Magnetosome Membrane Research Articles

A Versatile Magnetic Nanoplatform for Plug-and-Play Functionalization: Genetically Programmable Cargo Loading to Bacterial Magnetosomes by SpyCatcher "Click Biology".

Bacterial magnetosomes ("MAGs") represent a promising class of magnetic iron oxide nanoparticles with exceptional material characteristics and high application potential in the biomedical and biotechnological field. For the surface functionalization of MAGs with different protein cargos, their enveloping membrane can be addressed by genetic means. However, the expression of foreign polypeptides as translational fusion to magnetosome membrane proteins is still laborious and lacks versatility as the generated particles are monospecific and thus restricted to predetermined functions. Utilizing the SpyTag-SpyCatcher (ST-SC) bioconjugate system, we here establish a flexible platform for the targeted nanoassembly of multifunctional MAGs that combines the rapidity of chemical coupling (e.g., by cross-linking reactions) and the unmatched selectivity and controllability of in vivo functionalization. MAGs genetically engineered to display either SC- or ST-connectors are shown to efficiently bind a variety of complementary tagged (protein) cargo. Specifically, we cover a broad spectrum of representative functional moieties and foreign cargo (such as enzymes, antibodies, fluorophores, and silica beads) with relevance in biotechnology and biomedicine and demonstrate the interchangeability of the MAGs-adapted ST-SC system. For the controlled generation of artificial shells surrounding the particles, SC-MAGs are effectively coated by protein-corona proteins. The potential of the here-provided toolkit is even more enhanced by using SC-MAGs as an affinity tool for selective protein pulldown in vitro and in vivo. Overall, this innovative technology turns bacterial MAGs into a flexible magnetic nanoscaffold for the targeted plug-and-play display of virtually unlimited additional functionalities, thereby generating a multitude of magnetic hybrid materials that can be used in many applications.

Intrinsic and extrinsic determinants of conditional localization of Mms6 to magnetosome organelles in Magnetospirillum magneticum AMB-1.

Magnetotactic bacteria are a diverse group of microbes that use magnetic particles housed within intracellular lipid-bounded magnetosome organelles to guide navigation along geomagnetic fields. The development of magnetosomes and their magnetic crystals in Magnetospirillum magneticum AMB-1 requires the coordinated action of numerous proteins. Most proteins are thought to localize to magnetosomes during the initial stages of organelle biogenesis, regardless of environmental conditions. However, the magnetite-shaping protein Mms6 is only found in magnetosomes that contain magnetic particles, suggesting that it might conditionally localize after the formation of magnetosome membranes. The mechanisms for this unusual mode of localization to magnetosomes are unclear. Here, using pulse-chase labeling, we show that Mms6 translated under non-biomineralization conditions translocates to pre-formed magnetosomes when cells are shifted to biomineralizing conditions. Genes essential for magnetite production, namely mamE, mamM, and mamO, are necessary for Mms6 localization, whereas mamN inhibits Mms6 localization. MamD localization was also investigated and found to be controlled by similar cellular factors. The membrane localization of Mms6 is dependent on a glycine-leucine repeat region, while the N-terminal domain of Mms6 is necessary for retention in the cytosol and impacts conditional localization to magnetosomes. The N-terminal domain is also sufficient to impart conditional magnetosome localization to MmsF, altering its native constitutive magnetosome localization. Our work illuminates an alternative mode of protein localization to magnetosomes in which Mms6 and MamD are excluded from magnetosomes by MamN until biomineralization initiates, whereupon they translocate into magnetosome membranes to control the development of growing magnetite crystals.IMPORTANCEMagnetotactic bacteria (MTB) are a diverse group of bacteria that form magnetic nanoparticles surrounded by membranous organelles. MTB are widespread and serve as a model for bacterial organelle formation and biomineralization. Magnetosomes require a specific cohort of proteins to enable magnetite formation, but how those proteins are localized to magnetosome membranes is unclear. Here, we investigate protein localization using pulse-chase microscopy and find a system of protein coordination dependent on biomineralization-permissible conditions. In addition, our findings highlight a protein domain that alters the localization behavior of magnetosome proteins. Utilization of this protein domain may provide a synthetic route for conditional functionalization of magnetosomes for biotechnological applications.

Ferromagnetic resonance spectra of linear magnetosome chains.

The ferromagnetic resonance (FMR) spectra of oriented and non-oriented assemblies of linear magnetosome chains are calculated by solving the stochastic Landau-Lifshitz equation. The dependence of the shape of the FMR spectrum of a dilute assembly of chains on the particle diameter, the number of particles in a chain, the distance between the centers of neighboring particles, the mutual orientation of the cubic axes of particle anisotropy, and the value of the magnetic damping constant is studied. It is shown that FMR spectra of non-oriented chain assemblies depend on the average particle diameter at a fixed thickness of the lipid magnetosome membrane, as well as on the value of the magnetic damping constant. At the same time, they are practically independent of the number Np of particles in the chain under the condition Np ≥ 10. The FMR spectra of non-oriented assemblies of magnetosome chains are compared with that of random clusters of interacting spherical magnetite nanoparticles. The shape of FMR spectra of both assemblies is shown to differ appreciably even at sufficiently large values of filling density of random clusters.

Role of Water during the Early Stages of Iron Oxyhydroxide Formation by a Bacterial Iron Nucleator.

Understanding the nucleation of iron oxides and the underlying hydrolysis of aqueous iron species is still challenging, and molecular-level insights into the orchestrated response of water, especially at the hydrolysis interface, are lacking. We follow iron(III) hydrolysis in the presence of a synthetic bacterial iron nucleator, which is a magnetosome membrane specific peptide, by using a constant pH titration technique. Three distinct hydrolysis regimes were identified. Interface-selective sum frequency generation (SFG) spectroscopy was used to probe the interfacial reaction and water in direct contact with the peptide. SFG data reveal that iron(III) species react quickly with interfacial peptides while continuously enhancing water alignment into the later stages of hydrolysis. The gradually aligning water molecules are associated with initially promoted (regimes I and II) and later suppressed (regime III) hydrolysis after the saturation of water alignment has occurred until regime II. These interfacial insights are crucial for understanding the early stage of iron oxide biomineralization.

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.

Long-Term Stability, Biocompatibility, and Magnetization of Suspensions of Isolated Bacterial Magnetosomes.

Magnetosomes are magnetic nanoparticles biosynthesized by magnetotactic bacteria. Due to a genetically strictly controlled biomineralization process, the ensuing magnetosomes have been envisioned as agents for biomedical and clinical applications. In the present work, different stability parameters of magnetosomes isolated from Magnetospirillum gryphiswaldense upon storage in suspension (HEPES buffer, 4 °C, nitrogen atmosphere) for one year in the absence of antibiotics are examined. The magnetic potency, measured by the saturation magnetization of the particle suspension, drops to one-third of its starting value within this year-about ten times slower than at ambient air and room temperature. The particle size distribution, the integrity of the surrounding magnetosome membrane, the colloidal stability, and the biocompatibility turn out to be not severely affected by long-term storage.

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.

A Magnetosome-Based Platform for Flow Biocatalysis.

Biocatalysis in flow reactor systems is of increasing importance for the transformation of the chemical industry. However, the necessary immobilization of biocatalysts remains a challenge. We here demonstrate that biogenic magnetic nanoparticles, so-called magnetosomes, represent an attractive alternative for the development of nanoscale particle formulations to enable high and stable conversion rates in biocatalytic flow processes. In addition to their intriguing material characteristics, such as high crystallinity, stable magnetic moments, and narrow particle size distribution, magnetosomes offer the unbeatable advantage over chemically synthesized nanoparticles that foreign protein “cargo” can be immobilized on the enveloping membrane via genetic engineering and thus, stably presented on the particle surface. To exploit these advantages, we develop a modular connector system in which abundant magnetosome membrane anchors are genetically fused with SpyCatcher coupling groups, allowing efficient covalent coupling with complementary SpyTag-functionalized proteins. The versatility of this approach is demonstrated by immobilizing a dimeric phenolic acid decarboxylase to SpyCatcher magnetosomes. The functionalized magnetosomes outperform similarly functionalized commercial particles by exhibiting stable substrate conversion during a 60 h period, with an average space–time yield of 49.2 mmol L–1 h–1. Overall, our results demonstrate that SpyCatcher magnetosomes significantly expand the genetic toolbox for particle surface functionalization and increase their application potential as nano-biocatalysts.

A protease-mediated switch regulates the growth of magnetosome organelles in Magnetospirillum magneticum

Magnetosomes are lipid-bound organelles that direct the biomineralization of magnetic nanoparticles in magnetotactic bacteria. Magnetosome membranes are not uniform in size and can grow in a biomineralization-dependent manner. However, the underlying mechanisms of magnetosome membrane growth regulation remain unclear. Using cryoelectron tomography, we systematically examined mutants with defects at various stages of magnetosome formation to identify factors involved in controlling membrane growth. We found that a conserved serine protease, MamE, plays a key role in magnetosome membrane growth regulation. When the protease activity of MamE is disrupted, magnetosome membrane growth is restricted, which, in turn, limits the size of the magnetite particles. Consistent with this finding, the upstream regulators of MamE protease activity, MamO and MamM, are also required for magnetosome membrane growth. We then used a combination of candidate and comparative proteomics approaches to identify Mms6 and MamD as two MamE substrates. Mms6 does not appear to participate in magnetosome membrane growth. However, in the absence of MamD, magnetosome membranes grow to a larger size than the wild type. Furthermore, when the cleavage of MamD by MamE protease is blocked, magnetosome membrane growth and biomineralization are severely inhibited, phenocopying the MamE protease-inactive mutant. We therefore propose that the growth of magnetosome membranes is controlled by a protease-mediated switch through processing of MamD. Overall, our work shows that, like many eukaryotic systems, bacteria control the growth and size of biominerals by manipulating the physical properties of intracellular organelles.

In Situ Generation of Gold Nanoparticles on Bacteria‐Derived Magnetosomes for Imaging‐Guided Starving/Chemodynamic/Photothermal Synergistic Therapy against Cancer

AbstractThere are several attractive opportunities for using magnetic nanomaterials for anticancer applications. Herein, a magnetic nanomaterial platform is successfully developed based on natural Fe3O4 magnetosomes extracted from the bacterium Magnetospirillum magneticum AMB‐1 for anticancer therapy. The authors initially functionalize the magnetosome membranes in situ with gold nanoparticles to construct an attractive core‐satellite structure. Subsequently, the physical properties and application potentials of these structures are characterized as contrast agents for photoacoustic imaging and magnetic resonance imaging and as therapeutic agents with selective magnetic field guidance for diverse antitumor modalities, including starving, chemodynamic, and photothermal therapies. Owing to the high‐performance imaging‐guided synergistic effect, only a single injection and single laser irradiation result in excellent therapeutic efficacy against tumor growth in multiple cell‐derived xenograft tumor models and, most notably, patient‐derived organoid and patient‐derived xenograft tumor models. The demonstrations of the use of natural magnetic nanomaterials to achieve strong and synergistic antitumor performances highlight the promising application potential of this flexible and easy‐to‐prepare platform for developing innovative treatments for diseases in humans.

Localization of Native Mms13 to the Magnetosome Chain of Magnetospirillum magneticum AMB-1 Using Immunogold Electron Microscopy, Immunofluorescence Microscopy and Biochemical Analysis

Magnetotactic bacteria (MTB) biomineralize intracellular magnetite (Fe3O4) crystals surrounded by a magnetosome membrane (MM). The MM contains membrane-specific proteins that control Fe3O4 mineralization in MTB. Previous studies have demonstrated that Mms13 is a critical protein within the MM. Mms13 can be isolated from the MM fraction of Magnetospirillum magneticum AMB-1 and a Mms13 homolog, MamC, has been shown to control the size and shape of magnetite nanocrystals synthesized in-vitro. The objective of this study was to use several independent methods to definitively determine the localization of native Mms13 in M. magneticum AMB-1. Using Mms13-immunogold labeling and transmission electron microscopy (TEM), we found that Mms13 is localized to the magnetosome chain of M. magneticum AMB-1 cells. Mms13 was detected in direct contact with magnetite crystals or within the MM. Immunofluorescence detection of Mms13 in M. magneticum AMB-1 cells by confocal laser scanning microscopy (CLSM) showed Mms13 localization along the length of the magnetosome chain. Proteins contained within the MM were resolved by SDS-PAGE for Western blot analysis and LC-MS/MS (liquid chromatography with tandem mass spectrometry) protein sequencing. Using Anti-Mms13 antibody, a protein band with a molecular mass of ~14 kDa was detected in the MM fraction only. This polypeptide was digested with trypsin, sequenced by LC-MS/MS and identified as magnetosome protein Mms13. Peptides corresponding to the protein’s putative MM domain and catalytic domain were both identified by LC-MS/MS. Our results (Immunogold TEM, Immunofluorescence CLSM, Western blot, LC-MS/MS), combined with results from previous studies, demonstrate that Mms13 and homolog proteins MamC and Mam12, are localized to the magnetosome chain in MTB belonging to the class Alphaproteobacteria. Because of their shared localization in the MM and highly conserved amino acid sequences, it is likely that MamC, Mam12, and Mms13 share similar roles in the biomineralization of Fe3O4 nanocrystals.

Magnetosome membrane engineering to improve G protein-coupled receptor activities in the magnetosome display system

Magnetotactic bacterium, Magnetospirillum magneticum, produces biogenic magnetic nanoparticles termed magnetosomes, which are primarily composed of a magnetite core and a surrounding lipid bilayer membrane. We have fabricated human transmembrane protein-magnetosome complexes by genetic engineering with embedding the transmembrane proteins of interest, in particular G protein-coupled receptors (GPCRs), in the magnetosome membrane. The magnetosomes provide a promising platform for high throughput ligand screening towards drug discovery, and this is a critical advantage of the magnetosome display system beyond conventional membrane platforms such as liposomes and lipid nano-discs. However, the human GPCRs expressed on the magnetosomes were not fully functionalized in bacterial membranes the most probably due to the lack of essential phospholipids such as phosphatidylcholine (PC) for GPCR functionalization. To overcome this issue, we expressed two types of PC-producing enzymes, phosphatidylcholine synthase (PCS) and phosphatidylethanolamine N-methyltransferase (PMT) in M. magneticum. As a result, generation and incorporation of PC in cell- and magnetosome-membranes were demonstrated. To the best of our knowledge, M. magneticum is the second bacterial species which had the PC-incorporated lipid membrane by genetic engineering. Subsequently, a GPCR, thyroid-stimulating hormone receptor (TSHR) and PCS were simultaneously expressed. We found that PC in the magnetosome membrane assisted the binding of TSHR and its ligand, indicating that the genetic approach demonstrated in this study is useful to enhance the function of the GPCRs displayed on the magnetosomes.

Erratum to: Biomineralized and chemically synthesized magnetic nanoparticles: A contrast

Page 391, the caption of Fig. 3 “Biomineralization and role of magnetosome membrane proteins in synthesis of BM crystals in MTB (Magnetosome proteins: MamR, MamS, MamT, MamP, MamD, MamF, MamG, MamE (protease dependent), MamC, MamM, MamN, MamO, MamK, MamJ, MamE, MamI, MamL, MamQ, MamB).” should read “Biomineralization and role of magnetosome membrane proteins in synthesis of BM crystals in MTB (Magnetosome proteins: MamR, MamS, MamT, MamP, MamD, MamF, MamG, MamE (protease dependent), MamC, MamM, MamN, MamO, MamK, MamJ, MamE, MamI, MamL, MamQ, MamB). Reproduced with permission of PNAS from the reference as follows: Dorothee Murat, Anna Quinlan, Hojatollah Vali, Arash Komeili. Comprehensive genetic dissection of the magnetosome gene island reveals the step-wise assembly of a prokaryotic organelle. PNAS, 2010, 107(12): 5593–5598.” The authors apologize for any confusion.

Preparation and characterisation of magnetosomes based drug conjugates for cancer therapy.

The authors report a novel, effective and enhanced method of conjugating anticancer drug, paclitaxel and gallic acid with magnetosomes. Here, anticancer drugs were functionalised with magnetosomes membrane by direct and indirect (via crosslinkers: glutaraldehyde and 3-aminopropyltriethoxysilane) adsorption methods. The prepared magnetosome-drug conjugates were characterised by Fourier transform infrared, zeta potential, field-emission scanning electron microscope and thermogravimetric analysis/differential scanning calorimetry. The drug-loading efficiency and capacity were found to be 87.874% for paclitaxel (MP) and 71.3% for gallic acid (MG), respectively as calculated by ultraviolet spectroscopy and high-performance liquid chromatography. The drug release demonstrated by the diffusion method in phosphate buffer (PBS), showing a prolonged drug release for MP and MG, respectively. The cytotoxicity effect of the MP and MG displayed cytotoxicity of 69.71%, 55.194% against HeLa and MCF-7 cell lines, respectively. The reactive oxygen species, acridine orange and ethidium bromide and 4, 6-diamidino-2-phenylindole staining of the drug conjugates revealed the apoptotic effect of MP and MG. Further, the regulation of tumour suppressor protein, p53 was determined by western blotting which showed an upregulation of p53. Comparatively, the magnetosome-drug conjugates prepared by direct adsorption achieved the best effects on the drug-loading efficiency and the increased percentage of cancer cell mortality and the upregulation of P53. The proposed research ascertains that magnetosomes could be used as effective nanocarriers in cancer therapy.

Biomimetic synthesis of magnetic nanoparticles mediated by magnetosome proteins

Magnetotactic bacteria magnetosomes are magnetic nanoparticles enclosed by biofilms and arrange in the form of a chain. Magnetic nanoparticles formed by the biomineralization of magnetosome usually have regular shape, uniform particle size and high crystallinity, which have attracted extensive attention of researchers. The magnetosome membrane is composed of phospholipids and fatty acids, and the magnetosome membrane lipid vesicle acts as a nanoreactor which controls the precise synthesis of magnetic nanoparticles. A series of biomineralization proteins in the magnetosome membrane control the iron transport, redox reaction, nucleation and growth of the magnetic nanoparticles. At present, the specific biomineralization process of magnetosome is still unclear and the large-scale production of magnetosome is difficult, so the biomimetic synthesis of magnetosome has been initiated. In vivo studies have shown that magnetosome proteins of magnetotactic bacteria including Mms6, MamC, MmsF, MamG and MamD play an important role in regulating the size and morphology of the magnetosome, and they have been identified as the best candidates for the biomimetic synthesis of magnetosome. Biomimetic synthesis of magnetic nanoparticles mediated by recombinant magnetosome proteins such as Mms6, MamC and MmsF has been studied. These studies can not only help us better understand the biomineralization process of magnetosome, but also help us prepare high-quality magnetosome-like magnetic nanoparticles without the use of organic solvents, surfactants and high reaction temperature. This article mainly reviews the research progress of the biomimetic synthesis of magnetic nanoparticle mediated by several key magnetosome proteins and prospects its future development. Mms6 seems to mainly control over the growth kinetics of magnetic crystal by acting as an iron reservoir. MamC seems to preferably control the nucleation kinetics of magnetic crystals due to the ionotropic and template effects. MamP as an iron oxidase can support to form ferrihydrite which required for the formation of magnetite crystals in vivo . Although Mms6 is the most abundant magnetosome protein and there are the most researches on Mms6 mediated biomimetic synthesis of magnetic nanoparticles, the specific regulation mechanism of Mms6 is still unclear. In addition, the production cost of Mms6 is relatively high. These are all the shortcomings of Mms6 mediated biomimetic synthesis of magnetic nanoparticles. Therefore, in order to realize large scale biomimetic preparation of high-quality magnetic nanoparticles with controllable size and morphology, it is necessary to further clarify the regulation mechanism of Mms6 and develop cheaper and simpler methods to prepare Mms6 based functional polypeptides and additives. The biomineralization of magnetosome is a process regulated by a variety of mineralization proteins. Therefore, if several kinds of magnetosome proteins with different functions were used together to regulate the formation process of magnetic nanocrystal in the biomimetic synthesis methods, magnetic nanocrystals with more abundant morphology, size and surface crystal structure would be synthesized. Particularly, if MamP, MamE and other magnetosome proteins which control the redox reaction were used to assist the biomimetic synthesis of magnetic nanocrystals, the synthesis conditions might be further optimized, and the yield and performance of magnetic nanoparticles might be improved. In addition, constructing a micro lipid vesicle reactor which like the magnetosome membrane through the surface assembly of key magnetosome proteins and phospholipid molecules to control the biomimetic synthesis process is expected to introduce novel ideas and methods for the biomimetic synthesis of magnetic nanoparticles.

A Versatile Toolkit for Controllable and Highly Selective Multifunctionalization of Bacterial Magnetic Nanoparticles

Their unique material characteristics, i.e. high crystallinity, strong magnetization, uniform shape and size, and the ability to engineer the enveloping membrane in vivo make bacterial magnetosomes highly interesting for many biomedical and biotechnological applications. In this study, a versatile toolkit is developed for the multifunctionalization of magnetic nanoparticles in the magnetotactic bacterium Magnetospirillum gryphiswaldense, and the use of several abundant magnetosome membrane proteins as anchors for functional moieties is explored. High-level magnetosome display of cargo proteins enables the generation of engineered nanoparticles with several genetically encoded functionalities, including a core-shell structure, magnetization, two different catalytic activities, fluorescence and the presence of a versatile connector that allows the incorporation into a hydrogel-based matrix by specific coupling reactions. The resulting reusable magnetic composite demonstrates the high potential of synthetic biology for the production of multifunctional nanomaterials, turning the magnetosome surface into a platform for specific versatile display of functional moieties.

Probing the Nanostructure and Arrangement of Bacterial Magnetosomes by Small-Angle X-Ray Scattering.

Magnetosomes are membrane-enveloped single-domain ferromagnetic nanoparticles enabling the navigation of magnetotactic bacteria along magnetic field lines. Strict control over each step of biomineralization generates particles of high crystallinity, strong magnetization, and remarkable uniformity in size and shape, which is particularly interesting for many biomedical and biotechnological applications. However, to understand the physicochemical processes involved in magnetite biomineralization, close and precise monitoring of particle production is required. Commonly used techniques, such as transmission electron microscopy (TEM) or Fe measurements, allow only for semiquantitative assessment of the magnetosome formation without routinely revealing quantitative structural information. In this study, lab-based small-angle X-ray scattering (SAXS) is explored as a means to monitor the different stages of magnetosome biogenesis in the model organism Magnetospirillum gryphiswaldense SAXS is evaluated as a quantitative stand-alone technique to analyze the size, shape, and arrangement of magnetosomes in cells cultivated under different growth conditions. By applying a simple and robust fitting procedure based on spheres aligned in linear chains, it is demonstrated that the SAXS data sets contain information on both the diameter of the inorganic crystal and the protein-rich magnetosome membrane. The analyses corroborate a narrow particle size distribution with an overall magnetosome radius of 19 nm in Magnetospirillum gryphiswaldense Furthermore, the averaged distance between individual magnetosomes is determined, revealing a chain-like particle arrangement with a center-to-center distance of 53 nm. Overall, these data demonstrate that SAXS can be used as a novel stand-alone technique allowing for the at-line monitoring of magnetosome biosynthesis, thereby providing accurate information on the particle nanostructure.IMPORTANCE This study explores lab-based small-angle X-ray scattering (SAXS) as a novel quantitative stand-alone technique to monitor the size, shape, and arrangement of magnetosomes during different stages of particle biogenesis in the model organism Magnetospirillum gryphiswaldense The SAXS data sets contain volume-averaged, statistically accurate information on both the diameter of the inorganic nanocrystal and the enveloping protein-rich magnetosome membrane. As a robust and nondestructive in situ technique, SAXS can provide new insights into the physicochemical steps involved in the biosynthesis of magnetosome nanoparticles as well as their assembly into well-ordered chains. The proposed fit model can easily be adapted to account for different particle shapes and arrangements produced by other strains of magnetotactic bacteria, thus rendering SAXS a highly versatile method.

Structure–Function of MamC Loop and Its Effect on the in Vitro Precipitation of Biomimetic Magnetite Nanoparticles

MamC, an integral protein of the magnetosome membrane, has recently been proposed as a strong candidate to produce biomimetic (magnetosome-like) magnetite nanoparticles that could be used as an alternative to magnetosomes in different applications such as nanocarriers. The secondary structure of the protein contains two helical transmembrane domains connected by an α-helical loop oriented toward the magnetosome lumen. In this loop, the residues Glu66 and Asp70 seem to be responsible for a template effect that controls the nucleation and/or growth of biomimetic nanoparticles in vitro. In the present study, we have introduced a double mutation, E66A and D70A, in the sequence of MamC while working, for the first time, with the full-length protein. Our results show that this double mutation does not affect either the conformation or the stability of MamC, but it indeed makes the protein lose its functionality in terms of controlling the process of magnetite biomineralization in vitro. The present study shows ...

Generation of nanomagnetic biocomposites by genetic engineering of bacterial magnetosomes

Magnetosomes are magnetic nanoparticles biomineralized by magnetotactic bacteria. They consist of a monocrystalline magnetite core enveloped by the magnetosome membrane, which harbors a set of specialized proteins. For the alphaproteobacterium Magnetospirillum gryphiswaldense genetic techniques were developed for engineering both crystal morphology and the enveloping membrane, thereby generating building blocks for magnetic organic–inorganic hybrid materials. Genetic manipulation of magnetite biomineralization enabled the generation of core-engineered nanoparticles with adjusted magnetic and physicochemical properties. Functionalization of the particle surface was achieved by genetic expression of enzymes and peptides genetically fused to abundant magnetosome anchor proteins. High-level expression allowed the generation of multifunctional nanoparticles with maximized protein-to-particle ratios. This allowed for the tuning of surface properties (charge and hydrodynamic diameter), and the colloidal and enzymatic stability was improved by coating with inorganic and organic shells. The expression of molecular connectors might serve as scaffolds for the introduction of further functionalities. Overall, this demonstrates that the ‘synthetic biology’ approach enables the generation of multifunctional, magnetic hybrid materials with a tuned property spectrum exceeding those of conventional materials, and the combination of different biogenic materials generates fully genetically encoded biocomposites with enhanced potential for various biotechnological and biomedical applications.

Protein Conservation Method Affects MamC-Mediated Biomineralization of Magnetic Nanoparticles

Magnetite nanoparticles in the magnetosomes formed by magnetotactic bacteria present unique magnetic properties that make them the ideal nanoparticle with potential use in several biotechnological applications. These magnetoliposomes are organelles formed by crystals of magnetite (Fe3O4) or greigite (Fe3S4) surrounded by a lipid bilayer. However, scaling up the production of these nanoparticles cannot be achieved currently because of the slow growth and the strict physiological characteristics of the magnetotactic bacteria. An alternative to solve this problem is the biomimetic approach, which involves the in vitro production of magnetite nanoparticles mediated by magnetosome membrane proteins, expressed as recombinant. MamC-mediated magnetite nanoparticles (BMNPs) have been recently proposed as some of the best biomimetic (magnetosome-like) nanoparticles for potential use in targeted drug delivery and hyperthermia treatments. However, the production of these crystals still needs to be scaled up. One of t...