Collective magnetotaxis of microbial holobionts is optimized by the three-dimensional organization and magnetic properties of ectosymbionts.

Over the last few decades, symbiosis and the concept of holobiont-a host entity with a population of symbionts-have gained a central role in our understanding of life functioning and diversification. Regardless of the type of partner interactions, understanding how the biophysical properties of each individual symbiont and their assembly may generate collective behaviors at the holobiont scale remains a fundamental challenge. This is particularly intriguing in the case of the newly discovered magnetotactic holobionts (MHB) whose motility relies on a collective magnetotaxis (i.e., a magnetic field-assisted motility guided by a chemoaerotaxis system). This complex behavior raises many questions regarding how magnetic properties of symbionts determine holobiont magnetism and motility. Here, a suite of light-, electron- and X-ray-based microscopy techniques [including X-ray magnetic circular dichroism (XMCD)] reveals that symbionts optimize the motility, the ultrastructure, and the magnetic properties of MHBs from the microscale to the nanoscale. In the case of these magnetic symbionts, the magnetic moment transferred to the host cell is in excess (102 to 103 times stronger than free-living magnetotactic bacteria), well above the threshold for the host cell to gain a magnetotactic advantage. The surface organization of symbionts is explicitly presented herein, depicting bacterial membrane structures that ensure longitudinal alignment of cells. Magnetic dipole and nanocrystalline orientations of magnetosomes were also shown to be consistently oriented in the longitudinal direction, maximizing the magnetic moment of each symbiont. With an excessive magnetic moment given to the host cell, the benefit provided by magnetosome biomineralization beyond magnetotaxis can be questioned.

Wettability of Magnetite Nanoparticles Guides Growth from Stabilized Amorphous Ferrihydrite.

Crystal formation via amorphous precursors is a long-sought-after gateway to engineer nanoparticles with well-controlled size and morphology. Biomineralizing organisms, like magnetotactic bacteria, follow such a nonclassical crystallization pathway to produce magnetite nanoparticles with sophistication unmatched by synthetic efforts at ambient conditions. Here, using in situ small-angle X-ray scattering, we demonstrate how the addition of poly(arginine) in the synthetic formation of magnetite nanoparticles induces a biomineralization-reminiscent pathway. The addition of poly(arginine) stabilizes an amorphous ferrihydrite precursor, shifting the magnetite formation pathway from thermodynamic to kinetic control. Altering the energetic landscape of magnetite formation by catalyzing the pH-dependent precursor attachment, we tune magnetite nanoparticle size continuously, exceeding sizes observed in magnetotactic bacteria. This mechanistic shift we uncover here further allows for crystal morphology control by adjusting the pH-dependent interfacial interaction between liquidlike ferrihydrite and nascent magnetite nanoparticles, establishing a new strategy to control nanoparticle morphology. Synthesizing compact single crystals at wetting conditions and unique semicontinuous single-crystalline nanoparticles at dewetting conditions in combination with an improved control over magnetite crystallite size, we demonstrate the versatility of bio-inspired, kinetically controlled nanoparticle formation pathways.

Identification and elimination of genomic regions irrelevant for magnetosome biosynthesis by large-scale deletion in Magnetospirillum gryphiswaldense.

BACKGROUND: Magnetosome formation in the alphaproteobacterium Magnetospirillum gryphiswaldense is controlled by more than 30 known mam and mms genes clustered within a large genomic region, the 'magnetosome island' (MAI), which also harbors numerous mobile genetic elements, repeats, and genetic junk. Because of the inherent genetic instability of the MAI caused by neighboring gene content, the elimination of these regions and their substitution by a compact, minimal magnetosome expression cassette would be important for future analysis and engineering. In addition, the role of the MAI boundaries and adjacent regions are still unclear, and recent studies indicated that further auxiliary determinants for magnetosome biosynthesis are encoded outside the MAI. However, techniques for large-scale genome editing of magnetic bacteria are still limited, and the full complement of genes controlling magnetosome formation has remained uncertain. RESULTS: Here we demonstrate that an allelic replacement method based on homologous recombination can be applied for large-scale genome editing in M. gryphiswaldense. By analysis of 24 deletion mutants covering about 167 kb of non-redundant genome content, we identified genes and regions inside and outside the MAI irrelevant for magnetosome biosynthesis. A contiguous stretch of ~ 100 kb, including the scattered mam and mms6 operons, could be functionally substituted by a compact and contiguous ~ 38 kb cassette comprising all essential biosynthetic gene clusters, but devoid of interspersing irrelevant or problematic gene content. CONCLUSIONS: Our results further delineate the genetic complement for magnetosome biosynthesis and will be useful for future large-scale genome editing and genetic engineering of magnetosome biosynthesis.

Stokesian dynamics simulations of a magnetotactic bacterium.

The swimming of bacteria provides insight into propulsion and steering under the conditions of low-Reynolds number hydrodynamics. Here we address the magnetically steered swimming of magnetotactic bacteria. We use Stokesian dynamics simulations to study the swimming of single-flagellated magnetotactic bacteria (MTB) in an external magnetic field. Our model MTB consists of a spherical cell body equipped with a magnetic dipole moment and a helical flagellum rotated by a rotary motor. The elasticity of the flagellum as well as magnetic and hydrodynamic interactions is taken into account in this model. We characterized how the swimming velocity is dependent on parameters of the model. We then studied the U-turn motion after a field reversal and found two regimes for weak and strong fields and, correspondingly, two characteristic time scales. In the two regimes, the U-turn time is dominated by the turning of the cell body and its magnetic moment or the turning of the flagellum, respectively. In the regime for weak fields, where turning is dominated by the magnetic relaxation, the U-turn time is approximately in agreement with a theoretical model based on torque balance. In the strong-field regime, strong deformations of the flagellum are observed. We further simulated the swimming of a bacterium with a magnetic moment that is inclined relative to the flagellar axis. This scenario leads to intriguing double helical trajectories that we characterize as functions of the magnetic moment inclination and the magnetic field. For small inclination angles ([Formula: see text]) and typical field strengths, the inclination of the magnetic moment has only a minor effect on the swimming of MTB in an external magnetic field. Large inclination angles result in a strong reduction in the velocity in direction of the magnetic field, consistent with recent observations that bacteria with large inclination angles use a different propulsion mechanism.

Native-state imaging of calcifying and noncalcifying microalgae reveals similarities in their calcium storage organelles.

Calcium storage organelles are common to all eukaryotic organisms and play a pivotal role in calcium signaling and cellular calcium homeostasis. In most organelles, the intraorganellar calcium concentrations rarely exceed micromolar levels. Acidic organelles called acidocalcisomes, which concentrate calcium into dense phases together with polyphosphates, are an exception. These organelles have been identified in diverse organisms, but, to date, only in cells that do not form calcium biominerals. Recently, a compartment storing molar levels of calcium together with phosphorous was discovered in an intracellularly calcifying alga, the coccolithophore Emiliania huxleyi, raising a possible connection between calcium storage organelles and calcite biomineralization. Here we used cryoimaging and cryospectroscopy techniques to investigate the anatomy and chemical composition of calcium storage organelles in their native state and at nanometer-scale resolution. We show that the dense calcium phase inside the calcium storage compartment of the calcifying coccolithophore Pleurochrysis carterae and the calcium phase stored in acidocalcisomes of the noncalcifying alga Chlamydomonas reinhardtii have common features. Our observations suggest that this strategy for concentrating calcium is a widespread trait and has been adapted for coccolith formation. The link we describe between acidocalcisomal calcium storage and calcium storage in coccolithophores implies that our physiological and molecular genetic understanding of acidocalcisomes could have relevance to the calcium pathway underlying coccolithophore calcification, offering a fresh entry point for mechanistic investigations on the adaptability of this process to changing oceanic conditions.

Self-Confined Nucleation of Iron Oxide Nanoparticles in a Nanostructured Amorphous Precursor.

Crystallization from solution is commonly described by classical nucleation theory, although this ignores that crystals often form via disordered nanostructures. As an alternative, the classical theory remains widely used in a "multistep" variant, where the intermediate nanostructures merely introduce additional thermodynamic parameters. However, this variant still requires validation by experiments addressing indeed proper time and spatial scales (millisecond, nanometer). Here, we used in situ X-ray scattering to determine the mechanism of magnetite crystallization and, in particular, how nucleation propagates at the nanometer scale within amorphous precursors. We find that the self-confinement by an amorphous precursor slows down crystal growth by 2 orders of magnitude once the crystal size reaches the amorphous particle size (∼3 nm). Thus, not only the thermodynamic properties of transient amorphous nanostructures but also their spatial distribution determine crystal nucleation.

Chemotaxis in external fields: Simulations for active magnetic biological matter.

The movement of microswimmers is often described by active Brownian particle models. Here we introduce a variant of these models with several internal states of the swimmer to describe stochastic strategies for directional swimming such as run and tumble or run and reverse that are used by microorganisms for chemotaxis. The model includes a mechanism to generate a directional bias for chemotaxis and interactions with external fields (e.g., gravity, magnetic field, fluid flow) that impose forces or torques on the swimmer. We show how this modified model can be applied to various scenarios: First, the run and tumble motion of E. coli is used to establish a paradigm for chemotaxis and investigate how it is affected by external forces. Then, we study magneto-aerotaxis in magnetotactic bacteria, which is biased not only by an oxygen gradient towards a preferred concentration, but also by magnetic fields, which exert a torque on an intracellular chain of magnets. We study the competition of magnetic alignment with active reorientation and show that the magnetic orientation can improve chemotaxis and thereby provide an advantage to the bacteria, even at rather large inclination angles of the magnetic field relative to the oxygen gradient, a case reminiscent of what is expected for the bacteria at or close to the equator. The highest gain in chemotactic velocity is obtained for run and tumble with a magnetic field parallel to the gradient, but in general a mechanism for reverse motion is necessary to swim against the magnetic field and a run and reverse strategy is more advantageous in the presence of a magnetic torque. This finding is consistent with observations that the dominant mode of directional changes in magnetotactic bacteria is reversal rather than tumbles. Moreover, it provides guidance for the design of future magnetic biohybrid swimmers.

Decoding Biomineralization: Interaction of a Mad10-Derived Peptide with Magnetite Thin Films.

Protein-surface interactions play a pivotal role in processes as diverse as biomineralization, biofouling, and the cellular response to medical implants. In biomineralization processes, biomacromolecules control mineral deposition and architecture via complex and often unknown mechanisms. For studying these mechanisms, the formation of magnetite nanoparticles in magnetotactic bacteria has become an excellent model system. Most interestingly, nanoparticle morphologies have been discovered that defy crystallographic rules (e.g., in the species Desulfamplus magnetovallimortis strain BW-1). In certain conditions, this strain mineralizes bullet-shaped magnetite nanoparticles, which exhibit defined (111) crystal faces and are elongated along the [100] direction. We hypothesize that surface-specific protein interactions break the nanoparticle symmetry, inhibiting the growth of certain crystal faces and thereby favoring the growth of others. Screening the genome of BW-1, we identified Mad10 (Magnetosome-associated deep-branching) as a potential magnetite-binding protein. Using atomic force microscope (AFM)-based single-molecule force spectroscopy, we show that a Mad10-derived peptide, which represents the most conserved region of Mad10, binds strongly to (100)- and (111)-oriented single-crystalline magnetite thin films. The peptide-magnetite interaction is thus material- but not crystal-face-specific. It is characterized by broad rupture force distributions that do not depend on the retraction speed of the AFM cantilever. To account for these experimental findings, we introduce a three-state model that incorporates fast rebinding. The model suggests that the peptide-surface interaction is strong in the absence of load, which is a direct result of this fast rebinding process. Overall, our study sheds light on the kinetic nature of peptide-surface interactions and introduces a new magnetite-binding peptide with potential use as a functional coating for magnetite nanoparticles in biotechnological and biomedical applications.

Structural insight into magnetochrome-mediated magnetite biomineralization.

Magnetotactic bacteria align along the Earth's magnetic field using an organelle called the magnetosome, a biomineralized magnetite (Fe(II)Fe(III)2O4) or greigite (Fe(II)Fe(III)2S4) crystal embedded in a lipid vesicle. Although the need for both iron(II) and iron(III) is clear, little is known about the biological mechanisms controlling their ratio. Here we present the structure of the magnetosome-associated protein MamP and find that it is built on a unique arrangement of a self-plugged PDZ domain fused to two magnetochrome domains, defining a new class of c-type cytochrome exclusively found in magnetotactic bacteria. Mutational analysis, enzyme kinetics, co-crystallization with iron(II) and an in vitro MamP-assisted magnetite production assay establish MamP as an iron oxidase that contributes to the formation of iron(III) ferrihydrite eventually required for magnetite crystal growth in vivo. These results demonstrate the molecular mechanisms of iron management taking place inside the magnetosome and highlight the role of magnetochrome in iron biomineralization.

Biohybrid and Bioinspired Magnetic Microswimmers.

Many motile microorganisms swim and navigate in chemically and mechanically complex environments. These organisms can be functionalized and directly used for applications (biohybrid approach), but also inspire designs for fully synthetic microbots. The most promising designs of biohybrids and bioinspired microswimmers include one or several magnetic components, which lead to sustainable propulsion mechanisms and external controllability. This Review addresses such magnetic microswimmers, which are often studied in view of certain applications, mostly in the biomedical area, but also in the environmental field. First, propulsion systems at the microscale are reviewed and the magnetism of microswimmers is introduced. The review of the magnetic biohybrids and bioinspired microswimmers is structured gradually from mostly biological systems toward purely synthetic approaches. Finally, currently less explored parts of this field ranging from in situ imaging to swarm control are discussed.

Magnetosome Organization in Magnetotactic Bacteria Unraveled by Ferromagnetic Resonance Spectroscopy.

Magnetotactic bacteria form assemblies of magnetic nanoparticles called magnetosomes. These magnetosomes are typically arranged in chains, but other forms of assemblies such as clusters can be observed in some species and genetic mutants. As such, the bacteria have developed as a model for the understanding of how organization of particles can influence the magnetic properties. Here, we use ferromagnetic resonance spectroscopy to measure the magnetic anisotropies in different strains of Magnetosprillum gryphiswaldense MSR-1, a bacterial species that is amendable to genetic mutations. We combine our experimental results with a model describing the spectra. The model includes chain imperfections and misalignments following a Fisher distribution function, in addition to the intrinsic magnetic properties of the magnetosomes. Therefore, by applying the model to analyze the ferromagnetic resonance data, the distribution of orientations in the bulk sample can be retrieved in addition to the average magnetosome arrangement. In this way, we quantitatively characterize the magnetosome arrangement in both wild-type cells and ΔmamJ mutants, which exhibit differing magnetosome organization.

Lattice distortions in coccolith calcite crystals originate from occlusion of biomacromolecules.

During biomineralization, organisms control the formation and morphology of a mineral using biomacromolecules. The biomacromolecules that most strongly interact with the growing crystals frequently get occluded within. Such an observation has been recently obtained for the calcium carbonate producing coccolithophore species Pleurochrysis carterae. Coccolithophores are unicellular algae that produce calcified scales built from complex-shaped calcite crystals, termed coccoliths. It is unclear how widespread the phenomenon of biomacromolecular occlusion within calcite crystals is in calcifying haptophytes such as coccolithophores. Here, the coccoliths of biological replicates of the bloom forming Emiliania huxleyi are compared with that of Pleurochrysis carterae, two species with different coccolith morphologies and crystal growth mechanisms. From high-resolution synchrotron X-ray diffraction, changes in the lattice parameters of coccolith calcite, after heating to 450°C, are observed and associated with macrostrain originating from occluded biomacromolecules. We propose a mechanism governing the biomacromolecules' interaction with the growing coccolith crystals and their likely origin.

Structure-function studies of the magnetite-biomineralizing magnetosome-associated protein MamC.

Magnetotactic bacteria are Gram-negative bacteria that navigate along geomagnetic fields using the magnetosome, an organelle that consists of a membrane-enveloped magnetic nanoparticle. Magnetite formation and its properties are controlled by a specific set of proteins. MamC is a small magnetosome-membrane protein that is known to be active in iron biomineralization but its mechanism has yet to be clarified. Here, we studied the relationship between the MamC magnetite-interaction loop (MIL) structure and its magnetite interaction using an inert biomineralization protein-MamC chimera. Our determined structure shows an alpha-helical fold for MamC-MIL with highly charged surfaces. Additionally, the MamC-MIL induces the formation of larger magnetite crystals compared to protein-free and inert biomineralization protein control experiments. We suggest that the connection between the MamC-MIL structure and the protein's charged surfaces is crucial for magnetite binding and thus for the size control of the magnetite nanoparticles.

Magnetotactic bacteria form magnetite from a phosphate-rich ferric hydroxide via nanometric ferric (oxyhydr)oxide intermediates.

The iron oxide mineral magnetite (Fe3O4) is produced by various organisms to exploit magnetic and mechanical properties. Magnetotactic bacteria have become one of the best model organisms for studying magnetite biomineralization, as their genomes are sequenced and tools are available for their genetic manipulation. However, the chemical route by which magnetite is formed intracellularly within the so-called magnetosomes has remained a matter of debate. Here we used X-ray absorption spectroscopy at cryogenic temperatures and transmission electron microscopic imaging techniques to chemically characterize and spatially resolve the mechanism of biomineralization in those microorganisms. We show that magnetite forms through phase transformation from a highly disordered phosphate-rich ferric hydroxide phase, consistent with prokaryotic ferritins, via transient nanometric ferric (oxyhydr)oxide intermediates within the magnetosome organelle. This pathway remarkably resembles recent results on synthetic magnetite formation and bears a high similarity to suggested mineralization mechanisms in higher organisms.

Fast Magnetic Micropropellers with Random Shapes.

Studying propulsion mechanisms in low Reynolds number fluid has implications for many fields, ranging from the biology of motile microorganisms and the physics of active matter to micromixing in catalysis and micro- and nanorobotics. The propulsion of magnetic micropropellers can be characterized by a dimensionless speed, which solely depends on the propeller geometry for a given axis of rotation. However, this dependence has so far been only investigated for helical propeller shapes, which were assumed to be optimal. In order to explore a larger variety of shapes, we experimentally studied the propulsion properties of randomly shaped magnetic micropropellers. Surprisingly, we found that their dimensionless speeds are high on average, comparable to previously reported nanofabricated helical micropropellers. The highest dimensionless speed we observed is higher than that of any previously reported propeller moving in a low Reynolds number fluid, proving that physical random shape generation can be a viable optimization strategy.

The giant keyhole limpet radular teeth: A naturally-grown harvest machine.

The limpet radula is a feeding organ, which contains more than 100 rows of teeth. During their growth the teeth mature and advance in position along the radula. The simpler doccoglossan radulae operate by grinding rocky substrates, extracting the algae by rasping and scraping with the teeth functioning as shovels. Less is known about the rhipidoglossan radulae, used as rakes or brooms that brush and collect loose marine debris. This type of radula is found in the giant keyhole limpet (Megathura crenulata). The large size of this organism suggests that the rhipidoglossan radula entails a technological superiority for M. crenulata in its habitat. The structure and function of the radulae teeth have however not been reported in detail. Using a combination of 2D and 3D microscopy techniques coupled with amino acid analysis and X-ray scattering, we reveal the working components of M. crenulata's radula. It is characterized by numerous marginal teeth surrounding a pair of major hook-like lateral teeth, two pairs of minor lateral teeth and a large central tooth. The mature major lateral teeth show pronounced signs of wear, which gradually increase towards the very front end of the radula and are evidence for scraping. An abrupt change in the amino acid composition in the major lateral teeth and the concurrent formation of a chitinous fiber-network mark the onset of tooth maturation. In comparison to the simpler rock-scraping doccoglossate limpets, the radula of M. crenulata forms an elaborate feeding apparatus, which can be seen as a natural harvest machine.

Probing the mechanical properties of magnetosome chains in living magnetotactic bacteria.

The mechanical properties of cytoskeletal networks are intimately involved in determining how forces and cellular processes are generated, directed, and transmitted in living cells. However, determining the mechanical properties of subcellular molecular complexes in vivo has proven to be difficult. Here, we combine in vivo measurements by optical microscopy, X-ray diffraction, and transmission electron microscopy with theoretical modeling to decipher the mechanical properties of the magnetosome chain system encountered in magnetotactic bacteria. We exploit the magnetic properties of the endogenous intracellular nanoparticles to apply a force on the filament-connector pair involved in the backbone formation and stabilization. We show that the magnetosome chain can be broken by the application of external field strength higher than 30 mT and suggest that this originates from the rupture of the magnetosome connector MamJ. In addition, we calculate that the biological determinants can withstand in vivo a force of 25 pN. This quantitative understanding provides insights for the design of functional materials such as actuators and sensors using cellular components.

From bacteria to mollusks: the principles underlying the biomineralization of iron oxide materials.

Various organisms possess a genetic program that enables the controlled formation of a mineral, a process termed biomineralization. The variety of biological material architectures is mind-boggling and arises from the ability of organisms to exert control over crystal nucleation and growth. The structure and composition of biominerals equip biomineralizing organisms with properties and functionalities that abiotically formed materials, made of the same mineral, usually lack. Therefore, elucidating the mechanisms underlying biomineralization and morphogenesis is of interdisciplinary interest to extract design principles that will enable the biomimetic formation of functional materials with similar capabilities. Herein, we summarize what is known about iron oxides formed by bacteria and mollusks for their magnetic and mechanical properties. We describe the chemical and biological machineries that are involved in controlling mineral precipitation and organization and show how these organisms are able to form highly complex structures under physiological conditions.

Diversity of magneto-aerotactic behaviors and oxygen sensing mechanisms in cultured magnetotactic bacteria.

Microorganisms living in gradient environments affect large-scale processes, including the cycling of elements such as carbon, nitrogen or sulfur, the rates and fate of primary production, and the generation of climatically active gases. Aerotaxis is a common adaptation in organisms living in the oxygen gradients of stratified environments. Magnetotactic bacteria are such gradient-inhabiting organisms that have a specific type of aerotaxis that allows them to compete at the oxic-anoxic interface. They biomineralize magnetosomes, intracellular membrane-coated magnetic nanoparticles, that comprise a permanent magnetic dipole that causes the cells to align along magnetic field lines. The magnetic alignment enables them to efficiently migrate toward an optimal oxygen concentration in microaerobic niches. This phenomenon is known as magneto-aerotaxis. Magneto-aerotaxis has only been characterized in a limited number of available cultured strains. In this work, we characterize the magneto-aerotactic behavior of 12 magnetotactic bacteria with various morphologies, phylogenies, physiologies, and flagellar apparatus. We report six different magneto-aerotactic behaviors that can be described as a combination of three distinct mechanisms, including the reported (di-)polar, axial, and a previously undescribed mechanism we named unipolar. We implement a model suggesting that the three magneto-aerotactic mechanisms are related to distinct oxygen sensing mechanisms that regulate the direction of cells' motility in an oxygen gradient.