Magnetotactic Bacteria: From Evolution to Biomineralization and Biomedical Applications

Abstract

The synthesis of magnetosomes in magnetotactic bacteria (MTB) represents probably one of Earth’s most ancient forms of biomineralization. The evolution of magnetosomes and the origin of magnetotaxis date back to the Archean Eon, 4.4–2.5 Ga ago. Magnetosomes consist of fine magnetite nanocrystals coated with a lipidic envelope. Their findings in eukaryotic cells and animals support the evolutionary success of otherwise energetically very demanding biocrystallization. Moreover, the conservation of magnetite biomineralization genes in all domains of life has been proposed very recently. Therefore, it is not surprising that magnetosomes have attracted attention from various scientific fields, including mineralogy, microbiology, biochemistry, biophysics, and bioengineering. Here, we review the most recent iron flow findings that lead to magnetite nanocrystals’ biomineralization in MTB. We emphasize the historical milestones that formed the evolution of magnetosomes and magnetotaxis functionality. Finally, we discuss the usability of these unique structures in biomedical, biotechnological, environmental, and nutritional applications.

1. Magnetotactic Bacteria (MTB)

Biomineralization is widespread throughout the tree of life, offering protection, physical strength, or navigation capability to living systems [1]. It is characterized by the synthesis of mineral(s) within the body of a living organism. Currently, more than 60 minerals produced by organisms from various phyla are known [2]. Mineralization in living systems involves highly specialized processes, including ion uptake from the environment, transport, and accumulation of ions within the organism, and finally, mineralization to the required structure [3]. Usually, a vast network of organic molecules is needed to form highly ordered minerals [4]. It includes proteins, phospholipids, and polysaccharides. Proteins are genetically encoded and involved in biomineral formation, nucleation, and crystal growth. Magnetotactic bacteria (MTB) have a unique position among biomineralizing organisms mainly because of their ancient origins (Archeon Eon) and unique properties widely used in current biomedical applications, including MRI diagnostic, cancer therapy, or nutritional and water technological processes. MTB represent morphologically, metabolically, and phylogenetically diverse groups of Gram-negative motile prokaryotes, living in natural aquatic habitats [5,6]. MTB can be found globally in freshwater as well as marine environments in the oxic–anoxic transition zone (OATZ) regions (water column or sediments), since MTB are naturally microaerophiles or anaerobes [5,7]. MTB biomineralize iron from the environment into magnetic responsive magnetite [Fe²⁺Fe³⁺₂O⁻₄] or greigite [Fe²⁺Fe³⁺₂S⁻₄] nanoparticles [8]. MTB metabolites form an essential component of the iron biogeochemical cycle in these environments [9], also forming so-called magnetofossils [10]. Moreover, protozoans can consume the MTB, incorporating such magnetosomes into the food chain [11]. Furthermore, MTB are believed to play a significant role in the biogeochemical cycles of other biologically essential elements, including nitrogen, carbon, and sulfur [7]. Currently (since 2020), known MTB are phylogenetically associated with the sixteen phylum lineages, including Proteobacteria (Alpha, Beta, Gamma, and Delta classes), Nitrospirae, Latescibacteria, and Omnitrophica phyla [12]. It points to the wide distribution of magnetosome biomineralization in the Bacteria domain. From a morphological point of view, MTB involve various shapes, including cocci, rod, vibrio, spirilla, and multicellular morphotypes [12,13]. In the last 30 years, approximately 25 species of MTB have been isolated in axenic culture [14], with the Alphaproteobacteria Magnetospirillum magneticum strain AMB-1 and the Magnetospirillum gryphiswaldense strain MSR-1, as the most studied and cultivated strains [5,15]. The vast diversity within the different strains of MTB regarding MTB morphological properties and adjustments as swimmers supports the idea of the evolutionary advantage of navigation in the geomagnetic field. This advantage was recently quantified experimentally [16]. Smith et al. proposed that the key benefit of MTB navigation is to enhance the bacterium’s ability to detect oxygen and not its average speed increase to move away from high oxygen concentrations [17]. Navigation reduces the random three-dimensional movement of MTB to one dimension, allowing them a simplified movement toward optimal OATZ habitat [18]. This led to the spread of MTB to almost all known aquatic environments, including thermal and saline habitats [19,20]. The large-scale metagenomic survey of MTB revealed that genes responsible for encoding proteins for magnetosome biosynthesis are physically grouped in clusters and are more conserved within phylogenetically similar groups than between different taxonomic lineages [21]. The reason is probably that magnetosome genes were present in the ancestor of each of the phyla Proteobacteria, Nitrospirae, and Omnitorpica, or in the last common ancestor of the phyla Proteobacteria, Nitrospirae, Omnitrophica, Latescibacteria, and Planctomycetes, according to the findings of Lin et al., 2018. Subsequently, their pathways diverged, and the genomic islands responsible for magnetosome production evolved differently in each taxonomic lineage [21].

Due to its unusual magnetotactic behavior called magnetotaxis, MTB have become a phenomenon. Magnetotaxis is the ability to align passively and move along magnetic field lines [15,22]. Magnetotaxis in MTB is based on the capability to biomineralize intracellular, nano-sized magnetic crystals composed of magnetite (Fe₃O₄) and/or greigite (Fe₃S₄) that are encapsulated in protein-rich membrane organelles called magnetosomes (Figure 1) [5,6,15]. The size and shape of the particles are highly conserved within each strain but differ among various strains. The proteins required for magnetosome formation and biomineralization of magnetic nanoparticles are genetically controlled [14,23]. Genetic evidence exists that horizontal gene transfer could be responsible for the distribution of magnetosome genes among different phyla in the Bacteria domain [24]. This distribution is caused by the fact that the genes responsible for magnetosome synthesis are organized in clusters, forming what are called magnetosome genomic islands (MAI) [8,24,25] or magnetosome gene clusters (MGC) [26], which are interrupted or surrounded by specific genomic sequences. However, for example, the Magnetococcus marinus strain MC-1 is characteristic of its no “island” structure and genetic stability, indicating earlier magnetotaxis evolution. Based on phylogenetic analyses of magnetosome-related genes and proteins, Lefevre et al. proposed the monophyletic origin of magnetotaxis [27] instead of the polyphyletic scenario suggested by Delong et al. [28]. Recently Lin et al. suggested that a single common ancestor of MTB comes from the mid-Archean Eon, before the divergence between the Nitrospirae and Proteobacteria [26]. This suggestion supports the idea that the common ancestor of all Proteobacteria, Nitrospirae, Omnitrophica, and maybe also Latescibacteria and Planctomycetes [21] was magnetotactic and that navigation in the geomagnetic field represents an evolutionarily advantageous trait [27].

Bellinger et al. recently hypothesized that broad forms of life (including eukaryotes) use the genetic mechanism of biogenic magnetite formation for geomagnetic sensory perception [29]. Widespread biomineralization is supported by the findings of magnetite nanoparticles in algae [30], honeybee and dolphin [31], pigeon beak [32], and salmon [29]. Vali and Kirschvink proposed that the first eukaryotes inherited the ability to biomineralize magnetosomes from a magnetotactic α-proteobacterium by gene transfer during the symbiotic evolution of mitochondria [33]. No data support this ability in Archaea or viruses, making the MTB the most primitive magnetic-sensing organism [34].

However, we still lack a detailed understanding of the evolutionary and metabolic processes that lead to the production of magnetosomes and their use to navigate the Earth’s magnetic field. Therefore, this review summarizes the current state-of-the-art in the evolution of iron metabolic pathways in MTB that enables the production of energy-intensive nanocrystals in environments where free iron is a scarce commodity. To our knowledge, no such review deals with the iron metabolism-induced evolution of magnetosomes and magnetotaxis, which are crucial traits of MTB. Although, there are several valuable publications available regarding the biochemistry of magnetosome formation (e.g., [8,15,24]). Surprisingly, understanding (the evolution) of iron metabolic pathways in MTB is of fundamental importance to understand several pathological processes in humans, including neurodegeneration, cancer, etc. [35]. This connection results from the fact that these disorders are associated with the accumulation of biogenic iron and the mineralization of magnetite nanoparticles in tissue [36]. Due to disrupted iron homeostasis, ferritin particles are believed to be a precursor of iron accumulation and magnetite biomineralization in human tissue [37]. Understanding the iron accumulation and excretion pathways in MTB can help to understand the pathological biomineralization of magnetite in humans and to find a therapeutic approach to diseases associated with iron accumulation. However, unique MTB properties have already been used in various areas, including biotechnology, biomedicine, nutrition, and ecology [6,38,39]. Fine magnetic particles from magnetosomes are also employed as a drug delivery system, an agent for magnetic resonance (MRI) contrast enhancement, bioremediators of heavy metals, etc. [6]. Here, we focus only on the iron metabolism and evolution of magnetite-producing MTB as the only one MTB in axenic culture biomineralized greigite. However, its biomechanism has not been elucidated yet, and in general, only magnetite-based MTB have been used in studies investigating its usefulness for commercial practice [6]. Understanding the basic milestones in the evolution and biomineralization of magnetite in MTB gives us insight into the origin and adaptation of life from its early forms. Moreover, it has a very practical meaning for current biomedical research. Additionally, the main goal of this review is to contribute to this knowledge.

2. Magnetotaxis

2.1. Evolution of Magnetoreception

The only permanent condition under which life on Earth has evolved is the Earth’s magnetic field. A recent study revealed that the Earth’s magnetic field is at least 4 billion years old [40], while the oldest known microfossils are almost 3.5 [41], 3.7 [42], and potentially up to 4.1 billion years old [43]. Finding such old complex microbes indicates that life on our planet originated symbiotically with the origin of Earth’s magnetic field 4 billion years ago. This supports the idea that geomagnetic field formation was a crucial condition in the origin of life on our planet [44]. The main proposed contribution of the Earth’s magnetic field to the origin of life is protection against harmful solar (cosmic) radiation [45]. However, in addition to its “life-protective” function, it also offered a “navigation” function. Many living species currently evolved with the ability to navigate in the ambient magnetic field of our planet. Navigation in the Earth’s magnetic field is typical, especially for migratory species, including higher organisms, such as fish, birds, and mammals, and also involves the bacteria kingdom [46]. The existence of geomagnetic navigation in bacteria indicates that magnetoreception is archaic. Alternatively, the presence of magnetoreception in various organisms proves the evolutionary advantage of such a trait during the evolution of organisms. Many species, including humans, are believed to sense the geomagnetic field [47].

Currently, there exist two different ideas regarding magnetoreception. The first is the idea that the magnetic field triggers quantum chemical reactions in cryptochrome proteins, which can be found in retinas [48]. However, this approach is only related to evolutionary-advanced organisms and is still largely debated. The second concept is generally accepted and involves the idea of nanosized superparamagnetic magnetite crystals with the so-called inclination compass response [49]. The idea is supported by the finding that magnetite crystals produced by biogenic processes for navigation differ from magnetite formed through geological processes with five main features [50]. In general, magnetoreception is usually associated with higher organisms, and magnetotaxis is linked to microorganisms as a subgroup of magnetoreception.

2.2. Evolution of Magnetotaxis

The evolution of magnetotaxis is still a mystery. However, recent findings point to the monophyletic origin of magnetotaxis (single common ancestor) [21]. Regarding time, phylogenic and molecular clock analysis points to the origin of MTB and magnetotaxis in the mid-Archean Eon [26] or even earlier [51]. Two different mechanisms have been proposed: the nongenetically controlled process through photoferrotrophy-driven production of magnetite nanocrystals [52] and the genetically controlled mechanism through the process of exaptation [50] of biomineralized iron oxide nanoparticles as antioxidants to mitigate intracellular reactive oxygen species (ROS) toxicity in early MTB ancestors [53]. Both approaches assume at least local co-evolution of MTB and cyanobacteria ancestors since, from the current perspective, both magnetotaxis and ROS elimination are only needed in at least partly oxic environments. However, this is easily fulfilled from a time point of view because the emergence of both bacteria dates back to approximately the same time period: MTB with its origin in the mid-Archean Eon [26] or even earlier [51], and cyanobacteria with traces of oxygen throughout Archean eon (4–2.5 Ga) [54], with a well-established oxygenic photosynthesis of ≈3 Ga [55]. The sharing of the same environment by both species is debatable, although definitely not impossible.

Both proposals have in common that the structure of the formed magnetosomes (magnetite crystals) determined their new function (magnetotaxis). Furthermore, both mechanisms depend on the presence of low oxygen levels in the environment before the Great Oxygenation Event (GOE). Such microaerobic environments could originate from different chemical reactions (e.g., UV photolysis [56]) and were described as “oxygen whiffs” [57]. However, both proposals have their shortcomings. In the first proposal, the transition from extracellular non-genetically encoded magnetotaxis to intracellular genetically encoded magnetotaxis is debatable, although a niche construction origin of genes responsible for intracellular magnetosome formation was proposed [52]. On the other hand, a massive event of birth gene families in Archean life occurred at approximately 3.3–2.9 Ga, supporting the niche origin [58]. The second proposal assumes genetically controlled biomineralization of iron oxide nanoparticles to scavenge ROS. However, the rubrerythrin-like protein was proposed to have already existed in the Last Universal Common Ancestor (LUCA). Additionally, rubrerythrin molecules played and play a significant role in defense against toxic ROS, and current ferritins are believed to be derived from the simple rubrerythrin-like molecule [59]. From this point of view, it is questionable whether MTB ancestors would also need another mechanism to remove ROS in a microaerobic environment, with the help of a cumbersome and very energy-demanding nanosized particle production mechanism compared to simple protein generation. The second mechanism would probably have a post-GOE rationale; however, as discussed before, the evolution of magnetotaxis predates the GEO.

2.3. Relevance of Magnetotaxis

Until recently, MTB were thought to be the only known group of currently living microorganisms that use magnetotaxis for navigation in the ambient geomagnetic field. However, magnetotaxis has also recently been described in Protozoa, belonging to Eukaryotes [60], including unique eukaryotic magnetoreception acquired by symbiosis with magnetic Deltaproteobacteria [61]. Despite this, the MTB are still the only known prokaryotes capable of magnetotaxis. Magnetotaxis is a unique and the most primitive case of magnetoreception and is defined as the preferred motility of microorganisms based on the geomagnetic field. However, magnetotaxis is not truly a taxis, because it does not involve a sensing mechanism and does not result in an active cellular response to a stimulus. Magnetotaxis only passively aligns bacteria along the field lines, meaning dead MTB are also oriented [6,22]. In other words, the MTB behavior is similar to a magnetic compass needle, where bacteria use the magnetic torque of magnetosomes in the geomagnetic field for navigation [5,62]. In current MTB, only the linkage of magnetotaxis with chemotaxis and aerotaxis determines the active movement into the most appropriate OATZ environment [15,17]. MTB use magnetotaxis to direct themselves into OATZ, encompassing passive alignment and active migration along geomagnetic field lines [22]. OATZ separates the upper oxic layer from the lower anoxic layer in stratified columns in a water environment. The oxic layer is due to the oxygenic photosynthesis rich in oxygen, which enables the aerobic production of a high amount of energy. On the other hand, anaerobic processes produce only a limited amount of energy compared to the aerobic environment. However, mixing the energy-rich compounds (fermentation products) from the anoxic layer and low concentrations of molecular oxygen from the oxic environment occurs directly in the OATZ layer. This makes OATZ a very suitable environment for MTB as microaerophiles. As the OATZ layer is relatively narrow compared to other layers and can move within the water column, magnetotaxis and chemotaxis have evolved in the MTB symbiotically [63]. Both mechanisms help the MTB to find and swim toward the OATZ layer. In general, bacteria in the Northern Hemisphere swim to the magnetic north, while bacteria in the Southern Hemisphere swim to the magnetic south, pointing to the polarity of MTB [64]. However, several species of MTB possess the opposite polarity [65]. The associated “taxis” enable movement in all three directions to effectively reach and stay in the required microaerophile space: chemotaxis, aerotaxis [64], phototaxis, and redox taxis. In contrast, coupling with magnetotaxis allows only one-dimensional movement, which is very effective in finding the OATZ layer in the stratified water columns. Magnetotaxis was proposed to have developed before chemotaxis [52]. Based on the previous description, magnetotaxis can be regarded as a very primitive protosensory system that evolved on early Earth [45,53,62].

2.4. Magnetotaxis Functionality

The magnetotaxis functionality is solely based on the formation of intracellular “magnetosome” structure, which consists exclusively of magnetite (Fe₃O₄) or greigite (Fe₃S₄) nanocrystals coated by a lipid membrane with associated proteins [14]. In currently living MTB, the magnetosome formation, morphology, size, and arrangement within the cell are under specific biochemical and genetic control, resulting in a high functionality of magneto-navigation [17,22]. MTB produce stable single-domain ranged particles (≈35–120 nm) characterized by high coercivity [66]. The typical size of stable single-domain particles in magnetite typically ranges from 20 to 80 nm [67]. Above 80–100 nm, the particles divide into multiple domains, while below 20 nm, they exhibit superparamagnetic behavior. Thus, single-domain-sized particles possess optimized magnetic properties for navigation in the geomagnetic field. However, regarding the size of a single magnetic moment (one single-domain particle), the magnetic moment is insufficient to perform magnetotaxis [15,62]. Therefore, several magnetosomes are aligned in one or several chains to enable the passive rotation of the bacterium along the geomagnetic field [5,62]. In such an arrangement, the magnetic moments of the particles are parallel to each other, resulting in a dipole moment as the sum of individual dipoles [15,17]. Frankel et al. estimated that a chain of at least 20 magnetosomes would provide a sufficient magnetic moment to perform magnetotaxis in Magnetospirillum [68]. However, some species synthesize far more magnetosomes than are needed for magnetotaxis [69]. It points to a possible different function of magnetosomes than magnetotaxis. One of the possible explanations is the elimination of reactive oxygen species from the cell, since magnetosomes exhibit peroxidase-like activity [70]. A precise genetic and transport mechanism must be involved in the arrangement of the magnetosome chain, since they would naturally form rings to minimize the energy [5,52]. MTB use actin-like filaments for the chain arrangement of magnetosomes. Due to the strong correlation between MTB phylogeny and morphology of magnetosomes, Lefevre et al. proposed that bullet-shaped magnetite crystals were the first minerals to appear in magnetosomes [27]. This proposal is based on the findings of the biomineralization of bullet-shaped crystals biomineralization in the OP3 candidate division [71] and Nitrospirae [72] as the most deeply branching phylogenetic groups and in Deltaproteobacteria [73] as the most deeply diverging group of the Proteobacteria. On the contrary, MTB from later diverging Alpha- and Gammaproteobacteria only biomineralize well-defined crystals of cuboctahedral and elongated prismatic magnetite [27].

Similar magnetite nanoparticles and magnetosome-like structures have been found in many other living systems, including algae, fishes, termites, pigeons, bats, dolphins, and even humans [74]. Their widespread use suggests that iron-based magnetoreception could be a simple sensory system of various living organisms [53], including humans [75]. In fact, specific navigation skills in the geomagnetic field have also been observed in other animals, including desert ants, newts, snails, moths, wood mice, mole rats, deer, and dogs [12]. Although even in this case it is very probably about navigation in a geomagnetic field, it is not magnetotaxis.

3. Magnetosomes

3.1. Evolution of Magnetosomes

The spread and evolution of magnetosome biomineralization among bacteria is supposed to be a 2.7-billion-year process accelerated by horizontal gene transfers [76]. Currently, magnetosome synthesis in MTB is a highly controlled process comprising several proteins encoded by operons in MAI [8,24,25] or MGC [53]. Approximately 30 genes located in MAI are required for the successful formation of magnetosomes [77]. The proteins encoded by MAI are defined as “magnetosome membrane-associated (Mam) and magnetic particle membrane-specific (Mms) proteins. Interestingly, MAI, especially the mamAB operon, is conserved in all known MTB strains. Other operons are specific to different strains, such as mamCDFG, mamXY, and mms6 for Alphaproteobacteria [14]. In Deltaproteobacteria, Nitrospirae, and Omnitrophica, we recognize magnetosome-associated Deltaproteobacteria genes (mad) [78]. Nitrospirae possesses (man) genes [69]. Both (mad) and (man) are supposed to have the same role as the Mam and Mms proteins, but in different strains [15]. The overview of all the functions of Mam and Mms proteins involved in magnetosome biomineralization is clearly described in [76].

However, magnetosomes have been proposed to possess functions different from magnetotaxis, e.g., gravity detection units, iron sequestration and iron storage, or batteries that provide energy from the oxidation-reduction cycling [79]. Recently, Lin et al. proposed that the magnetosome’s initial role was to mitigate intracellular reactive oxygen species toxicity, and magnetotaxis is only the result of the later exaptation process [53].

On the other hand, Strbak and Dobrota proposed non-genetic magnetosome formation as a byproduct of Archean iron cycling [52]. They showed that coupling of anoxygenic photosynthesis with ferrous iron as an electron donor and with anaerobic respiration with ferric iron as an electron acceptor provided sufficient material for non-genetically controlled magnetite formation. Surprisingly, the metagenomic analysis revealed the similarity of MTB genes with the microbiomes of some other living organisms [80], including humans [81], suggesting possible symbiotic magnetoreception. Moreover, Bellinger et al., 2022 proposed a model of biogenic magnetite formation in eukaryotes and hypothesized that broad life forms use this genetic mechanism for geomagnetic sensory perception [29].

3.2. Magnetosomes Biomineralization

Magnetosomes originate from invaginations of the cytoplasmic membrane lipid bilayer (Figure 2) to create a separated environment from the cytoplasm characteristic of the specific chemical environment [15,82]. The phospholipid bilayer of magnetosomes varies from 3 nm to 6.7 nm [83]. The composition of the magnetosome membrane differs from the cytoplasmic membrane of bacteria [24]. Inside the magnetosomes are encapsulated magnetic nanoparticles and numerous strictly controlled proteins [6,15]. In the MTB genome, about thirty genes are associated with crystal biomineralization [84]. Around ten genes are considered crucial in crystal synthesis and magnetosome chain formation [85]. Magnetic particles produced by MTB are one of the most perfect in microbes. All known MTB from Alphaproteobacteria phyla biomineralize magnetite particles, while bacteria from Deltaproteobacteria synthesize the greigite magnetosomes [76]. MTB possess specific genes that, together with the pH and redox control system, are responsible for inner-cell membrane invagination, magnetosome chain formation, iron transport and chemical environment regulation, magnetic particle nucleation, crystal growth, size, and morphology ensuring [15,77,86]. Magnetosome formation is usually controlled by a set of specific genes (e.g., mam—magnetosome membrane-associated, mms—magnetosome membrane-specific, man—magnetosome genes in Nitrospirae, mad—magnetosome-associated Deltaproteobacteria), which are organized in MAI clusters [5,24]. However, for example, the Magnetococcus marinus strain MC-1 is characteristic of its no-island structure and genetic stability, indicating earlier magnetotaxis evolution [5,52]. It was further proven that some operons, such as mamAB, are found in all characterized MTB strains, although other operons, such as mamCDFG, mamXY, and mms6 are specific to Alphaproteobacteria [15]. These genes encoded appropriate Mam, Mms, Mad, and other related proteins that participate in particular processes that result in functioning magnetosome chains. Only then can magnetic particle nucleation start [24]. Specifically, the biomineralization process in Magnetospirillum magnetotacticum results in four final components: magnetosome crystal, magnetosome membrane, interparticle connections, and magnetosomal matrix surrounding the magnetosome [87]. However, Yamamoto et al. did not observe the magnetosomal matrix. Instead, they observed the lipid bilayer around the magnetite crystal, surrounded by a layer of MamA protein [88]. It is generally accepted that magnetosomes can only be synthesized in the presence of sufficient amounts of ferrous iron [15]. Uebe et al. showed that empty but functional vesicles are present in iron-starved cells, and magnetite synthesis is recovered immediately after adding iron [89]. Ferrous iron accumulates inside magnetosome vesicles to be later oxidized by magnetochromes (oxidizing domains of MamE, MamP, MamT, and MamX) [90].

Despite significant progress in the study of MTB, the understanding of iron biochemistry responsible for magnetite biomineralization is insufficient. Current knowledge of magnetosome formation is derived from two of the most studied species of Alphaproteobacteria: Magnetospirillum magneticum and Magnetospirillum gryphiswaldense [8]. Several more or less identical mechanisms of magnetosome formation have been proposed in MTB, four of which are listed in

Table 1. Proposed mechanisms of magnetosome formation.

Step	Murat et al., 2010 [77]	Komeili, 2012 [24]	Crichton 2016 [74]	Ben-Shimon et al., 2021 [8]
1.	invagination of the inner membrane (regulated by MamI, MamL, MamQ, MamB)	creation of the magnetosome membrane by invagination of the inner cell membrane (mediated by MamI, MamL, MamQ, MamB)	a reductive uptake of iron from the external environment of the bacterial cell and its transport (ferritin?) across the magnetosome membrane	protein sorting and membrane invagination
2.	targeting the magnetosome proteins to the newly created vesicles (regulated by MamE)	MamE is incorporated into the newly created magnetosome membrane	an accumulation of iron within the precursor of the vesicular structure of the magnetosome	magnetosomes alignment into a single or multiple chains
3.	release of vesicles from the inner membrane and their alignment along the created actin-like filament (regulated by MamJ and MamK)	MamE recruits other proteins to the newly created magnetosome membrane	transformation of the initial iron deposit (ferrihydrite) into magnetite	ion transport and magnetosome inner environment control
4.	iron uptake into aligned vesicles and initiation of crystal formation (regulated by MamM, MamN, and MamO)	MamK and MamJ organize newly created magnetosomes into chains (it can take place before or after crystal formation)	crystallization of the magnetite mineral to give a particle within the vesicle of a specific size and orientation	iron nucleation and crystal shape and size control
5.	crystal maturation (regulated by MamA, MamGFDC, MamP, MamR, MamS, MamT)	initiation of biomineralization—formation of small crystals of magnetite (mediated by MamM, MamN, MamO, MagA, MamA)
6.		crystal growing (MamR, MamS, MamT, MamP, MamE, Mms6, MamC, MamD, MamF, MamG are employed)

. A detailed description of individual steps is given in the references provided.

Formed magnetosomes are believed to include fully or partially open channels that allow the exchange of at least iron compounds with the periplasmic environment [24,91]. Furthermore, this iron distribution per-channel could be facilitated by the MamB protein family [62,89]. However, several effective methods of iron transport into magnetosomes were observed, including ferrous cell accumulation in the form of FeP granules and subsequent conversion to other iron forms such as ferritin, magnetite, and mainly ferrous FeS, which allowed intramembrane accumulation [62,92]. It is generally assumed that iron is transported to the magnetosomes in the form of ferrous rather than ferric ions [15]. It is the reducing nature of the cytoplasm that causes the presence of only ferrous ions. Amor et al. showed by green fluorescence that Fe²⁺ is distributed to magnetosomes during biomineralization [93]. They speculated that the ferrous ions are present in magnetosomes whether they contain magnetite or not. Iron accumulation in the magnetosome membrane before magnetite biomineralization was already proposed in [83]. Using energy-dispersive X-ray (EDS) mapping and electron energy loss spectroscopy (EELS), they confirmed the presence of iron close to and inside the lipid bilayer of the magnetosome membrane. This was also confirmed by Amor et al., 2020 [93]. In addition, they also showed the ferrous state of the accumulated iron. However, both forms of iron ions are required for magnetite formation in a 1:2 ratio [52,62]. From this point of view, within the magnetosomes or on their surface, a precisely controlled oxidation mechanism is required to oxidize exactly two-thirds of ferrous ions. If all the ions were oxidized, hematite would be formed instead of magnetite, which does not show magnetic activity [94].

3.3. Magnetosomes Properties

Magnetosomes can vary significantly in size and morphology, which is surprising with regard to genetic control mechanisms [13,95]. It points to the strong environmental influence on particle biomineralization [96]. Iron and oxygen concentration, pH, temperature, nutrient concentrations, and external magnetic fields are important environmental factors affecting magnetosome characteristics [97,98].

Magnetosomes are/were produced in a microaerophilic environment. The process requires the oxidation of biologically available ferrous iron to its ferric form. Currently, there are three known anaerobic mechanisms of oxidation of ferrous iron that produce ferric ions, ultraviolet photooxidation [99], phototropic Fe²⁺ [100], and nitrate-dependent Fe²⁺ oxidation [101]. For magnetite-based magnetosome biomineralization, dominant precursors such as ferrihydrite, hematite, or high-spin-reduced iron complexes were proposed [6]. For greigite biomineralization, mackinawite (tetragonal FeS) or cubic FeS were suggested [6]. Several methods enable the production of abiotic magnetites, such as oxidative precipitation [102], thermal decomposition [103], microemulsion [104], Sol-Gel method [105], and solvothermal method [106]. Additionally, specific proteins are being investigated, suggesting their biotechnological usability. For example, one of the most abundant proteins in the MTB membrane, MamC (encoded by the mamC gene), is associated with magnetite crystals and has been proven to be a stable anchor for many molecules [77,86]. The greigite magnetosome crystals are often shape-irregular with a wrinkled surface [5,24]. On the other hand, magnetite nanocrystals exhibit consistent crystal morphology (usually cuboctahedral, elongated prismatic and anisotropic [24,107], a narrow crystal size range (usually 35–120 nm [15]), high chemical purity, and few crystallographic defects [6,24,62]. As described previously, biomineralizing single-domain-sized magnetite nanoparticles enable the most effective magnetotaxis. Smaller particles (<30 nm) fall into the superparamagnetic domain and are not stable at room temperature, while particles below 10 nm cannot be fully saturated under the geomagnetic field. However, a small portion of superparamagnetic particles (≈5%) was observed in wild-type cultures. It points to defects in magnetite biomineralization since the mixture of single-domain and superparamagnetic-sized particles lead to lower remanent to saturation magnetization ratios (M_rs/M_s) [93].

Furthermore, magnetosome crystals are permanently magnetic at room temperature without being placed in an external magnetic field [22]. The combination of these physical, chemical and magnetic properties provide highly valued potential in chemical, medical, biotechnological, nanotechnological, and other technical applications. However, a general problem in the production of biogenic magnetite nanoparticles is MTB’s fastidious and microaerophilic growth characteristics, leading to MTB’s low and synthetic time-consuming production [22]. Therefore, different strategies for MTB cultivation have been suggested that most often use Magnetospirillum strains that grow in batches, fed-batches, and semi-continuous cultures [6]. In any case, the methods published so far guarantee the production of a large quantity (up to 170 mg/L/day) of magnetosomes [108].

4. Iron Flow in MTB

4.1. Evolution of Iron Metabolism

Magnetite biomineralization has its roots in ancient biochemical and biogeological processes on the Archean Earth and is probably related to the origin of banded iron formations (BIF) [52]. Broadly speaking, before iron’s involvement in magnetite biomineralization, it also played an essential role in the origin and evolution of life on Earth. Several reasons can be recognized for why early life was dependent on iron and why the evolution of MTB is a direct consequence of this dependence [52]:

(1) Abundance. Ferrous iron was an abundant element in early Earth, making it an easily accessible source for the biochemistry of primitive life forms. Bau and Moller (1993) proposed that hydrothermal activity was the dominant source of ferrous iron in the Archean Ocean [109]. However, the weathering of the continental crust has also been suggested as a significant source of Archean iron [110]. It was probably a combination of both that led to an ubiquitous distribution of ferrous iron in the ocean during the first two billion years of the Earth [111]. The dramatic decrease in the concentration of soluble ferrous iron occurred ~2.5 billion years ago due to the increase in oxygen level due to the evolution of oxic photosynthesis (Great Oxidation Event) [51,112]. Ferrous iron readily reacts with oxygen, leading to widespread oxidation and precipitation, forming insoluble iron (hydr)oxide minerals. The most common minerals are ferrihydrite, hematite (Fe₂O₃), and magnetite (Fe₃O₄). However, ferric iron precipitation due to an increase in oxygen level resulted in a dramatic loss of iron bioavailability (from 10⁻⁶ M of Fe²⁺ to 10⁻¹⁷ M Fe³⁺ [113]).

(2) Geomagnetic shield. The spinning iron inside the Earth’s core created (and still creates) the geomagnetic field, which protects the planet from harmful solar (cosmic) radiation. Without iron, our planet would be far less habitable. The stronger the magnetic field of the Earth’s dynamo, the thicker the atmosphere created, which better protects the Earth’s surface from high-energy gamma and ultraviolet radiation [45]. Based on simulations of solar irradiation and early Earth atmosphere absorption during the Archean Eon, Cnossen et al. concluded that almost all very harmful radiation with a wavelength shorter than 200 nm was attenuated effectively [114], even by a very tenuous atmosphere of approximately only ~50% of the present value [115].

(3) Various oxidation states. Iron can exist in several oxidation states, ranging from −2 to +6. As a result, it coordinates with inorganic and organic ligands, forming a wide variety of complexes that are incorporated into biochemical processes. The most frequent complexes are with coordination number 6, i.e., octahedral complexes [116]. The nature of the complexing ligands, pH, and temperature influence the reactivity of iron. An interesting feature of iron is that it is usually not found as a free metal in the environment.

(4) Fe²⁺/Fe³⁺ redox couple. The most widespread forms of iron, which occur naturally, are ferrous (Fe²⁺) and ferric (Fe³⁺) ions. Ferric iron is a hard acid that prefers hard oxygen ligands, while ferrous iron is near the borderline between a hard and a soft acid, favoring nitrogen and sulfur ligands [116]. The Fe²⁺/Fe³⁺ couple is characterized by great variability in its redox potential, which is ligand-dependent and can meet the requirements of many biological processes. The two different and easily interchangeable oxidation states make iron a potential electron donor as well as an electron acceptor in different dissimilatory reactions. This dimorphism is apparently the main reason for the success of iron in living systems and, simultaneously, very probably also led to the origin of magnetotaxis in the Archean environment.

(5) Ferrous salt solubility. The process of incorporating ferrous iron into the biochemistry of early life forms was fueled by the easy solubility of ferrous salts.

The ubiquity of ferrous iron during the period when early life was created led to the incorporation of iron in the proto-metabolism of the earliest life forms. It resulted in the widespread incorporation of iron into the metabolic pathways of living systems. In addition to its role as a cofactor for metalloproteins, it also plays a central role as an electron acceptor (Fe³⁺) for heterotrophic bacterial growth and as an electron donor (Fe²⁺) for the chemotrophic and phototrophic growth of bacteria and archaea [117]. It is generally believed that during the evolution of early life forms, ferrous iron (together with H₂ and S⁰) served as an electron donor [111]. Fe-S clusters embedded in the primitive proteins of the membranes of very early cells are considered the first carriers of external electron donors inside the membrane [113]. After the origin of oxic photosynthesis, the release of oxygen also depended on the iron electron chains in the membrane. However, the increase in oxygen concentration caused the irreversible oxidation of soluble ferrous iron and the precipitation of ferric iron in the form of oxides and hydroxides. As a result, the bioavailability of soluble ferrous iron decreased dramatically. Despite this, iron is preserved in many essential biological processes, highlighting its crucial properties for life. Indeed, except for Lactobacillus and some Bacillus strains [118], iron is an essential compound for living organisms from all three domains [117]. Despite natural selection and limited bioavailability, iron is still present in many essential metalloproteins, proving its unique properties for the evolution of life on Earth. Iron is usually an integral part of various proteins and enzymes in currently living organisms. It involves many critical metabolic processes, including photosynthesis, respiration, DNA synthesis, cell proliferation and differentiation, and methanogenesis. However, iron is also involved in navigation in the Earth’s ambient magnetic field, called magnetoreception [46].

4.2. Iron Uptake in MTB

Several complex processes are required to transform the biologically available iron from the environment into fine and functional magnetite nanoparticles. A magnetosome membrane with associated proteins forms a suitable environment for biomineralizing magnetite nanoparticles. They nucleate and grow using biologically available iron from the environment, which is transported to the magnetosome vesicle by specific proteins. The synthesis of magnetic nanocrystals (magnetite or greigite) in magnetosomes requires a supply of a large amount of iron from the environment [15]. Lin et al., therefore, hypothesized that the presence of MTB could inhibit the bioavailability of iron to other organisms [20]. Dissolved iron trapped in magnetosomes of MTB ends up as sedimentary rocks after the death of bacteria, preventing the required iron cycling. However, the findings of Amor et al. question this since they showed that most iron in MTB exists as soluble species (Fe²⁺, soluble Fe³⁺ organic compounds) rather than magnetite [93]. In another study, Amor et al. showed that the bacterial iron content depends on the external iron concentration and reaches a maximum value of ≈10⁻⁶ ng of iron per cell [9]. They estimated the flux of dissolved iron from the environment to MTB and concluded that MTB could mineralize a significant fraction of dissolved iron into crystals. Except for iron, in the case of magnetite (greigite), the oxygen (sulfur) must also be transported into the magnetosome vesicle. At least in Magnetospirillum and Magnetovibrio strains, oxygen comes from water molecules.

The recent model of iron uptake suggests the incorporation of dissolved Fe²⁺ and Fe³⁺ into the bacterium and its storage in Fe³⁺ form [119]. The stored iron is then partially reduced for transport to magnetosomes and again partially oxidized to form a magnetite crystal [120]. Mass spectrometry data showed that most iron in MTB is not contained in magnetosomes, but bacteria accumulate iron in reservoirs distinct from magnetite crystals [93]. The data revealed that magnetite in magnetosomes represents only 30% of total cellular iron, and iron accumulates predominantly in reservoirs (partially reduced large iron pools with approximately 75% of total cell iron), different from magnetosomes [93]. Based on these findings, Amor et al. proposed a two-step process for magnetite biomineralization: (i) iron is stored in the noncrystalline fraction of the cell, and (ii) magnetite precipitation [93]. In fact, these high-resolution mass spectrometry results contradict previous X-ray absorption data on the same strain (AMB-1), revealing magnetite as the sole iron carrier in bacteria, with several iron phases during magnetite biomineralization [121]. As the main iron pool in MTB, magnetite was also supported by Mossbauer spectroscopy [122]. It would support the idea of magnetite precipitation to prevent iron toxicity since it was also proposed to be the primary role of magnetite biomineralization in MTB ancestors [50]. On the other hand, in the case of a large iron pool in an intracellular medium, the presence of efficient iron storage is required since an excess of free Fe²⁺ ions is toxic to the cell. It supports the findings of Amor et al., who proposed ferritins, bacterioferritins, and Dps proteins as the primary iron storage and detoxifying pathways [93]. However, this discrepancy points to a more complex picture of iron biochemistry and homeostasis in MTB.

It seems that the MamM and MamB proteins are somehow connected to the transport of ferrous iron to the newly created magnetosome vehicles. Uebe et al. proposed that both might sense the concentration of ferrous iron in the cytoplasm and allow iron transport to magnetosomes only if there is enough iron in the cytoplasm [89]. Moreover, they showed that single amino acid alterations in MamB prevent the formation of magnetite. On the other hand, the same amino acid change in MamM causes the production of particles with different sizes and mixed phases (magnetite and hematite) compared to wild-type cells. Their experiments reveal that the nonmodified MamBM complex transports ferrous ions into the vesicles in exchange for protons and that oxidation of two out of three ferrous ions occurs. Hydrogen peroxide is proposed as a possible electron acceptor, and two oxidized iron (ferric) ions react with the remaining ferrous ion and water to form a magnetite crystal. Changes in the MamM protein do not affect iron transport but are responsible for the correct biomineralization of magnetite. If the binding site of MamM is modified, haematite (Fe₂O₃) is mineralized instead of magnetite (Fe₃O₄). It is caused by the oxidation of too many ferrous ions to ferric ions because the oxidation protection of the third ferrous iron is unreliable. Uebe et al. characterized D50 as the essential amino acid that prevents the oxidation of incoming ferrous ions [89].

4.3. Iron Transport in MTB

Iron transport and accumulation in specific precursors precede the nucleation and growth of magnetite crystals in MTB [83]. After iron enters the cell through the outer membrane, it is stored in the supposed precursors, which involve hematite, high-spin reduced Fe complexes, ferritins, bacterioferritins, and Dps proteins [93,122,123]. A study by Fdez-Gubieda et al. using Fe K-edge X-ray absorption near-edge structure (XANES) and high-resolution transmission electron microscopy (TEM) confirmed ferrihydrite in the form of bacterial ferritin core as the source of iron for magnetite formation [121]. Ferrihydrite from bacterial ferritin is characterized by a poorly crystalline structure and high phosphorus content. Phase transformation from the highly disordered phosphate-rich ferric hydroxide ferritin-like phase to the highly ordered pure magnetite phase through transient nanometric ferric (oxyhydr)oxide intermediates within the magnetosome organelle has also been confirmed by Baumgartner et al. [67].

Werckmann et al. analyzed iron transport in MTB using a combination of analytical scanning transmission electron microscopy (ASTEM), energy dispersive X-ray spectroscopy (EDS), and electron energy loss spectroscopy (EELS) with an electron beam spot size less than 0.2 nm [83]. Using this sub-nanometric analysis, they found that magnetite-containing magnetosomes are surrounded by a matrix sequestering significant amounts of iron and that iron ions accumulate around all faces outside the magnetite crystal and inside the lipid bilayer of magnetosomes before their transfer and incorporation into the forming crystal. Furthermore, EDS mapping observed a 20 nm thick magnetosomal matrix with iron inside and outside the membrane, probably in the MamA-containing layer [83]. It suggests a crucial role for the iron-rich magnetosomal matrix in directing and trapping iron to the required location for the rapid accumulation of iron around magnetosomes [83]. However, it contradicts previous findings, proposing magnetite formation without previous iron accumulation in the magnetosome membrane [124].

MTB generally contains more iron (10–100 times) than E. coli as a bacterial model system [9]. It points to a more effective iron uptake and storage system. Amor et al. found that magnetite represents approximately 25–30% of the bulk cellular iron in the AMB-1 cells [9]. It would require a sufficient amount of soluble iron at MTB disposal from external sources. On the other hand, MTB was proposed to accumulate iron in a special reservoir [93]. Amor et al. quantified the total cellular iron contained in the magnetic crystals of the magnetotactic strain Magnetospirillum magneticum AMB-1 [9]. Compared with the iron mass from different cellular pools, they showed that most of the bacterial iron in AMB-1 is not contained in crystals. It reveals another picture of iron homeostasis in MTB than previously thought [119,120].

The biomineralization of magnetite and greigite nanoparticles requires a specific chemical environment characterized by a slightly alkaline pH [125] and a high iron supply [15]. Only after the formation of the magnetosome membrane does the nucleation of minerals begin, as empty magnetosome vesicles can be found in nonmagnetic MTB species [126] or starving bacteria [89]. The formation of the lipidic magnetosome membrane produces the appropriate environment for nucleation and mineral maturation. It also protects the cell from unwanted toxic by-products, often associated with high iron concentration [127]. In some strains, magnetosome vesicles are attached to the cell membrane, facilitating the orientation of the bacterium and facilitating the iron supply from the external environment easier [85]. However, in other species, they are not attached to the cytoplasmatic membrane [128]. The crucial protein for magnetosome formation appears to be MamB, which participates in the delivery of ferrous ions into the magnetosome vesicle [129]. MamB, together with MamQ, MamL, and MamY are believed to be responsible for the invagination of magnetosome membrane [62]. After the invagination of the magnetosome membrane from the cytoplasmatic membrane, the crucial step is selecting and activating proteins required for mineral nucleation [130]. At first, only a magnetosome membrane of a certain (smaller) size is produced to allow the supersaturation of iron [91]. Only the start of nucleation and biomineralization initiates magnetosome membrane growth [91]. MamE was suggested as the proteolytic switcher of biomineralization and membrane growth [131]. The created magnetosomes must be arranged in a line to ensure magnetotaxis functionality [15,62]. MamK, an actin-like protein that extends through the entire cell, is believed to be associated with the formation of a magnetosome chain [132].

In general, the biomineralization process will only begin when the following conditions are met: (i) a suitable chemical environment (pH) and (ii) a high concentration of iron [15]. MTB had to develop an effective iron uptake and transportation mechanism. It may differ in different species. For example, in Magnetotacticum spirillum AMB-1, iron is stored in a ferric form in a ferritin-like structure (a phosphate-rich ferric hydroxide) [67]. In contrast, in Deltaproteobacteria strain RS-1, iron is stored in ferrous form as iron-phosphate granules, which are later transformed into other iron forms (e.g., ferritin) [133]. It is widely accepted that ferrous ions rather than ferric ions are transported into magnetosomes, although the precise molecular mechanism is still unknown [15]. Uebe et al. proposed MamB and MamM proteins as active iron transporters into the magnetosome interior [89]. Both are part of the Cation Diffusion Facilitator (CDF) protein family, which participates in active iron transport (proton motive force) from the cytoplasm to the extracellular space or intracellular organelles [134]. They also probably indirectly increase the pH of the magnetosome interior, as magnetite formation releases more protons than iron is consumed [122]. However, an internal oxidation process must be involved to ensure magnetite nucleation consisting of ferrous and ferric ions. The MamP protein probably plays a fundamental role in this case since it can oxidize ferrous ions to ferric form in alkaline pH in vitro [135]. Spinonen et al. proposed the oxidation of ferrous ions to ferric iron in ferrihydrite minerals as a precursor of magnetite [136]. The subsequent transformation to magnetite starts after the delivery of new ferrous ions.

5. Applications of MTB

During the last two decades, MTB, or the magnetosomes produced by MTB, have found use in biotechnological, medical, nutritional, and other potential applications [6,22,108,137]. Because only one MTB in the axenic culture biomineralizes greigite, the biomechanism of which has not been elucidated, only magnetite-producing MTB were used in studies investigating the utility of MTB for commercial practice [14,23]. Magnetite-producing MTB were studied in a wide range of potential applications such as: drug delivery systems [108,138], tissue contrast agents [139,140,141], hyperthermia [108,142]) or nanotechnology tools (cell separation [39,143], DNA and antigen retrieval/detection [138,144], toxin scavenging [145,146], electrochemical batteries [147] and the source of microlocal strong magnetic fields [148]. Despite many indisputable advantages of MTB or magnetosomes in these studied applications, some limitations prevent their mass use (Table 2). The most striking limitation is the complexity of effective laboratory cultivation, proliferation, and magnetosome isolation [39,144]. On the other hand, the most important advantages related to biomedical applications are non-toxicity and immunogenicity, biocompatibility and tissue stability, the potential ability for aggregation, proper distribution in tissue, resistance to modifications, and body excretion systems [22,102,137].

MTB could be applied as small therapeutics, so-called nanorobots, that enable efficient drug delivery under magnetic field navigation to target tissue [39,140]. The Magnetococcus marinus strain MC-1 or the Magnetospirillum gryphyswaldense strain MSR-1 have been tested as a potential drug-mediated treatment method [6,39]. Several studies introducing MTB into living organisms guarantee the safety of use, non-pathogenicity, and biocompatibility of MTB [6,138]. From this point of view, MTB have been discussed to have great potential in targeted cancer therapy as carriers of substances with antitumor activity in combination with ligands that recognize specific molecular targets of the cancer cell [39,140]. Since neoplastic tumors are characterized by irregular zones characterized by chaotic vasculature, altered hypoxic, metabolic, and chemoresistant environments, the most important characteristic of MTB as applied nanorobots is penetration into these tumor regions [39,140]. Several investigators studied new treatment strategies using MTB in combination with anthracycline antibiotics isolated from Streptococcus peucetius and their synthetic analogs [149,150]. This combination eliminated the extensive side effects of these drugs and could therefore find wide application in cancer treatment, especially in breast and liver cancer [39]. Another tested approach is targeted molecular therapy that takes advantage of the junction of MTB-encapsulated iron-binding proteins (e.g., transferrin) and their receptors (e.g., transferrin receptor CD71) highly expressed by cancer cells. Based on this principle, increased uptake of nanocarriers by tumor cells was achieved, which subsequently induced much greater apoptosis in the tumor than in normal tissue [151]. Furthermore, transferrin-conjugated MTB can also be considered effective cancer surveillance and treatment tool, as it targets transferrin receptors expressed by circulating tumor cells responsible for metastasis and recurrence [151]. In the next study, a combination of MTB with bifunctional linkers (e.g., genipin [152]) and cytosine arabinoside, a chemotherapy drug used for non-Hodgkin’s lymphoma and a wide range of leukemia treatments, allowed direct binding to target cells and elimination of side effects of chemotherapy drugs [152]. Another form of cancer treatment that is still under development is immunotherapy, which stimulates the immune response directed against cancer cells [39,140]. The promising approach involves influencing T lymphocyte activation pathways with magnetosome-enriched antigens (e.g., 4–1BB antigen). Co-stimulatory receptors are found on the surface of T lymphocytes and natural killer cells and induce apoptosis [153]. Another promising nanorobot anticancer strategy is gene therapy, applying engineered MTB conjugates with gene silencing or regulatory transcription factors for genes involved in cell survival, proliferation, chemoresistance, and angiogenesis [39,143,149]. From a modification point of view, magnetosomes are very useful because they are easily functionalized because of the different chemical groups on their surface.

In addition, the magnetic properties of the encapsulated magnetite crystals could also be modified by other encapsulated transition metal elements such as Au, Cu, Se, Mn, Cd, or Co [6,86]. Such metal-doped MTB significantly influence therapeutic effects in cancer therapy [140,144,154]. Furthermore, magnetite nanoparticles are used as a hyperthermic tumor treatment method by changing the magnetic field [39,140]. Increasing the temperature in tumor tissue up to 47 °C [6,140] can reduce or completely eliminate cancer without the toxic side effects of standard cancer therapy methods [108,140]. The use of MTB in cancer therapy using magnetic hyperthermia has been approved in Europe since 2011 as a recombined method with chemotherapy and radiotherapy, thus increasing the efficiency of treatment [140]. In 2014, the US Food and Drug Administration approved this procedure for the treatment of glioblastoma and the treatment of prostate cancer [140].

MTBs were also tested for tumor imaging and localization. Several studies have shown that magnetosomes are suitable as superparamagnetic contrast agents for magnetic resonance imaging (MRI) or magnetic particle imaging (MPI) [e.g., 141,155]. The most commonly used MRI contrast agents, gadolinium-based metal chelates, enhance the contrast of the image between normal and diseased tissues due to their ability to shorten the longitudinal relaxation time T₁ of the surrounding water molecules [155]. On the other hand, superparamagnetic iron oxide-based nanoparticles (SPIONs), including magnetosomes, significantly reduce the transverse T₂ relaxation time due to their high magnetic moment. This creates the so-called negative contrast [140,155]. An additional aspect of small (~25 nm) magnetosomes is the generation of a T₁-weighted positive MRI contrast, which improves the visualization of MTB in the tumors. The T₁-weighted MRI signal increases with increasing bacterial concentrations until it reaches a visualization-prospective threshold of 0.5 × 10¹⁰ cells/mL, when the competing T₂-weighted MRI contrast effect is enhanced [108]. In general, the MRI-effective contrast agents achieve very short T₂ relaxation time values and, therefore, very high relaxivities (r₂ > 133 mM/s), the inverse of the T₂ value [108,155]. Bacterial magnetosomes possess values larger than the value of chemically synthesized magnetic nanoparticles [108]. In addition, due to its nonlinear magnetization response, MPI allows tracking and quantifying MTB in the body with high spatial and temporal resolution [140]. SPIONs mainly contain magnetite, which can be synthesized with uniform sizes, diameters, and lengths. The Magnetospirillum magneticum AMB-1 strain is broadly acceptable for specific magnetosome generation [155]. Recently, MTB have been applied for in vivo MRI monitoring of transplanted stem cells [156]. Another example of a tumor detection method is the application of genetically modified magnetosomes with surface peptide expression, which allows MRI to monitor their targeting to integrin-overexpressing tumors [140,155].

Table 2. Comparison of advantages and disadvantages of magnetosome production for biomedical applications.

	Advantages	Disadvantages
Drug delivery	bio-properties (membrane encapsulation, composites modification, uniform shape and size, high surface-to-volume ratio, dispersibility, blood-brain-barrier crossing) [22,102,138] magnetic properties (paramagnetism, aerotaxis, magnetotaxis) [66,67] biocompatibility and low toxicity conditions [6,139]	complex preparation technologies (cultivation, growing methodologies, magnetosomes isolation) [39,145] unexplored properties (imuno- and geno-toxicity, pharmacokinetics, alternation due modification) limited penetration through biological barriers (intra-tumoral injection) [141]
MRI contrast agents	high sensitivity [108,142,156] high affinity to target tissue [141,156,157] high spatial and temporal resolution [141] the heating efficiency of MTBs under appropriate magnetic field amplitudes enables simultaneous magnetic hyperthermia treatment [39,141]	unexplored biological fate and endotoxicity difficult to differentiate signal loss induced by B0 inhomogeneity/susceptibility artifacts from the effect of MTB contrast agents [156]

Table 2. Comparison of advantages and disadvantages of magnetosome production for biomedical applications.

	Advantages	Disadvantages
Drug delivery	bio-properties (membrane encapsulation, composites modification, uniform shape and size, high surface-to-volume ratio, dispersibility, blood-brain-barrier crossing) [22,102,138] magnetic properties (paramagnetism, aerotaxis, magnetotaxis) [66,67] biocompatibility and low toxicity conditions [6,139]	complex preparation technologies (cultivation, growing methodologies, magnetosomes isolation) [39,145] unexplored properties (imuno- and geno-toxicity, pharmacokinetics, alternation due modification) limited penetration through biological barriers (intra-tumoral injection) [141]
MRI contrast agents	high sensitivity [108,142,156] high affinity to target tissue [141,156,157] high spatial and temporal resolution [141] the heating efficiency of MTBs under appropriate magnetic field amplitudes enables simultaneous magnetic hyperthermia treatment [39,141]	unexplored biological fate and endotoxicity difficult to differentiate signal loss induced by B0 inhomogeneity/susceptibility artifacts from the effect of MTB contrast agents [156]

MTB can be used for many other health-promoting applications. A large number of studies show the high potential of magnetosomes for cell marking [6]. Labeling red blood cells by cell fusion using polyethylene glycol and labeling leukocytes based on MTB phagocytosis have been particularly successful [157]. After applying the targeting magnetic field, exposed-labeled cells can be efficiently separated from those without the MTB label. Magnetic cell separation using magnetosomes and specific antibodies (e.g., anti-mouse G immunoglobulins) has also been achieved for sorting peripheral blood cells [157]. The literature describes the possibility of using MTB as vectors for vaccine DNA carriers for experimental immunization against various viruses such as hand, foot, and mouth disease, secondary lymphoid virus, and human papillomavirus [39]. Magnetosomes appear to be helpful in the detection of nucleotide polymorphism, for which submicron-sized (~100 nm) fluorescent-dye-labeled nanoparticles are used [158]. This method uncovered several human genetic polymorphisms, such as B. Single-nucleotide polymorphisms responsible for cancer, hypertension, and diabetes. Similarly, an epidermal growth factor receptor gene and a beta-1 transforming growth factor gene, a risk factor for bone loss, have been successfully identified [144]. In addition, MTB can be helpful in the extraction of DNA from blood samples using the neodymium-iron-boron magnet to collect and transfer magnetic particles. This form of extraction is based on DNA adhesion to the surface of aminosilane-modified MTB, resulting in highly accurate DNA recovery for subsequent analysis [137]. Several researchers have focused on the use of microorganisms in food and water technology processes. The ideal adepts for this practice appear to be MTB based on the ability to absorb and magnetically remove metals from the culture medium [145]. The functionalized magnetosomes of the Magnetospirillum gryphiswaldense MSR-1 strain have efficiently detected pathogens in water and food, such as Vibrio parahaemolyticus, Salmonella, or Staphylococcus aureus [6,146]. MTB thus represent a suitable biological tool for scientific solutions in various technological applications.

6. Conclusions

MTB represent unique ancient microorganisms whose remarkable properties also find applicability in current biomedical research. This review analyses the origin of magnetosome biomineralization and magnetotaxis from an evolutionary and iron metabolism point of view. Magnetosomes and magnetotaxis form two basic characteristics of MTB. MTB are believed to have already evolved in the mid-Archean Eon in a predominantly anoxic environment. Their evolution must therefore indicate the existence of at least a local microaerophilic environment(s) in this period. Therefore, we focused on the description of the environmental factors that led to the emergence of iron-based metabolism, which directly impacted almost all living organisms on Earth, including humans. Thus, iron became an essential element for various crucial cell functions. The fact that another element has not replaced iron in these functions after becoming a scarce commodity after the GOE confirms its unique properties. Based on its scarcity, a specific mechanism for the excretion of excess iron did not evolve in living systems. This results in the accumulation of excess iron in organisms with aging and the development of pathology. Additionally, surprisingly, understanding iron metabolism and magnetite biomineralization in MTB can help reveal iron flow in various disorders associated with the pathological iron accumulation and magnetite biomineralization, including neurodegenerative diseases, cancer, etc. Therefore, this review focused on the MTB’s evolutionary and magnetosome biomineralization milestones related to their iron metabolism. We discussed magnetotaxis evolution, relevance, and functionality as the most primitive (first?) sensory system that evolved on Earth. We also reviewed the evolution and biomineralization of magnetosomes, which are mandatory for magnetotaxis functionality. At the same time, they have a wide range of applications in current biomedical applications, which we discuss in the final chapter. We believe that this evolutionary (re)view of iron flow and metabolism in MTB can contribute to the overall picture of these unique organisms.