Download Microscopy studies on uncultivated magnetotactic bacteria

Modern Research and Educational Topics in Microscopy.
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A. Méndez-Vilas and J. Díaz (Eds.)
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Microscopy studies on uncultivated magnetotactic bacteria
T. S. Silveira, J. L. Martins, K. T. Silva, F. Abreu & U. Lins∗
Instituto de Microbiologia Professor Paulo de Góes, Universidade Federal do Rio de Janeiro, 21941-590,
Rio de Janeiro, RJ, Brazil
Magnetotactic bacteria are characterized by the magnetosome, a membrane-bound, nano-sized organelle
containing a magnetic crystal. This organelle is believed to help the bacteria to navigate along the
geomagnetic field lines and find a preferred habitat for survival. The magnetosome is a biomineral
structure highly controlled by the bacteria, composed of either magnetite (Fe3O4) or greigite (Fe3S4), and
arranged in one or more linear chains in the cell. Most magnetotactic bacteria are not cultivated and
studies rely on environmental samples, where microscopy techniques are powerful tools for the structural
characterization of the bacteria and their magnetosomes. Here, we show examples of the application of
microscopy techniques to the study of uncultivated magnetotactic bacteria.
Keywords magnetosome; magnetotactic bacteria; magnetotaxis; biominerals.
1. Introduction
Magnetotactic bacteria (MB) are prokaryotes that align and migrate along the lines of a magnetic field.
All MB are associated to the domain Bacteria, are gram-negative and motile by flagella. MB generally
live in aquatic habitats and respire microaerobically or anaerobically. The hallmark of all magnetotactic
bacteria is the unique ability to produce magnetosomes. Magnetosomes are organelles that consist of a
magnetic nanoparticle enveloped by a membrane. Each MB species or strain can produce only one type
of magnetosome of a specific shape, size and composition [1] with very few exceptions [2, 3]. The
production of magnetosomes is encoded and regulated at gene level [1]. The magnetosome membrane is
supposed to originate from the cytoplasmic membrane based on its lipid and protein composition [4] at
least in the cultivated Magnetospirillum species [5]. Some recent studies suggest they are structurally
contiguous with the plasma membrane, like invaginations, in Magnetospirillum magneticum [6]. The
magnetosome membrane contains exclusive proteins that are thought to have a crucial importance in the
magnetic particle nucleation, growth and arrangement [7]. Advances in this subject depend on the
establishment of a genetic and biochemical model, which is gradually been achieved with the increasing
characterization of some cultivated species.
Two types of magnetic minerals are found in magnetosomes: the iron oxide magnetite (Fe3O4) and the
iron sulfide greigite (Fe3S4). The crystalline part of the magnetosome has been extensively studied with
microscopy, especially high resolution transmission electron microscopy (HRTEM). Bacterial magnetite
typically has cube {100}, octahedron {111}, and dodecahedron {110} faces in crystals that are
commonly elongated [8] or bullet-shaped [9, 10, 11]. In contrast most greigite magnetosomes occur in
parallelepiped or cuboctahedral with irregular border [12]. A good review on magnetosome
crystallographic properties can be found in [13]. Other non-magnetic minerals, like tetragonal or cubic
FeS, were reported as precursors of greigite [14]. Magnetite-containing MB can be collected in
freshwater, brackish or marine environments, whereas greigite-containing MB seems to be restricted to
anoxic environments [14]. The number of magnetosomes varies from a few to thousands per cell [15].
The magnetosomes are generally in the range of 35-120 nm [1], which corresponds to the single-domain
size range for magnetite or greigite. However, magnetosomes up to 250 nm were reported [15, 16].
∗
Corresponding author: e-mail: [email protected], Phone: +55212562-6738
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The magnetosomes form chains within the cell, which impart the cell body a magnetic moment that
overcomes the viscous drag of the environment where the bacteria live. This allows the efficient
orientation of the cell to the magnetic field. When magnetosomes are arranged in a linear chain within
the bacterial cell, the total magnetic moment is the highest for that assembly [17, 18]. Biophysical
arguments [19] indicate that supporting structures are necessary to prevent the chains from collapsing
into a disordered clump of particles and a cytoskeleton connection between the cell envelope and the
magnetosome chains was proposed for the efficient transfer of torque to the cell body [20, 21]. There are
at least two hypotheses to explain how the magnetic torque is transmitted from magnetosomes to the cell.
In cocci, the magnetotactic repulsion between two or more magnetosome chains drives them apart from
each other. As a consequence, the chains touch the cytoplasmic membrane. In this case, the torque
generated by a situation of field modification is efficiently transferred from the chain to the whole cell by
mechanical contact [22, 23]. The second hypothesis proposes the existence of specific molecules to
anchor magnetosomes in the proper place in the cell [20, 21]. Recently evidences of the presence of such
molecules have been described in Magnetospirillum species [6, 24, 25].
The model of passive orientation of MB along the lines of a magnetic field combined with the active
migration because of the flagella, the so-called magnetotaxis, is maintained until today. Magnetotaxis is
considered an advantage in finding the optimum position in the environment because MB would have the
geomagnetic poles as fixed references to swim, unlike other bacteria. So, if a magnetotactic bacterium is
moved away from its niche, it would use the lines of a magnetic field to return to its preferred habitat,
whereas other bacteria would randomly swim until it reached the habitat, thus, expending more time and,
probably, more energy. A major improvement in the magnetotaxis model was the combination of
magnetotaxis and aerotaxis, the so-called magneto-aerotaxis. Based on experimental findings of MC-1, a
cultivated coccus [26], the magneto-aerotaxis model states that MB can be either in an oxidized or
reduced state. Considering that MB are in a vertical gradient of oxygen, with the saturated O2 waters in
the upper layers, they swim downwards to reach an optimum position (remember: MB are
microaerophiles or anaerobes). MB do it by rotating their flagella counterclockwise, because they are in
the oxidized state. However in the reduced state, their flagella would rotate clockwise and the bacteria
should move upwards, towards the preferred oxygenated conditions. In this model, MB still benefits
from having a fixed axis for swimming and do not need to “run and tumble” as non-magnetotactic
bacteria. There is evidence that magneto-aerotaxis cannot explain the behavior of all MB populations
[27], but other models to extend or substitute magneto-aerotaxis were not suggested yet.
The vertical chemical gradient used in experiments for the magneto-aerotaxis was verified in some
MB natural habitats, generally associated to an opposite sulfide gradient. Carefull measurements showed
that MB occur in the oxic-anoxic interface (OAI) or slightly above or below this transition zone [28, 29].
The OAI can be established in the water column or in the first centimeters of the sediment. Iron gradients
can also influence their distribution and peaks of dissolved and particulated iron can be related to their
presence [28]. Other gradients are not known to influence MB local distribution, but sources of carbon
are thought to be important to their positioning [30]. Cultivated strains indicate that short chain fatty
acids, tricarboxylic acid cycle intermediates and acetate, can be used as carbon sources. These molecules
are typical products of fermentation and are generally present in the suboxic zone [30].
MB are distributed worldwide and the most common morphotypes are spirilla, cocci, rods, vibrios and
the unusual multicellular form, the multicellular magnetotactic prokaryote (MMP). MB differ in flagella
arrangement, placed monotrichously or lophotrichously in the cells; cytoplasmic contents, like carbon,
sulfur or phosphorous inclusions; and magnetosome number, shape, size and composition. There are
sampling sites where only one morphotype can be recovered [29, 31] but this is not common. There is no
consistent relationship among a morphotype, a phylotype and/or the mineral produced. MB are related to
the alphaproteobacteria, such as the genus Magnetospirillum, MC-1 and MV-1 [32]; others are related to
the deltaproteobacteria, like some uncultured multicellular forms, the recently named Candidatus
Magnetoglobus multicellularis [29] and Desulfovibrio magneticus; an uncultured rod is affiliated to
gammaproteobacteria group [33]; and the Nitrospira phylum which includes an uncultured MB, the
tentatively named “Magnetobacterium bavaricum” [15]. Few MB were phylogenetically studied so far
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and not even a fraction of their potential habitats was explored. Therefore, the phylogenetic diversity is
largely unknown.
As few species or strains can be cultured nowadays, most of the studies rely on uncultured MB. But
how to detect and recognize MB in these samples? Although simple, the question prompts for criteria to
identify the magnetotactic behavior and to distinguish the magnetosomes from other commonly found
inclusion in prokaryotes. Because the currently available culture media are not selective enough to
guarantee retrieving only magnetotactic bacteria, light and electron microscopy are still the most
important and straightforward culture-independent techniques to determine if a bacterium is
magnetotactic. Here, we discuss some uses of microscopy to detect uncultured MB. Two examples of
MB are illustrated.
2. Light microscopy
Light microscopy is the most used technique to detect MB in environmental samples. Water and
sediment samples can be observed between a slide and coverslip or in a hanging drop. If MB are
expected to be in the sediment, water and sediment from the sampling site is placed on a slide and
observed preferably with a long working distance objective. At that time, one must look for swimming
bacteria and response to magnetic field. The discovery of MB by R. Blakemore was based on the
observation of the motility of bacteria in the local magnetic field of his laboratory [34]. Nowadays, an
ordinary magnet is placed near the slide for few minutes. The light microscopy observation leads to a
first recognition of MB and their magneto-aerotactic behavior. Under a magnetic field lines, most
bacteria will accumulate at one edge of the drop and the bacteria are said to have polar magnetotactic
behaviour. If the magnetic field lines define only the axis of swimming, changes in the magnetic field
direction will cause bacteria only to rotate, not accumulate at the edge of the drop [26]. This behaviour is
known as axial magnetotaxis and it is far less common than polar magnetotaxis [26].
Light microscopy lacks the resolution to individualize each magnetosome, but an accurate observation
in bright field can reveal the magnetosome chain as a dark dot or a small segment within the cells (Fig.
1a). Some MB contain other inclusions that can be seen by light microscopy. Sulphur inclusions appear
as birefringent spheres when imaged with phase contrast microscopy and colourless inclusions are easily
observed by differential interferential contrast. Flagella bundles can be observed with dark field
microscopy. Fluorescence in situ hybridization (FISH) has been used to study the population structure
and function. The rRNA-targeted probes used in this method allow the identification and quantification
of bacteria [35]. The association of FISH with fluorescence microscopy is frequently used to access the
phylogenetic relationships between MB because it overcomes the difficulty of cultivation and confirms
the origin of the sequences retrieved from magnetic enrichments (Figs. 2b-d). Individual magnetosome
chains were observed by reflectance confocal laser scanning microscopy [36]. This technique allows the
rapid detection and location of magnetosomes even in small quantities of material. Details of
magnetosome size or shape cannot be observed with this technique. Confocal laser scanning microscopy
was also used to show the arrangement of living cells in “Candidatus M. multicelularis” using a
fluorescent lipophilic dye [37]. It has also been used to demonstrate that all cells within a MMP belong
to the same species [38] and to measure the acidic nature of grazing protozoa that ingested MB [39].
3. Transmission electron microscopy
Transmission electron microscope (TEM) is the technique of choice for observing individual
magnetosomes and to absolutely classify a bacterium as capable of producing magnetosomes. The
simplest preparation method consists of placing a suspension of bacteria on a Formvar or carbon coated
grid and checking for magnetosome positioning in chains within the cell (Fig. 3a). Magnetosomes can
occur in single, double or multiple chains, peripherally or along the major axis of the cell. Clusters of
magnetosomes arranged in a disordered pattern are occasionally observed. This configuration is
interpreted as a consequence of the disorganization caused by the loss of chain stability. The
magnetosome shape can be determined on TEM whole-mount cell preparation or ultra-thin sections.
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TEM also allows counting and measuring of the magnetosomes. Magnetosome size distribution is used
as a marker of the biological origin of the crystals [40]. Other intracellular structures such as carbon,
sulphur, or phosphorous inclusions are observed [41]. Electron-dense inclusions can be sometimes
difficult to distinguish from magnetosomes, because of the similar morphology and size. X-ray
microanalysis is the first approach to elucidate the composition of the mineral phase of magnetosomes
and to check for the purity of the crystal. A typical spectrum shows peaks for oxygen and iron or sulphur
and iron, which indicates that magnetite (Fe3O4) or greigite (Fe3S4), respectively, are present (Fig. 3b). A
conclusive classification needs electron diffraction analysis or HRTEM, two techniques that can
differentiate among minerals and their possible precursors. Recently, several authors claimed to have
isolated new strains of MB [42, 43]. However, the authors fail to demonstrate that the electron-dense
structures are magnetosomes. The individual “crystal” morphology is difficult to see and unusually
irregular for magnetosomes. The irregular inclusions within the cytoplasm of the isolated strains are
more likely amorphous inclusions bodies, instead of magnetosomes, which are not irregular particles, but
ordered crystalline nanostructures. Even greigite pleiomorphic magnetosomes show a high degree of
structural organization. As no crystallographic data on the “magnetosomes” (electron diffraction or
HRTEM) is presented it is difficult to conclude that the newly described structures are magnetosomes.
Recently, non-crystalline magnetic inclusions were reported in prokaryotic cells [44], but they cannot be
considered true magnetosomes.
Negative staining contributes to the observation of flagella (Fig. 2i), which are fragile structures
frequently lost during simple preparation, probably because of mechanical stress. Differently from other
bacteria, MB produce short flagella, with filaments less than one helix turn [45]. The short flagella force
the bacterium body to rotate around its axis while it swims. Under the geomagnetic field, the beating of
the short flagella causes a misalignment of the trajectories [46] that enables the cell to change the
trajectory direction if it hits an obstacle in the sediment.
Ultra-thin sections are used to observe distinct internal details of the cell, including magnetosomes.
The magnetosome membrane is recognized to have a critical importance in biomineralization [1].
However, a trilaminar appearance of membranes, typical of osmium stained preparations, is difficult to
be visualized, mainly because of the diffraction conditions of the crystal. Not all magnetosomes are
believed to be enveloped by lipid membranes, but protein [22] or a matrix of unknown composition [47].
Freeze-fracture and freeze-etching have been used to study the properties of the magnetosome
membranes in cultivated species [24] and the surface organization of the cells [48, 49]. Structures
connecting the cell surface of Magnetospirillum cells to the magnetosome chains [24] as well as
numerous membranes vesicles were described with these cryotechniques [48]. Uncultured magnetotactic
cocci have a complex surface composed of microdomains of proteins (S-layers) which covers the cell
(Fig. 1e) and the flagella tufts were observed to emerge from a depression in the cell surface [49].
The association of gold labelling and phylogenetic affiliation with in situ hybridization in TEM
identified uncultured cocci isolated from Itaipu lagoon, in Brazil (Fig. 1f). Polyribonucleotides probes
were labelled with digoxigenin or fluorescein and detected by immunolabelling with anti-fluorescein and
anti-digoxigenin antibodies labelled with 10 and 15 nm gold-particles. This is possibly the only
technique that can associate the phylogenetic positioning of a bacterium with its magnetosome
morphology [50].
4. Scanning electron microscopy
Scanning electron microscopy (SEM) is a powerful technique for studying biomineralization products.
Their surface and crystallographic features can be studied. In unicellular MB, SEM has limited use as
most bacteria are morphologically simple. An exception is the MMP which is morphologically unique
among prokaryotes and can be easily distinguished in a sample because of its “mulberry” appearance in
SEM (Figs 2e-g) and in light microscopy (Fig. 2a). Estimated cell numbers and volumes were key
parameters for suggesting an exclusively multicellular life cycle for this microorganism and SEM images
greatly illustrated this hypothesis [31].
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Isolated magnetosomes have been imaged with field emission scanning electron microscopy (Fig. 1d)
and the results compared to scanning probe microscopy [51] and energy-filtered TEM of platinum
shadowed isolated crystals [52]. All techniques are useful for studying the surface of magnetosome and
other biominerals. Depending on the treatment used to isolate the magnetosomes, some organic material
remains (probably membrane proteins and lipids) attached to the crystals and the surfaces seem irregular.
5. Illustrated examples
5.1 Cocci from Itaipu lagon
Itaipu Lagoon (43o04’W, 22o57’S), is located on the coast of Brazil north of Rio de Janeiro. This lagoon
communicates with the sea through an artificial channel. At least four coccoid morphotypes with
magnetite magnetosomes, Itaipu-1, -2, -3 and -4 [50], a rarely observed rod-shaped bacterium [53], and
MMPs [3], occur in the lagoon. Itaipu-1, the largest coccoid microorganism, contains two chains of
magnetosomes. The magnetosome crystals are the largest-volume magnetosome crystals yet reported
with lengths up to 250 nm and width-to-length ratios of about 0.9 [16]. Itaipu-2 and -4 are smaller cocci
containing magnetosome crystals that are smaller, but with similar projected shapes to those in Itaipu-1.
Itaipu-3 has magnetosome crystals with lengths up to 120 nm and prominent corner facets. Whereas
magnetosomes in most magnetotactic bacteria, including Itaipu-3, are permanent single-magnetic
domains, it has been previously reported that the large magnetosomes in Itaipu-1 are metastable, singlemagnetic domains [18]. TEM microdiffraction combined with HRTEM was used to provide information
on the crystallography of individual magnetosomes isolated from the cells [16]. It is remarkable that the
crystals in the chain of Itaipu-1 remain in a chainlike disposition after isolation (Fig. 1c). The crystals
align with their [111] elongation axes parallel to the chain direction and all in the [1-10] zone, and are
also ordered with like corner faces of adjacent crystals facing each other [16]. This particular feature may
be used as a potential biomarker for magnetite formation in MB.
Electron spectroscopic imaging was used to directly image magnetosome size and distribution within
Itaipu cocci (Fig 1b) as well as other uncultured bacteria [9]. With this technique, the magnetosome
arrangement and the disposition of flagella were associated with unique clarity. Also, the magnetosome
substructure was analyzed. Distortions in magnetosomes caused by strong strain magnetic fields and
twinned crystals were also observed [9]. Electron spectroscopic imaging is a powerful and efficient tool
for studying the diversity of MB and magnetosomes biomineralization because of the minimal
requirements for specimen preparation, and because of the acquisition of detailed information through
the simultaneous visualization of bacterial morphology, crystal morphology, and chain alignment. This
would be difficult to be achieved, at least with the same efforts, by other microscopic techniques. One
major disadvantage is that it consists of a rather specialized technique which requires sophisticated
instrumentation. Electron spectroscopic images were used to calculate high-resolution maps of the
elements of magnetosomes in plastic resin embedded and ultra-thin sectioned Itaipu cocci. Iron (Fig. 3c)
and oxygen (Fig. 3d) maps were used to identify the crystals of the magnetosomes as containing
magnetite. Electron energy-loss spectroscopy of the magnetosomes confirmed the oxygen and iron
elements (Fig. 3e). Similarly sulfur maps and spectra can be produced for greigite magnetosomes.
5.2 Candidatus Magnetoglobus multicelularis
Recently, an intriguing MMP has been described as “Candidatus Magnetoglobus multicelularis” [29].
This curious multicellular form of MB (Fig. 2a) is found in a hypersaline lagoon in Rio de Janeiro and
showed many characteristics that are unique among prokaryotes. Phylogenetic analysis of “Candidatus
Magnetoglobus multicelularis” and FISH associated this microorganism to the deltaproteobacterium
group of Bacteria (Figs. 2b and 2c). “Candidatus Magnetoglobus multicelularis” have an unusual
multicellular life cycle, cells precisely organized, polarized and unable to live individually. It is
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composed of gram-negative bacterial cells which are genetically identical and arranged side by side in a
sphere that moves as a unity in straight or helicoidal trajectories. Each cell faces both the external
environment and an acellular compartment in the middle of microorganism (Fig. 2h). This acellular
compartment is maintained during the multicellular life cycle of “Candidatus Magnetoglobus
multicelularis”, in which one microorganism (a) grows, (b) doubles the volume and the number of
constituent cells, (c) reorganizes the cells and then (d) divides into two new and identical
microorganisms. On the surface of the microorganism, cells are covered by a capsule and numerous
flagella (Fig 2i). The flagella are responsible for its coordinated complex movement, including rotation
and the so-called escape motility. “Candidatus Magnetoglobus multicelularis” contain thousands of
magnetosomes (Fig. 3a) composed of iron sulfide (Fig. 3b). The magnetosomes are enveloped by a
membrane and are distributed in planar groups near the periphery of the microorganism.
Unfortunately, “Candidatus Magnetoglobus multicelularis” is an uncultured microorganism and many
interesting questions involving multicellularity and magnetosome formation are still unsolved. Recently,
we demonstrated that grazing ciliates can ingest this microorganism (Fig. 2j) and dissolve the iron
sulfide magnetosomes in the ciliate acid digestive vacuoles [39]. We do not know the impact of the
magnetosomes digestion by the ciliates in the environment. Previous estimates have shown that marine
MB can reach populations densities of 104 cells/ml [54]. The large amount of iron concentrated in
magnetosomes (10-13 to 10-15 g iron per cell), considering their estimated population density, indicates
that nanomolar concentrations of iron may be sequestered in the biomass. The ingestion of “Candidatus
Magnetoglobus multicelularis” by ciliates shows the possibility of recycling of the iron trapped within
magnetosomes. Thus, depending of the rate consumption part of the iron trapped within the
magnetosomes is recycled to the environment in a more soluble form. Each microorganism contains
about 30 cells with up to 100 magnetosomes, which adds up to 3000 magnetosomes per microorganism.
For an average magnetosome size of 100nm, which results in a cubic volume of 106nm3, an estimated
total amount of 1.2 x 10-11g of greigite per microorganism (greigite density = 4.1g/cm3) [55] can be
solubilized within the ciliates and eventually returned to the environment. For a population of 6.2 x 103
“Candidatus Magnetoglobus multicelularis”/ml in the sediment, nanomolar concentrations of iron can be
recycled.
6. Perspectives
Magnetotactic bacteria and magnetosomes are subject of an active and interdisciplinary field of research
which includes not only microscopy but other topics such as cell biology, geomicrobiology,
palaeomagnetism, biomineralization, biophysics and biochemistry to name a few. New and exciting
findings are constantly being reported on these microorganisms. Of course, these breakthroughs do not
always include, but usually benefit from all aspects of microscopy. New directions will come that will
inspire research groups in this highly multidisciplinary field of microbiological research; future
developments in light and electron microscopy will be certainly very helpful.
Acknowledgements: U.L thanks Prof. Marcos Farina and Carolina N. Keim for previous collaborative work. This
work was supported by CNPq, CAPES, FAPERJ (Pronex) Brazilian agencies.
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Fig 1. Microscopy of Itaipu cocci. a) Bright-field image where cocci with magnetosome chains are observed. The
inset shows the magnetosome chains (arrow), bar: 5µm and 500 nm in the inset; b) Electron spectroscopic image
showing the magnetosomes with Itaipu-4 morphotype, bar: 500 nm. c) Transmission electron microscopy image of
isolated magnetosomes. The largest particles come from Itaipu-1 morphotype, bar: 600 nm. d) Scanning electron
microscopy image of isolated magnetosomes, bar: 200 nm. e) Freezing-etching image of a Itaipu coccus. Note the
magnetosome chains and the domains on the surface of the cell., bar: 500 nm, f) In situ hybridization of an ultra-thin
section of Itaipu-1 with a specific 16S rDNA -digoxigenin (DIG) probe labelled with anti-DIG antibody-gold
particle, bar: 300 nm.
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Fig. 2 Microscopy of “Candidatus Magnetoglobus multicellularis”. a) Nomarski interferential contrast image of
several microorganisms, bar: 5 µm; b-d) Fluorescence micrographs of DAPI-stained microorganisms (b), and
fluorescent in situ hybridization with the probes for multicellular forms of MB: MMP [56] (c) and “Candidatus
Magnetoglobus multicellularis” [29] (d), bar: 5 µm. e-f) SEM of three microorganisms, bar: 1 µm. h) Ultra-thin
section of “Candidatus Magnetoglobus multicellularis”, bar: 1µm. i) TEM of a whole-mount microorganism
showing its short flagella (arrow), bar: 250 nm; j) TEM image of a degraded “Candidatus Magnetoglobus
multicellularis” ingested by a ciliate. The inset shows a high magnification image where magnetosomes can be seen,
bars: 2 µm and 50 nm in the inset.
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Fig. 3: a) TEM image of a whole-mount “Candidatus Magnetoglobus multicellularis” where numerous cells are
observed. A “Candidatus Magnetoglobus multicellularis” can contain more than 5000 magnetosomes. The inset
shows chains of magnetosomes (arrowhead) within a cell, bar: 2µm and 300 nm in the inset; b) X-ray microanalysis
spectrum of a magnetosome (asterisk) from a “Candidatus Magnetoglobus multicellularis”. Note the presence of
sulphur and iron, indicating iron sulphides as the crystalline part of the organelle. All other elements come from the
cytoplasm or the grid bar (Cu); c-d) Elemental mapping of magnetosomes from a thin-sectioned Itaipu coccus
calculated by the three window power-low method. Both iron (c) and oxygen (d) co-localize within the crystalline
part of the magnetosomes, indicating that the iron oxide is present, bar: 50 nm. e) Electron energy loss spectrum of a
magnetite (Fe3O4) magnetosome from a Itaipu coccus where the OK and Fe L2,3 edges are observed. The edges were
used to calculate the maps in (c and d).
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