Magnetotactic bacteria are widespread aquatic microorganisms that use unique intracellular organelles to navigate along the Earth's magnetic field. These organelles, called magnetosomes, consist of membrane-enclosed magnetite crystals that are thought to help to direct bacterial swimming towards growth-favouring microoxic zones at the bottom of natural waters. Questions in the study of magnetosome formation include understanding the factors governing the size and redox-controlled synthesis of the nano-sized magnetosomes and their assembly into a regular chain in order to achieve the maximum possible magnetic moment, against the physical tendency of magnetosome agglomeration. A deeper understanding of these mechanisms is expected from studying the genes present in the identified chromosomal 'magnetosome island', for which the connection with magnetosome synthesis has become evident. Here we use gene deletion in Magnetospirillum gryphiswaldense to show that magnetosome alignment is coupled to the presence of the mamJ gene product. MamJ is an acidic protein associated with a novel filamentous structure, as revealed by fluorescence microscopy and cryo-electron tomography. We suggest a mechanism in which MamJ interacts with the magnetosome surface as well as with a cytoskeleton-like structure. According to our hypothesis, magnetosome architecture represents one of the highest structural levels achieved in prokaryotic cells.

Frequent spontaneous loss of the magnetic phenotype was observed in stationary-phase cultures of the magnetotactic bacterium Magnetospirillum gryphiswaldense MSR-1. A nonmagnetic mutant, designated strain MSR-1B, was isolated and characterized. The mutant lacked any structures resembling magnetosome crystals as well as internal membrane vesicles. The growth of strain MSR-1B was impaired under all growth conditions tested, and the uptake and accumulation of iron were drastically reduced under iron-replete conditions. A large chromosomal deletion of approximately 80 kb was identified in strain MSR-1B, which comprised both the entire mamAB and mamDC clusters as well as further putative operons encoding a number of magnetosomeassociated proteins. A bacterial artificial chromosome clone partially covering the deleted region was isolated from the genomic library of wild-type M. gryphiswaldense. Sequence analysis of this fragment revealed that all previously identified mam genes were closely linked with genes encoding other magnetosome-associated proteins within less than 35 kb. In addition, this region was remarkably rich in insertion elements and harbored a considerable number of unknown gene families which appeared to be specific for magnetotactic bacteria. Overall, these findings suggest the existence of a putative large magnetosome island in M. gryphiswaldense and other magnetotactic bacteria.Magnetotactic bacteria are capable of forming magnetosomes, which are specific intracellular structures that enable the cells to orient along magnetic field lines (3, 4, 41). The superior crystalline and magnetic properties of magnetosomes make them potentially useful as a highly ordered biomaterial in a number of applications, e.g., in the immobilization of bioactive compounds, magnetic drug targeting, or as a contrast agent for magnetic resonance imaging (24,41). Recently, the characteristics of bacterial magnetosomes have even been considered for use as biosignatures to identify presumptive Martian magnetofossils (49). Moreover, understanding bacterial magnetosome formation is expected to provide insights into more complex biomineralization systems in higher organisms (19). The biomineralization of magnetosome particles is achieved by a complex mechanism with control over the uptake, accumulation, and precipitation of iron, which, however, is poorly understood at the molecular and biochemical level.The magnetotactic ␣-proteobacterium Magnetospirillum gryphiswaldense microaerobically produces up to 60 cubo-octahedral magnetosomes, which are approximately 45 nm in size and consist of membrane-bounded crystals of the iron mineral magnetite (Fe 3 O 4 ) (34,42). In contrast to most other magnetotactic bacteria, methods for mass culture and genetic manipulation of M. gryphiswaldense are available (17,38,44), which has facilitated its analysis in a number of studies (37,39,40,43).In Magnetospirillum species, the deposition of the mineral particle occurs within a specific compartment, which is provided by the magnetosome membrane ...

Diatoms are eukaryotic microalgae that produce species-specifically structured cell walls made of SiO 2 (silica). Formation of the intricate silica structures of diatoms is regarded as a paradigm for biomolecule-controlled self-assembly of three-dimensional, nano-to microscale-patterned inorganic materials. Silica formation involves long-chain polyamines and phosphoproteins (silaffins and silacidins), which are readily soluble in water, and spontaneously form dynamic supramolecular assemblies that accelerate silica deposition and influence silica morphogenesis in vitro. However, synthesis of diatom-like silica structure in vitro has not yet been accomplished, indicating that additional components are required. Here we describe the discovery and intracellular location of six novel proteins (cingulins) that are integral components of a silica-forming organic matrix (microrings) in the diatom Thalassiosira pseudonana. The cingulin-containing microrings are specifically associated with girdle bands, which constitute a substantial part of diatom biosilica. Remarkably, the microrings exhibit protein-based nanopatterns that closely resemble characteristic features of the girdle band silica nanopatterns. Upon the addition of silicic acid the microrings become rapidly mineralized in vitro generating nanopatterned silica replicas of the microring structures. A silica-forming organic matrix with characteristic nanopatterns was also discovered in the diatom Coscinodiscus wailesii, which suggests that preassembled protein-based templates might be general components of the cellular machinery for silica morphogenesis in diatoms. These data provide fundamentally new insight into the molecular mechanisms of biological silica morphogenesis, and may lead to the development of self-assembled 3D mineral forming protein scaffolds with designed nanopatterns for a host of applications in nanotechnology.biomineralization | GFP | silica deposition vesicle | bioinformatics

Magnetospirillum gryphiswaldense and related magnetotactic bacteria form magnetosomes, which are membrane-enclosed organelles containing crystals of magnetite (Fe 3 O 4 ) 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 ability of magnetotactic bacteria (MTB) to orient in the earth's magnetic field is based on specific organelles, the magnetosomes, which are membrane-enveloped crystals of a magnetic mineral that are arranged in chain-like structures within the cell (5). Magnetosome crystals display a variety of species-specific shapes and sizes, which for most MTB are between 35 and 120 nm (3, 4). In MTB of the genus Magnetospirillum, cubo-octahedral nanocrystals of the magnetic mineral magnetite (Fe 3 O 4 ) are synthesized within magnetosome membrane (MM) vesicles, which are roughly spherical and are formed by invagination from the cytoplasmic membrane prior to magnetite biomineralization (2, 15, 16). The MM is a phospholipid bilayer with a distinctive biochemical composition (8,29,35). In Magnetospirillum gryphiswaldense, a specific set of 20 MM proteins (MMPs) was identified by biochemical and proteomic approaches (9,10,22), and these proteins were speculated to be involved in magnetosome biomineralization (29). However, the individual functions of M...

SummaryMagnetotactic bacteria synthesize magnetosomes, which are unique organelles consisting of membraneenclosed magnetite crystals. For magnetic orientation individual magnetosome particles are assembled into well-organized chains. The actin-like MamK and the acidic MamJ proteins were previously implicated in chain assembly. While MamK was suggested to form magnetosome-associated cytoskeletal filaments, MamJ is assumed to attach the magnetosome vesicles to these structures. Although the deletion of either mamK in Magnetospirillum magneticum, or mamJ in Magnetospirillum gryphiswaldense affected chain formation, the previously observed phenotypes were not fully consistent, suggesting different mechanisms of magnetosome chain assembly in both organisms. Here we show that in M. gryphiswaldense MamK is not absolutely required for chain formation. Straight chains, albeit shorter, fragmented and ectopic, were still formed in a mamK deletion mutant, although magnetosome filaments were absent as shown by cryo-electron tomography. Loss of MamK also resulted in reduced numbers of magnetite crystals and magnetosome vesicles and led to the mislocalization of MamJ. In addition, extensive analysis of wild type and mutant cells revealed previously unidentified ultrastructural characteristics in M. gryphiswaldense. Our results suggest that, despite of their functional equivalence, loss of MamK proteins in different bacteria may result in distinct phenotypes, which might be due to a species-specific genetic context.

Magnetotactic bacteria navigate along the earth's magnetic field using chains of magnetosomes, which are intracellular organelles comprising membrane-enclosed magnetite crystals. The assembly of highly ordered magnetosome chains is under genetic control and involves several specific proteins. Based on genetic and cryo-electron tomography studies, a model was recently proposed in which the acidic MamJ magnetosome protein attaches magnetosome vesicles to the actin-like cytoskeletal filament formed by MamK, thereby preventing magnetosome chains from collapsing. However, the exact functions as well as the mode of interaction between MamK and MamJ are unknown. Here, we demonstrate that several functional MamJ variants from Magnetospirillum gryphiswaldense and other magnetotactic bacteria share an acidic and repetitive central domain, which displays an unusual intra-and interspecies sequence polymorphism, probably caused by homologous recombination between identical copies of Glu-and Pro-rich repeats. Surprisingly, mamJ mutant alleles in which the central domain was deleted retained their potential to restore chain formation in a ⌬mamJ mutant, suggesting that the acidic domain is not essential for MamJ's function. Results of two-hybrid experiments indicate that MamJ physically interacts with MamK, and two distinct sequence regions within MamJ were shown to be involved in binding to MamK. Mutant variants of MamJ lacking either of the binding domains were unable to functionally complement the ⌬mamJ mutant. In addition, two-hybrid experiments suggest both MamK-binding domains of MamJ confer oligomerization of MamJ. In summary, our data reveal domains required for the functions of the MamJ protein in chain assembly and maintenance and provide the first experimental indications for a direct interaction between MamJ and the cytoskeletal filament protein MamK.

Coccoliths are calcitic particles produced inside the cells of unicellular marine algae known as coccolithophores. They are abundant components of sea-floor carbonates, and the stoichiometry of calcium to other elements in fossil coccoliths is widely used to infer past environmental conditions. Here we study cryo-preserved cells of the dominant coccolithophore Emiliania huxleyi using state-of-the-art nanoscale imaging and spectroscopy. We identify a compartment, distinct from the coccolith-producing compartment, filled with high concentrations of a disordered form of calcium. Co-localized with calcium are high concentrations of phosphorus and minor concentrations of other cations. The amounts of calcium stored in this reservoir seem to be dynamic and at a certain stage the compartment is in direct contact with the coccolith-producing vesicle, suggesting an active role in coccolith formation. Our findings provide insights into calcium accumulation in this important calcifying organism.

Many organisms form elaborate mineralized structures, constituted of highly organized arrangements of crystals and organic macromolecules. The localization of crystals within these structures is presumably determined by the interaction of nucleating macromolecules with the mineral phase. Here we show that, preceding nucleation, a specific interaction between soluble organic molecules and an organic backbone structure directs mineral components to specific sites. This strategy underlies the formation of coccoliths, which are highly ordered arrangements of calcite crystals produced by marine microalgae. On combining the insoluble organic coccolith scaffold with coccolith-associated soluble macromolecules in vitro, we found a massive accretion of calcium ions at the sites where the crystals form in vivo. The in vitro process exhibits profound similarities to the initial stages of coccolith biogenesis in vivo.