Biomineralization of Magnetite5/12/11

Advanced Biochemistry

AbstractMany organisms precipitate minerals to form a useable product. This process,

biomineralization, produces minerals that assist in survival, usually through skeleton

building with calcium carbonate or calcium phosphate. Magnetite, the most magnetic

mineral, is precipitated in eukaryotes and bacteria throughout the domains. Magnetite

precipitation is proposed to allow organisms to detect magnetic fields, still controversial in

eukaryotes but widely accepted in magnetotactic bacteria. Magnetotactic bacteria utilize

magnetite through the creation of unique membrane bound magnetosomes. Current

research focuses on the properties and formation of magnetosomes, to better understand

their ability to detect magnetic fields and to utilize in industrial applications.

Biomineralization is the process of living organisms precipitating minerals. The

organism can either induce biomineralization or control biomineralization. Induced

biomineralization is generally caused by organisms shifting conditions to favor

precipitation of a mineral, while controlled biomineralization is the formation of a mineral

product under direction of an organism. Precipitation is induced through chemical shifts

caused by metabolism or by cells developing reactive surfaces. Metal sulfides precipitate

when sulfate reducers excrete sulfides, and calcium carbonates form as pH changes during

photosynthesis. Cells can induce mineralization of extracellular skeletons through strategic

spacing of ligands on membranes that fit targeted metals. This spacing lowers the energy

of precipitation by creating a nucleation surface. As the organisms do not actively select or

recruit the metal or silicate ions, this type of mineralization is still considered induced.

Controlled biomineralization involves more input of the cell towards crystallization in

recruiting the right ions and organizing the mineral product. Common biominerals are

aragonite (calcium carbonate), calcite (calcium carbonate), opal (silicon dioxide), and

hydroxyapatite (calcium phosphate), which form skeleton structures in many organisms

(Figure 1).

Figure 1. Skeletonizing biominerals. From top left clockwise: Mammoth bone (hydroxyapatite), Elkhorn coral (CaCO3), diatom (opal), and coccolithophore (CaCO3).

Mineralization is generally energetically favorable. Precipitates form more stable

phases than the solutes, despite the decrease in entropy. Energy input is required to

overcome the activation energy of precipitation, mainly to create a new interface between

reactants, remove chelating agents from cations, and to shed hydration shells so the ligands

and cations can react. The two steps from solutes to a crystal are nucleation, then growth of

the crystal. The first step is to create a nucleation surface, usually a seed crystal that can

then grow larger and guide further precipitation in the growth stage. In homogenous

solutions, nucleation occurs when the solution is supersaturated. In heterogeneous

solutions, nucleation starts on a foreign surface that acts as a catalyst for crystal growth.

The energy required for nucleation is given by GΔ n= GΔ bulk+ GΔ min, where GΔ n is the Gibbs

free energy for nucleation, equal to the Gibbs free energy of the solution plus the interfacial

free energy. The free energy of the solution is decreased by higher temperatures and by a

greater saturation of the solution. The interfacial free energy is determined by surface

energy, the disruption of intermolecular bonds on a surface. Minerals often go through an

intermediate amorphous or hydrated stage before forming the final product because the

surface energy for an amorphous or hydrated crystal is lower than that of an ordered or

dehydrated crystal. The intermediate forms disrupt less intermolecular bonds between the

solution and solid than the final form does, so it is favored kinetically (Figure 2).

Figure 2. Gibbs free energy plotted against chemical state for silicon dioxide crystallization. The first stage is the solute (dissolved silica) then hydrated silica (amorphous), and then quartz, pure SiO2. This reaction scheme is common throughout biomineralization processes.

Konhauser, 2007

Magnetite, Fe3O4, is found throughout life including plants, bacteria, birds, bees,

humans, and fish, except in archeabacteria. Magnetite is the most magnetic mineral,

composing two ferric ions, one ferrous ion, and four oxygens (Figure 3). The unpaired

electrons in the iron give the magnetite its magnetic properties. In order for the magnetite

to form a functioning magnet, its size must be constrained so that the magnetite contains a

single magnetic domain. If the crystal is too small, it does not form a magnetic domain

(superparamagnetic). If the crystal is too large, multiple domains of magnetism can form,

reducing the overall magnetic moment through interfering domains. The optimal size is

greater than 30 nanometers to form single magnetic domains. Magnetite found in

magnetotactic bacteria and in animals are larger than 30 nanometers (Figure 4), and

chemically pure. This is unusual for magnetite crystallized by non-biotic means or by

nonmagnetic bacteria. The bacterium Geobacter metallireducens reduces Fe(III), creating

conditions that favor magnetite precipitation. The magnetite precipitates outside the cell,

has irregular structures, is not magnetic, and is from 1-100 nanometers long. The

differences between the magnetite produced by magnetic bacteria and nonmagnetic

bacteria were hints that magnetotactic bacteria control magnetite crystallization to detect

the Earth’s magnetic field.

Figure 3. Magnetite as found in non-organism derived outcrops. The left image is of a cuboidal magnetite crystal, and the right is of magnetite crystals (black) mixed with pyrite crystals (gold, FeS2).

Figure 4. Magnetite crystal sizes found in organisms (dotted lines are for magnetotactic bacteria) as well as magnetic domain classes (superparamagnetic is not magnetic, single domain is strongly magnetic, two and multi-domain is less magnetic).

Magnetotactic bacteria are a diverse collection of gram-negative bacteria (Figure 5).

All magnetotactic bacteria have membrane bound magnetic crystals called magnetosomes.

The magnetic mineral in the magnetosome can be either magnetite, or a less magnetic iron

sulfide greigite (Fe3S4). Unfortunately, a pure culture has not been established for any

greigite crystallizing magnetotactic bacteria, while several magnetite-utilizing bacteria

have pure cultures, so this report will mainly focus on magnetite bacteria. Magnetite

crystals in the magnetosome are 30 to 120 nanometers, with various morphologies that are

consistent within species (Figure 6). Each bacterium usually hosts twenty or more

magnetosomes linked into one or more magnetosome chains (Figure 6). These chains act

like a compass needle and align the bacteria to the magnetic field of Earth. Magnetotactic

Kirschvink, 1989

bacteria are either microaerophilic or anaerobic and usually live in the transition zone

between environments with no oxygen and oxygenated environments. As such, they

occupy a unique environmental niche— their environment has too much oxygen for purely

anaerobic organisms and too little oxygen for aerobic organisms. The proposed mechanism

for the magnetosome chain “compass” is that it allows bacteria to navigate oxygen and

redox gradients in one dimension instead of in three, giving them a navigational boost over

other microaerophilic bacteria without magnetosomes (Figure 7). This adaptation allows

magnetotactic microaerophilic bacteria to find and stay in shifting oxic/ anoxic boundaries,

giving them an edge over non-magnetic bacteria.

Figure 5. Phylogenetic tree for magnetotactic bacteria and related species. Horizontal gene transfers (HGT) are marked for events, with hypothetical transfers marked with a question mark. MTB stands for magnetotactic bacteria and MMP for magnetotactic prokaryote. Magnetotactic bacteria are spread through several groups of Protobacter and Nitrospira.

Jogler and Schuler, 2009

Figure 6. Magnetite crystal morphologies (top) and magnetosome membranes (bottom). Magnetite crystals can come in bullet shaped (left), elongated prism (middle), or cubo-octahedral (right) morphologies, and in either a single chain (left top, bottom) to multiple chains (middle top). The bottom image is a bacterium with a close up of three magnetosomes (inset).

Figure 7. Magnetotactic bacteria environment and proposed magnetite function in navigation. Magnetotactic bacteria use magnetite to align parallel to the Earth’s magnetic field, helping the bacteria move up and down oxygen and redox gradients to the interface region, the oxic-anoxic transition zone (or interface), where they occupy an ecological niche. Alignment with the Earth’s magnetic field allows for movement along one axis instead of three when navigating gradients.

Jogler and Schuler, 2009

Schuler, 2008

Konhauser, 2007

The base unit for functional magnetotaxis is the magnetosome. The magnetosome

comprises of an individual single domain crystal of magnetite surrounded by the

magnetosome membrane. This membrane is crucial in controlling precipitation and in

organizing magnetite. Isolated magnetosomes treated with detergent result in clumps of

magnetite forming (Figure 8). The magnetosome membrane has roughly the same lipid and

fatty acid content as the cytoplasmic membrane. The difference arises in protein content:

the magnetosome has around twenty magnetosome-specific proteins. These proteins fall

into various protein classes: actin-like, generic transporters, TPR proteins, CDF

transporters, HtrA-like serine proteases, and proteins not in any class (unknown proteins).

The genes for these proteins are mostly found in a 130kb magnetosome island in the

genome, found through comparing the sequences of four magnetotactic bacteria (Figure 9).

The hypothesized method for magnetosome formation is first an invagination of the

cytoplasmic membrane, then linking magnetosomes to form a magnetosome chain, and

finally to precipitate magnetite in the magnetosome (Figure 10A). The sequence of

magnetosome formation is somewhat implied from TEM images of magnetotactic bacteria

(Figure 10B)— magnetosomes linked in chains with magnetite are observed, as are linked

empty magnetosomes and budding membrane pockets (implied starting magnetosome

membranes). The controls for magnetosome formation are still being investigated, aided by

advancements in cryo-electron tomography (CET), a way to generate three-dimensional

images of cells in a near natural state (hydrated and frozen) by taking two-dimensional

images and fitting them. CET is useful in investigating the structure of the magnetosomes.

The theorized start of magnetosome formation is the formation of a separate

membrane to surround magnetite particles. One magnetosome membrane GTPase found,

dubbed Mms16, the most abundant magnetosome protein out of five isolated from

Magnetospirillum magneticum AMB-1, shows similarity to eukaryotic small GTPases that

prime vesicle formation (Matsunada and Okamura, 2003). To test Mms16 potential as a

magnetosome membrane trigger, cells were cultured and AlF4-, a GTPase inhibitor, was

added. Cells experience constant growth but gradually lost magnetism due to

magnetosome chains breaking and fewer magnetosomes than control bacteria (Figure 11).

It is not clear from these effects whether Mms16 has a role in priming invagination,

although it is clear than GTPase activity is necessary for normal magnetosome chain

formation. The magnetosome chain breaks could be due to fewer magnetosomes being

added to sustain the chain, or GTPases could be important in forming the chain, a separate

process from invagination.

Figure 8. Magnetosome chains (left) and detergent treatment of magnetosome chains (right). The magnetosome membrane prevents the magnetite from clumping in solution.

The magnetosome chain is integral for magnetic sensing for the cell. In order for

magnetite crystals to align with the Earth’s field, the crystals have to have enough magnetic

energy to overcome Brownian motion (random motion of particles). To do this, individual

magnetosomes are linked in a chain to create a greater magnetic moment, and to allow the

cell to align with the field. The backbone of the chain is a cytoskeletal filament that

connects the magnetosomes together (Figure 12). Both filled and empty magnetosomes are

attached to the membrane, indicating that mineralization is the last step to forming a

magnetosome chain. Two magnetosome specific proteins, MamJ and MamK have been

implicated in chain formation through mutant studies in Magnetospirillum gryphiswaldense.

Cells with mutated MamJ have fine individual magnetosomes, but they form clumps of

magnetosomes instead of chains (Figure 13), linking MamJ with attaching the

magnetosomes to the cytoskeleton filament to form the chain (Figure 14). MamK mutants

are not as drastic, but there are noticeable irregularities in the mutants— the

magnetosome chain was considerable looser, with more space inbetween magnetosomes

on the chain and breaks in the chain as well (Figure 13). The role for MamK is not as

certain, although it is proposed that MamK stabilizes the chain, perhaps by preventing

alterations due to lipid insertion in the chain (Komeili, 2007). MamK mutants also have

problems splitting magnetosomes during division, lending credance to the proposition of

cytoskeletal filament stabilization.

Figure 9. The magnetosome island, a 130kb genetic region in magnetotactic bacteria. Open reading frames are shown as arrows.

Schuler, 2008

Figure 10. Theorized magnetosome formation pathway. A) a schematic pathway for magnetosome formation, while B) is a series of TEM images of (a) magnetotactic bacterium, (b) a closeup of the cytoplasmic membrane (CM) and outer membrane (OM) of the cell with budding and complete magnetosomes, and (c) is a series of forming magnetosome membranes at different timepoints in their development, from budding membrane to magnetite filled magnetosome.

Figure 11. Cells without AlF4

- added (top) and with AlF4- added (bottom). The cells with

inhibited GTPase had fewer magnetosomes compared to control (top) and the magnetosome chain was disturbed, implicating GTPase activity is crucial to proper magnetosome formation.

Schuler, 2008

Komeili, 2007

Matsunaga and Okamura, 2003

A) B)

Figure 12. Cryo-electron tomography (CET) image of magnetosome chain. Blue are magnetosomes without magnetite, connected to the inner membrane, while the orange are mature magnetosomes with magnetite and appear to be disconnected. Both empty and full magnetosomes are connected to the cytoskeletal filament.

Figure 13. Mutant studies and role of MamJ and MamK. From left to right: TEM image of MamJ mutant with inset of clumps of magnetosomes, CET of MamJ mutant, and CET of MamK mutant. For the CET images, blue is the cytoplasmic membrane, green is the cytoskeletal filament, yellow are magnetosome membranes, and red is magnetite. The MamK mutant still retains a loosely affiliated chain.

Komeili, 2007

Schuler, 2008

Figure 14. Proposed actions of MamJ and MamK. MamJ is the green protein, proposed to connect the magnetosome to the cytoskeletal filament (CF). MamK is the red protein, proposed to affiliate with the CF to either help stabilize and guide magnetosomes (a) or prevent membrane lipids from interfering with the CF (b).

The last step of forming the magnetosome is the precipitation of magnetite in the

magnetosome. The general reaction that takes place is Fe2++2OH+2Fe(OH)3àFe3O4+4H2O,

requiring high iron uptake and reduction. The intracellular iron of magnetotactic bacteria is

170 times that of E. coli, with a concentration of 10mM. Both general and specialized forms

of iron transport coexist in the cell. Magnetotactic bacteria utilize siderophores, proteins

that catch and bind to iron, as well as transport proteins for Fe(II) and Fe (III). Specialized

forms of iron transport include magnetosome specific proteins in the cation diffusion

facilitator family (CDF). The cation diffusion family proteins transport toxic metals in other

organisms, but could be adapted to carry high concentrations of iron across through an

antiporter function. Another specific protein is MagA, a transporter nearly exclusive to the

magnetosome membranes. Most of the MagA is on the magnetosome membrane, but some

is on the cytoplasmic membrane, inversly oriented than those on the magnetosome

membrane. MagA is a suggested Fe(II)/ H+ antiporter as it is analogus to the Na+/H+

antiporter. Several proteins have been implicated in the process of controlling

crystallization. An in vitro solution of magnetite precipiates magnetite from 1-100 nm

heterogenous crystals, but when Mms6 is added, the crystals are 20- 30 nm cuboidal

Komeili, 2007

crystals, strongly suggesting a similar role in vivo. Another four proteins, MamC, MamD,

MamF, and MamG account for 35% of all magnetosome membrane proteins, and two,

MamD and MamG, have a Leu-Gly repeat found in Mms6. Mutant studies with a

magnetotactic bacteria lacking all four Mams (MamCDFG) resulted in a cell that still grew

crystals, but they were smaller and less constrained than in wild type cells.

Complementation with any one to three genes produced control type crystals, but

complementation with all four produced crystals bigger than wild type crystals, indicating

possibly redundant genes regulated tightly (Figure 15). The exact timing for crystallization

is not known, and there is a debate over wheter magnetite crystallizes when the

magnetosome is attached to the inner membrane or if the magnetite only starts to

crystallize once the membrane is separate. Growing crystals of magnetite were found in a

membrane invagination (Schuler 2008), accessible to the periplasmic space. Mineralization

requires precise conditions not obtainable when the membrane is not sealed from the

periplasmic space, leading to the question of how does the cell maintain the right

conditions? Current theories include transport protein structure in the gap, or that the

nucleation crystal is created, but growth is restricted until the magnetosome is seperated

fully from the membrane.

Figure 15. MamGFDC protein domains, molecular weight, isoelectric point, relative abundance in the membrane, and a TEM image of a mamGFDC mutant, with insets of the magnetosome chain and magnetosomes. The mamGFDC mutant shows irregular crystals when compared to the wild type, but not as drastic as MamJ or MamK mutants and easily rescued by complementation with any one to three Mam G-C genes.

There are still so many questions left in the field of magnetic biominerals. The role

of magnetic biominerals in eurkaryotes is still contentious, as it is not known whether the

biominerals detect the magnetic field, and if so, how does the organism interpret their

signal? Current research involves testing animals navigation through magnetized and

demagnitized mazes, with various results (Kirschivink, 1989). It is still not quite clear how

bacteria use magnetic signals and oxygen sensing together, much less how animals use

magnetic signals and navigation. Another open topic is the use of other magnetic minerals

besides magnetite in biomagnetic organisms, and how the systems differ. A magnetotatic

bacterium found in an estuary was discovered to utilize both magnetite and greigite in

magnetosomes depending on the depth in the water column (Bazylinski et al, 1995). Depth

corresponded to either oxic or anoxic and sulfidic conditions, and bacteria from the surface

sometimes had only magnetite whereas bacteria from the bottom of the sample had only

greigite, the mineralization changing based on environmental conditions. The study of

magnetic biominerals is an exciting field with much left to do.

Sources

Bazylinski, D, Frankel, R, Heywood, B, Mann, S, King, J, Donaghay, L, and Hanson, A. “Controlled Biomineralization of Magnetite (Fe3O4) and Greigite (Fe3S4) in a Magnetotactic Bacterium.” Applied and Environmental Microbiology (1995): 3232–3239.

Gorby, Y, R Blakemore, and T Beveridge. "Characterization of the Bacterial Magnetosome Membrane ." Journal of Bacteriology 170.2 (1988): 834.

Jogler, C, and D Schuler. "Genomics, Genetics, and Cell Biology of Magnetosome Formation." Annual Review of Microbiology 63 (2009): 501-521.

Kirschivink, J. "Magnetite Biomineralization and Geomagnetic Sensitivity in Higher Animals." Bioelectromagnetics 10 (1989): 239.

Komeili, A. "Molecular mechanisms of magnetosome formation ." Annual Review of Biochemistry 76 (2007): 351.

Konhauser, Kurt. "Biomineralization." Introduction to geomicrobiology . Malden, MA: Blackwell Pub., 2007. 139-141.

Matsunaga, T, and Y Okamura. "Genes and proteins involved in bacterial magnetic particle formation ." Trends in Microbiology 11.11 (2003): 536.

Schuler, D. "Genetics and cell biology of magnetosome formation in magnetotactic." FEMS Microbiology Reviews 32 (2008): 654.