The cation diffusion facilitator proteins MamB and MamM of Magnetospirillum gryphiswaldense have distinct and complex functions, and are involved in magnetite biomineralization and magnetosome membrane assembly

Magnetotactic bacteria form chains of intracellular membrane-enclosed, nanometre-sized magnetite crystals for navigation along the earth's magnetic field. The assembly of these prokaryotic organelles requires several specific polypeptides. Among the most abundant proteins associated with the magnetosome membrane of Magnetospirillum gryphiswaldense are MamB and MamM, which were implicated in magnetosomal iron transport because of their similarity to the cation diffusion facilitator family. Here we demonstrate that MamB and MamM are multifunctional proteins involved in several steps of magnetosome formation. Whereas both proteins were essential for magnetite biomineralization, only deletion of mamB resulted in loss of magnetosome membrane vesicles. MamB stability depended on the presence of MamM by formation of a heterodimer complex. In addition, MamB was found to interact with several other proteins including the PDZ1 domain of MamE. Whereas any genetic modification of MamB resulted in loss of function, site-specific mutagenesis within MamM lead to increased formation of polycrystalline magnetite particles. A single amino acid substitution within MamM resulted in crystals consisting of haematite, which coexisted with magnetite crystals. Together our data indicate that MamM and MamB have complex functions, and are involved in the control of different key steps of magnetosome formation, which are linked by their direct interaction.     

The dual role of MamB in magnetosome membrane assembly and magnetite biomineralization

Magnetospirillum gryphiswaldense MSR‐1 synthesizes membrane‐enclosed magnetite (Fe₃O₄) nanoparticles, magnetosomes, for magnetotaxis. Formation of these organelles involves a complex process comprising key steps which are governed by specific magnetosome‐associated proteins. MamB, a cation diffusion facilitator (CDF) family member has been implicated in magnetosome‐directed iron transport. However, deletion mutagenesis studies revealed that MamB is essential for the formation of magnetosome membrane vesicles, but its precise role remains elusive. In this study, we employed a multi‐disciplinary approach to define the role of MamB during magnetosome formation. Using site‐directed mutagenesis complemented by structural analyses, fluorescence microscopy and cryo‐electron tomography, we show that MamB is most likely an active magnetosome‐directed transporter serving two distinct, yet essential functions. First, MamB initiates magnetosome vesicle formation in a transport‐independent process, probably by serving as a landmark protein. Second, MamB transport activity is required for magnetite nucleation. Furthermore, by determining the crystal structure of the MamB cytosolic C‐terminal domain, we also provide mechanistic insight into transport regulation. Additionally, we present evidence that magnetosome vesicle growth and chain formation are independent of magnetite nucleation and magnetic interactions respectively. Together, our data provide novel insight into the role of the key bifunctional magnetosome protein MamB, and the early steps of magnetosome formation.     

Bacterial Magnetosome Biomineralization - A Novel Platform to Study Molecular Mechanisms of Human CDF-Related Type-II Diabetes

Cation diffusion facilitators (CDF) are part of a highly conserved protein family that maintains cellular divalent cation homeostasis in all organisms. CDFs were found to be involved in numerous human health conditions, such as Type-II diabetes and neurodegenerative diseases. In this work, we established the magnetite biomineralizing alphaproteobacterium Magnetospirillum gryphiswaldense as an effective model system to study CDF-related Type-II diabetes. Here, we introduced two ZnT-8 Type-II diabetes-related mutations into the M. gryphiswaldense MamM protein, a magnetosome-associated CDF transporter essential for magnetite biomineralization within magnetosome vesicles. The mutations'' effects on magnetite biomineralization and iron transport within magnetosome vesicles were tested in vivo. Additionally, by combining several in vitro and in silico methodologies we provide new mechanistic insights for ZnT-8 polymorphism at position 325, located at a crucial dimerization site important for CDF regulation and activation. Overall, by following differentiated, easily measurable, magnetism-related phenotypes we can utilize magnetotactic bacteria for future research of CDF-related human diseases.     

Formation of magnetosomes in magnetotactic bacteria

The ability of magnetotactic bacteria to orient and migrate along geomagnetic field lines is based on intracellular magnetic structures, the magnetosomes, which comprise nano-sized, membrane bound crystals of magnetic iron minerals. The formation of magnetosomes is achieved by a biological mechanism that controls the accumulation of iron and the biomineralization of magnetic crystals with a characteristic size and morphology within membrane vesicles. This paper focuses on the current knowledge about magnetotactic bacteria and will outline aspects of the physiology and molecular biology of magnetosome formation. The biotechnological potential of the biomineralization process is discussed.     

Effect of Polyethylene Glycol on the Formation of Magnetic Nanoparticles Synthesized by Magnetospirillum magnetotacticum MS-1

Magnetotactic bacteria (MTB) synthesize intracellular magnetic nanocrystals called magnetosomes, which are composed of either magnetite (Fe₃O₄) or greigite (Fe₃S₄) and covered with lipid membranes. The production of magnetosomes is achieved by the biomineralization process with strict control over the formation of magnetosome membrane vesicles, uptake and transport of iron ions, and synthesis of mature crystals. These magnetosomes have high potential for both biotechnological and nanotechnological applications, but it is still extremely difficult to grow MTB and produce a large amount of magnetosomes under the conventional cultural conditions. Here, we investigate as a first attempt the effect of polyethylene glycol (PEG) added to the culture medium on the increase in the yield of magnetosomes formed in Magnetospirillum magnetotacticum MS-1. We find that the yield of the formation of magnetosomes can be increased up to approximately 130 % by adding PEG200 to the culture medium. We also measure the magnetization of the magnetosomes and find that the magnetosomes possess soft ferromagnetic characteristics and the saturation mass magnetization is increased by 7 %.     

细菌纳米磁小体的合成机制及应用

综述了近年趋磁细菌纳米磁小体生物合成的分子机制及应用进展。磁小体的合成涉及磁小体膜的形成、铁的吸收和转运、磁小体晶体的矿化、成熟以及磁小体的链状排列等。其中Mam J和Mam K互作并丝状排列,固定磁小体使其链状排列及磁小体膜由细胞质膜内陷而形成是两个令人注目的成就。我们也提出了关于磁小体的生理意义及合成机制的假说:细胞在低氧浓度下由于氧胁迫大量吸收铁,Fe³⁺/Fe²⁺电子对可起到类似O₂/H₂O的作用,产生能量并作为电子受体;Fe3+得到电子还原成的Fe²⁺可引起Fenton反应,此反应产生的活性氧可影响到生物体的正常生理代谢,细胞为降低Fe²⁺浓度,将其与Fe³⁺一同转化为Fe₃O₄颗粒;磁小体的生理功能之一是降低胞内的活性氧。     

The cation diffusion facilitator protein MamM's cytoplasmic domain exhibits metal-type dependent binding modes and discriminates against Mn2+

Cation diffusion facilitator (CDF) proteins are a conserved family of divalent transition metal cation transporters. CDF proteins are usually composed of two domains: the transmembrane domain, in which the metal cations are transported through, and a regulatory cytoplasmic C-terminal domain (CTD). Each CDF protein transports either one specific metal or multiple metals from the cytoplasm, and it is not known whether the CTD takes an active regulatory role in metal recognition and discrimination during cation transport. Here, the model CDF protein MamM, an iron transporter from magnetotactic bacteria, was used to probe the role of the CTD in metal recognition and selectivity. Using a combination of biophysical and structural approaches, the binding of different metals to MamM CTD was characterized. Results reveal that different metals bind distinctively to MamM CTD in terms of their binding sites, thermodynamics, and binding-dependent conformations, both in crystal form and in solution, which suggests a varying level of functional discrimination between CDF domains. Furthermore, these results provide the first direct evidence that CDF CTDs play a role in metal selectivity. We demonstrate that MamM''s CTD can discriminate against Mn²⁺, supporting its postulated role in preventing magnetite formation poisoning in magnetotactic bacteria via Mn²⁺ incorporation.     

The HtrA/DegP family protease MamE is a bifunctional protein with roles in magnetosome protein localization and magnetite biomineralization

Magnetotactic bacteria contain nanometre-sized, membrane-bound organelles, called magnetosomes, which are tasked with the biomineralization of small crystals of the iron oxide magnetite allowing the organism to use geomagnetic field lines for navigation. A key player in this process is the HtrA/DegP family protease MamE. In its absence, Magnetospirillum magneticum str AMB-1 is able to form magnetosome membranes but not magnetite crystals, a defect previously linked to the mislocalization of magnetosome proteins. In this work we use a directed genetic approach to find that MamE, and another predicted magnetosome-associated protease, MamO, likely function as proteases in vivo. However, as opposed to the complete loss of mamE where no biomineralization is observed, the protease-deficient variant of this protein still supports the initiation and formation of small, 20 nm-sized crystals of magnetite, too small to hold a permanent magnetic dipole moment. This analysis also reveals that MamE is a bifunctional protein with a protease-independent role in magnetosome protein localization and a protease-dependent role in maturation of small magnetite crystals. Together, these results imply the existence of a previously unrecognized 'checkpoint' in biomineralization where MamE moderates the completion of magnetite formation and thus committal to magneto-aerotaxis as the organism's dominant mode of navigating the environment.     

Common ancestry of iron oxide- and iron-sulfide-based biomineralization in magnetotactic bacteria

Magnetosomes are prokaryotic organelles produced by magnetotactic bacteria that consist of nanometer-sized magnetite (Fe₃O₄) or/and greigite (Fe₃S₄) magnetic crystals enveloped by a lipid bilayer membrane. In magnetite-producing magnetotactic bacteria, proteins present in the magnetosome membrane modulate biomineralization of the magnetite crystal. In these microorganisms, genes that encode for magnetosome membrane proteins as well as genes involved in the construction of the magnetite magnetosome chain, the mam and mms genes, are organized within a genomic island. However, partially because there are presently no greigite-producing magnetotactic bacteria in pure culture, little is known regarding the greigite biomineralization process in these organisms including whether similar genes are involved in the process. Here using culture-independent techniques, we now show that mam genes involved in the production of magnetite magnetosomes are also present in greigite-producing magnetotactic bacteria. This finding suggest that the biomineralization of magnetite and greigite did not have evolve independently (that is, magnetotaxis is polyphyletic) as once suggested. Instead, results presented here are consistent with a model in which the ability to biomineralize magnetosomes and the possession of the mam genes was acquired by bacteria from a common ancestor, that is, the magnetotactic trait is monophyletic.     

The major magnetosome proteins MamGFDC are not essential for magnetite biomineralization in Magnetospirillum gryphiswaldense but regulate the size of magnetosome crystals

Magnetospirillum gryphiswaldense and related magnetotactic bacteria form magnetosomes, which are membrane-enclosed organelles containing crystals of magnetite (Fe₃O₄) that cause the cells to orient in magnetic fields. The characteristic sizes, morphologies, and patterns of alignment of magnetite crystals are controlled by vesicles formed of the magnetosome membrane (MM), which contains a number of specific proteins whose precise roles in magnetosome formation have remained largely elusive. Here, we report on a functional analysis of the small hydrophobic MamGFDC proteins, which altogether account for nearly 35% of all proteins associated with the MM. Although their high levels of abundance and conservation among magnetotactic bacteria had suggested a major role in magnetosome formation, we found that the MamGFDC proteins are not essential for biomineralization, as the deletion of neither mamC, encoding the most abundant magnetosome protein, nor the entire mamGFDC operon abolished the formation of magnetite crystals. However, cells lacking mamGFDC produced crystals that were only 75% of the wild-type size and were less regular than wild-type crystals with respect to morphology and chain-like organization. The inhibition of crystal formation could not be eliminated by increased iron concentrations. The growth of mutant crystals apparently was not spatially constrained by the sizes of MM vesicles, as cells lacking mamGFDC formed vesicles with sizes and shapes nearly identical to those formed by wild-type cells. However, the formation of wild-type-size magnetite crystals could be gradually restored by in-trans complementation with one, two, and three genes of the mamGFDC operon, regardless of the combination, whereas the expression of all four genes resulted in crystals exceeding the wild-type size. Our data suggest that the MamGFDC proteins have partially redundant functions and, in a cumulative manner, control the growth of magnetite crystals by an as-yet-unknown mechanism.     

The MagA protein of Magnetospirilla is not involved in bacterial magnetite biomineralization

Magnetotactic bacteria have the ability to orient along geomagnetic field lines based on the formation of magnetosomes, which are intracellular nanometer-sized, membrane-enclosed magnetic iron minerals. The formation of these unique bacterial organelles involves several processes, such as cytoplasmic membrane invagination and magnetosome vesicle formation, the accumulation of iron in the vesicles, and the crystallization of magnetite. Previous studies suggested that the magA gene encodes a magnetosome-directed ferrous iron transporter with a supposedly essential function for magnetosome formation in Magnetospirillum magneticum AMB-1 that may cause magnetite biomineralization if expressed in mammalian cells. However, more recent studies failed to detect the MagA protein among polypeptides associated with the magnetosome membrane and did not identify magA within the magnetosome island, a conserved genomic region that is essential for magnetosome formation in magnetotactic bacteria. This raised increasing doubts about the presumptive role of magA in bacterial magnetosome formation, which prompted us to reassess MagA function by targeted deletion in Magnetospirillum magneticum AMB-1 and Magnetospirillum gryphiswaldense MSR-1. Contrary to previous reports, magA mutants of both strains still were able to form wild-type-like magnetosomes and had no obvious growth defects. This unambiguously shows that magA is not involved in magnetosome formation in magnetotactic bacteria.     

Molecular analysis of a subcellular compartment: the magnetosome membrane in<Emphasis Type="Italic"> Magnetospirillum gryphiswaldense</Emphasis>

The ability of magnetotactic bacteria (MTB) to orient and migrate along magnetic field lines is based on magnetosomes, which are membrane-enclosed intracellular crystals of a magnetic iron mineral. Magnetosome biomineralization is achieved by a process involving control over the accumulation of iron and deposition of the magnetic particle, which has a specific morphology, within a vesicle provided by the magnetosome membrane. In Magnetospirillum gryphiswaldense, the magnetosome membrane has a distinct biochemical composition and comprises a complex and specific subset of magnetosome membrane proteins (MMPs). Classes of MMPs include those with presumed function in magnetosome-directed uptake and binding of iron, nucleation of crystal growth, and the assembly of magnetosome membrane multiprotein complexes. Other MMPs comprise protein families of so far unknown function, which apparently are conserved between all other MTB. The mam and mms genes encode most of the MMPs and are clustered within several operons, which are part of a large, unstable genomic region constituting a putative magnetosome island. Current research is directed towards the biochemical and genetic analysis of MMP functions in magnetite biomineralization as well as their expression and localization during growth.Abbreviations MM Magnetosome membrane - MMP Magnetosome membrane protein - MTB Magnetotactic bacteria     

Bacterial magnetosomes: microbiology, biomineralization and biotechnological applications

Magnetotactic bacteria orient and migrate along geomagnetic field lines. This ability is based on intracellular magnetic structures, the magnetosomes, which comprise nanometer-sized, membrane-bound crystals of the magnetic iron minerals magnetite (Fe₃O₄) or greigite (Fe₃S₄). Magnetosome formation is achieved by a mineralization process with biological control over the accumulation of iron and the deposition of the mineral particle with specific size and orientation within a membrane vesicle at specific locations in the cell. This review focuses on the current knowledge about magnetotactic bacteria and will outline aspects of the physiology and molecular biology of the biomineralization process. Potential biotechnological applications of magnetotactic bacteria and their magnetosomes as well as perspectives for further research are discussed. Received: 2 December 1998 / Received revision: 2 March 1999 / Accepted: 5 March 1999     

Molecular mechanisms of compartmentalization and biomineralization in magnetotactic bacteria

Magnetotactic bacteria (MB) are remarkable organisms with the ability to exploit the earth's magnetic field for navigational purposes. To do this, they build specialized compartments called magnetosomes that consist of a lipid membrane and a crystalline magnetic mineral. These organisms have the potential to serve as models for the study of compartmentalization as well as biomineralization in bacteria. Additionally, they offer the opportunity to design applications that take advantage of the particular properties of magnetosomes. In recent years, a sustained effort to identify the molecular basis of this process has resulted in a clearer understanding of the magnetosome formation and biomineralization. Here, I present an overview of MB and explore the possible molecular mechanisms of membrane remodeling, protein sorting, cytoskeletal organization, iron transport, and biomineralization that lead to the formation of a functional magnetosome organelle.     

趋磁细菌的磁小体

趋磁细菌是一类对磁场有趋向性反应的细菌,其菌体能吸收外界环境中铁元素并在体内合成包裹有膜的纳米磁性颗粒Fe₃O₄或Fe₃O_{3S4晶体即磁小体。综述了趋磁细菌的磁小体生物矿化的条件,以及趋磁细菌的铁离子吸收、磁小体囊泡的形成、铁离子的转运到磁小体囊泡及囊泡中受控的Fe3O4生物矿化的分子生物学和生物化学等方面的研究进展,重点介绍了趋磁细菌磁小体合成机制的研究进展及未来研究磁小体的发展方向。}     

FeoB2 Functions in magnetosome formation and oxidative stress protection in Magnetospirillum gryphiswaldense strain MSR-1

Magnetotactic bacteria (MTB) synthesize unique organelles, the magnetosomes, which are intracellular nanometer-sized, membrane-enveloped magnetite. The biomineralization of magnetosomes involves the uptake of large amounts of iron. However, the iron metabolism of MTB is not well understood. The genome of the magnetotactic bacterium Magnetospirillum gryphiswaldense strain MSR-1 contains two ferrous iron transport genes, feoB1 and feoB2. The FeoB1 protein was reported to be responsible mainly for the transport of ferrous iron and to play an accessory role in magnetosome formation. To determine the role of feoB2, we constructed an feoB2 deletion mutant (MSR-1 ΔfeoB2) and an feoB1 feoB2 double deletion mutant (MSR-1 NfeoB). The single feoB2 mutation did not affect magnetite crystal biomineralization. MSR-1 NfeoB had a significantly lower average magnetosome number per cell (～65%) than MSR-1 ΔfeoB1, indicating that FeoB2 plays a role in magnetosome formation when the feoB1 gene is deleted. Our findings showed that FeoB1 has a greater ferrous iron transport ability than FeoB2 and revealed the differential roles of FeoB1 and FeoB2 in MSR-1 iron metabolism. Interestingly, compared to the wild type, the feoB mutants showed increased sensitivity to oxidative stress and lower activities of the enzymes superoxide dismutase and catalase, indicating that the FeoB proteins help protect bacterial cells from oxidative stress.     

Genetics and cell biology of magnetosome formation in magnetotactic bacteria

The ability of magnetotactic bacteria (MTB) to orient in magnetic fields is based on the synthesis of magnetosomes, which are unique prokaryotic organelles comprising membrane-enveloped, nano-sized crystals of a magnetic iron mineral that are aligned in well-ordered intracellular chains. Magnetosome crystals have species-specific morphologies, sizes, and arrangements. The magnetosome membrane, which originates from the cytoplasmic membrane by invagination, represents a distinct subcellular compartment and has a unique biochemical composition. The roughly 20 magnetosome-specific proteins have functions in vesicle formation, magnetosomal iron transport, and the control of crystallization and intracellular arrangement of magnetite particles. The assembly of magnetosome chains is under genetic control and involves the action of an acidic protein that links magnetosomes to a novel cytoskeletal structure, presumably formed by a specific actin-like protein. A total of 28 conserved genes present in various magnetic bacteria were identified to be specifically associated with the magnetotactic phenotype, most of which are located in the genomic magnetosome island. The unique properties of magnetosomes attracted broad interdisciplinary interest, and MTB have recently emerged as a model to study prokaryotic organelle formation and evolution.     

MMS6 protein regulates crystal morphology during nano-sized magnetite biomineralization in vivo

Biomineralization, the process by which minerals are deposited by organisms, has attracted considerable attention because this mechanism has shown great potential to inspire bottom-up material syntheses. To understand the mechanism for morphological regulation that occurs during biomineralization, many regulatory proteins have been isolated from various biominerals. However, the molecular mechanisms that regulate the morphology of biominerals remain unclear because there is a lack of in vivo evidence. Magnetotactic bacteria synthesize intracellular magnetosomes that comprise membrane-enveloped single crystalline magnetite (Fe(3)O(4)). These nano-sized magnetite crystals (<100 nm) are bacterial species dependent in shape and size. Mms6 is a protein that is tightly associated with magnetite crystals. Based on in vitro experiments, this protein was first implicated in morphological regulation during nano-sized magnetite biomineralization. In this study, we analyzed the mms6 gene deletion mutant (Δmms6) of Magnetospirillum magneticum (M. magneticum) AMB-1. Surprisingly, the Δmms6 strain was found to synthesize the smaller magnetite crystals with uncommon crystal faces, while the wild-type and complementation strains synthesized highly ordered cubo-octahedral crystals. Furthermore, deletion of mms6 gene led to drastic changes in the profiles of the proteins tightly bound to magnetite crystals. It was found that Mms6 plays a role in the in vivo regulation of the crystal structure to impart the cubo-octahedral morphology to the crystals during biomineralization in magnetotactic bacteria. Magnetotactic bacteria synthesize magnetite crystals under ambient conditions via a highly controlled morphological regulation system that uses biological molecules.     

The magnetosome membrane protein, MmsF, is a major regulator of magnetite biomineralization in Magnetospirillum magneticum AMB-1

Magnetotactic bacteria (MTB) use magnetosomes, membrane-bound crystals of magnetite or greigite, for navigation along geomagnetic fields. In Magnetospirillum magneticum sp. AMB-1, and other MTB, a magnetosome gene island (MAI) is essential for every step of magnetosome formation. An 8-gene region of the MAI encodes several factors implicated in control of crystal size and morphology in previous genetic and proteomic studies. We show that these factors play a minor role in magnetite biomineralization in vivo. In contrast, MmsF, a previously uncharacterized magnetosome membrane protein encoded within the same region plays a dominant role in defining crystal size and morphology and is sufficient for restoring magnetite synthesis in the absence of the other major biomineralization candidates. In addition, we show that the 18 genes of the mamAB gene cluster of the MAI are sufficient for the formation of an immature magnetosome organelle. Addition of MmsF to these 18 genes leads to a significant enhancement of magnetite biomineralization and an increase in the cellular magnetic response. These results define a new biomineralization protein and lay down the foundation for the design of autonomous gene cassettes for the transfer of the magnetic phenotype in other bacteria.     

Identification and functional characterization of liposome tubulation protein from magnetotactic bacteria

Magnetotactic bacteria synthesize intracellular magnetosomes that are comprised of membrane‐enveloped magnetic crystals. In this study, to identify the early stages of magnetosome formation, we isolated magnetosomes containing small magnetite crystals and those containing regular‐sized magnetite crystals from Magnetospirillum magneticum AMB‐1. This was achieved by using a novel size fractionation technique, resulting in the identification of a characteristic protein (Amb1018/MamY) from the small magnetite crystal fraction. The gene encoding MamY was located in the magnetosome island. Like the previously reported membrane deformation proteins, such as bin/amphiphysin/Rvs (BAR) and the dynamin family proteins, recombinant MamY protein bound directly to the liposomes, causing them to form long tubules. We established a mamY gene deletion mutant (ΔmamY) and analysed MamY protein localization in it for functional characterization of the protein in vivo. The ΔmamY mutant was found to have expanded magnetosome vesicles and a greater number of small magnetite crystals relative to the wild‐type strain, suggesting that the function of the MamY protein is to constrict the magnetosome membrane during magnetosome vesicle formation, following which, the magnetite crystals grow to maturity within them.