From the Department of Biotechnology, Tokyo
University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan and the § Division of Science and Engineering, Murdoch
University, Perth, Western Australia 6150, Australia
Received for publication, November 18, 2002
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ABSTRACT |
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Magnetic bacteria synthesize magnetite crystals
with species-dependent morphologies. The molecular
mechanisms that control nano-sized magnetite crystal formation
and the generation of diverse morphologies are not well understood.
From the analysis of magnetite crystal-associated proteins,
several low molecular mass proteins tightly bound to bacterial
magnetite were obtained from Magnetospirillum magneticum
strain AMB-1. These proteins showed common features in their amino acid
sequences, which contain hydrophobic N-terminal and hydrophilic
C-terminal regions. The C-terminal regions in Mms5, Mms6, Mms7, and
Mms13 contain dense carboxyl and hydroxyl groups that bind iron ions.
Nano-sized magnetic particles similar to those in magnetic bacteria
were prepared by chemical synthesis of magnetite in the presence of the
acidic protein Mms6. These proteins may be directly involved in
biological magnetite crystal formation in magnetic bacteria.
Magnetic bacteria synthesize nano-sized magnetic particles of an
iron oxide, magnetite (Fe3O4); an iron sulfide,
greigite (Fe3S4); or a combination of greigite
and iron pyrite (Fe2S) (1-3). These particles are
individually covered with a stable lipid bilayer membrane that mainly
consists of lipid and protein (4). The mineral size, type, and
morphology of bacterial magnetic particles (BMPs)1 are highly controlled
within bacterial species or strains (5). The species-specific control
of BMP formation has focused attention on the possible roles of the
surrounding membrane structures.
The molecular mechanism of BMP synthesis is a multistep process,
including vesicle formation, iron transport, and magnetite crystallization (5, 6). Recent molecular studies have postulated the
steps of BMP synthesis (7-9). Several proteins located on or in the
BMP membrane have been isolated and analyzed in Magnetospirillum magneticum strain AMB-1. The first event of BMP synthesis is the formation of vesicles. Invagination of the cytoplasmic membrane is
primed by a BMP membrane-specific GTPase (Mms16) to form the intracellular vesicle (7). MpsA, a homolog of an acetyltransferase containing a CoA-binding motif, is also considered to be involved in
this process (8). The second process in BMP synthesis is iron transport
into the BMP vesicles. It appears that ferric iron is reduced on the
cell surface, taken into the cytoplasm, transported into the BMP
vesicle, and finally oxidized to produce magnetite. The magA
gene was isolated through transposon mutagenesis in strain AMB-1 (9).
This gene encodes an integral membrane protein that is involved in the
transport of iron into the BMP vesicles. The last process is
crystallization of magnetite within the vesicle, but the process
remains unclear.
Other proteins associated with the BMP membrane have been partially
characterized in magnetic bacteria to date. Gorby et al. (4)
observed two specific proteins in the BMP membrane from Magnetospirillum magnetotacticum MS-1. Okuda et
al. (10) identified three additional specific proteins and
determined the nucleotide/amino acid sequence of a 22-kDa protein. From
motif analysis, this is considered to function as a receptor
interacting with associated cytoplasmic proteins (11). Recently,
Grünberg et al. (12) cloned and sequenced four genes
that were assigned to two different genomic regions coding for BMP
membrane-specific proteins in Magnetospirillum gryphiswaldense MSR-1.
To understand the molecular mechanism of magnetite crystallization in
M. magneticum AMB-1, several proteins tightly bound to the
bacterial magnetite crystals were isolated and characterized. A new
class of mineral-associated proteins that may have important roles in
the initiation of nucleation and magnetite crystal growth is described.
Strains and Growth Conditions--
Escherichia coli
strains DH5 Isolation of Proteins Tightly Bound to BMPs--
BMPs were
extracted from 8 liters of fed-batch culture and washed 20 times with
HEPES (pH 7.0). The other cell fractions were also prepared as
described previously (8). The purified BMPs were treated three times
with 800 µl of 7 M urea, 2 M thiourea, 4%
(w/v) CHAPS, and 40 mM Tris base solution with weak
sonication to remove membrane-associated proteins and washed with HEPES
several times. To isolate the tightly bound proteins, BMPs were treated three times with 800 µl of 1% (w/v) SDS in a 100 °C water bath for 30 min. During this process, the sample was treated briefly in an
ultrasonic bath. After several washings, BMPs were treated with 20 ml
of 2 M hydrofluoric acid plus 8 M ammonium
hydrofluoride solution (pH 5.0). The dissolved material was dialyzed
several times against 4 liters of fresh HEPES. The sample was
precipitated by the same volume of 20% (w/v) trichloroacetic acid and
resuspended in 400 µl of 0.1% SDS solution. The amount of protein on
BMPs was evaluated using a modified Lowry method (14). One-hundred microliters of 1 N NaOH and 0.02% (w/v) SDS solution was
added to 1 mg of BMPs and incubated in a 100 °C water bath for 10 min. After the BMPs were removed by centrifugation, the supernatant was
used to measure the protein amount using bovine serum albumin as a standard.
Gel Electrophoresis--
Protein concentrations in solution were
measured using a protein assay kit (Bio-Rad) and adjusted before
electrophoresis. Each fraction was mixed in the same volume of 2×
sample buffer (0.125 M Tris-HCl (pH 6.8), 10%
mercaptoethanol, 4% SDS, 10% sucrose, and 0.004% bromphenol blue)
and denatured by boiling. A 3% stacking gel and a 15% resolving gel
were used according to the method of Laemmli (15). Tricine/SDS-PAGE was
performed according to the method of Schägger and von Jagow (16).
Gels were stained with Coomassie Brilliant Blue R-250 or SYPRO
Ruby (Molecular Probes, Inc., Eugene, OR). The molecular mass was
calculated from a standard linear regression curve using a low
molecular mass calibration kit (Amersham Biosciences, Uppsala, Sweden)
and polypeptide SDS-PAGE molecular mass standards (Bio-Rad). Western
analysis was performed by electroblotting polyacrylamide gels onto
polyvinylidene difluoride membranes (ImmobilonTM-P,
Millipore Corp., Bedford, MA). The membranes were immunostained with
mouse anti-His monoclonal antibody (cat no. 34670, QIAGEN GmbH)
at a 1:5000 dilution and developed with alkaline phosphatase-coupled goat anti-mouse IgG secondary antibody (cat no. 59296, ICN Biomedicals Inc., Aurora, OH).
Two-dimensional PAGE was carried out by the method of O'Farrel
(17). The first dimension was carried out with immobilized dry strip
gels (pH 3-10; 130 mm) using the electrophoresis apparatus immobilized pH gradients Phor II (Amersham Biosciences). The
strips were rehydrated with buffer containing of 7 M urea,
2 M thiourea, 4% (w/v) CHAPS, 40 mM Tris base,
19.4 mM dithiothreitol, 0.5% (v/v) carrier ampholytes
(Amersham Biosciences), and traces of bromphenol blue at 20 °C for
12 h. The strips were run with the following program: step 1, 500 V for 2 h; step 2, 1000 V for 2 h; step 3, 8000 V (gradient)
for 4 h; and step 4, 8000 V (hold) for 2 h. The second
dimension was carried out with Tricine/SDS gels as described
above. The two-dimensional gels were stained with Coomassie Brilliant
Blue R-250. CarbamylyteTM calibration markers
(Amersham Biosciences) were used to estimate the pI of the proteins.
Amino Acid and DNA Sequencing--
After gel electrophoresis,
gels were electroblotted onto ImmobilonTM-PSQ (Millipore
Corp.). The membrane was stained with Coomassie Brilliant Blue R-250,
and identified protein spots were excised. Amino acid sequencing of the
proteins/peptides was performed by automated Edman degradation using a
Shimadzu PPSQ-1 amino acid sequencing system.
Oligonucleotide primers were designed using the N-terminal amino acid
sequence. The codon usage pattern of previously reported proteins in
strain AMB-1 was used (7-9). Gene walking was performed to obtain the
entire gene encoding the Mms5 and Mms6 proteins. Primer
S1 (CAGGCCCTTGCCGGTCCAGATGGT) and primer S2 (ATCATCCTGGGCGTTGTTGGCGCC) were used to amplify the mms6 region. Primer S3
(GTGCTGCTGGGCGTGGTCGGCGTG) and primer S4 (CACGCCGACCACGCCCAGCAGCAC)
were designed for the mms5 region. To sequence the
mms7-13 region, primer mms7-13F (GCCTAACCAAATCCAGATGAG) and primer mms7-13R (CCGTAAGGAAAGACAGACACG) were designed from the genome sequence data of M. magneticum
AMB-1.2 The other primers for
sequencing the region containing mam7, mam13, and mms6 were designed from the same data
base. The preliminary sequence data of M. magnetotacticum
MS-1 obtained from the DOE Joint Genome
Institute3 was used for
comparison. The amplified PCR fragment was cloned into the pGEM-T-easy
vector (pGEM-T-easy vector system, Promega, Madison, WI) and sequenced
using ABI PRISM 377 (PerkinElmer Life Sciences).
The computer software package LASERGENE (DNASTAR, Inc. Madison, WI) was
used for DNA and protein sequence analyses. The sequence was further
analyzed by performing homology searches using the programs FASTA and
BLAST against the GenBankTM/EBI DNA Data Bank.
Expression and Purification of Recombinant Protein--
The
recombinant plasmid pET15b-mms6, containing the sequence
coding the mature Mms6 protein, was constructed by cloning PCR products into the expression vector pET15b. The primer set
5'-GGGGGACATATGGTCGGTGGAACCATCTGGACCGGTAAG-3' and
5'-GGGGGATCCAAATCAGGCCAGCGCGTCGCGCAGTTCGAC-3' was used for amplification of the mms6 gene from AMB-1 genomic DNA.
E. coli BL21 cells were cultured in 200 ml of Luria broth at
37 °C under isopropyl-1-thio- Iron Binding Analysis--
The blotting technique for detecting
the iron-binding ability of Mms6 was performed according to Chen and
Drysdale (18). 55FeCl3 was used instead of
59FeCl3 in the same procedure. After blotting
0.3 nmol of proteins, the nitrocellulose membrane was rinsed with
metal-binding buffer (0.02 M Tris-HCl and 0.15 M NaCl (pH 7.0)) for 30 min. The membrane was then immersed
in 5 ml of the same buffer containing 0.05 mCi/ml 55FeCl3 (PerkinElmer Life Sciences). The total
iron concentration in the solution was adjusted to 8.16 µg/ml by
adding FeCl3. Unbound iron ion was removed by washing the
membrane with metal-binding buffer three times for 30 min. The membrane
was wrapped and exposed to x-ray film. Equine spleen apoferritin
(Calbiochem) and bovine serum albumin were used as positive and
negative controls, respectively. Competitive iron binding was performed
using 0.1 nmol of proteins. The membrane was immersed in 2 ml of
metal-binding buffer with 0.05 mCi/ml 55FeCl3
in the absence or presence of competing ions, including 1 mM FeCl3, 10 mM CaCl2,
10 mM MgCl2, 10 mM
CuCl2, 10 mM MgCl2, 10 mM NiCl2, and 10 mM
ZnCl2. Autoradiography was done for 3 h.
Magnetite Synthesis in the Presence of Proteins Tightly Bound to
BMPs--
Purified recombinant Mms6 was used for magnetite formation
by coprecipitation (19). Recombinant Mms6 (2 µg) was added to 100 µl of solution containing 33 mM ferrous sulfate and 33 mM ferric chloride. The solution was titrated very slowly
using 0.1 N NaOH solution with sparging argon gas.
Transmission Electron Microscopy and Electron Diffraction
Analysis of Magnetic Particles--
A transmission electron microscope
(H700-H, Hitachi, Tokyo) was used to observe the particle size and
morphology. The sample was applied to carbon-coated 150-mesh copper
grids (Nisshin EM Co., Ltd., Tokyo), dried overnight at room
temperature, and stored with silica gel desiccant. The magnetic
particles were visualized at 150 keV. Electron diffraction was
performed using an analytical microscope (JEM-2000FX, Jeol Ltd., Tokyo).
Isolation of Proteins Tightly Bound to BMPs--
After extraction
and purification, ~200 mg of BMPs was obtained from an 8-liter
fermentor. The presence of membranes encapsulating the BMPs was
confirmed by transmission electron microscopy (Fig. 1A). Results from the protein
assay showed that the membrane proteins constituted 3% of the total
mass weight of BMPs. By treating BMPs with a solution containing 7 M urea, 2 M thiourea, and 4% CHAPS with weak
sonication, 60% of the proteins were extracted. As previously reported, Mms24 (identical to MAM22) (20), MpsA (8), and Mms16
(7) were observed in this fraction (Fig.
2A, lane 1). The
residual 40% of the proteins associated with BMPs could not be
extracted using the same solution. To strip these tightly associated proteins, BMPs were immersed three times in boiling 1% SDS solution. Over 95% of the proteins were removed, and aggregations of naked BMPs
were observed (Fig. 1B). The protein profile of the 1% SDS fraction showed that it consisted mainly of low molecular mass proteins
(<15 kDa) (Fig. 2A, lane 4). For amino acid
sequencing, the same protein sample was subjected to two-dimensional
gel electrophoresis. Four prominent spots were identified in the gel
(Fig. 2B). The residual magnetite was dissolved in 2 M hydrofluoric acid plus 8 M ammonium
hydrofluoride solution (pH 5.0) to identify any embedded proteins in
the bacterial magnetite after protein removal with boiling 1% SDS
solution. The magnetite was dissolved completely, and some compounds
were observed by transmission electron microscopy (Fig. 1D).
No protein bands were observed by SDS-PAGE (Fig. 2A, lane 5), suggesting that proteins exist on or near the
surface of the crystals, but not within the magnetites. The
proteins tightly bound to BMPs were further characterized to
understand their roles in magnetite crystallization.
N-terminal Amino Acid Sequencing--
The amino acid sequences of
the small proteins obtained by Edman degradation are shown in Table
I. From the obtained sequences, a spot
with an approximate molecular mass of 13 kDa (pI 7.2) was determined as
a homolog of MamC in M. gryphiswaldense (12). The molecular
size visualized by Tricine/SDS matched the reported size of MamC. The
protein was designated as Mms13. The N-terminal amino acid sequences of
the spots at 5 kDa (pI 6.1), 6 kDa (pI 4.5), and 7 kDa (pI 5.9) showed
high degrees of homology to each other and were designated as Mms5,
Mms6, and Mms7, respectively. These proteins have the common sequence
LGLGLGLGAWGPXXLGXXGXAGA. However, from
several sequencing trials, we concluded that these are different
proteins. Mms7 is homologous to the C-terminal part of MamD in M. gryphiswaldense. It was reported that the apparent mass of MamD on
the SDS gel and the molecular mass calculated from the predicted
mamD gene are different (12). These results suggest the
existence of proteolytic cleavage of MamD. The obtained sequence most
probably is the remnant of MamD after proteolytic digestion.
Isolation of Genes Encoding Proteins Tightly Bound to
BMPs--
Based on the obtained amino acid sequences, primers
were designed for the gene sequencing of Mms5 and Mms6. Complete DNA
fragments coding open reading frames were obtained from the AMB-1
genome. Putative sequence for the ribosome-binding site and
several promoter regions were found upstream of the start codon. FASTA
and BLAST searches showed that the obtained nucleotide and deduced
amino acid sequences of Mms5 and Mms6 have no significant similarity, except to MamD. Interestingly, mms6, mms7, and
mms13 are located closely (within a 3.2-kilobase pair
region) in the AMB-1 genome (Fig. 3). The
gene homolog of mms6 was also found in the genome sequence
of MS-1 obtained from the data base. The amino acid sequence alignment
of Mms6, Mms7, and Mms13 between M. magneticum AMB-1 and
M. magnetotacticum MS-1 revealed 100, 81, and 80%
similarities, respectively. No motifs were identified in all four
proteins. The gene encoding Mms5 was not found in the genome
sequence of MS-1 obtained from the data base.
The amino acid sequence of Mms6 deduced from the full-length
399-bp gene is shown in Fig. 4.
The sequence encodes a 12.5-kDa premature polypeptide. The N-terminal
sequence obtained directly from the purified protein is completely
contained within this deduced sequence. A predicted signal peptide
presumed to mediate secretion, followed by a propeptide, was found
using the SOSUI program (21). However, there was a gap between the
signal peptidase cleavage site and the N-terminal sequence obtained by
Edman degradation. This may be due to digestion by some specific
proteases after protein secretion. The N-terminal region contains
hydrophobic amino acids, and computer analysis suggests that this is a
transmembrane region. However, the C-terminal region of Mms6 is highly
acidic, consistent with a pI of 4.5. Amino acids containing hydroxyl
groups were also observed. Furthermore, the region between the middle and C-terminal regions contains basic amino acids such as Lys, Tyr, and
Arg. These structural features were also observed in Mms5, Mms7, and
Mms13. The consensus sequences among these proteins are shown in Fig.
5. The hydrophilic domains in
mineral-associated proteins capture metal ions (22, 23) or interact
with the mineral phase (24), as previously reported.
Purification of Recombinant Mms6--
Recombinant Mms6 was
produced in E. coli BL21. The gene encoding the mature
peptide of Mms6 (6 kDa) was amplified by PCR and cloned into the pET15b
vector. The pET15b-mms6 plasmid was then transformed into
E. coli and overexpressed. The His-tagged protein was
purified from the cell lysate using a nickel-nitrilotriacetic acid
column. The N-terminal amino acid of the purified protein was sequenced
and confirmed that the desired protein was expressed in E. coli. The protein was reconstituted, and the His tag was removed
by thrombin digestion. The protein sample after each step was checked
by gel electrophoresis and Western blotting using anti-His antibody
(Fig. 6). The expected sizes of
His-tagged (7.5 kDa) and thrombin-digested (6 kDa) proteins were
determined.
Iron-binding Activity and Specificity of Mms6--
The
iron-binding ability of Mms6 was found both in the presence and absence
of the His tag (Fig. 7A). This
indicates that the observed iron binding is mainly derived from Mms6,
not from the His tag. In contrast, bovine serum albumin did not show
iron-binding capability. Addition of 10 mM nonradioactive
Fe3+ blocked the binding of radioactive Fe3+ to
both Mms6 and ferritin (Fig. 7B). Inhibition of binding of radioactive Fe3+ to Mms6 (but not to ferritin) was also
observed in the presence of Ca2+ and Mg2+. This
binding inhibition was scarcely observed in the presence of
Ni2+, Cu2+, and Zn2+.
Chemical Magnetite Synthesis in the Presence of Mms6--
To
determine the effect of Mms6 on crystal formation, artificial magnetite
was synthesized. At the beginning of titration, we observed a
yellow-to-white precipitate, which changed to dark green and finally to
black at neutral pH. The magnetic iron precipitates produced in the
presence of Mms6 showed cuboidal morphology, with sizes ranging from 20 to 30 nm (Fig. 8A). The
results of electron diffraction analysis indicated that the black
particles were composed mainly of magnetites (data not shown). The
shape of the crystalline magnetites was similar to that of BMPs
synthesized in M. magneticum AMB-1. Furthermore, the
magnetic particles produced in the absence of Mms6 were non-homogeneous
in size (1-100 nm) and shape (Fig. 8B). The observed
needle-shaped crystals are similar to Organic molecules acting as templates that facilitate crystal
formation have been isolated from demineralized materials (25-27) and
organic matrix associated with mineral surfaces (28, 29). Direct
evidence of biomineral formations by proteins has been reported in
calcium carbonate (26, 27), silica (25, 30), and hydroxylapatite (24).
Although extraction of the proteins associated with BMPs has been
examined using SDS (4, 10, 12) and urea-based (8, 20) solutions, no
direct evidence of magnetite crystal formation by the proteins has been
shown. In this study, several different protein fractions were
separated by sequential treatment with urea, boiling SDS, and
hydrofluoric acid. Peripheral proteins such as Mms24 (MAM22) (10, 20), MpsA (8), and Mms16 (7) and transmembrane proteins were removed by
treatment with a urea-based solution. Four proteins (Mms5, Mms6, Mms7,
and Mms13) were observed in the same fraction as minor components upon
two-dimensional gel electrophoresis. The four protein spots were
dominant in the fraction obtained by boiling SDS treatment. The protein
solution showed a yellowish color, and the presence of iron was
confirmed. Transmission electron microscopy showed no differences in
the size and shape of BMPs before and after this treatment, indicating
that only the surfaces of the particles were degraded. The presence of
some specific interaction is suggested between these proteins and BMP
surfaces. Furthermore, because protein was not observed in the BMP
crystal core, the proteins bound to the surface play important roles in crystal formation.
The four proteins (Mms5, Mms6, Mms7, and Mms13) showed no sequence
similarities to known functional proteins. The only observed similarity
was to the BMP membrane proteins MamC and MamD, reported in M. gryphiswaldense (12). Common features observed within their
sequences may describe a structural property. The proteins are mainly
composed of two domains, hydrophobic N-terminal and hydrophilic
C-terminal regions. The organic matrix surrounding the BMP crystal
consists of a lipid bilayer membrane (4, 7). The hydrophobic peptide of
the N-terminal transmembrane region may be integrated into the lipid
bilayer. The dense carboxyl and hydroxyl groups in the C-terminal
regions may interact directly with the mineral surface.
For the proteins that directly interact with biominerals, two plausible
functions are suggested: (i) initiation of crystal nucleation (25, 27,
30) and (ii) inhibition and regulation of crystal growth (24, 31) and
determination of morphology (32, 33). The initiation starts from the
interaction and accumulation of metal ions on or in the organic
molecules. The acidic groups (carboxylate derived from aspartate and
glutamate) are known to have strong affinity with metal ions and to act
as bridging ligands. The hydroxyl groups in serine, threonine, and
tyrosine also possess metal-binding capability. These side chains of
amino acids preferentially bind to metal ions such as Fe3+,
Ca2+, and Mg2+ (23). The binding of these
groups to Cu2+, Ni2+, and Zn2+ is
unlikely. The competitive iron binding assay supports that the observed
iron binding is derived from the carboxyl and hydroxyl groups.
Therefore, the hydrophilic C-terminal region in Mms6 is considered to
initiate crystal nucleation in magnetite formation. It has been
reported that a small acidic protein called statherin possesses dense
negative charges in the N-terminal region that are responsible for the
mineral-adsorbing ability, inhibiting crystal growth (24). Although the
interaction between organic molecules and solid phases is dependent
upon the mineral (34), the observed iron-binding ability of Mms6 is
also thought to inhibit iron mineralization, controlling morphology.
However, a control mechanism of morphology is still unknown.
In magnetic bacteria, the origin of BMP membrane vesicles has been
suggested to arise through invagination of the cytoplasmic membrane (4,
7). The biological compartmentalization through the formation of
enclosed vesicles enables the chemical process of magnetite formation
to be optimized and regulated. On the basis of the high resolution
transmission electron microscopy and Mössbauer spectroscopy
results, mineral transformation through BMP synthesis was hypothesized
(35). The first step involves the accumulation of iron, followed by the
precipitation of hydrated iron oxide and, finally, phase transformation
of amorphous iron to magnetite during the nucleation stage and
surface-controlled growth similar to the formation of magnetoferritin
cores (36). Magnetite films have been synthesized using -OH groups of
arrayed lipid layers as molecular templates for crystal synthesis by
controlling gas-phase oxygen concentrations at room temperature (37).
The densely arrayed -OH groups derived from carboxylate adsorb iron
ions and stimulate magnetite growth. Similarly, magnetite has been
synthesized in iron solutions by adding recombinant Mms6. The observed
precipitate color change suggests the phase transformation of iron
oxides, Fe(OH)2 and In this study, the existence of proteins tightly associated with BMP
crystals and their iron-binding ability have been shown. Mms6 provides
nucleation sites for precipitation of iron oxide in the BMP vesicle.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and BL21 were used for gene cloning and protein
expression, respectively. E. coli cells were cultured in
Luria broth at 37 °C after adding appropriate antibiotics. M. magneticum AMB-1 (ATCC700264) was grown anaerobically in an 8-liter fermentor as described previously (13).
-D-galactopyranoside
induction. The recombinant protein was purified under denaturing
conditions using a nickel-nitrilotriacetic acid column (QIAGEN). The
eluted protein was diluted in the same volume of refolding buffer (50 mM Tris-HCl, 1 mM EDTA, 0.1 M
L-arginine, 1 mM reduced glutathione, 10%
(v/v) glycerol, and 0.8 mM oxidized glutathione (pH 8.0)).
The purified protein was renatured by dialysis in 0.5 liter of buffer
(0.01 M Tris-HCl, 10% glycerol, and 0.01 M
EDTA (pH 8.0)) containing 4 and 2 M urea for 3 h each.
Final dialysis was performed overnight in 1 liter without urea. The protein was further dialyzed for 3 h several times against 1 liter of fresh Tris-HCl (pH 8.0). All dialysis steps were performed at
4 °C. The His tag of the recombinant protein was digested with thrombin (QIAGEN) and then removed by filtration using a miniprep Microcon YM-3 membrane (Millipore Corp.).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Morphological changes in BMPs during protein
extraction. A, no treatment; B-D, after
treatment with urea, SDS, and hydrofluoride solutions,
respectively.
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Fig. 2.
Identification of proteins tightly bound to
BMPs by SDS-PAGE. A, protein profile of cell
fractions. Lane 1, BMP protein fraction extracted by a
solution containing 7 M urea, 2 M thiourea, and
4% CHAPS; lane 2, cell membrane lysates; lane 3,
cytoplasmic fraction; lane 4, BMP protein fraction extracted
with boiling 1% SDS solution; lane 5, demineralized
material of BMPs treated with 2 M hydrofluoric acid plus 8 M ammonium hydrofluoride solution (pH 5.0). Ten micrograms
of proteins for lanes 1-4 and 15 µl of sample for
lane 5 were loaded. The gel was stained with SYPRO Ruby.
B, two-dimensional gel electrophoresis of the BMP protein
fraction extracted with boiling 1% SDS solution. Twenty micrograms of
protein was loaded. The gel was stained with Coomassie
Brilliant Blue R-250. The identified proteins are indicated by
arrows. Numbers to the left of A and
B indicate molecular mass markers.
N-terminal amino acid sequences of tightly bound BMP proteins
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Fig. 3.
Molecular organization of the
genes encoding proteins tightly bound to BMPs in the
genome.
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Fig. 4.
Amino acid sequence of Mms6 deduced from the
DNA sequence. The arrow indicates the putative signal
peptidase site. The arrowhead shows the boundary observed
between the propeptide and mature peptide. Amino acid residues that
possibly bind metal ions are boxed.
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Fig. 5.
Sequence alignment and comparison of Mms5,
Mms6, Mms7, and Mam13. A, hydrophobic region;
B, hydrophilic region. Conserved residues are boxed. The
numbers represent amino acid positions in the mature
proteins.
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Fig. 6.
Purification of recombinant Mms6 produced in
E. coli. A, profile of the
purified protein stained with Coomassie Brilliant Blue R-250;
B, Western blot stained with anti-His antibody. Lane
M, molecular mass markers; lanes 1 and 2,
E. coli cell lysate before and after
isopropyl-1-thio- -D-galactopyranoside induction,
respectively; lane 3, purified His-tagged Mms6; lane
4, thrombin-digested His-tagged Mms6.
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Fig. 7.
Iron binding analysis of recombinant Mms6
(A) and competition for 55Fe binding by
metal ions (B). Proteins were blotted at 0.1 nmol
(A) and 0.3 nmol (B). BSA, bovine
serum albumin.
-FeOOH.
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Fig. 8.
Electron micrographs of magnetic particles
synthesized in the presence (A) and absence
(B) of Mms6. Arrowheads indicate
needle-shaped crystals. Bars indicate 100 nm.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-FeOOH, through magnetite.
Significant shape similarity was observed between BMP and the crystals
obtained from in vitro mineralization using Mms6. The
-OH-rich C-terminal parts of the proteins tightly bound to BMPs
might act as templates for magnetite crystal formation and direct the
shape of magnetite crystals formed.
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ACKNOWLEDGEMENTS |
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We thank S. Kamiya (Tokyo Denkikagaku Kogyo Akita Laboratory Corp.) and S. Uchida (Corporate Research and Development Center, Tokyo Denkikagaku Kogyo K.K. (TDK Electronics Co., Ltd.)) for electron diffraction analysis.
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FOOTNOTES |
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* This work was supported in part by Grant-in-aid for Specially Promoted Research 13002005 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by a visiting fellowship from the Japan Society for the Promotion of Science (to J. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number(s) AB096081 and AB096082.
¶ To whom correspondence should be addressed: Dept. of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan. Tel.: 81-42-388-7020; Fax: 81-42-385-7713; E-mail: tmatsuna@cc.tuat.ac.jp.
Published, JBC Papers in Press, December 19, 2002, DOI 10.1074/jbc.M211729200
2 T. Matsunaga, Y. Okamura, A. T. Wahyudi, Y. Fukuda, and H. Takeyama, manuscript in preparation.
3 Available at www.jgi.doe.gowv/tempweb/JGI_microbial/html/index.html.
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ABBREVIATIONS |
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The abbreviations used are: BMPs, bacterial magnetic particles; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
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