Lipid membrane modulated control of magnetic nanoparticles within bacterial systems.

Bacterial magnetosomes synthesized by the magnetotactic bacterium Magnetospirillum magneticum are suitable for biomedical and biotechnological applications because of their high level of chemical purity of mineral with well-defined morphological features and a biocompatible lipid bilayer coating. However, utilizations of native magnetosomes are not sufficient for maximum effectiveness in many applications as the appropriate particle size differs. In this study, a method to control magnetosome particle size is developed for integration into targeted technological applications. The size and morphology of magnetosome crystals are highly regulated by the complex interactions of magnetosome synthesis-related genes; however, these interactions have not been fully elucidated. In contrast, previous studies have shown a positive correlation between vesicle and crystal sizes. Therefore, control of the magnetosome vesicle size is tuned by modifying the membrane lipid composition. Exogenous phospholipid synthesis pathways have been genetically introduced into M. magneticum. The experimental results show that these phospholipids altered the properties of the magnetosome membrane vesicles, which yielded larger magnetite crystal sizes. The genetic engineering approach presented in this study is shown to be useful for controlling magnetite crystal size without involving complex interactions of magnetosome synthesis-related genes.

Adsorption of Biomineralization Protein Mms6 on Magnetite (Fe3O4) Nanoparticles.

Biomineralization is an elaborate process that controls the deposition of inorganic materials in living organisms with the aid of associated proteins. Magnetotactic bacteria mineralize magnetite (Fe3O4) nanoparticles with finely tuned morphologies in their cells. Mms6, a magnetosome membrane specific (Mms) protein isolated from the surfaces of bacterial magnetite nanoparticles, plays an important role in regulating the magnetite crystal morphology. Although the binding ability of Mms6 to magnetite nanoparticles has been speculated, the interactions between Mms6 and magnetite crystals have not been elucidated thus far. Here, we show a direct adsorption ability of Mms6 on magnetite nanoparticles in vitro. An adsorption isotherm indicates that Mms6 has a high adsorption affinity (Kd = 9.52 µM) to magnetite nanoparticles. In addition, Mms6 also demonstrated adsorption on other inorganic nanoparticles such as titanium oxide, zinc oxide, and hydroxyapatite. Therefore, Mms6 can potentially be utilized for the bioconjugation of functional proteins to inorganic material surfaces to modulate inorganic nanoparticles for biomedical and medicinal applications.

Nanoarchitected Tough Biological Composites from Assembled Chitinous Scaffolds.

Over hundreds of millions of years, organisms have derived specific sets of traits in response to common selection pressures that serve as guideposts for optimal biological designs. A prime example is the evolution of toughened structures in disparate lineages within plants, invertebrates, and vertebrates. Extremely tough structures can function much like armor, battering rams, or reinforcements that enhance the ability of organisms to win competitions, find mates, acquire food, escape predation, and withstand high winds or turbulent flow. From an engineering perspective, biological solutions are intriguing because they must work in a multifunctional context. An organism rarely can be optimally designed for only one function or one environmental condition. Some of these natural systems have developed well-orchestrated strategies, exemplified in the biological tissues of numerous animal and plant species, to synthesize and construct materials from a limited selection of available starting materials. The resulting structures display multiscale architectures with incredible fidelity and often exhibit properties that are similar, and frequently superior, to mechanical properties exhibited by many engineered materials. These biological systems have accomplished this feat through the demonstrated ability to tune size, morphology, crystallinity, phase, and orientation of minerals under benign processing conditions (i.e., near-neutral pH, room temperature, etc.) by establishing controlled synthesis and hierarchical 3D assembly of nano- to microscaled building blocks. These systems utilize organic-inorganic interactions and carefully controlled microenvironments that enable kinetic control during the synthesis of inorganic structures. This controlled synthesis and assembly requires orchestration of mineral transport and nucleation. The underlying organic framework, often consisting of polysaccharides and polypeptides, in these composites is critical in the spatial and temporal regulation of these processes. In fact, the organic framework is used not only to provide transport networks for mineral precursors to nucleation sites but also to precisely guide the formation and phase development of minerals and significantly improve the mechanical performance of otherwise brittle materials.Over the past 15 years, we have focused on a few of these extreme performing organisms, (Wang , Adv. Funct. Mater. 2013, 23, 2908; Weaver , Science 2012, 336, 1275; Huang , Nat. Mater. 2020, 19, 1236; Rivera , Nature 2020, 586, 543) investigating not only their ultrastructural features and mechanical properties but in some cases, how these assembled structures are mineralized. In specific instances, comparative analyses of multiscale structures have pinpointed which design principles have arisen convergently; when more than one evolutionary path arrives at the same solution, we have a good indication that it is the best solution. This is required for survival under extreme conditions. Indeed, we have found that there are specific architectural features that provide an advantage toward survival by enabling the ability to feed effectively or to survive against predatory attacks. In this Account, we describe 3 specific design features, nanorods, helicoids, and nanoparticles, as well as the interfaces in fiber-reinforced biological composites. We not only highlight their roles in the specific organisms but also describe how controlled syntheses and hierarchical assembly using organic (i.e., often chitinous) scaffolds lead to these integrated macroscale structures. Beyond this, we provide insight into multifunctionality: how nature leverages these existing structures to potentially add an additional dimension toward their utility and describe their translation to biomimetic materials used for engineering applications.

Single-cell genotyping of phytoplankton from ocean water by gel-based cell manipulation.

A comprehensive understanding of phytoplankton diversity is valuable for assessing an environment of interest as phytoplankton are primary producers in various aquatic food webs. Microscopic analyses are useful for diversity assessment based on characteristic cell morphologies. However, phylogenetic classification based solely on morphology requires an extremely high level of expertise. The genetic approach is another option for evaluating phytoplankton diversity; however, it cannot reveal morphological information. To integrate these two approaches, an original technology was developed, that is referred to as microcavity array (MCA)/gel-based cell manipulation (GCM). The model experiments using monocultures of various phytoplankton indicated that the efficiencies of cell recovery and isolation of single-cell plankton were dependent on cell size and shape. Cells with widths larger than the cavity width showed high level of recovery and isolation efficiency. Subsequent whole-genome amplification (WGA) of isolated single-cell plankton provided a sufficient amount (≈30 µg) of WGA products for genetic analyses. Furthermore, it is showed that MCA/GCM could directly analyze phytoplankton in ocean water obtained from Suruga Bay, Japan, without any cumbersome pretreatment. These results indicate that MCA/GCM technology is a powerful tool for elucidating the phytoplankton diversity in marine environment.

Unveiling characteristic proteins for the structural development of beetle elytra.

Beetles possess a set of highly modified and tanned forewings, elytra, which are lightweight yet rigid and tough. Immediately after eclosion, the elytra are initially thin, pale and soft. However, they rapidly expand and subsequently become hardened and often dark, resulting from both pigmentation and sclerotization. Here, we identified changes in protein composition during the developmental processes of the elytra in the Japanese rhinoceros beetle, Trypoxylus dichotomus. Using mass spectrometry, a total of 414 proteins were identified from both untanned and tanned elytra, including 31 cuticular proteins (CPs), which constitute one of the major components of insect cuticles. Moreover, CPs containing Rebers and Riddiford motifs (CPR), the most abundant CP family, were separated into two groups based on their expression and amino acid sequences, such as a Gly-rich sequence region and Ala-Ala-Pro repeats. These protein groups may play crucial roles in elytra formation at different time points, likely including self-assembly of chitin nanofibers that control elytral macro and microstructures and dictate changes in other properties (i.e., mechanical property). Clarification of the protein functions will enhance the understanding of elytra formation and potentially benefit the development of lightweight materials for industrial and biomedical applications. STATEMENT OF SIGNIFICANCE: The beetle elytron is a light-weight natural bio-composite which displays high stiffness and toughness. This structure is composed of chitin fibrils and proteins, some of which are responsible for architectural development and hardening. This work, which involves insights from molecular biology and materials science, investigated changes in proteomic, architectural, and localized mechanical characteristics of elytra from the Japanese rhinoceros beetle to understand molecular mechanisms driving elytra development. In the present study, we identified a set of new protein groups which are likely related to the structural development of elytra and has potential for new pathways for processing green materials.

Toughening mechanisms of the elytra of the diabolical ironclad beetle.

Joining dissimilar materials such as plastics and metals in engineered structures remains a challenge1. Mechanical fastening, conventional welding and adhesive bonding are examples of techniques currently used for this purpose, but each of these methods presents its own set of problems2 such as formation of stress concentrators or degradation under environmental exposure, reducing strength and causing premature failure. In the biological tissues of numerous animal and plant species, efficient strategies have evolved to synthesize, construct and integrate composites that have exceptional mechanical properties3. One impressive example is found in the exoskeletal forewings (elytra) of the diabolical ironclad beetle, Phloeodes diabolicus. Lacking the ability to fly away from predators, this desert insect has extremely impact-resistant and crush-resistant elytra, produced by complex and graded interfaces. Here, using advanced microscopy, spectroscopy and in situ mechanical testing, we identify multiscale architectural designs within the exoskeleton of this beetle, and examine the resulting mechanical response and toughening mechanisms. We highlight a series of interdigitated sutures, the ellipsoidal geometry and laminated microstructure of which provide mechanical interlocking and toughening at critical strains, while avoiding catastrophic failure. These observations could be applied in developing tough, impact- and crush-resistant materials for joining dissimilar materials. We demonstrate this by creating interlocking sutures from biomimetic composites that show a considerable increase in toughness compared with a frequently used engineering joint.

Radular stylus of Cryptochiton stelleri: A multifunctional lightweight and flexible fiber-reinforced composite.

Chitons are herbivorous invertebrates that use rows of ultrahard magnetite-based teeth connected to a flexible belt (radula) to rasp away algal deposits growing on and within rocky outcrops along coastlines around the world. Each tooth is attached to the radula by an organic structure (stylus) that provides mechanical support during feeding. However, the underlying structures within the stylus, and their subsequent function within the chiton have yet to be investigated. Here, we investigate the macrostructural architecture, the regional material and elemental distribution and subsequent nano-mechanical properties of the stylus from the Northern Pacific dwelling Cryptochiton stelleri. Using a combination of µ-CT imaging, optical and electron microscopy, as well as elemental analysis, we reveal that the stylus is a highly contoured tube, mainly composed of alpha-chitin fibers, with a complex density distribution. Nanoindentation reveals regiospecific and graded mechanical properties that can be correlated with both the elemental composition and material distribution. Finite element modeling shows that the unique macroscale architecture, material distribution and elemental gradients have been optimized to preserve the structural stability of this flexible, yet robust functionally-graded fiber-reinforced composite tube, providing effective function during rasping. Understanding these complex fiber-based structures offers promising blueprints for lightweight, multifunctional and integrated materials.

Restoration and Modification of Magnetosome Biosynthesis by Internal Gene Acquisition in a Magnetotactic Bacterium.

Integration of a large-sized DNA fragment into a chromosome is an important strategy for characterization of cellular functions in microorganisms. Magnetotactic bacteria synthesize intracellular organelles comprising membrane-bound single crystalline magnetite, also referred to as magnetosomes. Magnetosomes have gained interest in both scientific and engineering sectors as they can be utilized as a material for biomedical and nanotechnological applications. Although genetic engineering of magnetosome biosynthesis mechanism has been investigated, the current method requires cumbersome gene preparation processes. Here, the chromosomal integration of a plasmid containing ≈27 magnetosome genes (≈26 kbp region) in a non-magnetic mutant of Magnetospirillum magneticum AMB-1 using a broad-host-range plasmid is shown. The genome sequencing of gene-complemented strains reveals the chromosomal integration of the plasmid with magnetosome genes at a specific site, most likely by catalysis of an endogenous transposase. Magnetosome production is successfully enhanced by integrating a variation of magnetosome gene operons in the chromosome. This chromosomal integration mechanism will allow the design of functional magnetosomes de novo and M. magneticum AMB-1 may be used as a chassis for the designed magnetosome production.

Crystallization by particle attachment is a colloidal assembly process.

The nucleation of crystals has long been thought to occur through the stochastic association of ions, atoms or molecules to form critical nuclei, which will later grow out to crystals1. Only in the past decade has the awareness grown that crystallization can also proceed through the assembly of different types of building blocks2,3, including amorphous precursors4, primary particles5, prenucleation species6,7, dense liquid droplets8,9 or nanocrystals10. However, the forces that control these alternative pathways are still poorly understood. Here, we investigate the crystallization of magnetite (Fe3O4) through the formation and aggregation of primary particles and show that both the thermodynamics and the kinetics of the process can be described in terms of colloidal assembly. This model allows predicting the average crystal size at a given initial Fe concentration, thereby opening the way to the design of crystals with predefined sizes and properties.

Enhanced Tubulation of Liposome Containing Cardiolipin by MamY Protein from Magnetotactic Bacteria.

Lipid tubules are of particular interest for many potential applications in nanotechnology. Among various lipid tubule fabrication techniques, the morphological regulation of membrane structure by proteins mimicking biological processes may provide the chances to form lipid tubes with highly tuned structures. Magnetotactic bacteria synthesize magnetosomes (a unique prokaryotic organelle comprising a magnetite crystal within a lipid envelope). MamY protein is previously identified as the magnetosome protein responsible for magnetosome vesicle formation and stabilization. Furthermore, MamY is shown in vitro liposome tubulation activity. In this study, the interaction of MamY and phospholipids is investigated by using a lipids-immobilized membrane strip and a peptide array. Here, the binding of MamY to the anionic phospholipid, cardiolipin, is found and enhanced liposome tubulation efficiency. The authors propose the interaction is responsible for recruiting and locating cardiolipin to elongate liposome in vitro. The authors also suggest a similar mechanism for the invagination site in magnetosomes vesicle formation, where the lipid itself contributes further to increasing the curvature. These findings are highly important to develop an effective biomimetic synthesis technique of lipid tubules and to elucidate the unique prokaryotic organelle formation in magnetotactic bacteria.

Integrated molecular analysis of the inactivation of a non-enveloped virus, feline calicivirus, by UV-C radiation.

UV-C treatment has been shown to be a powerful way to inactivate non-enveloped viruses in water samples. However, little is known about how the viruses were inactivated by UV-C radiation. In this study, we investigated the inactivation mechanism of a single-stranded RNA (ssRNA) non-enveloped virus, feline calicivirus (FCV), as a surrogate for the human norovirus, using UV-C radiation with different wavelengths. Integrated molecular analyses using RT-qPCR, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and mass spectrometry were employed to evaluate the extent of ssRNA genome and protein degradation. UV-C radiation of FCV efficiently impaired the infectivity of FCV in mammalian cells. We also identified degradation of the RNA genome, whose copy numbers decreased from 48% to 56% following UV255 or UV281 radiation. Significant degradation of capsid protein was not observed, whereas oxidation of amino acid residues in the major capsid protein VP-1 was determined. Our results suggest that damage to the RNA genome is primarily responsible for the observed decrease in FCV infectivity of CRFK cells. This study provides not only relevant baseline data but also an overview and possible mechanism for the disinfection of non-enveloped ssRNA viruses using UV-C radiation.

Quantitative and time-course analysis of microbial degradation of 1H,1H,2H,2H,8H,8H-perfluorododecanol in activated sludge.

A methylene group in the fluorinated carbon backbone of 1H,1H,2H,2H,8H,8H-perfluorododecanol (degradable telomer fluoroalcohol, DTFA) renders the molecule cleavable by microbial degradation into two fluorinated carboxylic acids. Several biodegradation products of DTFA are known, but their rates of conversion and fates in the environment have not been determined. We used liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) to quantitatively investigate DTFA biodegradation by the microbial community in activated sludge in polyethylene terephthalate (PET) flasks, which we also determined here showed least adsorption of DTFA. A reduction in DTFA concentration in the medium was accompanied by rapid increases in the concentrations of 2H,2H,8H,8H-perfluorododecanoic acid (2H,2H,8H,8H-PFDoA), 2H,8H,8H-2-perfluorododecenoic acid (2H,8H,8H-2-PFUDoA), and 2H,2H,8H-7-perfluorododecenoic acid and 2H,2H,8H-8-perfluorododecenoic acid (2H,2H,8H-7-PFUDoA/2H,2H,8H-8-PFUDoA), which were in turn followed by an increase in 6H,6H-perfluorodecanoic acid (6H,6H-PFDeA) concentration, and decreases in 2H,2H,8H,8H-PFDoA, 2H,8H,8H-2-PFUDoA, and 2H,2H,8H-7-PFUDoA/2H,2H,8H-8-PFUDoA concentrations. Accumulation of perfluorobutanoic acid (PFBA), a presumed end product of DTFA degradation, was also detected. Our quantitative and time-course study of the concentrations of these compounds reveals main routes of DTFA biodegradation, and the presence of new biodegradation pathways.

UV-C irradiation accelerates neutral lipid synthesis in the marine oleaginous diatom Fistulifera solaris.

This study investigated the induction of oil synthesis in the oleaginous diatom, Fistulifera solaris, following irradiation with small doses of UV-C. A rapid induction of oil accumulation was confirmed within 6h following UV-C radiation of the diatom cells, with increases in cell oil body volumes after 24h of approximately 4- to 6-fold from the initial volume. Reactive oxygen species (ROS), which can be generated by a UV-C-mediated reaction, were detected in irradiated cells and the correlation between ROS generation and oil accumulation was confirmed. The smallest UV-C intensity required for oil induction in the cells was 10mJ/cm2. Based on the ideal biodiesel profile, the most suitable FAME composition was obtained when UV255 was used to irradiate the cells. The UV-C radiation method is therefore a solution for shortening the oil accumulation period and improving biodiesel productivity.

Core Amino Acid Residues in the Morphology-Regulating Protein, Mms6, for Intracellular Magnetite Biomineralization.

Living organisms produce finely tuned biomineral architectures with the aid of biomineral-associated proteins. The functional amino acid residues in these proteins have been previously identified using in vitro and in silico experimentation in different biomineralization systems. However, the investigation in living organisms is limited owing to the difficulty in establishing appropriate genetic techniques. Mms6 protein, isolated from the surface of magnetite crystals synthesized in magnetotactic bacteria, was shown to play a key role in the regulation of crystal morphology. In this study, we have demonstrated a defect in the specific region or substituted acidic amino acid residues in the Mms6 protein for observing their effect on magnetite biomineralization in vivo. Analysis of the gene deletion mutants and transformants of Magnetospirillum magneticum AMB-1 expressing partially truncated Mms6 protein revealed that deletions in the N-terminal or C-terminal regions disrupted proper protein localization to the magnetite surface, resulting in a change in the crystal morphology. Moreover, single amino acid substitutions at Asp123, Glu124, or Glu125 in the C-terminal region of Mms6 clearly indicated that these amino acid residues had a direct impact on magnetite crystal morphology. Thus, these consecutive acidic amino acid residues were found to be core residues regulating magnetite crystal morphology.

Comparative Subcellular Localization Analysis of Magnetosome Proteins Reveals a Unique Localization Behavior of Mms6 Protein onto Magnetite Crystals.

UNLABELLED: The magnetosome is an organelle specialized for inorganic magnetite crystal synthesis in magnetotactic bacteria. The complex mechanism of magnetosome formation is regulated by magnetosome proteins in a stepwise manner. Protein localization is a key step for magnetosome development; however, a global study of magnetosome protein localization remains to be conducted. Here, we comparatively analyzed the subcellular localization of a series of green fluorescent protein (GFP)-tagged magnetosome proteins. The protein localizations were categorized into 5 groups (short-length linear, middle-length linear, long-length linear, cell membrane, and intracellular dispersing), which were related to the protein functions. Mms6, which regulates magnetite crystal growth, localized along magnetosome chain structures under magnetite-forming (microaerobic) conditions but was dispersed in the cell under nonforming (aerobic) conditions. Correlative fluorescence and electron microscopy analyses revealed that Mms6 preferentially localized to magnetosomes enclosing magnetite crystals. We suggest that a highly organized spatial regulation mechanism controls magnetosome protein localization during magnetosome formation in magnetotactic bacteria. IMPORTANCE: Magnetotactic bacteria synthesize magnetite (Fe3O4) nanocrystals in a prokaryotic organelle called the magnetosome. This organelle is formed using various magnetosome proteins in multiple steps, including vesicle formation, magnetosome alignment, and magnetite crystal formation, to provide compartmentalized nanospaces for the regulation of iron concentrations and redox conditions, enabling the synthesis of a morphologically controlled magnetite crystal. Thus, to rationalize the complex organelle development, the localization of magnetosome proteins is considered to be highly regulated; however, the mechanisms remain largely unknown. Here, we performed comparative localization analysis of magnetosome proteins that revealed the presence of a spatial regulation mechanism within the linear structure of magnetosomes. This discovery provides evidence of a highly regulated protein localization mechanism for this bacterial organelle development.

Control of magnetite nanocrystal morphology in magnetotactic bacteria by regulation of mms7 gene expression.

Living organisms can produce inorganic materials with unique structure and properties. The biomineralization process is of great interest as it forms a source of inspiration for the development of methods for production of diverse inorganic materials under mild conditions. Nonetheless, regulation of biomineralization is still a challenging task. Magnetotactic bacteria produce chains of a prokaryotic organelle comprising a membrane-enveloped single-crystal magnetite with species-specific morphology. Here, we describe regulation of magnetite biomineralization through controlled expression of the mms7 gene, which plays key roles in the control of crystal growth and morphology of magnetite crystals in magnetotactic bacteria. Regulation of the expression level of Mms7 in bacterial cells enables switching of the crystal shape from dumbbell-like to spherical. The successful regulation of magnetite biomineralization opens the door to production of magnetite nanocrystals of desired size and morphology.

Biomagnetic Recovery and Bioaccumulation of Selenium Granules in Magnetotactic Bacteria.

UNLABELLED: Using microorganisms to remove waste and/or neutralize pollutants from contaminated water is attracting much attention due to the environmentally friendly nature of this methodology. However, cell recovery remains a bottleneck and a considerable challenge for the development of this process. Magnetotactic bacteria are a unique group of organisms that can be manipulated by an external magnetic field due to the presence of biogenic magnetite crystals formed within their cells. In this study, we demonstrated an account of accumulation and precipitation of amorphous elemental selenium nanoparticles within magnetotactic bacteria alongside and independent of magnetite crystal biomineralization when grown in a medium containing selenium oxyanion (SeO3 (2-)). Quantitative analysis shows that magnetotactic bacteria accumulate the largest amount of target molecules (Se) per cell compared with any other previously reported nonferrous metal/metalloid. For example, 2.4 and 174 times more Se is accumulated than Te taken up into cells and Cd(2+) adsorbed onto the cell surface, respectively. Crucially, the bacteria with high levels of Se accumulation were successfully recovered with an external magnetic field. The biomagnetic recovery and the effective accumulation of target elements demonstrate the potential for application in bioremediation of polluted water. IMPORTANCE: The development of a technique for effective environmental water remediation is urgently required across the globe. A biological remediation process of waste removal and/or neutralization of pollutant from contaminated water using microorganisms has great potential, but cell recovery remains a bottleneck. Magnetotactic bacteria synthesize magnetic particles within their cells, which can be recovered by a magnetic field. Herein, we report an example of accumulation and precipitation of amorphous elemental selenium nanoparticles within magnetotactic bacteria independent of magnetic particle synthesis. The cells were able to accumulate the largest amount of Se compared to other foreign elements. More importantly, the Se-accumulating bacteria were successfully recovered with an external magnetic field. We believe magnetotactic bacteria confer unique advantages of biomagnetic cell recovery and of Se accumulation, providing a new and effective methodology for bioremediation of polluted water.

Biomineralization-inspired synthesis of functional organic/inorganic hybrid materials: organic molecular control of self-organization of hybrids.

Organisms produce various organic/inorganic hybrid materials, which are called biominerals. They form through the self-organization of organic molecules and inorganic elements under ambient conditions. Biominerals often have highly organized and hierarchical structures from nanometer to macroscopic length scales, resulting in their remarkable physical and chemical properties that cannot be obtained by simple accumulation of their organic and inorganic constituents. These observations motivate us to create novel functional materials exhibiting properties superior to conventional materials--both synthetic and natural. Herein, we introduce recent progress in understanding biomineralization processes at the molecular level and the development of organic/inorganic hybrid materials by these processes. We specifically outline fundamental molecular studies on silica, iron oxide, and calcium carbonate biomineralization and describe material synthesis based on these mechanisms. These approaches allow us to design a variety of advanced hybrid materials with desired morphologies, sizes, compositions, and structures through environmentally friendly synthetic routes using functions of organic molecules.

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 (Fe3 O4 ). 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.