Anisotropy of Bullet-Shaped Magnetite Nanoparticles in the Magnetotactic Bacteria Desulfovibrio magneticus sp. Strain RS-1

Magnetotactic bacteria (MTB) build magnetic nanoparticles in chain configuration to generate a permanent dipole in their cells as a tool to sense the Earth’s magnetic field for navigation toward favorable habitats. The majority of known MTB align their nanoparticles along the magnetic easy axes so that the directions of the uniaxial symmetry and of the magnetocrystalline anisotropy coincide. Desulfovibrio magneticus sp. strain RS-1 forms bullet-shaped magnetite nanoparticles aligned along their (100) magnetocrystalline hard axis, a configuration energetically unfavorable for formation of strong dipoles. We used ferromagnetic resonance spectroscopy to quantitatively determine the magnetocrystalline and uniaxial anisotropy fields of the magnetic assemblies as indicators for a cellular dipole with stable direction in strain RS-1. Experimental and simulated ferromagnetic resonance spectral data indicate that the negative effect of the configuration is balanced by the bullet-shaped morphology of the nanoparticles, which generates a pronounced uniaxial anisotropy field in each magnetosome. The quantitative comparison with anisotropy fields of Magnetospirillum gryphiswaldense, a model MTB with equidimensional magnetite particles aligned along their (111) magnetic easy axes in well-organized chain assemblies, shows that the effectiveness of the dipole is similar to that in RS-1. From a physical perspective, this could be a reason for the persistency of bullet-shaped magnetosomes during the evolutionary development of magnetotaxis in MTB.     

Chains of single-domain magnetite particles in chinook salmon,Oncorhynchus tshawytscha

Summary Although the presence of magnetite in their tissues is correlated with the ability of different species to detect magnetic fields, proof that the magnetite is involved in magnetoreception has not yet been provided. Using the approach employed to localize and isolate magnetic particles in the yellowfin tuna, we found that single-domain magnetite occurs in chains of particles in tissue contained within the dermethmoid cartilage of adult chinook salmon,Oncorhynchus tshawytscha. The particles are present in sufficient numbers to provide the adult fish with a very sensitive magnetoreceptor system. Magnetite in the chinook can be correlated with responses to magnetic fields in a congeneric species, the sockeye salmon. Based on the presence of the chains of particles, we propose behavioral experiments that exploit the responses of sockeye salmon fry to magnetic fields to test explicit predictions of the ferromagnetic magnetoreception hypothesis.     

Magnetite-based magnetoreception

Orientation, navigation, and homing are critical traits expressed by organisms ranging from bacteria through higher vertebrates. Sensory systems that aid such behavior have provided key selective advantages to these groups over the past 4 billion years, and are highly evolved; magnetoreception is no exception. Across many species and groups of organisms, compelling evidence exists that the physical basis of this response is tiny crystals of single-domain magnetite (Fe3O4). It is the opinion of the authors that all magnetic field sensitivity in living organisms, including elasmobranch fishes, is the result of a highly evolved, finely-tuned sensory system based on single-domain, ferromagnetic crystals.     

Encouragement of Enzyme Reaction Utilizing Heat Generation from Ferromagnetic Particles Subjected to an AC Magnetic Field

We propose a method of activating an enzyme utilizing heat generation from ferromagnetic particles under an ac magnetic field. We immobilize α-amylase on the surface of ferromagnetic particles and analyze its activity. We find that when α-amylase/ferromagnetic particle hybrids, that is, ferromagnetic particles, on which α-amylase molecules are immobilized, are subjected to an ac magnetic field, the particles generate heat and as a result, α-amylase on the particles is heated up and activated. We next prepare a solution, in which α-amylase/ferromagnetic particle hybrids and free, nonimmobilized chitinase are dispersed, and analyze their activities. We find that when the solution is subjected to an ac magnetic field, the activity of α-amylase immobilized on the particles increases, whereas that of free chitinase hardly changes; in other words, only α-amylase immobilized on the particles is selectively activated due to heat generation from the particles.     

Magnetic particle motions within living cells. Physical theory and techniques.

Body tissues are not ferromagnetic, but ferromagnetic particles can be present as contaminants or as probes in the lungs and in other organs. The magnetic domains of these particles can be aligned by momentary application of an external magnetic field; the magnitude and time course of the resultant remanent field depend on the quantity of magnetic material and the degree of particle motion. The interpretation of magnetometric data requires an understanding of particle magnetization, agglomeration, random motion, and both rotation and translation in response to magnetic fields. We present physical principles relevant to magnetometry and suggest models for intracellular particle motion driven by thermal, elastic, or cellular forces. The design principles of instrumentation for magnetizing intracellular particles and for detecting weak remanent magnetic fields are described. Such magnetic measurements can be used for noninvasive studies of particle clearance from the body or of particle motion within body tissues and cells. Assumptions inherent to this experimental approach and possible sources of artifact are considered and evaluated.     

A Tailored galK Counterselection System for Efficient Markerless Gene Deletion and Chromosomal Tagging in Magnetospirillum gryphiswaldense

Magnetotactic bacteria have emerged as excellent model systems to study bacterial cell biology, biomineralization, vesicle formation, and protein targeting because of their ability to synthesize single-domain magnetite crystals within unique organelles (magnetosomes). However, only few species are amenable to genetic manipulation, and the limited methods for site-specific mutagenesis are tedious and time-consuming. Here, we report the adaptation and application of a fast and convenient technique for markerless chromosomal manipulation of Magnetospirillum gryphiswaldense using a single antibiotic resistance cassette and galK-based counterselection for marker recycling. We demonstrate the potential of this technique by genomic excision of the phbCAB operon, encoding enzymes for polyhydroxyalkanoate (PHA) synthesis, followed by chromosomal fusion of magnetosome-associated proteins to fluorescent proteins. Because of the absence of interfering PHA particles, these engineered strains are particularly suitable for microscopic analyses of cell biology and magnetosome biosynthesis.     

Avian orientation: the pulse effect is mediated by the magnetite receptors in the upper beak

Migratory silvereyes treated with a strong magnetic pulse shift their headings by approximately 90°, indicating an involvement of magnetite-based receptors in the orientation process. Structures containing superparamagnetic magnetite have been described in the inner skin at the edges of the upper beak of birds, while single-domain magnetite particles are indicated in the nasal cavity. To test which of these structures mediate the pulse effect, we subjected migratory silvereyes, Zosterops l. lateralis, to a strong pulse, and then tested their orientation, while the skin of their upper beak was anaesthetized with a local anaesthetic to temporarily deactivate the magnetite-containing structures there. After the pulse, birds without anaesthesia showed the typical shift, whereas when their beak was anaesthetized, they maintained their original headings. This indicates that the superparamagnetic magnetite-containing structures in the skin of the upper beak are most likely the magnetoreceptors that cause the change in headings observed after pulse treatment.     

Microwave absorption by magnetite: A possible mechanism for coupling nonthermal levels of radiation to biological systems

The presence of trace amounts of biogenic magnetite (Fe₃O₄) in animal and human tissues and the observation that ferromagnetic particles are ubiquitous in laboratory materials (including tissue culture media) provide a physical mechanism through which microwave radiation might produce or appear to produce biological effects. Magnetite is an excellent absorber of microwave radiation at frequencies between 0.5 and 10.0 GHz through the process of ferromagnetic resonance, where the magnetic vector of the incident field causes precession of Bohr magnetons around the internal demagnetizing field of the crystal. Energy absorbed by this process is first transduced into acoustic vibrations at the microwave carrier frequency within the crystal lattice via the magnetoacoustic effect; then, the energy should be dissipated in cellular structures in close proximity to the magnetite crystals. Several possible methods for testing this hypothesis experimentally are discussed. Studies of microwave dosimetry at the cellular level should consider effects of biogenic magnetite. © 1996 Wiley-Liss, Inc.     

Use of magnetic particles isolated from magnetotactic bacteria for enzyme immobilization

Summary We report the novel use of magnetic particles isolated from magnetotactic bacteria. Magnetotactic bacteria were collected from enriched sludge by use of a magnetic harvesting apparatus. Magnetic particles separated from magnetotactic bacteria were shown to be pure magnetite. Glucose oxidase and uricase were immobilized on magnetic particles. The activity of glucose oxidase immobilized on biogenic magnetites was 40 times that immobilized on artificial magnetites or Zn-ferrite particles. Both glucose oxidase and uricase coupled with biogenic magnetic particles retained their activities when they were reused 5 times.     

Superparamagnetic Magnetite in the Upper Beak Tissue of Homing Pigeons

Homing pigeons have been subject of various studies trying to detect magnetic material which might be involved in magnetic field perception. Here we focus on the upper-beak skin of homing pigeons, a region that has previously been shown to contain nerves sensitive to changes of the ambient magnetic field. We localized Fe³⁺ concentrations in the subcutis and identified the material by transmission electronmicroscopy (TEM) as aggregates of magnetite nanocrystals (with grain sizes between 1 and 5 nm). The particles form clusters of 1–3 m diameter, which are arranged in distinct coherent elongated structures, associated with nervous tissue and located between fat cells. Complementary low-temperature magnetic measurements confirm the microscopic observations of fine-grained superparamagnetic particles in the tissue. Neither electron-microscopic nor magnetic measurements revealed any single-domain magnetite in the upper-beak skin tissue.     

Magnetosome magnetite biomineralization in a flagellated protist: evidence for an early evolutionary origin for magnetoreception in eukaryotes

The most well-recognized magnetoreception behaviour is that of the magnetotactic bacteria (MTB), which synthesize membrane-bounded magnetic nanocrystals called magnetosomes via a biologically controlled process. The magnetic minerals identified in prokaryotic magnetosomes are magnetite (Fe₃O₄) and greigite (Fe₃S₄). Magnetosome crystals, regardless of composition, have consistent, species-specific morphologies and single-domain size range. Because of these features, magnetosome magnetite crystals possess specific properties in comparison to abiotic, chemically synthesized magnetite. Despite numerous discoveries regarding MTB phylogeny over the last decades, this diversity is still considered underestimated. Characterization of magnetotactic microorganisms is important as it might provide insights into the origin and establishment of magnetoreception in general, including eukaryotes. Here, we describe the magnetotactic behaviour and characterize the magnetosomes from a flagellated protist using culture-independent methods. Results strongly suggest that, unlike previously described magnetotactic protists, this flagellate is capable of biomineralizing its own anisotropic magnetite magnetosomes, which are aligned in complex aggregations of multiple chains within the cell. This organism has a similar response to magnetic field inversions as MTB. Therefore, this eukaryotic species might represent an early origin of magnetoreception based on magnetite biomineralization. It should add to the definition of parameters and criteria to classify biogenic magnetite in the fossil record.     

Expression of Green Fluorescent Protein Fused to Magnetosome Proteins in Microaerophilic Magnetotactic Bacteria

The magnetosomes of magnetotactic bacteria are prokaryotic organelles consisting of a magnetite crystal bounded by a phospholipid bilayer that contains a distinct set of proteins with various functions. Because of their unique magnetic and crystalline properties, magnetosome particles are potentially useful as magnetic nanoparticles in a number of applications, which in many cases requires the coupling of functional moieties to the magnetosome membrane. In this work, we studied the use of green fluorescent protein (GFP) as a reporter for the magnetosomal localization and expression of fusion proteins in the microaerophilic Magnetospirillum gryphiswaldense by flow cytometry, fluorescence microscopy, and biochemical analysis. Although optimum conditions for high fluorescence and magnetite synthesis were mutually exclusive, we established oxygen-limited growth conditions, which supported growth, magnetite biomineralization, and GFP fluorophore formation at reasonable rates. Under these optimized conditions, we studied the subcellular localization and expression of the GFP-tagged magnetosome proteins MamC, MamF, and MamG by fluorescence microscopy and immunoblotting. While all fusions specifically localized at the magnetosome membrane, MamC-GFP displayed the strongest expression and fluorescence. MamC-GFP-tagged magnetosomes purified from cells displayed strong fluorescence, which was sensitive to detergents but stable under a wide range of temperature and salt concentrations. In summary, our data demonstrate the use of GFP as a reporter for protein localization under magnetite-forming conditions and the utility of MamC as an anchor for magnetosome-specific display of heterologous gene fusions.     

Detection of submicroscopic magnetite particles using reflectance mode confocal laser scanning microscopy

Light microscopy and transmission electron microscopy work at such different scales that some components of cells may be too small to detect using light microscopy but too dispersed among cells within tissues to be discovered using electron microscopy. We have used reflectance mode confocal laser scanning microscopy to detect single-domain magnetite crystals in both live and resin-embedded preparations of magnetotactic bacteria. We show that reflections from bacterial cells are uniquely associated with the magnetite, which underpins the magnetotactic response of the bacteria. En bloc viewing shows that relatively large volumes of material can be searched with sufficient resolution to enable detection of submicroscopic particles. The techniques reported here may be of interest to others wishing to detect submicroscopic objects dispersed in large volumes of tissue.     

Sub-Micrometer-Scale Mapping of Magnetite Crystals and Sulfur Globules in Magnetotactic Bacteria Using Confocal Raman Micro-Spectrometry

The ferrimagnetic mineral magnetite is biomineralized by magnetotactic microorganisms and a diverse range of animals. Here we demonstrate that confocal Raman microscopy can be used to visualize chains of magnetite crystals in magnetotactic bacteria, even though magnetite is a poor Raman scatterer and in bacteria occurs in typical grain sizes of only 35–120 nm, well below the diffraction-limited optical resolution. When using long integration times together with low laser power (<0.25 mW) to prevent laser induced damage of magnetite, we can identify and map magnetite by its characteristic Raman spectrum (303, 535, 665 ) against a large autofluorescence background in our natural magnetotactic bacteria samples. While greigite (cubic ; Raman lines of 253 and 351 ) is often found in the Deltaproteobacteria class, it is not present in our samples. In intracellular sulfur globules of Candidatus Magnetobacterium bavaricum (Nitrospirae), we identified the sole presence of cyclo-octasulfur (: 151, 219, 467 ), using green (532 nm), red (638 nm) and near-infrared excitation (785 nm). The Raman-spectra of phosphorous-rich intracellular accumulations point to orthophosphate in magnetic vibrios and to polyphosphate in magnetic cocci. Under green excitation, the cell envelopes are dominated by the resonant Raman lines of the heme cofactor of the b or c-type cytochrome, which can be used as a strong marker for label-free live-cell imaging of bacterial cytoplasmic membranes, as well as an indicator for the redox state.     

ZFC/FC of oriented magnetic material in the Solenopsis interrupta head with antennae: Characterization by FMR and SQUID

Ferromagnetic resonance and SQUID magnetometry have been used to study magnetic material in the head with antennae, thorax, and abdomen of Solenopsis interrupta ants. The temperature dependence of the head with antennae using both techniques was measured. Room-temperature spectra and saturation magnetization were used to compare the magnetic material amount in the ant body parts. Both techniques show that the highest magnetic material fraction is in the head with antennae. The ordering temperature is observed at 100 ± 20 K for the ferromagnetic resonance spectra HF component. The estimated magnetic anisotropy constant K and g-values at room temperature are in good agreement with magnetite, supporting this material as the main magnetic particle constituent in the Solenopsis interrupta head with antenna. Particle diameters of 26 ± 2 nm and smaller than 14 nm were estimated. This work suggests that the head with antenna of the Solenopsis interrupta ant contains organized magnetic material and points to it as a good candidate as a magnetic sensor.     

Biogenic magnetite as a basis for magnetic field detection in animals

Bacteria, sharks, honey bees, and homing pigeons as well as other organisms seem to detect the direction of the earth's magnetic field. Indirect but reproducible evidence suggests that the bees and birds can also respond to very minute changes in its intensity. The mechanisms behind this sensitivity are not known. Naturally magnetic, biologically precipitated magnetite (Fe₃O₄) has been found in chitons, magnetotactic bacteria, honey bees, homing pigeons, and dolphins. Its mineralization in localized areas may be associated with the ability of these animals to respond to the direction and intensity of the earth's magnetic field. The presence of large numbers (～10⁸) of superparamagnetic magnetite crystals in honey bees and similar numbers of single-domain magnetite grains in pigeons suggests that there may be at least two basic types of ferrimagnetic magnetoreceptive organelles. Theoretical calculations show that ferrimagnetic organs using either type of grain when integrated by the nervous system are capable of accounting for even the most extreme magnetic field sensitivities reported. Indirect evidence suggests that organic magnetite may be a common biological component, and may account for the results of numerous high field and electromagnetic experiments on animals.     

Contrast agents for nuclear magnetic resonance imaging

Nuclear magnetic resonance imaging relies upon differences in relaxation times for much of its ability to resolve anatomical structures and to detect changes in tissue. The natural differences can be changed by the administration of paramagnetic substances, such as metal complexes and stable organic free radicals, and ferromagnetic materials, such as small particles of magnetite. Detailed studies of the chemistry and biophysics of such substances in the body are required if they are to become safe and effective contrast agents for use in medical NMR imaging.     

Formation of Magnetite Nanoparticles at Low Temperature: From Superparamagnetic to Stable Single Domain Particles

The room temperature co-precipitation of ferrous and ferric iron under alkaline conditions typically yields superparamagnetic magnetite nanoparticles below a size of 20 nm. We show that at pH  =  9 this method can be tuned to grow larger particles with single stable domain magnetic (> 20–30 nm) or even multi-domain behavior (> 80 nm). The crystal growth kinetics resembles surprisingly observations of magnetite crystal formation in magnetotactic bacteria. The physicochemical parameters required for mineralization in these organisms are unknown, therefore this study provides insight into which conditions could possibly prevail in the biomineralizing vesicle compartments (magnetosomes) of these bacteria.     

Anomalous magnetic susceptibility values and traces of subsurface microbial activity in carbonate banks on San Salvador Island,Bahamas

Pure limestones beneath the paleosols on San Salvador Island, Bahamas, contain strong positive magnetic susceptibility anomalies, although the iron content is generally very low. These magnetic phenomena differ from those associated with disconformities, which are marked by accumulation of paramagnetic airborne dust deposits with relatively high iron content. The strength and characters of the magnetic response in these subsurface zones correspond to the presence of magnetite, particularly small single-domain magnetite crystals of microbial origin. These crystals are not present elsewhere in the intergranular rock pores or microvugs. They are preferentially concentrated in capillary microborings, which developed concurrently with formation of calcite cements that have soil-related C and O isotope compositions. These magnetic zones occur several meters below the overlying soil horizons. Very thin and long linear microborings may be attributable to cyanobacterial microborers. The single-domain magnetites in these micrometer-size tunnels plugged by calcite appear to result from later occupation of these tiny holes by magnetotactic bacteria. Inorganic origin of the magnetite seems unlikely. Numerous traces that suggest subsurface microbial activity provide evidence that may be used to develop possible scenarios for subsequent biological studies of the precise bacteria involved.