André Scheffel

An acidic protein aligns magnetosomes along a filamentous structure in magnetotactic bacteria.

Magnetotactic bacteria are widespread aquatic microorganisms that use unique intracellular organelles to navigate along the Earths 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.

Characterization of a Spontaneous Nonmagnetic Mutant of Magnetospirillum gryphiswaldense Reveals a Large Deletion Comprising a Putative Magnetosome Island

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 magnetosome-associated 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.

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.

Nanopatterned protein microrings from a diatom that direct silica morphogenesis

Diatoms are eukaryotic microalgae that produce species-specifically structured cell walls made of SiO2 (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.

Loss of the actin-like protein MamK has pleiotropic effects on magnetosome formation and chain assembly in Magnetospirillum gryphiswaldense

Magnetotactic bacteria synthesize magnetosomes, which are unique organelles consisting of membrane‐enclosed 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.

The acidic repetitive domain of the Magnetospirillum gryphiswaldense MamJ protein displays hypervariability but is not required for magnetosome chain assembly.

Magnetotactic bacteria navigate along the earths 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 DeltamamJ mutant, suggesting that the acidic domain is not essential for MamJs 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 DeltamamJ 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.

A vacuole-like compartment concentrates a disordered calcium phase in a key coccolithophorid alga

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.

Live Diatom Silica Immobilization of Multimeric and Redox-Active Enzymes

ABSTRACT Living organisms are adept in forming inorganic materials (biominerals) with unique structures and properties that exceed the capabilities of engineered materials. Biomimetic materials syntheses are being developed that aim at replicating the advantageous properties of biominerals in vitro and endow them with additional functionalities. Recently, proof-of-concept was provided for an alternative approach that allows for the production of biomineral-based functional materials in vivo. In this approach, the cellular machinery for the biosynthesis of nano-/micropatterned SiO2 (silica) structures in diatoms was genetically engineered to incorporate a monomeric, cofactor-independent (“simple”) enzyme, HabB, into diatom silica. In the present work, it is demonstrated that this approach is also applicable for enzymes with “complex” activity requirements, including oligomerization, metal ions, organic redox cofactors, and posttranslational modifications. Functional expression of the enzymes β-glucuronidase, glucose oxidase, galactose oxidase, and horseradish peroxidase in the diatom Thalassiosira pseudonana was accomplished, and 66 to 78% of the expressed enzymes were stably incorporated into the biosilica. The in vivo incorporated enzymes represent approximately 0.1% (wt/wt) of the diatom biosilica and are stabilized against denaturation and proteolytic degradation. Furthermore, it is demonstrated that the gene construct for in vivo immobilization of glucose oxidase can be utilized as the first negative selection marker for diatom genetic engineering.

Pentalysine clusters mediate silica targeting of silaffins in Thalassiosira pseudonana

Background: Morphogenesis of diatom biosilica depends on a silaffin-dependent organic matrix inside intracellular vesicles (SDVs). Results: Silaffin-derived 12–14-mer peptides containing five modified lysines and several phosphoserines (pentalysine clusters) are sufficient for silica targeting in vivo. Conclusion: Pentalysine clusters function as address tags for SDV targeting of silaffins. Significance: Elucidating the molecular mechanisms for biogenesis of mineral-forming vesicles is essential for understanding biomineral morphogenesis. The biological formation of inorganic materials (biomineralization) often occurs in specialized intracellular vesicles. Prominent examples are diatoms, a group of single-celled eukaryotic microalgae that produce their SiO2 (silica)-based cell walls within intracellular silica deposition vesicles (SDVs). SDVs contain protein-based organic matrices that control silica formation, resulting in species specifically nanopatterned biosilica, an organic-inorganic composite material. So far no information is available regarding the molecular mechanisms of SDV biogenesis. Here we have investigated by fluorescence microscopy and subcellular membrane fractionation the intracellular transport of silaffin Sil3. Silaffins are a group of phosphoproteins constituting the main components of the organic matrix of diatom biosilica. We demonstrate that the N-terminal signal peptide of Sil3 mediates import into a specific subregion of the endoplasmic reticulum. Additional segments from the mature part of Sil3 are required to reach post-endoplasmic reticulum compartments. Further transport of Sil3 and incorporation into the biosilica (silica targeting) require protein segments that contain a high density of modified lysine residues and phosphoserines. Silica targeting of Sil3 is not dependent on a particular peptide sequence, yet a lysine-rich 12–14-amino acid peptide motif (pentalysine cluster), which is conserved in all silaffins, strongly promotes silica targeting. The results of the present work provide the first insight into the molecular mechanisms for biogenesis of mineral-forming vesicles from an eukaryotic organism.

Macromolecular recognition directs calcium ions to coccolith mineralization sites

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.