pJG4-5 derivative containing AD-mopAdi-MOP pEG202 derivative containing DBD-mopAdi-MOP Km A. Baslis and B. Masepohl, Bochum A. Baslis and B. Masepohl, Bochum IBA, Göttingen, Germany This study Stratagene, Amsterdam, The Netherlands 6 Novagen, Darmstadt, Germany Novagen, Darmstadt, Germany 6 This study 32 32 This study This study This study This study This study This study This study This study This study This study This study This study This study This study 16 6 This study This study This study This study 28 a Ap, ampicillin; Km, kanamycin; Sm, streptomycin; Tc, tetracycline. and a C-terminal Mo-binding di-MOP domain. In addition to MopA and MopB, R. capsulatus synthesizes two further MOP domain proteins, the Mo transport protein ModC and the molbindin Mop (Fig. 1). Homologous molbindins from other bacteria form homohexamers, suggesting that MOP domains are sufficient for protein-protein interaction (23). We therefore asked whether the above-mentioned four R. capsulatus MOP domain proteins form homomeric and/or heteromeric structures. In a first attempt to analyze protein-protein interactions between MopA, MopB, Mop, and ModC, Y2H studies were carried out. For this purpose, appropriate DBD and AD fusions were constructed. All DBD fusions were tested for selfactivation. None of them showed significant background activity (data not shown), and therefore, all of them were suitable for Y2H studies. The results of Y2H studies (Fig. 2) may be summarized as follows. (i) As expected, homomer formation was found for MopA, MopB, and Mop. (ii) MopA and MopB formed heteromers. (iii) Strong interactions were observed for the protein pair MopB-Mop. (iv) A weak but reproducible interaction was detected between ModC and MopA when ModC was used as bait. Copurification experiments were performed to verify interactions identified by Y2H studies. ModC was excluded from these studies, as overexpression and purification of HisModC resulted in very small amounts of soluble protein that were insufficient for this method. Suitable combinations of His- and Strep-tagged MopA, MopB, and Mop proteins were coexpressed in E. coli and subsequently purified by Ni-NTA chromatography. After elution from the Ni-NTA column, His- Downloaded from jb.asm.org at North Carolina State University Libraries on October 5, 2009 E. coli DH5␣ BL21(DE3) 5208 WIETHAUS ET AL. J. BACTERIOL. TABLE 2. Primers used for PCR amplification of selected DNA fragments Oligonucleotide sequence (5⬘ 3 3⬘) Relevant characteristics mopB-up mopB-down CGAATTCCCGGTTTGCGCCACAATGGCGGC GCAGGTCGACGGGCAGGGCCAGGATCACATGGC mopB coding region (purification of MopBStrep) UP-mopA LP-mopA GAATTCCTATATAACGATCCACCT GTCGACGGGCATCGCCAGGATGAC mopA coding region (purification of MopAStrep) PJW23-U PJW23-L GGCCAATTGATGATCTCGGCGCGGTTC GAACAATTGCTACCCTCCGGTTTGCGC modC coding region (Y2H) PJW56-U PJW56-L GACCAATTGATGAAACTCAGCGCACGC GTCCAATTGTCAGTTCTTGCCGACGAT mop coding region (Y2H) PJW66-U PJW66-L GCAGAATTCGACTCAATCGTTCCGGGA GTTGTCGACGTTCTTGCCGACGATGAC mop coding region (purification of MopHis) PJW67-U PJW67-L ACCCATATGAAACTCAGCGCACGCAAT CGACTCGAGGTTCTTGCCGACGATGAC mop coding region (purification of MopStrep) PJW68-U SN2 GACGAATTCATGAACGAACAGCCCCTC GAACTCGAGTCACGTCAGACTCCACCA mopAHTH coding region (Y2H) PJW69-U PJW68_L GACGAATTCATGCGCACTTCGAACCGC GAACTCCAGTCAGGGCATCGCCAGGAT mopAdi-MOP coding region (Y2H) PJW71-U PJW71-L GCGCATATGATCTCGGCGCGGTTC GAACGGATCCTACCCTCCGGTTTG modC coding region (purification of up-mop-uni PJW12-L CCGCCGTCTGGATCTGCCGCTCTC TCGGCGGCGGCTTCGTTGGTGAT mop promoter region (lacZ fusion) tagged proteins and associated Strep-tagged proteins were separated by SDS-PAGE and detected by Western blotting with His- or Strep-specific HRP conjugates. As controls, crude extracts with the individual Strep-tagged proteins were loaded HISModC) on Ni-NTA columns. The results obtained in the presence of Mo (Fig. 3 and 4) were essentially the same as those in the absence of Mo (data not shown). As expected, His-tagged proteins were retained by the Ni-NTA columns, whereas nei- FIG. 2. Protein-protein interactions identified by Y2H studies. DBD fusion proteins are indicated for each diagram, while AD fusion proteins are indicated on the x axis. Corresponding -galactosidase activities are given in Miller units (20). Results represent the means and standard deviations for three independent yeast transformants. Note the different scale (y axis) for each diagram. Downloaded from jb.asm.org at North Carolina State University Libraries on October 5, 2009 Primer VOL. 191, 2009 GENE REGULATION BY MOLYBDENUM 5209 ther of the Strep-tagged proteins bound unspecifically (Fig. 3). Surprisingly, when MopHis (7.5 kDa) and MopStrep (9 kDa) were purified in the presence of Mo, additional bands of approximately 35 kDa or 55 kDa appeared (Fig. 3). Most likely these bands correspond to the hexameric form of Mop as indicated by cross-linking experiments (see below). Mop oligomers were stable even under the harsh conditions during SDS-PAGE. Copurification experiments showed binding of MopAStrep (29.3 kDa) to MopAHis (28.2 kDa) as well as binding of MopBStrep (29 kDa) to MopBHis (27.9 kDa), verifying homomer formation (Fig. 4). Heteromer formation of the regulatory proteins was tested in both possible combinations. Consistently, binding of MopAStrep to MopBHis as well as binding of MopBStrep to MopAHis was observed. In line with the Y2H studies, MopStrep copurified with MopBHis but did not interact with MopAHis (Fig. 4). Interestingly, MopStrep was mainly monomeric after pulldown. Therefore, Mop might interact with MopB in a lower-oligomerization state that is not as stable as the Mop homohexamer shown in Fig. 3. Taken together, the Y2H studies and copurification experi- ments are consistent in showing that MopA and MopB form homomers as well as heteromers (Fig. 1). Moreover, the two regulatory proteins differ in their ability to interact with either the Mo transport protein ModC (MopA) or the molbindin protein Mop (MopB). Although crystal structures of two ABCtype molybdate importers from Archaeglobus fulgidus and Methanosarcina acetivorans revealed a dimeric state of the ATPases (4, 12), homomer formation of R. capsulatus ModC was not detected in Y2H studies. The attached DBD and AD domains might interfere with ModC interaction. Contribution of the DNA- and Mo-binding domains to MopA homomer formation. The E. coli ModE crystal structure revealed that about 70% of the dimer interface is generated by the DNA-binding HTH domain (10). In contrast, the crystal structures of the ModE di-MOP domain and chimeric ModE proteins indicated that the di-MOP domains are primarily responsible for dimerization (7, 19). To investigate the role of individual domains in dimerization of MopA, Y2H studies with the isolated HTH and di-MOP domains of MopA were carried out (Fig. 5A). As a basis for these studies, appropriate fusions of MopAHTH (amino acid residues 1 to 124) and MopAdi-MOP Downloaded from jb.asm.org at North Carolina State University Libraries on October 5, 2009 FIG. 3. Binding of His- and Strep-tagged proteins to Ni-NTA agarose. As a control for copurification experiments (Fig. 4), His- and Strep-tagged versions of MopA, MopB, and Mop were used for Ni-NTA affinity chromatography in the presence of Mo. Crude extracts of soluble proteins (S) and eluates (E) were analyzed by SDS-PAGE (A) and Western blotting (B and C). Either His-tagged (B) or Strep-tagged (C) proteins were detected. His-tagged proteins in panel A are marked by dashed ovals. The PageRuler prestained protein ladder (Fermentas, St. Leon-Rot, Germany) was used as a molecular mass standard. 5210 WIETHAUS ET AL. J. BACTERIOL. (amino acid residues 125 to 265) to the AD and DBD were constructed. High reporter gene activities demonstrated that the isolated HTH and di-MOP domains are sufficient for homomer formation (Fig. 5B). In contrast, no heteromer formation between the two domains was observed. These findings indicate that both the HTH domain and the di-MOP domain contribute to MopA oligomerization. Based on homology between MopA and MopB, we assume similar dimerization properties for the HTH and di-MOP domains of MopB. Formation of MopA dimers, MopB dimers, and Mop hexamers. While E. coli ModE is dimeric, the oligomeric state of molbindins differs substantially (7). Mono-MOP molbindins such as MopII from Clostridium pasteurianum are organized as hexamers, whereas di-MOP molbindins such as ModG from Azotobacter vinelandii are trimeric (2, 26). As Mo is bound at the interface of MOP domains, oligomerization occurs prior to Mo binding. MopA, MopB, and Mop form homomeric structures as demonstrated by Y2H studies. To determine the precise oligomer- ization state of these homomers, glutaraldehyde cross-linking and size exclusion chromatography with either MopAHis, MopBHis, or MopHis was performed. To address the question of whether Mo influences oligomer formation, these studies were performed in the presence and absence of Mo. MopAHis and MopBHis showed comparable cross-linking products independent of Mo availability (Fig. 6). In addition to the monomeric forms, a second band corresponding to MopAHis dimers (56.4 kDa) or MopBHis dimers (55.8 kDa) appeared after cross-linking. In line with these findings gel filtration profiles of MopAHis and MopBHis, obtained in the presence (Fig. 7) and absence (data not shown) of Mo were indistinguishable from each other. MopAHis and MopBHis eluted as complexes of about 61 kDa, which correlates well with the calculated masses of MopAHis and MopBHis homodimers. Since peaks corresponding to monomeric or higher-oligomeric forms were missing, it seems likely that both MopA and MopB preferentially exist in the dimeric state in solution. Dimerization occurs independent of Mo, as in case for E. coli ModE (1). Cross-linking experiments and gel filtration chromatography Downloaded from jb.asm.org at North Carolina State University Libraries on October 5, 2009 FIG. 4. Oligomer formation by MopA, MopB, and Mop. Copurification experiments were performed with His- and Strep-tagged MopA, MopB, and Mop proteins coexpressed in E. coli BL21(DE3). The respective combinations of coexpressed proteins are indicated above the gels. After Ni-NTA chromatography in the presence of Mo, crude extracts of soluble proteins (S) and eluates (E) were analyzed by SDS-PAGE (A) and Western blotting (B and C). Either His-tagged (B) or Strep-tagged (C) proteins were detected. Bands in panel C corresponding to copurified Strep-tagged proteins are labeled. The PageRuler prestained protein ladder (Fermentas, St. Leon-Rot, Germany) was used as a molecular mass standard. VOL. 191, 2009 GENE REGULATION BY MOLYBDENUM 5211 with a mixture of MopA and MopB resulted in elution profiles indistinguishable from those obtained with either MopA or MopB alone (data not shown). Given that heteromers were formed under these conditions, they do not form structures of higher order than the homodimers. In contrast to MopAHis and MopBHis, MopHis caused complex cross-linking and gel filtration profiles. The size of crosslinked MopHis complexes increased with increasing incubation time in the absence of Mo (Fig. 6). After 20 min, MopHis FIG. 6. Homomer formation by MopA, MopB, and Mop. Purified proteins were cross-linked with glutaraldehyde. The reactions were carried out in either the absence (⫺ Mo) or presence (⫹ Mo) of Mo for the indicated time intervals. Proteins incubated without glutaraldehyde served as controls (C). Homomer formation was analyzed by SDS-PAGE followed by Western blotting and detection of His-tagged proteins. Oligomeric states ranging from monomer (1 ⫻) to hexamer (6 ⫻) were calculated using the PageRuler prestained ladder (Fermentas, St. Leon-Rot, Germany). FIG. 7. Gel filtration profiles of MopA, MopB, and Mop. Purified proteins were analyzed by size exclusion chromatography on a Superdex HR 10/30 column. Proteins were detected by absorbance at 280 nm. Oligomeric states are indicated. particles ranging from monomers up to small amounts of hexamers (47.4 kDa) occurred. In the presence of Mo, the hexameric form appeared even without cross-linking and resisted the harsh SDS-PAGE conditions. Intermediate complexes were not detected, and after 5 min of incubation with glutaraldehyde, all monomers disappeared. Four peaks with calculated masses of about 92 kDa, 46 kDa, 16 kDa, and 10 kDa were detected by size exclusion chromatography in the presence of Mo (Fig. 7). The larger peaks most likely represent the MopHis dodecamer (94.8 kDa) and the hexamer (47.4 kDa), while the smaller peaks may correspond to the dimer (15.8 kDa) and the monomer (7.9 kDa). A similar pattern was obtained in the absence of Mo (data not shown). These findings suggest that R. capsulatus Mop is composed of dimeric building blocks like other mono-MOP molbindins, which organize as trimers of dimers. It is likely that the hexamer is the physiological state of Mop, since cross-linking experiments identified no complexes of higher order than the hexamer. Dodecamer formation during gel filtration chromatography might be the result of unphysiologically high protein concentrations. As expected, Mop oligomerization occurred without Mo binding. Downloaded from jb.asm.org at North Carolina State University Libraries on October 5, 2009 FIG. 5. Interaction of individual MopA domains identified by Y2H studies. MopA consists of an N-terminal DNA-binding HTH and a C-terminal Mo-binding di-MOP domain (A). Both domains were tested for interaction in Y2H studies (B). DBD fusion proteins are indicated in both diagrams, while AD fusion proteins are given on the x axis. Corresponding -galactosidase activities are given in Miller units (20). Results represent the means and standard deviations for three independent yeast transformants. Note the different scales (y axis) for the two diagrams. 5212 WIETHAUS ET AL. J. BACTERIOL. TABLE 3. Mo-binding properties of R. capsulatus MOP domain proteins Protein Bmaxa, mean ⫾ SD KDb (M), mean ⫾ SD MopA MopB Mop ModC 1.83 ⫾ 0.07 1.69 ⫾ 0.13 1.10 ⫾ 0.07 1.08 ⫾ 0.09 0.68 ⫾ 0.06 0.31 ⫾ 0.09 1.80 ⫾ 0.50 2.47 ⫾ 0.73 a Bmax describes the maximum saturation with molybdenum per monomer revealed by a nonlinear fitting procedure (Origin 6.0; Microcal) following the law of mass action. b KD values were obtained by ultrafiltration as described previously (21). Molybdate was used in a concentration range of 0 to 16 M in the presence of 4 M of MopA, MopB, Mop, or ModC and quantified as described in Materials and Methods. FIG. 8. Alignment of MOP domains from R. capsulatus MopA, MopB and Mop, and E. coli ModE. Amino acid residues are aligned for maximal matching, and residues involved in Mo binding are highlighted (23). Vertical arrows on top of the alignment mark type 2 Mo-binding sites, while arrows below indicate type 1 Mo-binding sites. Note that type 1 binding sites occur exclusively in molbindins. Downloaded from jb.asm.org at North Carolina State University Libraries on October 5, 2009 However, the cross-linking data strongly suggest that Mo stabilizes the Mop hexamer. MopA and MopB dimers bind four Mo oxyanions with high affinity. The crystal structures of several molbindins, E. coli ModE, and Methanosarcina acetivorans ModC demonstrate that MOP domains are sufficient for Mo binding (2, 4, 10, 26, 30). To determine the dissociation constants and the binding ratio of Mo to MopAHis, MopBHis, MopHis, and HisModC, the purified proteins were incubated with sodium molybdate for 15 min at room temperature. Unbound Mo was separated by ultrafiltration using a membrane with a molecular weight cutoff of 10,000. The Mo concentration in the flowthrough was determined by inductively coupled plasma optical emission spectroscopy as described in Materials and Methods. All four MOP domain proteins were able to bind Mo (Table 3), with dissociation constants of 0.68 M for MopA, 0.31 M for MopB, 1.80 M for Mop, and 2.47 M for ModC. The binding stoichiometry showed that MopA and MopB coordinate two Mo anions per monomer, while Mop and ModC bind one Mo per monomer. We believe that the lower binding stoichiometry value of two for the MopA and MopB monomer is due to the experimental error based on the method, which might be based on unstable or incorrectly folded recombinant protein. Curve fitting of the Mo binding to MopA and MopB revealed no biphasic binding, implying that the two Mo binding sites have similar values which could not be distinguished within the experimental error during curve fitting. Cooperative binding of molybdate was not observed for MopA and MopB. In contrast to MopA and MopB, E. coli ModE binds two Mo anions at the interface of the dimer as shown by crystal structure analysis (7). Like MopA and MopB, ModE contains two C-terminal MOP domains, referred to as MOP1 and MOP2 below (Fig. 5A). Only MOP1 of each ModE subunit participates in Mo binding. The failure of MOP2 to bind Mo correlates with a degenerated Mo-binding motif in MOP2 (Fig. 8). In contrast to the case for E. coli ModE, amino acid residues involved in ligand binding are well conserved in both MOP domains of R. capsulatus MopA and MopB, suggesting Mobinding capability of both MOP1 and MOP2. However, Mo binding by MOP2 domains would require an arrangement of di-MOP domains in MopA and MopB homodimers different from that of the ModE dimer, in which the MOP2 domains would be too distant to form a ligand-binding site. The dissociation constants of R. capsulatus MopAHis and MopBHis are comparable to the dissociation constant of E. coli ModE of ⬃0.8 M (1). The lower Mo affinity of MopA compared to MopB might be due to the Arg-202-Cys substitution in the Mo-binding motif of the MOP2 domain (Fig. 8). R. capsulatus MopHis binds Mo with a stoichiometry of six oxyanions per hexamer (Table 3). In contrast, structural data for all molbindins studied so far demonstrate that eight Mo anions are coordinated by a hexameric arrangement of MOP domains (2, 18, 26, 30). Mo-binding sites are formed by cooperation of either two type 2 or three type 1 binding sites (23). While six oxyanions are bound by type 2 binding sites, two further Mo oxyanions are coordinated by type 1 binding sites. Since amino acid residues contributing to type 1 and type 2 Mo-binding sites of the R. capsulatus Mop protein are conserved (Fig. 8), it would result in a binding of eight Mo oxyanions per Mop hexamer. However, since binding of only six oxyanions was detected, it is possible that some binding sites were not occupied under our test conditions. Type 1 sites of Haemophilus influenzae Mop exhibit lower Mo affinity than type 2 sites, whereas type 2 sites of C. pasteurianum MopII have lower affinity than type 1 sites (18, 26). As R. capsulatus Mop bound six oxyanions, we assume that exclusively type 2 binding sites were occupied. These type 2 sites would bind Mo with high affinity (KD of ⬃1.8 M) (Table 3), while type 1 sites should exhibit much lower affinity, which was not detectable under our assay conditions. In total, no heterogeneous binding was observed by nonlinear fitting of the data, underlining that the type 2 binding sites were not occupied under our assay conditions. Additionally, cooperative binding was not observed from our data. A binding ratio of one Mo oxyanion per HisModC monomer was detected in our experiments (Table 3). In agreement with VOL. 191, 2009 GENE REGULATION BY MOLYBDENUM 5213 that, the crystal structure of the M. acetivorans molybdate ABC importer revealed the presence of two Mo oxyanions bound at the interface of the C-terminal MOP domains of the ATPase dimer (4). Analogously, Mo binding by R. capsulatus ModC is likely to require homodimer formation. In its Mo-bound state, the M. acetivorans ModC dimer is stabilized mainly by contact to the integral membrane protein ModB. Purified R. capsulatus ModC exhibited lower Mo affinity (KD of ⬃2.47 M) than MopA, MopB, or Mop, which is a prerequisite to distribute the metal to cytoplasmic proteins. One candidate to receive Mo directly from ModC is MopA, as suggested by Y2H studies (see above). Fine-tuning of Mo-dependent gene regulation by formation of MopA-MopB heteromers. The regulator proteins MopA and MopB form heteromers as shown by Y2H studies and copuri- fication assays. While mopA expression is repressed by Mo, the mopB gene is transcribed independent of Mo availability (32). As a consequence, the MopA/MopB ratio is thought to change in response to the intracellular Mo level, raising the question of whether MopA-MopB heteromer formation influences Modependent gene regulation. We examined expression of the mop gene, which provides an optimal test system to follow up on this question. The mop gene is dependent solely on activation by MopA (Fig. 9A), while all other Mo-regulated genes are repressed by MopA or MopB (32). Activation of the mop gene is achieved by direct binding of MopA to a conserved DNA element in the mop promoter region, while MopB does not bind to the mop promoter. As expected from previous studies, mop transcription was activated by MopA in the presence of Mo (Fig. 9B). Interest- Downloaded from jb.asm.org at North Carolina State University Libraries on October 5, 2009 FIG. 9. Mo-dependent mop-lacZ expression in wild-type R. capsulatus and in mutant strains defective for mopA, mopB, or both. (A) MopA (but not MopB) binds to the mop promoter and activates transcription. A bent arrow (marked ⫹1) indicates the mop transcription start site. (B) Abilities of wild-type and mutant strains to form MopA homodimers (open ovals), MopB homodimers (filled ovals), and MopA-MopB heterodimers (combined open and filled ovals). (C) Wild-type and mutant strains containing mop-lacZ reporter plasmid pLPRUB12 were grown in AK-NL medium with increasing Mo concentrations (0, 10 nM, 100 nM, 1 M, 10 M, and 100 M Na2MoO4) prior to determination of -galactosidase activity. -Galactosidase activities are given in Miller units (20). Results represent the means and standard deviations of three independent measurements. 5214 WIETHAUS ET AL. DBD (7, 25). These changes are thought to improve DNA binding by ModE and therefore adapt gene expression to the Mo status of the cell. Since in vitro binding of MopA and MopB to their target promoters is clearly enhanced by Mo (32), we propose similar conformational changes upon Mo binding. In addition to homodimer formation, MopA and MopB were shown to form heteromers. As mentioned above, the MopA/MopB ratio is thought to vary in response to Mo availability (32). Accordingly, the relative amounts of MopA and MopB homo- and heteromers will differ. Since MopA-dependent mop gene expression was much higher in a mopB deletion mutant than in the wild type, we propose that formation of MopA-MopB heteromers prevents formation of MopA homodimers, leading to reduced mop gene expression. Most likely this type of control mainly concerns mop expression. Both MopA and MopB are able to repress transcription of the anfA gene (32). Hence, it is likely that MopA-MopB heteromers are as active as MopA and MopB homodimers at the anfA promoter. Fine-tuning of mop gene activation might therefore be very relevant in an aquatic environment with ever-changing metal concentrations. ACKNOWLEDGMENTS We thank Antonios Baslis and Lucia Püttmann for constructing plasmids pAB4II, pAB5II, and pLPRUB12 and Nicole FrankenbergDinkel for critical reading of the manuscript. This work was supported by grants from the Deutsche Forschungsgemeinschaft (Ma 1814/3-3 and Le1171/3-3) and the Ruhr University Research School. REFERENCES 1. Anderson, L. A., T. Palmer, N. C. Price, S. Bornemann, D. H. Boxer, and R. N. Pau. 1997. Characterisation of the molybdenum-responsive ModE regulatory protein and its binding to the promoter region of the modABCD (molybdenum transport) operon of Escherichia coli. Eur. J. Biochem. 246: 119–126. 2. Delarbre, L., C. E. Stevenson, D. J. White, L. A. Mitchenall, R. N. Pau, and D. M. Lawson. 2001. 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Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166:557–580. 12. Hollenstein, K., D. C. Frei, and K. P. Locher. 2007. Structure of an ABC transporter in complex with its binding protein. Nature 446:213–216. Downloaded from jb.asm.org at North Carolina State University Libraries on October 5, 2009 ingly, much stronger mop activation was observed in the mopB mutant than in the wild-type. Therefore, it seems that the formation of MopA-MopB heteromers in the wild-type background reduces activation of the MopA-specific promoter. One can assume three different scenarios: (i) MopA-MopB heteromers are not able to bind to the mop promoter at all, (ii) heteromers exhibit lower affinity to the mop promoter than MopA homodimers, or (iii) MopA-MopB heteromers bind to the mop promoter but are not able to interact with the transcription machinery. In each scenario, formation of MopAMopB heteromers will lead to lower levels of transcriptionally active MopA homodimers. Alternatively, enhanced mop transcription in the mopB mutant might be explained by increased mopA expression in this background. In the wild type, MopB represses mopA transcription, thereby indirectly inhibiting mop transcription, which requires MopA. Conclusions. R. capsulatus encodes two ModE-like Mo-responsive regulators, MopA and MopB, whereas E. coli and all other bacteria analyzed to date synthesize only one such regulator. This enables R. capsulatus to control target gene expression very precisely in response to Mo availability. As shown previously, MopA and MopB exhibit partially overlapping and specialized functions (15, 32). In the present study, we analyzed protein-protein interactions between MopA, MopB, the Mo transport protein ModC, and the molbindin Mop, all of which contain one or two MOP domains implicated in Mo binding. MopA was shown to interact with ModC, while no interaction between MopB and the transport protein was observed. This interaction is likely to be specific, since mopA-modABC form an operon which is expressed only under Mo limitation, while mopB is constitutively expressed from a separate transcription unit. One may speculate about three different roles for MopA-ModC interaction. (i) MopA activity may be controlled by membrane sequestration. Such a mechanism is wellknown for different regulator proteins which reversibly bind to the ammonia transporter AmtB (29). (ii) ModC might transfer Mo to ModA, which exhibits higher Mo affinity than the transport protein. (iii) Activity of the Mo transporter may be controlled by MopA. This assumption is strengthened by the finding that Mo binding by M. acetivorans ModC results in allosteric inhibition of ATPase activity and hence interruption of Mo import (4). Only if Mo is passed on to another protein is ATPase activity restored. MopB (but not MopA) was shown to interact with the molbindin Mop. 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