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.
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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.
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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.
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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
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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
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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.
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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
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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-
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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.
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