A novel protein tightly bound to bacterial magnetic particles in Magnetospirillum magneticum strain AMB-1

Magnetic bacteria synthesize magnetite crystals with species-dependent morphologies. The molecular mechanisms that control nano-sized magnetite crystal formation and the generation of diverse morphologies are not well understood. From the analysis of magnetite crystal-associated proteins, several low molecular mass proteins tightly bound to bacterial magnetite were obtained from Magnetospirillum magneticum strain AMB-1. These proteins showed common features in their amino acid sequences, which contain hydrophobic N-terminal and hydrophilic C-terminal regions. The C-terminal regions in Mms5, Mms6, Mms7, and Mms13 contain dense carboxyl and hydroxyl groups that bind iron ions. Nano-sized magnetic particles similar to those in magnetic bacteria were prepared by chemical synthesis of magnetite in the presence of the acidic protein Mms6. These proteins may be directly involved in biological magnetite crystal formation in magnetic bacteria.     

Co‐ordinated functions of Mms proteins define the surface structure of cubo‐octahedral magnetite crystals in magnetotactic bacteria

Magnetotactic bacteria synthesize magnetosomes comprised of membrane‐enveloped single crystalline magnetite (Fe₃O₄). The size and morphology of the nano‐sized magnetite crystals (< 100 nm) are highly regulated and bacterial species dependent. However, the control mechanisms of magnetite crystal morphology remain largely unknown. The group of proteins, called Mms (Mms5, Mms6, Mms7, and Mms13), was previously isolated from the surface of cubo‐octahedral magnetite crystals in Magnetospirillum magneticum strain AMB‐1. Analysis of an mms6 gene deletion mutant suggested that the Mms6 protein plays a major role in the regulation of magnetite crystal size and morphology. In this study, we constructed various mms gene deletion mutants and characterized the magnetite crystals formed by the mutant strains. Comparative analysis showed that all mms genes were involved in the promotion of crystal growth in different manners. The phenotypic characterization of magnetites also suggested that these proteins are involved in controlling the geometries of the crystal surface structures. Thus, the co‐ordinated functions of Mms proteins regulate the morphology of the cubo‐octahedral magnetite crystals in magnetotactic bacteria.     

Functional analysis of the magnetosome island in Magnetospirillum gryphiswaldense: the mamAB operon is sufficient for magnetite biomineralization

Bacterial magnetosomes are membrane-enveloped, nanometer-sized crystals of magnetite, which serve for magnetotactic navigation. All genes implicated in the synthesis of these organelles are located in a conserved genomic magnetosome island (MAI). We performed a comprehensive bioinformatic, proteomic and genetic analysis of the MAI in Magnetospirillum gryphiswaldense. By the construction of large deletion mutants we demonstrate that the entire region is dispensable for growth, and the majority of MAI genes have no detectable function in magnetosome formation and could be eliminated without any effect. Only <25% of the region comprising four major operons could be associated with magnetite biomineralization, which correlated with high expression of these genes and their conservation among magnetotactic bacteria. Whereas only deletion of the mamAB operon resulted in the complete loss of magnetic particles, deletion of the conserved mms6, mamGFDC, and mamXY operons led to severe defects in morphology, size and organization of magnetite crystals. However, strains in which these operons were eliminated together retained the ability to synthesize small irregular crystallites, and weakly aligned in magnetic fields. This demonstrates that whereas the mamGFDC, mms6 and mamXY operons have crucial and partially overlapping functions for the formation of functional magnetosomes, the mamAB operon is the only region of the MAI, which is necessary and sufficient for magnetite biomineralization. Our data further reduce the known minimal gene set required for magnetosome formation and will be useful for future genome engineering approaches.     

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.     

Origin of magnetosome membrane: proteomic analysis of magnetosome membrane and comparison with cytoplasmic membrane

Prokaryotes are known to have evolved one or more unique organelles. Although several hypotheses have been proposed concerning the biogenesis of these intracellular components, the majority of these proposals remains unclear. Magnetotactic bacteria synthesize intracellular magnetosomes that are enclosed by lipid bilayer membranes. From the identification and characterization of several surface and transmembrane magnetosome proteins, we have postulated that magnetosomes are derived from the cytoplasmic membrane (CM). To confirm this hypothesis, a comparative proteomic analysis of the magnetosome membrane (MM) and CM of the magnetotactic bacterium, Magnetospirillum magneticum AMB-1, was undertaken. Based on the whole genome sequence of M. magneticum AMB-1, 78 identified MM proteins were also found to be prevalent in the CM, several of which are related to magnetosome biosynthesis, such as Mms13, which is tightly bound on the magnetite surface. Fatty acid analysis was also conducted, and showed a striking similarity between the CM and MM profiles. These results suggest that the MM is derived from the CM.     

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.     

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。}     

A magnetosome-specific GTPase from the magnetic bacterium Magnetospirillum magneticum AMB-1

Magnetic bacteria produce intracellular vesicles that envelope single domain magnetite crystals. Although many proteins are present in this intracellular vesicle membrane, five are specific to this membrane. A 16-kDa protein, designated Mms16, is the most abundant of the magnetosome-specific proteins, and to establish its function we cloned and sequenced its gene from Magnetospirillum magneticum AMB-1. This was achieved by determination of the N-terminal amino acid sequence of the protein following two dimensional polyacrylamide gel electrophoresis, and sequencing of the gene was performed by gene walking using anchored polymerase chain reaction. Mms16 contains a putative ATP/GTP binding motif (P-loop). Recombinant Mms16 with a hemagglutinin tag, was expressed in Escherichia coli and purified. Recombinant Mms16 protein could bind GTP and showed GTPase activity. GTP was the preferred substrate for Mms16-catalyzed nucleotide triphosphate hydrolysis. These results suggest that a novel protein specifically localized on the magnetic particle membrane, Mms16, is a GTPase. Mms16 protein showed similar characteristics to small GTPases involved in the formation of intracellular vesicles. Furthermore, addition of the GTPase inhibitor AlF(4)- also inhibited magnetic particle synthesis, suggesting that GTPase is required for magnetic particles synthesis.     

Synthesis of Magnetic Nanoparticles Stabilized by Magnetite-Binding Protein for Targeted Delivery to Cancer Cells

A new method for obtaining biomodified magnetite nanoparticles for targeted delivery to cells was developed. The method is based on the use of the C-terminal fragment of the Mms6 protein, which is involved in the magnetite biomineralization during the synthesis of magnetosomes in magnetotactic bacteria Magnetospirillum magneticum AMB-1, and the barnase*barstar high-affinity protein pair. The Mms6 protein fragment is required for stabilizing magnetite, and the barnase*barstar pair mediates the interaction between nanoparticles and the component for modification. The efficiency of this method was confirmed in the synthesis of magnetite nanoparticles recognizing the HER2/neu tumor marker and in the selective labeling of HER2/neu with these nanoparticles on the surface of cancer cells.     

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.     

Structural and functional analyses of organic molecules regulating biomineralization

ABSTRACT

Biomineralization by living organisms are common phenomena observed everywhere. Molluskan shells are representative biominerals that have fine microstructures with controlled morphology, polymorph, and orientation of CaCO₃ crystals. A few organic molecules involved in the biominerals play important roles in the formation of such microstructures. Analyses of structure–function relationships for matrix proteins in biominerals revealed that almost all matrix proteins have an acidic region for the binding of calcium ion in CaCO₃ crystals and interaction domains for other organic molecules. On the other hand, biomineralization of metal nanoparticles by microorganisms were also investigated. Gold nanoparticles and quantum dots containing cadmium were successfully synthesized by bacteria or a fungus. The analyses of components revealed that glycolipids, oligosaccharides, and lactic acids have key roles to synthesize the gold nanoparticle in Lactobacillus casei as reductants and dispersants. These researches about biomineralization will give new insights for material and environmental sciences in the human society.     

Efficient and stable display of functional proteins on bacterial magnetic particles using mms13 as a novel anchor molecule

Magnetic particles are increasingly used for various biomedical applications because they are easy to handle and separate from biological samples. In this work, a novel anchor molecule was used for targeted protein display onto magnetic nanoparticles. The magnetic bacterium Magnetospirillum magneticum AMB-1 synthesizes intracellular bacterial magnetic particles (BMPs) covered with a lipid bilayer membrane. In our recent research, an integral BMP membrane protein, Mms13, was isolated and used as an anchor molecule to display functional proteins onto BMPs. The anchoring properties of Mms13 were confirmed by luciferase fusion studies. The C terminus of Mms13 was shown to be expressed on the surface of BMPs, and Mms13 was bound to magnetite directly and tightly permitting stable localization of a large protein, luciferase (61 kDa), on BMPs. Consequently, luminescence intensity obtained from BMPs using Mms13 as an anchor molecule was >400 or 1,000 times higher than Mms16 or MagA, which previously were used as anchor molecules. Furthermore, the immunoglobulin G-binding domain of protein A (ZZ) was displayed uniformly on BMPs using Mms13, and antigen was detected by transmission electron microscopy using antibody-labeled gold nanoparticles on a single BMP displaying the ZZ-antibody complex. The results of this study demonstrated the utility of Mms13 as a molecular anchor, which will facilitate the assembly of other functional proteins onto BMPs in the near feature.     

Interplay of magnetic interactions and active movements in the formation of magnetosome chains

Magnetotactic bacteria assemble chains of magnetosomes, organelles that contain magnetic nano-crystals. A number of genetic factors involved in the controlled biomineralization of these crystals and the assembly of magnetosome chains have been identified in recent years, but how the specific biological regulation is coordinated with general physical processes such as diffusion and magnetic interactions remains unresolved. Here, these questions are addressed by simulations of different scenarios for magnetosome chain formation, in which various physical processes and interactions are either switched on or off. The simulation results indicate that purely physical processes of magnetosome diffusion, guided by their magnetic interactions, are not sufficient for the robust chain formation observed experimentally and suggest that biologically encoded active movements of magnetosomes may be required. Not surprisingly, the chain pattern is most resembling experimental results when both magnetic interactions and active movement are coordinated. We estimate that the force such active transport has to generate is compatible with forces generated by the polymerization or depolymerization of cytoskeletal filaments. The simulations suggest that the pleiotropic phenotypes of mamK deletion strains may be due to a defect in active motility of magnetosomes and that crystal formation in magneteosome vesicles is coupled to the activation of their active motility in M. gryphiswaldense, but not in M. magneticum.     

Mms1 and Mms22 stabilize the replisome during replication stress

Mms1 and Mms22 form a Cul4(Ddb1)-like E3 ubiquitin ligase with the cullin Rtt101. In this complex, Rtt101 is bound to the substrate-specific adaptor Mms22 through a linker protein, Mms1. Although the Rtt101(Mms1/Mms22) ubiquitin ligase is important in promoting replication through damaged templates, how it does so has yet to be determined. Here we show that mms1Δ and mms22Δ cells fail to properly regulate DNA replication fork progression when replication stress is present and are defective in recovery from replication fork stress. Consistent with a role in promoting DNA replication, we find that Mms1 is enriched at sites where replication forks have stalled and that this localization requires the known binding partners of Mms1-Rtt101 and Mms22. Mms1 and Mms22 stabilize the replisome during replication stress, as binding of the fork-pausing complex components Mrc1 and Csm3, and DNA polymerase ε, at stalled replication forks is decreased in mms1Δ and mms22Δ. Taken together, these data indicate that Mms1 and Mms22 are important for maintaining the integrity of the replisome when DNA replication forks are slowed by hydroxyurea and thereby promote efficient recovery from replication stress.     

Measuring magnetosomal pH of the magnetotactic bacterium Magnetospirillum magneticum AMB-1 using pH-sensitive fluorescent proteins

Magnetotactic bacteria synthesize uniform-sized and regularly shaped magnetic nanoparticles in their organelles termed magnetosomes. Homeostasis of the magnetosome lumen must be maintained for its role accomplishment. Here, we developed a method to estimate the pH of a single living cell of the magnetotactic bacterium Magnetospirillum magneticum AMB-1 using a pH-sensitive fluorescent protein E²GFP. Using the pH measurement, we estimated that the cytoplasmic pH was approximately 7.6 and periplasmic pH was approximately 7.2. Moreover, we estimated pH in the magnetosome lumen and cytoplasmic surface using fusion proteins of E²GFP and magnetosome-associated proteins. The pH in the magnetosome lumen increased during the exponential growth phase when magnetotactic bacteria actively synthesize magnetite crystals, whereas pH at the magnetosome surface was not affected by the growth stage. This live-cell pH measurement method will help for understanding magnetosome pH homeostasis to reveal molecular mechanisms of magnetite biomineralization in the bacterial organelle.     

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.     

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.     

Dynamics of Iron Uptake and Fe3O4 Biomineralization during Aerobic and Microaerobic Growth of Magnetospirillum gryphiswaldense

Iron uptake and magnetite (Fe₃O₄) crystal formation could be studied in the microaerophilic magnetic bacterium Magnetospirillum gryphiswaldense by using a radioactive tracer method for iron transport and a differential light-scattering technique for magnetism. Magnetite formation occurred only in a narrow range of low oxygen concentration, i.e., 2 to 7 μM O₂ at 30°C. Magnetic cells stored up to 2% iron as magnetite crystals in intracytoplasmic vesicles. This extraordinary uptake of iron was coupled tightly to the biomineralization of up to 60 magnetite crystals with diameters of 42 to 45 nm.     

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.