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.     

Fur in Magnetospirillum gryphiswaldense influences magnetosomes formation and directly regulates the genes involved in iron and oxygen metabolism

Magnetospirillum gryphiswaldense strain MSR-1 has the unique capability of taking up large amounts of iron and synthesizing magnetosomes (intracellular magnetic particles composed of Fe(3)O(4)). The unusual high iron content of MSR-1 makes it a useful model for studying biological mechanisms of iron uptake and homeostasis. The ferric uptake regulator (Fur) protein plays a key role in maintaining iron homeostasis in many bacteria. We identified and characterized a fur-homologous gene (MGR_1314) in MSR-1. MGR_1314 was able to complement a fur mutant of E. coli in iron-responsive manner in vivo. We constructed a fur mutant strain of MSR-1. In comparison to wild-type MSR-1, the mutant strain had lower magnetosome formation, and was more sensitive to hydrogen peroxide and streptonigrin, indicating higher intracellular free iron content. Quantitative real-time RT-PCR and chromatin immunoprecipitation analyses indicated that Fur protein directly regulates expression of several key genes involved in iron transport and oxygen metabolism, in addition it also functions in magnetosome formation in M. gryphiswaldense.     

A novel Porphyromonas gingivalis FeoB plays a role in manganese accumulation

FeoB is an atypical transporter that has been shown to exclusively mediate ferrous ion transport in some bacteria. Unusually the genome of the periodontal pathogen Porphyromonas gingivalis has two genes (feoB1 and feoB2) encoding FeoB homologs, both of which are expressed in bicistronic operons. Kinetic analysis of ferrous ion transport by P. gingivalis W50 revealed the presence of a single, high affinity system with a K(t) of 0.31 microM. FeoB1 was found to be solely responsible for this transport as energized cells of the isogenic FeoB1 mutant (W50FB1) did not transport radiolabeled iron, while the isogenic FeoB2 mutant (W50FB2) transported radiolabeled iron at a rate similar to wild type. This was reflected in the iron content of W50FB1 grown in iron excess conditions which was approximately half that of the wild type and W50FB2. The W50FB1 mutant had increased sensitivity to both oxygen and hydrogen peroxide and was avirulent in an animal model of infection whereas W50FB2 exhibited the same virulence as the wild type. Analysis of manganous ion uptake using inductively coupled plasma-mass spectrometry revealed a greater than 3-fold decrease in intracellular manganese accumulation in W50FB2 which was also unable to grow in manganese-limited media. The protein co-expressed with FeoB2 appears to be a novel FeoA-MntR fusion protein that exhibits homology to a manganese-responsive, DNA-binding metalloregulatory protein. These results indicate that FeoB2 is not involved in iron transport but plays a novel role in manganese transport.     

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.     

The magnetosome proteins MamX,MamZ and MamH are involved in redox control of magnetite biomineralization in Magnetospirillum gryphiswaldense

Magnetospirillum gryphiswaldense uses intracellular chains of membrane‐enveloped magnetite crystals, the magnetosomes, to navigate within magnetic fields. The biomineralization of magnetite nanocrystals requires several magnetosome‐associated proteins, whose precise functions so far have remained mostly unknown. Here, we analysed the functions of MamX and the Major Facilitator Superfamily (MFS) proteins MamZ and MamH. Deletion of either the entire mamX gene or elimination of its putative haem c‐binding magnetochrome domains, and deletion of either mamZ or its C‐terminal ferric reductase‐like component resulted in an identical phenotype. All mutants displayed WT‐like magnetite crystals, flanked within the magnetosome chains by poorly crystalline flake‐like particles partly consisting of haematite. Double deletions of both mamZ and its homologue mamH further impaired magnetite crystallization in an additive manner, indicating that the two MFS proteins have partially redundant functions. Deprivation of ΔmamX and ΔmamZ cells from nitrate, or additional loss of the respiratory nitrate reductase Nap from ΔmamX severely exacerbated the magnetosome defects and entirely inhibited the formation of regular crystals, suggesting that MamXZ and Nap have similar, but independent roles in redox control of biomineralization. We propose a model in which MamX, MamZ and MamH functionally interact to balance the redox state of iron within the magnetosome compartment.     

FeoB is not required for ferrous iron uptake in Campylobacter jejuni

Among strains of Campylobacter jejuni, levels of ferrous iron (Fe2+) uptake was comparable. However, C. jejuni showed a lower level of ferrous iron uptake than Escherichia coli. Consistent with studies of E. coli, Fe2+ uptake in C. jejuni was significantly enhanced by low Mg2+ concentration. The C. jejuni genome sequence contains a single known ferrous iron uptake gene, feoB, whose product shares 50% amino acid identity to Helicobacter pylori FeoB and 29% identity to E. coli FeoB. However, Fe2+ uptake could not be attributed to FeoB for several reasons. Site-directed mutations in feoB caused no defect in 55Fe2+ uptake. Among C. jejuni strains, various nucleotide alterations were found in feoB, indicating that some C. jejuni feoB genes are defective. In addition, uptake could not be attributed to the magnesium transporter CorA, since no reduction in 55Fe2+ uptake was observed in the presence of a CorA-specific inhibitor.     

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 %.     

From invagination to navigation: The story of magnetosome‐associated proteins in magnetotactic bacteria

Magnetotactic bacteria (MTB) are a group of Gram‐negative microorganisms that are able to sense and change their orientation in accordance with the geomagnetic field. This unique capability is due to the presence of a special suborganelle called the magnetosome, composed of either a magnetite or gregite crystal surrounded by a lipid membrane. MTB were first detected in 1975 and since then numerous efforts have been made to clarify the special mechanism of magnetosome formation at the molecular level. Magnetosome formation can be divided into several steps, beginning with vesicle invagination from the cell membrane, through protein sorting, followed by the combined steps of iron transportation, biomineralization, and the alignment of magnetosomes into a chain. The magnetosome‐chain enables the sensing of the magnetic field, and thus, allows the MTB to navigate. It is known that magnetosome formation is tightly controlled by a distinctive set of magnetosome‐associated proteins that are encoded mainly in a genomically conserved region within MTB called the magnetosome island (MAI). Most of these proteins were shown to have an impact on the magnetism of MTB. Here, we describe the process in which the magnetosome is formed with an emphasis on the different proteins that participate in each stage of the magnetosome formation scheme.     

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。}     

Iron acquisition and virulence in Helicobacter pylori: a major role for FeoB, a high-affinity ferrous iron transporter

The genome sequence of Helicobacter pylori suggests that this bacterium possesses several Fe acquisition systems, including both Fe2+- and Fe3+-citrate transporters. The role of these transporters was investigated by generating insertion mutants in feoB, tonB, fecA1 and fecDE. Fe transport in the feoB mutant was approximately 10-fold lower than in the wild type (with 0.5 microM Fe), irrespective of whether Fe was supplied in the Fe2+ or Fe3+ form. In contrast, transport rates were unaffected by the other mutations. Complementation of the feoB mutation fully restored both Fe2+ and Fe3+ transport. The growth inhibition exhibited by the feoB mutant in Fe-deficient media was relieved by human holo-transferrin, holo-lactoferrin and Fe3+-dicitrate, but not by FeSO4. The feoB mutant had less cellular Fe and was more sensitive to growth inhibition by transition metals in comparison with the wild type. Biphasic kinetics of Fe2+ transport in the wild type suggested the presence of high- and low-affinity uptake systems. The high-affinity system (apparent Ks = 0.54 microM) is absent in the feoB mutant. Transport via FeoB is highly specific for Fe2+ and was inhibited by FCCP, DCCD and vanadate, indicating an active process energized by ATP. Ferrozine inhibition of Fe2+ and Fe3+ uptake implied the concerted involvement of both an Fe3+ reductase and FeoB in the uptake of Fe supplied as Fe3+. Taken together, the results are consistent with FeoB-mediated Fe2+ uptake being a major pathway for H. pylori Fe acquisition. feoB mutants were unable to colonize the gastric mucosa of mice, indicating that FeoB makes an important contribution to Fe acquisition by H. pylori in the low-pH, low-O2 environment of the stomach.     

The FtsZ-Like Protein FtsZm of Magnetospirillum gryphiswaldense Likely Interacts with Its Generic Homolog and Is Required for Biomineralization under Nitrate Deprivation

Midcell selection, septum formation, and cytokinesis in most bacteria are orchestrated by the eukaryotic tubulin homolog FtsZ. The alphaproteobacterium Magnetospirillum gryphiswaldense (MSR-1) septates asymmetrically, and cytokinesis is linked to splitting and segregation of an intracellular chain of membrane-enveloped magnetite crystals (magnetosomes). In addition to a generic, full-length ftsZ gene, MSR-1 contains a truncated ftsZ homolog (ftsZm) which is located adjacent to genes controlling biomineralization and magnetosome chain formation. We analyzed the role of FtsZm in cell division and biomineralization together with the full-length MSR-1 FtsZ protein. Our results indicate that loss of FtsZm has a strong effect on microoxic magnetite biomineralization which, however, could be rescued by the presence of nitrate in the medium. Fluorescence microscopy revealed that FtsZm-mCherry does not colocalize with the magnetosome-related proteins MamC and MamK but is confined to asymmetric spots at midcell and at the cell pole, coinciding with the FtsZ protein position. In Escherichia coli, both FtsZ homologs form distinct structures but colocalize when coexpressed, suggesting an FtsZ-dependent recruitment of FtsZm. In vitro analyses indicate that FtsZm is able to interact with the FtsZ protein. Together, our data suggest that FtsZm shares key features with its full-length homolog but is involved in redox control for magnetite crystallization.     

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     

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.     

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.     

Identification of Iron Transporters Expressed in the Magnetotactic Bacterium <Emphasis Type="Italic">Magnetospirillum magnetotacticum</Emphasis>

The magnetotactic bacterium Magnetospirillum magnetotacticum MS-1 mineralizes the magnetite (Fe₃O₄) crystal and organizes a highly ordered intracellular structure, called the magnetosome. However, the iron transport system, which supports the biogenesis of magnetite, is not fully understood. In this study, we first identified the expressions of both the ferric and the ferrous iron transporter proteins in M. magnetotacticum. The cellular protein compositions of ferric and ferrous iron-rich cultures were examined using two-dimensional electrophoresis. According to the gel patterns, two outer-membrane ferric-siderophore receptor homologues were identified as proteins strongly induced in the ferrous iron-rich condition. Also, we identified for the first time that the ferrous iron transport protein, FeoB, is expressed in the M. magnetotacticum cytoplasmic membrane using immunoblotting.     

Diverse phylogeny and morphology of magnetite biomineralized by magnetotactic cocci

Magnetotactic bacteria (MTB) are diverse prokaryotes that produce magnetic nanocrystals within intracellular membranes (magnetosomes). Here, we present a large-scale analysis of diversity and magnetosome biomineralization in modern magnetotactic cocci, which are the most abundant MTB morphotypes in nature. Nineteen novel magnetotactic cocci species are identified phylogenetically and structurally at the single-cell level. Phylogenetic analysis demonstrates that the cocci cluster into an independent branch from other Alphaproteobacteria MTB, that is, within the Etaproteobacteria class in the Proteobacteria phylum. Statistical analysis reveals species-specific biomineralization of magnetosomal magnetite morphologies. This further confirms that magnetosome biomineralization is controlled strictly by the MTB cell and differs among species or strains. The post-mortem remains of MTB are often preserved as magnetofossils within sediments or sedimentary rocks, yet paleobiological and geological interpretation of their fossil record remains challenging. Our results indicate that magnetofossil morphology could be a promising proxy for retrieving paleobiological information about ancient MTB.     

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

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