Visualization and structural analysis of the bacterial
magnetic organelle magnetosome using atomic
force microscopy
Daisuke Yamamotoa,b,1, Azuma Taokac,1, Takayuki Uchihashia,b, Hideaki Sasakic, Hiroki Watanabea, Toshio Andoa,b,d,and Yoshihiro Fukumoric,2aDepartment of Physics, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan; bCore Research for Evolutional Science and Technology, Japan
Science and Technology Agency, Sanban-cho, Chiyoda-ku, Tokyo 102-0075, Japan; cDepartment of Life Science, Graduate School of Natural Science andTechnology, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan; and dFrontier Science Organization, Kanazawa University, Kakuma-machi,
Kanazawa 920-1192, Japan

Edited by Dianne K. Newman, Caltech/ Howard Hughes Medical Institute, Pasadena, CA, and accepted by the Editorial Board April 8, 2010 (received for review
February 16, 2010)

The unique ability of magnetotactic bacteria to navigate along
a geomagnetic ﬁeld is accomplished with the help of prokaryoticorganelles, magnetosomes. The magnetosomes have well-ordered
chain-like structures, comprising membrane-enveloped, nanosized magnetic crystals, and various types of speciﬁcally associatedproteins. In this study, we applied atomic force microscopy (AFM)
to investigate the spatial conﬁguration of isolated magnetosomesfrom Magnetospirillum magneticum AMB-1 in near-native bufferconditions. AFM observation revealed organic material with a ∼7nm thickness surrounding a magnetite crystal. Small globular proteins, identiﬁed as magnetosome-associated protein MamA, were
distributed on the mica surface around the magnetosome.
Immuno-labeling with AFM showed that MamA is located on themagnetosome surface. In vitro experiments showed that MamA
proteins interact with each other and form a high molecular masscomplex. These ﬁndings suggest that magnetosomes are covered
with MamA oligomers in near-native environments. Furthermore,
nanodissection revealed that magnetosomes are built with heterogeneous structures that comprise the organic layer. This study
provides important clues to the supramolecular architecture of
the bacterial organelle, the magnetosome, and insight into thefunction of the proteins localized in the organelle.

|magnetotactic bacteria scanning probe microscopy
TPR protein protein localization|| nanoparticle |Magnetosomes are unique prokaryotic organelles synthesized in magnetotactic bacteria, which function as a cellular compass to navigate along the Earth’s magnetic ﬁeld (1–4).
Proteomic analyses of the isolated magnetosomes indicate thatthe magnetosome contains various types of speciﬁc associatedproteins (5–7). Most of the magnetosome-associated proteins are
encoded in gene clusters within a genetic “magnetosome island,”
which is essential for the synthesis of magnetosomes (8–11).
These proteins are thought to function in magnetite biomineralization, magnetic sensing, formation of the magnetosome
vesicle, and construction of magnetosomal structures.
Insights into the magnetosome structure were provided usingtransmission electron microscopic (TEM) techniques such as
negative staining, freeze-etching, and cryo-electron microscopy(12–17). These studies demonstrated that magnetosomes arehighly ordered structures. Magnetosomes comprise a chain of
regular-sized bio-mineralized magnetite crystals, each of which issurrounded by a lipid bilayer membrane and organic components. Also, individual magnetosome particles are connected byinterparticle structures. Furthermore, most magnetosomes arearranged intimately along novel cytoskeletal ﬁlaments as visualized by cryo-electron tomography (14, 15, 18–20).
Although a number of important ﬁndings about the magnetosome structure have been provided by TEM, there are somedisadvantages associated with TEM techniques. TEM techniques
9382–9387 | PNAS | May 18, 2010 | vol. 107 | no. 20

require sample preparation methods such as ﬁxation, staining,dehydration, embedding, and thin sectioning, all of which may
potentially damage or alter the native structure of biologic
specimens. Cryo-electron microscopy does not have these disadvantages, and allows visualization of cellular structures ina near-native, frozen hydrated states. Using this method, 3- to 4nm resolution has been achieved for putative cytoskeletal ﬁlaments in magnetotactic bacteria (14). However, in most of theprecedent studies, extraction of ﬁne geometries is prevented forlow electron density materials in a crowded environment, such as
membrane-embedded proteins surrounded by lipid molecules.
This is probably because of the low electron dose that must be
used with frozen hydrated materials, which results in a low signal-to-noise ratio of the projection images (21). As a complementary technique, atomic force microscopy (AFM) has beenused to visualize organic samples ranging from single molecules
to living cells under physiologic conditions (22–24). In AFM, thesurface proﬁle of the sample is imaged by detecting the interaction between the sample and the AFM stylus during theraster scanning of the sample. With this imaging technique, biologic molecules can be visualized with a high signal-to-noise
ratio. Remarkably, AFM allows molecular resolution imaging oforganelles such as bacterial photosynthetic membranes (25) anddisk membranes (26). These AFM studies elucidated the organization of networks of constituent molecules in the native
membranes, which has been difﬁcult using other methods.
Of particular importance is the identiﬁcation of the proteinaceous supramolecular structure of the magnetosome. Because of AFM’s ability to visualize biologic specimens in theirnear-native conditions with a high signal-to-noise ratio, AFM canfeasibly be used to visualize the constitutions of submicron-sizedbacterial organelles at molecular resolution. Here, we applied
AFM to investigate the spatial conﬁguration of magnetosomesfrom Magnetospirillum magneticum AMB-1. AFM observations
indicated that the thickness of the organic layer wrapped aroundthe magnetite crystal was ∼7 nm, and magnetosome-associatedprotein MamA was localized at the surface of the organic layer.
In vitro experiments revealed that MamA proteins interact with

Author contributions: D.Y., A.T., T.A., and Y.F. designed research; D.Y., A.T., T.U., H.S., and
H.W. performed research; D.Y. and A.T. analyzed data; and D.Y., A.T., T.U., T.A., and Y.F.
wrote the paper.
The authors declare no conﬂict of interest.
This article is a PNAS Direct Submission. D.K.N. is a guest editor invited by the Editorial
Board.
1

D.Y. and A.T. contributed equally to this work.

2

To whom correspondence should be addressed. E-mail: fukumor@kenroku.kanazawa-u.
ac.jp.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1001870107/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1001870107

Results
Structure of the Puriﬁed Magnetosome. In the present study, hydrophilic bare mica and hydrophobilized mica were served as substrates for AFM observations. These surfaces have different
afﬁnities for the magnetosomes and magnetosome-associated proteins, as described below. Thus, we used both substrates dependingon the object of interest. Although magnetosomes were observed on
both substrates, magnetosomes were more efﬁciently attached tothe hydrophobilized mica surface than the bare mica surface.
Figure 1A shows an AFM image of the puriﬁed magnetosomes
adsorbed on the hydrophobilized mica. The chain-like structureof magnetosomes observed by AFM was consistent with thatobserved by TEM (16). To estimate the organic layer surrounding the magnetite crystals, the height of the magnetosomes
and the size of the magnetite crystals were measured verticallyalong the magnetosome chains using AFM and TEM, respectively. The height of each magnetosome particle was 60.8 ±7.1 nm (n = 404), whereas the crystal size of the magnetite was
46.9 ± 6.9 nm (n = 298) in diameter. This ﬁnding indicated thatthe individual magnetite crystal is surrounded with ∼7 nm of anelectron permeable layer composed of organic components.
Regular-sized globular particles were found to be dispersed on
the bare mica (Fig. 1B), whereas these particles were not observed
around magnetosomes on the hydrophobilized mica. Removal of
the particles could not be achieved by further puriﬁcation of themagnetosomes. Moreover, the particles were not observed
around the magnetosomes when the magnetosomes were chemically cross-linked with glutaraldehyde before deposition onto thebare mica. These results strongly suggest that the particles originated from the magnetosomes. Another architectural featureobserved by AFM was a sheet-like structure in the proximity of themagnetosomes (Fig. 1B, asterisk). This sheet-like structure wasobserved on both bare mica and hydrophobilized mica. The
thickness of the sheet-like structures was ≈3 nm.

Fig. 1. AFM observations of magnetosomes adsorbed on the mica surfaces.
(A) AFM image of the magnetosomes chain adsorbed on a hydrophobilized
mica surface. (B) AFM image of magnetosomes adsorbed on a bare mica
surface. Small particles (arrows) and a sheet-like structure (asterisk) wereobserved in the proximity of the magnetosomes. High-magniﬁcation (C)topographic and (D) phase contrast images of the magnetosome particles onhydrophobilized mica. AFM images were recorded at imaging rates of (A)2.1, (B) 1.0, and (C and D) 4.0 s/frame, and the number of pixels of (A and B)
256 × 256 and (C and D) 150 × 150.

Yamamoto et al.

The surface of the magnetosome was closely examined by simultaneously obtaining topographic and phase images (Fig. 1 C
and D). In the topographic image, detailed surface structures
were difﬁcult to visualize. In contrast, a clear contrast was
obtained in the phase image. The phase image showed texture
with granular and wrinkled lines on the magnetosome vesicle.
The phase contrast of AFM is strongly relevant to several surface
properties such as viscoelasticity, elasticity and surface adhesionenergy (27). The phase imaging mode allows one to visualize
compositional variation, even for the sample that the ﬁne
structures are difﬁcult to visualize in the topographic images.
Therefore, the phase contrast shown in Fig. 1D should represent
heterogeneity in the sample, and suggests that the outermost
layer of magnetosomes is formed by an amorphous layer ofmagnetosome-associated proteins.
Identiﬁcation of Globular Particles Observed on Bare Mica. To understand the origination of the small particles (Fig. 1B and Fig.
2A), we treated magnetosomes with alkaline buffer. As reported
previously, magnetosomal protein MamA (Mam22) and cytochrome cd1 are efﬁciently solubilized from magnetosomes by alkaline buffer (13). When the alkaline-treated magnetosomes were
loaded onto the bare mica, the small particles were not observed(Fig. 2B), whereas a number of particles were observed on thebare mica when the spent alkaline solution was used as a sample(Fig. 2C). In contrast, the sheet-like structures were not removedby the alkaline treatment (Fig. S1). Before the alkaline treatment,
the height distribution of the particles showed two clear peaks on
the histogram (Fig. 2E). Most of the particles were ∼3 nm inheight, and 6- to 8-nm particles were also detected. The meanheight of the particles solubilized from magnetosomes (Fig. 2F)
was in good agreement with the major distribution of the particles
observed before the alkaline treatment.
To identify the small particles, proteins attached to the baremica were analyzed. A 24-kDa protein band was detected by SDS/
PAGE analysis of samples extracted from the mica (Fig. 2H), andwas positively recognized by anti-MamA antibody (Fig. 2I). To
further examine the correlation of MamA protein with theglobular particles, we observed the puriﬁed magnetosomes fromthe ΔmamA mutant of M. magneticum AMB-1 (28). In this case,the number density of the particles observed on the bare micasigniﬁcantly reduced (Fig. 2 D and G). This clearly indicates thatmajority of the particles observed around magnetosomes on baremica are MamA molecules.
In addition to structural imaging, the AFM stylus can be used asa manipulator to dissect individual biologic samples (29, 30).
Figure 3 and the supplemental movie show high-speed AFM
images of the dissection process of the magnetosomes observed on
the bare mica. While the magnetosomes were being imaged, additional tapping force was applied (20–33 frames). The magnetosomes were removed by the scanning stylus. Consequently, sheetlike structures appeared at the initial position of the magnetosome(Fig. 3, asterisks). The appearance and thickness (3 nm) of thesesheets were consistent with those of the sheets observed in theproximity of magnetosomes (Fig. 1B). These sheets seem to belipid bilayers, based on their featureless surface structure andthickness that is comparable to the typical value of lipid membranes (3–4 nm) measured by x-ray scattering (31) and AFM (26).
Together with the sheet-like structures, additional small particles,
which were 3 nm in height, were observed around the magnetosomes after the dissection (Fig. 3, arrowheads). These ﬁndings
indicated that magnetosomes contain heterogeneous structuresthat should comprise the organic layer.
Oligomerization of MamA. MamA, one of the most abundant proteins in the magnetosome, contains ﬁve or six tetratrico-peptide
repeat (TPR) motifs (32, 33) that mediate the protein–protein
interactions to assemble the multiprotein complexes (34). TherePNAS | May 18, 2010 | vol. 107 | no. 20 | 9383

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each other to form a high–molecular-mass complex. Moreover,reconstruction experiment of MamA showed a possibility that
MamA may contributes to stabilize the magnetosome chain
structure as observed using AFM.

Fig. 2. Identiﬁcation of small globular proteins found on bare mica. AFM images of small particles observed on bare mica (A) before and (B) after alkaline
treatment of puriﬁed magnetosomes. (C) AFM micrograph of the particles removed from the puriﬁed magnetosomes with alkaline buffer. (D) AFM image ofparticles observed on bare mica around magnetosomes from ΔmamA mutant. (E) Histogram for heights of small particles observed around the magnetosomes. Black curve represents the ﬁt to the sum of two Gaussians (shown individually in white lines; 3.0 ± 0.6 nm and 6.6 ± 0.8 nm). (F) Histogram for heightsof the particles removed from magnetosomes by alkaline treatment. Curve represents the ﬁt to a Gaussian distribution (2.6 ± 0.6 nm). (G) Histogram forheights of particles observed around magnetosomes from ΔmamA mutant. (H) Silver-stained SDS/PAGE gel of magnetosomal protein absorbed on bare mica(lane 1), and proteins extracted from the puriﬁed magnetosomes (lane 2). Precision Plus protein standard was used (lane M). (I) Immunoblot analysis with antiMamA antibody. The proteins extracted from bare mica and magnetosomes were loaded on lanes 1 and 2, respectively. Magnetosomes were prepared fromwild-type M. magneticum AMB-1 except for D and G.

fore, MamA may function as a receptor for the protein–protein
interaction in magnetosomes. We examined the partner protein of
MamA using M. magnetotacticum MS-1. M. magnetotacticum MS-1
is very closely related with M. magneticum AMB-1. The amino acidsequences of M. magnetotacticum MamA (MamAMS-1: known as
Mam22: BAA11643) and M. magneticum AMB-1 MamA
(MamAAMB-1: known as Mms24: BAE49775) are identical.
For the isolation of MamA-associated proteins, the recombinant N-terminal his-tagged MamAMS-1 (His-MamA) was chemically conjugated with the resin to prepare an MamA-afﬁnity
column. We subjected solubilized magnetosome-associatedproteins from M. magnetotacticum MS-1 to the MamA-afﬁnity
column. MamA afﬁnity column chromatography showed that
one major protein band (23.6 kDa) and four minor protein bands(26.8 kDa, 31.6 kDa, 54.0 kDa, and 63.5 kDa) were eluted (Fig.
4A). The 23.6-kDa protein was identiﬁed to be MamAMS-1 byimmunoblotting (Fig. S2). Unfortunately, the N-terminal aminoacid sequences of the four minor protein bands were not determined because the amounts of these proteins were not sufﬁcient to analyze. To conﬁrm this result, a pull-down assay was
performed. MamAMS-1 was coprecipitated with His-MamA (Fig.
4B). These results indicated that MamA proteins interact witheach other and form an oligomeric complex.
We examined the oligomerization status of the recombinant
MamA. Puriﬁed His-MamA was separated by gel ﬁltration intoa single peak, which was estimated to be 560 kDa (Fig. S3). To
further analyze the oligomeric status of His-MamA, we examined the peak fractions of the gel ﬁltration by AFM. On the baremica, we observed small particles (∼3 nm in height), similar tothat shown in Fig. 2A, instead of a large complex (Fig. S4). In
contrast, His-MamA oligomers were visualized on aminosilanefunctionalized mica (AP-mica). The AFM image of oligomerized
His-MamA revealed a unique conﬁguration: a regular-sized,
rugged-shaped globular structure (Fig. 4 C and D). The size ofthe observed complex ranged from 4.5 to 6.5 nm in height and
14–20 nm in diameter. This agrees with the molecular mass estimated from the gel ﬁltration column chromatography.
Localization of MamA Protein Complexes in the Magnetosomes.

Immuno-labeling was performed to identify the location of
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MamAAMB-1 in the magnetosomes using AFM. Figure 5A shows an
AFM image of the magnetosomes labeled with anti-MamA antibodies. After labeling, antibodies bound densely to magnetosomes.
By contrast, preimmune serum, which has no signiﬁcant afﬁnity for
MamA, had no effect on the appearance of the magnetosomes(Fig. 5B). The dense packing of the antibodies on the magnetosomes indicated that a considerable amount of MamAAMB-1 waslocated at the outermost layer of the magnetosomes. The dimension of magnetosomes signiﬁcantly increased from 57.2 ± 7.8
nm (n = 25) to 72.7 ± 10.8 nm (n = 69) in height, and from 59.2 ±7.6 nm (n = 25) to 90.7 ± 15.8 nm (n = 69) in width after labelingwith anti-MamA antibody (Fig. 5 C and D). The height difference
between labeled and nonlabeled magnetosomes (15 nm) was ingood agreement with the diameter of the antibody (35).
A previous study has indicated that the recombinant HisMamA can attach to the MamA-eliminated magnetosomes
prepared by the alkaline treatment (13). To elucidate the location of reconstructed His-MamA in the magnetosomes, immunolabeling was performed for both alkaline-treated and MamAreconstructed magnetosomes. The anti-MamA antibody failed tobind to the alkaline-treated magnetosomes (Fig. 5E). This shows
the depletion of MamAAMB-1 from the magnetosomes by thetreatment. After the reconstruction of the His-MamA to thealkaline-treated magnetosomes, antibodies densely bound tomagnetosomes (Fig. 5F). The appearance of the immuno-labeled
His-MamA-reconstructed magnetosomes was very similar to that

Fig. 3. Dissection of magnetosomes adsorbed on bare mica. The surface wasscanned at 1.0 s/frame with the number of pixels of 256 × 256. This nanodissection treatment exposed the underlying sheet-like structure (asterisks).
Also, small particles (which were measured ∼3 nm in height) appeared on themica surface (arrowheads). Numbers indicate frame number.

Yamamoto et al.

Fig. 4. Oligomerization of MamA. (A) SDS/PAGE analysis of binding magnetosomal proteins on the His-MamA afﬁnity column. Protein bands were visualized by silver staining. Lane 1, solubilized magnetosome-associated proteins.
Lane 2, eluted proteins from BSA column. Lane 3, eluted proteins from HisMamA afﬁnity column. The 24-kDa protein (arrow) was identiﬁed as MamAMS-1
by immunoblotting. Arrowhead shows that the His-MamA came off from thecolumn. (B) Pull-down assays. MamAMS-1 (arrow) was coprecipitated from solubilized magnetosome-associated proteins with His-MamA (arrowhead) binding to Ni-NTA resin. Gel was stained with Coomassie Brilliant Blue G-250. (C)AFM image of His-MamA oligomer on AP-mica. (D) High-magniﬁcation imageof His-MamA oligomer on AP-mica. AFM images were recorded at an imaging
rate of 1 s/frame; the number of pixels was 256 × 256.

of the untreated magnetosomes. This result suggests that theendogenous MamAAMB-1 and the recombinant His-MamA share
the binding site on the magnetosomes.
Effect of MamA Elimination on the Chain Structure. To examine theeffect of MamA elimination on the chain structure of magnetosomes, spacing between magnetosome particles was compared
between the intact magnetosomes and MamA-eliminated magnetosomes prepared by alkaline treatment (Fig. 6A). The aver-Fig. 5. AFM images of immuno-labeled magnetosomes. Magnetosomeslabeled with (A) anti-MamA antibodies and (B) preimmuno serum. (C and D)
Surface proﬁle along the lines indicated in A and B. Magnetosomes labeledwith anti-MamA antibodies after (E) alkaline treatment and (F) His-MamA
reconstruction. AFM images were recorded at an imaging rate of 3 s/frame;
number of pixels was (A and B) 200 × 200 and (E and F) 256 × 256.

Yamamoto et al.

Discussion
The aim of the present study is to characterize the structures andcompositional organization of magnetosomes in an aqueous
environment using AFM. The spatial localization, supramolecular organization, and functions of the individual components
within the magnetosome must be determined to understand how
this bacterial organelle functions as a magnetic compass. To
date, AFM visualization of prokaryotic intramembrane structures at a spatial resolution close to 1 nm has been achieved for
ﬂat membranes such as purple membrane (36), chromatophore(25, 37), and outer membrane (38). It remains challenging,
however, to obtain high-resolution images for a whole organellethat contains various molecular species and has a complex 3D
structure. Here, we visualized the near-native hemisphericalconﬁguration of the isolated magnetosomes (Fig. 1). In the phase
image, we were able to discern the surface structure at a lateral
resolution of 4–8 nm, which should represent the molecular organization at the surface of the magnetosomes. Although thisresolution is insufﬁcient to determine the precise molecular organization, the lateral resolution obtained in this study is the best
achieved so far for magnetosomal membrane in buffer condition.
The AFM showed that the magnetosome was ∼61 nm inheight. On the other hand, the crystal size of the magnetite was∼47 nm in diameter. Therefore, the thickness of the electron
permeable organic layer was calculated to be 7 nm. Althoughmagnetite crystal is enveloped by a lipid membrane, the thickness of the organic layer is signiﬁcantly larger than single bilayer
membrane. This means that the magnetic particle is surroundedby other organic components that may be composed of magnetosome-associated proteins. A previous TEM observation has
identiﬁed the organic layer, termed the magnetosomal matrix(13). Based on the TEM observation, the magnetosomal matrix
spread around the magnetosome vesicles several tens of nanometers in width. Instead of this huge structure, our AFM study
revealed a thin organic layer. The possible reason for this difference is the variation in surface properties of the substrates orthe imaging conditions between AFM and TEM.
The previous TEM observation has revealed also a ﬁbroustexture that connects the ﬂanking magnetosome particles (13). In
the present AFM study, however, this structure was not observedin the magnetosome chains because of the difﬁculty of AFM to
precisely trace deep features. To proﬁle surface morphologies innarrow spaces, the AFM stylus must have both a high aspectratio and a small apex radius. Otherwise, the apex of the AFM
stylus cannot access the ﬁne structures at the bottom of thetrough. Although our AFM styli were sufﬁciently sharp to visualize the structure of the magnetosomes, an extremely high aspect ratio will be needed to deﬁne the interparticle connection.
As shown in Fig. 4 C and D, we were able to visualize HisMamA oligomers on the AP-mica, whereas small particles 3 nm
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aged center-to-center distance between the adjacent particles in
the intact magnetosomes (59.4 ± 6.2 nm, n = 364) was consistent
with the spacing between magnetite crystals observed in cell using
cryo-TEM (28). Interestingly, the averaged distance between theparticles of alkaline-treated magnetosomes signiﬁcantly increased(P < 0.0001, estimated using F test) by 3–4 nm (62.8 ± 7.8 nm,
n = 336). On the other hand, the alkaline treatment had no
signiﬁcant effect on the distance between the magnetosomes
puriﬁed from ΔmamA mutant (Fig. 6B). When the His-MamA
was rebound to the alkaline-treated magnetosomes, the spacing
between the magnetosome particles decreased (58.5 ± 5.8 nm[n = 172]) to the value consistent with that of the untreatedmagnetosomes (Fig. 6A). The interparticle spacings of the untreated magnetosomes and the MamA reconstructed magnetosomes showed no signiﬁcant difference (P = 0.35, estimated using
F test). These results indicate that MamA have an effect on the
distance between magnetosomes.

Fig. 6. Histogram of center-to-center distance between adjacent magnetosomes. (A) From wild type: intact; magenta,
alkaline treated; green, MamA reconstructed; cyan. (B) From
ΔmamA mutant: intact, magenta; alkaline-treated, green.
Curves indicate ﬁt to the Gaussian distributions.

in height instead of large complexes were observed on the baremica (Fig. S4). This may be due to differences in the interaction
between the proteins and substrates. In addition to the particles
3 nm in height, the particles 6–8 nm in height were also observedon the bare mica (Fig. 2E). These 6- to 8-nm particles are attributed to partially deoligomerized MamA complex, because
they were not observed around the magnetosomes from mamA
mutant (Fig. 2G). Moreover, these particles were not observed
when the magnetosome was chemically ﬁxed before depositing
onto the bare mica. Therefore, the MamA proteins easily detached from magnetosomes in buffer conditions, indicating that
MamA was loosely bound by the magnetosomes.
The most striking ﬁnding in this study was that the magnetosome vesicles were surrounded by MamA protein. The subcellular localization of MamA has been previously demonstrated.
MamA-green ﬂuorescent protein was observed to localize asa patchy line within the cell (3, 28). Also, immunogold staining
with TEM showed that MamA associates with the magnetosomal
matrix (13). In this study, the AFM imaging of the immunolabeled magnetosomes (Fig. 5) clearly indicated that MamA was
located at the surface of the organelle. The close packing of theanti-MamA antibodies on the magnetosomes indicated that
MamA protein densely covers the entire outer surface of themagnetosome chain. As described above, the thickness of theorganic layer covering the magnetite is 7 nm. This value approximately coincides with the sum of the thickness of the bilayer
lipid membrane and the height of the MamA oligomer. Thisﬁnding supports the view that the magnetosome membrane
vesicle is coated with MamA oligomers. Although we attempted
to visualize MamA complexes on the surface of magnetosomes
using bare mica and AP-mica as the substrates, we could not
identify individual MamA oligomers on the magnetosome. Thisis likely due to the texture of the magnetosome surface, which isamorphous and closely packed with various types of protein.
The TPR protein MamA most probably functions as a receptor
that interacts with a partner protein in the magnetosome. Our
results showed that MamA interacts with MamA itself to form
oligomer (Fig. 4), and binds to the surface of magnetosomes(Fig. 5). In addition, MamA further interacts with unidentiﬁedproteins that were extracted from the magnetosomes (Fig. 4A).
These results suggest that MamA oligomers are anchored on themagnetosome membrane through magnetosome membraneassociated proteins, and partially through lipids of the magnetosome membrane. Because MamA is abundant relative to othermagnetosome-associated proteins, MamA oligomers would besparsely bound by the anchor proteins in the organic layer.
A previous study on ΔmamA mutant showed that the cells
produce regular number of magnetosome vesicles. However, not
all these vesicles are functional for the production of magnetite.
Based on these observations, Komeili et al. proposed that MamA
is part of the magnetosome assembly and maintenance processes
such as protein sorting or activation of the magnetosome vesicles
in response to external signals (28). In this study, we presented
that MamA is located at the outermost layer of magnetosomes.
With this spatial conﬁguration of MamA in the magnetosomes, it
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is possible that MamA act as a scaffold that links between themagnetosome vesicles and cytoplasmic factors that activate themagnetite formation. Although our study showed a possibility
that MamA contributes to the stabilization of magnetosome
chain (Fig. 6), it is unclear how this stabilizing effect associateswith magnetosome formation processes in vivo. Further studies
on molecular assembly and function of MamA would expand ourunderstanding of magnetosome formation.
It is now clear that bacteria are highly organized, possessing
cytoskeletons, internal compartments, and carefully positioned
macromolecular machines. To understand how they are organized and express their function, it is essential to unveil theultrastructures under near-native conditions. Here, we visualized
one of the most complex bacterial organelles, the magnetosome,in near-native conditions. To this end, AFM-based techniquessuch as immuno-labeling and nanodissection procedures are
powerful approaches, as evidenced in this study. Combined withthe possibility to reveal surface structures with high lateral resolution, AFM will exploit this unique avenue for the investigation of the ultrastructures of prokaryotic organelles.
Materials and Methods
Microorganisms and Cultures. M. magneticum AMB-1 (ATCC 700264), mamAdeletion mutant of AMB-1 (28), and M. magnetotacticum MS-1 (ATCC 31632)
were cultured in a liquid media under an O2 (1%)–N2 (99%) atmosphere at
25 °C in the dark (39). Escherichia coli strain BL21(DE3) (Novagen), containing
pET15b-mam22 (32), was used for overproduction of His-tagged MamA.
E. coli was cultivated as described (13).
Puriﬁcation of Recombinant His-MamA. His-MamA was puriﬁed as previously
described (32). Puriﬁed His-MamA was subjected to gel ﬁltration column chromatography (Sephacryl S-300, GE Healthcare). The apparent molecular mass wascalculated using a Gel Filtration Calibration Kit (GE Healthcare) as a standard.
Magnetosome Preparation. Magnetosomes were puriﬁed as previously described (13) and used immediately or stored at 4 °C without freezing. Alkaline treatment of the puriﬁed magnetosomes with 0.1 M Caps–NaOH
buffer (pH 11.0) was performed as described elsewhere (13). TEM observation of the puriﬁed magnetosomes was performed using a JEOL JEM 2000EX
TEM operating at 120 kV in bright-ﬁeld mode. For reconstruction with HisMamA, the alkaline treated magnetosomes (3 mg wet weight) were incubated with the His-MamA (20 μM) in 200 μL of 10 mM Tris·HCl (pH 8.0) at
25 °C for 16 h, and then centrifuged at 8,000 × g for 5 min. The pellets
obtained were washed with 1 mL of 10 mM Tris·HCl buffer (pH 8.0) andcentrifuged at 8,000 × g for 5 min. The supernatant containing the unboundHis-tagged His-MamA was removed by aspiration and resuspended in 10 mM
Tris·HCl buffer (pH 8.0). This washing step was repeated three times.
Atomic Force Microscopy. Imaging was performed with a laboratory-built highspeed AFM, an extensively improved version of the previously reported AFM(40, 41). The high-speed AFM was equipped with small cantilevers (k = 0.1–
0.2 N/m, f = 800–1,200 kHz in water) and operated in tapping mode. A lock-in
ampliﬁer (SR844-RF, Stanford Research Systems) was used to detect the phasedifference between the cantilever oscillation and the excitation signal. The
AFM styli were grown on each cantilever by electron beam deposition. Freshly
cleaved mica, AP-mica, and hydrophobilized mica were used as substrates. APmica was prepared by depositing 0.05% 3-aminopropyltriethoxysilane (ShinEtsu Chemical) on freshly cleaved mica and left for 3 min. Hydrophobilizationof mica was performed using a vapor deposition method, in which hexame-

Yamamoto et al.

Identiﬁcation of Mica Binding Protein. The magnetosome suspension (OD600nm= 7) was loaded onto a bare mica (76 × 26 mm), which was ﬁxed on slide
glass with double-stick tape and then incubated for 3 min. After the mica
was washed three times with 10 mM Tris·HCl (pH 8.0), the mica was sonicated with an ultrasonic oscillator (Branson model 450; 20 kHz 10 W) toremove bound magnetosomes from the mica. AFM conﬁrmed that only
a few magnetosomes were present on the mica surface, and a large amountof the globular particles and a small amount of sheet-like structures
remained on the mica. The resulting mica was incubated with 2% SDS
containing 10 mM Tris·HCl (pH 8.0). Based on AFM observation, most of theparticles were removed from the mica surface by SDS treatment. The proteins extracted from the mica were analyzed by SDS/PAGE.
MamA-afﬁnity Chromatography. To prepare His-MamA afﬁnity resin, 1 mL of
CNBr-activated Sepharose 4B (GE Healthcare) was coupled with the puriﬁed HisMamA (1.2 mg). The His-MamA resin column (0.5 × 4 cm) was equilibrated with
10 mM Tris·HCl (pH 8.0) containing 0.1% sucrose monocaprate (equilibrationbuffer). To solubilize magnetosome-associated proteins, the magnetosomes

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Yamamoto et al.

puriﬁed from M. magnetotacticum MS-1 (∼0.6 g, wet weight) were incubatedwith 10 mL of 10 mM Tris·HCl (pH 8.0) containing 2% sucrose monocaprate at
4 °C for 16 h. The suspension was then centrifuged at 10,000 × g for 15 min
at 4 °C. After the resultant supernatant was dialyzed against the equilibration buffer, the protein solution (12 mL) was subjected to the His-MamAcolumn with a ﬂow rate of 1 mL/h. After that, the column was washed withthe equilibration buffer containing 1 M NaCl. Then the binding proteins were
eluted with 0.1 M Caps-NaOH buffer (pH 11.0) containing 0.1% sucrose monocaprate from the column.
Pull-Down Assay. The solution of the His-MamA (50 μL, 1 mg/mL) was mixed
with 25 μL of the Ni-NTA resin (Qiagen), which had been equilibrated withthe pull-down buffer (20 mM Tris·HCl [pH 8.0], 0.2 M NaCl, 5 mM imidazole).
The Ni-NTA resin with His-MamA was incubated with 350 μL of the solubilized magnetosome-associated proteins from M. magnetotacticum MS-1
(0.14 mg/mL), in pull-down buffer at 25 °C for 1 h. After the resin waswashed three times with 1 mL of the washing buffer (20 mM Tris·HCl, pH 8.0,
0.2 M NaCl, 60 mM imidazole), the binding protein was eluted from the resinwith 30 μL of elution buffer (20 mM Tris·HCl, pH 8.0, 0.2 M NaCl, 250 mM
imidazole) and analyzed by SDS/PAGE.
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BSA asastandard.SDS/PAGEwasperformedaccording tothemethodofLaemmli(42). Immunoblotting analysis was performed as described elsewhere (13).
ACKNOWLEDGMENTS. We thank A. Komeili (University of California, Berkeley,CA) for providing the mamA deletion mutant of M. magneticum AMB-1.

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PNAS | May 18, 2010 | vol. 107 | no. 20 | 9387

MICROBIOLOGY

thyldisilazane (Shin-Etsu Chemical) and the freshly cleaved mica were placed
simultaneously in a sealed container and incubated at 60 °C for 30 min. The
puriﬁed magnetosomes (OD600nm= 7) were adsorbed on the substrates in
10 mM Tris·HCl (pH 8.0). After 3 min, the sample was rinsed with 10 mM Tris·HCl(pH 8.0). For chemical ﬁxation, the sample was incubated with 1% glutaraldehyde for 3 min. For immuno-labeling of MamA in the puriﬁed magnetosomes, the magnetosomes were adsorbed on the hydrophobilized mica andthen incubated with 1% BSA in PBS for 5 min. Subsequently, the sample wasincubated with anti-MamA rabbit polyclonal antibodies or preimmuno serum
as described (13). After rinsing with PBS two times for 1 min each, the specimens were chemically ﬁxed with 1% glutaraldehyde for 3 min in PBS.