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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.

Magnetic nanoparticles offer many exciting possibilities in biomedicine, from cell imaging to cancer treatment. One of the currently researched nanoparticles are magnetosomes, magnetite nanoparticles of high chemical purity synthesized by magnetotactic bacteria. Despite their therapeutic potential, very little is known about their degradation in human cells, and even less so of their degradation within tumours. In an effort to explore the potential of magnetosomes for cancer treatment, we have explored their degradation process in a 3D human lung carcinoma model at the subcellular level and with nanometre scale resolution. We have used state of the art hard X-ray probes (nano-XANES and nano-XRF), which allow for identification of distinct iron phases in each region of the cell. Our results reveal the progression of magnetite oxidation to maghemite within magnetosomes, and the biosynthesis of magnetite and ferrihydrite by ferritin. [ABSTRACT FROM AUTHOR]

Simple Summary: Cancer cells display elevated reactive oxygen species (ROS) and an altered redox status. For this study, taking advantage of these characteristics, we designed a multifunctional MRI-visualizable multi-drug delivery platform, AMB@PDAP-Fe (APPF), based on magnetotactic bacteria (AMB) for MRI-visualized tumor photo-thermotherapy (PTT) and enhanced chemodynamic therapy (CDT). Upon magnetic navigation, APPF accumulated in the tumor microenvironment. The PDAP-Fe in the composite was reduced by glutathione (GSH) in the tumor microenvironment and produced Fe2+, which catalyzed the generation of reactive oxygen species (ROS) through the Fenton reaction and induced tumor cell apoptosis. The magnetosomes in AMB displayed good dual-mode contrast capability in an MRI, which was later used to visualize the process of tumor treatment. Upon NIR light irradiation, the magnetosomes in magnetotactic bacteria immediately converted the light energy into heat energy and the tumor tissue was heated up, thus causing the death of tumor cells. It is noteworthy that the heat generated during the synergistic PTT process (to which PDAP partly contributed) accelerated the releasing of Fe2+ from PDAP-Fe, which enhanced the catalytic conversion of endogenous H2O2 into ·OH, and, thus, achieved synergistic photothermal-enhanced Fenton-reaction-mediated cancer therapy. This study demonstrates the feasibility of developing an MRI-visualizable multi-drug delivery platform from magnetic bacteria, which, compared with other manmade inorganic multi-drug delivery platforms, are lively, self-propelled, and magnetically navigated. This study also highlights the synergistic tumor-curing effect between magnetic bacteria and PDAP-Fe and the importance of understanding the underlying cooperative molecular mechanism between different drugs when scheduling combination therapy. Cancer cells display elevated reactive oxygen species (ROS) and altered redox status. Herein, based on these characteristics, we present a multi-drug delivery platform, AMB@PDAP-Fe (APPF), from the magnetotactic bacterium AMB-1 and realize MRI-visualized tumor-microenvironment-responsive photothermal–chemodynamic therapy. The Fe3+ in PDAP-Fe is reduced by the GSH at the tumor site and is released in the form of highly active Fe2+, which catalyzes the generation of ROS through the Fenton reaction and inhibits tumor growth. At the same time, the significant absorption of the mineralized magnetosomes in AMB-1 cells in the NIR region enables them to efficiently convert near-infrared light into heat energy for photothermal therapy (PTT), to which PDAP also contributes. The heat generated in the PTT process accelerates the process of Fe2+ release, thereby achieving an enhanced Fenton reaction in the tumor microenvironment. In addition, the magnetosomes in AMB-1 are used as an MRI contrast agent, and the curing process is visualized. This tumor microenvironment-responsive MTB-based multi-drug delivery platform displays the potency to combat tumors and demonstrates the utility and practicality of understanding the cooperative molecular mechanism when designing multi-drug combination therapies. [ABSTRACT FROM AUTHOR]

AbstractMagnetosomes are iron oxide or iron sulphide nano-sized particles surrounded by a lipid bilayer synthesised by a group of bacteria known as magnetotactic bacteria (MTB). Magnetosomes have become a promising candidate for biomedical applications and could be potentially used as a drug-carrier. However, pharmacokinetics and immunogenicity of the magnetosomes have not been understood yet which preclude its clinical applications. Herein, we investigated the pharmacokinetics of magnetosomes including Absorption, Distribution, Metabolism, and Elimination (ADME) along with its immunogenicity in vitro and in vivo. The magnetosomes were conjugated with fluorescein isothiocyanate (Mag-FITC) and their conjugation was confirmed through fluorescence microscopy and its absorption in HeLa cell lines was evaluated using flow cytometry analysis. The results revealed a maximum cell uptake of 97% at 200 µg/mL concentration. Further, the biodistribution of Mag-FITC was investigated in vivo by a bioimaging system using BALB/c mice as a subject at different time intervals. The Mag-FITC neither induced death nor physical distress and the same was eliminated post 36 h of injection with meagre intensities left behind. The metabolism and elimination analysis were assessed to detect the iron overload which revealed that magnetosomes were entirely metabolised within 48-h interval. Furthermore, the histopathology and serum analysis reveal no histological damage with the absence of any abnormal biochemical parameters. The results support our study that magnetosomes were completely removed from the blood circulation within 48-h time interval. Moreover, the immunogenicity analysis has shown that magnetosomes do not induce any inflammation as indicated by reduced peaks of immune markers such as IL 1β, IL 2, IL 6, IL8, IFN γ, and TNF α estimated through Indirect ELISA. The normal behaviour of animals with the absence of acute or chronic toxicities in any organs declares that magnetosomes are safe to be injected. This shows that magnetosomes are benign for biological systems enrouting towards beneficial biomedical applications. Therefore, this study will advance the understanding and application of magnetosomes for clinical purposes.

Magnetotactic bacteria produce chains of nanoscopic iron minerals used for navigation, which can be preserved over geological timescales in the form of magnetofossils. Micrometer-sized magnetite crystals with unusual shapes suggesting a biologically controlled mineralization have been found in the geological record and termed giant magnetofossils. The biological origin and function of giant magnetofossils remains unclear, due to the lack of modern analogues to giant magnetofossils. Using distinctive Ptychographic nanotomography data of Precambrian (1.88 Ga) rocks, we recovered the morphology of micrometric cuboid grains of iron oxides embedded in an organic filamentous fossil to construct synthetic magnetosomes. Their morphology is different from that of previously found giant magnetofossils, but their occurrence in filamentous microfossils and micromagnetic simulations support the hypothesis that they could have functioned as a navigation aid, akin to modern magnetosomes. [ABSTRACT FROM AUTHOR]

Bacterial species referred to as magnetotactic bacteria (MTB) biomineralize iron oxides and iron sulphides inside the cell. Bacteria can arrange themselves passively along geomagnetic field lines with the aid of these iron components known as magnetosomes. In this study, magnetosome nanoparticles, which were obtained from the taxonomically identified MTB isolate Providencia sp. PRB-1, were characterized and their antibacterial activity was evaluated. An in vitro test showed that magnetosome nanoparticles significantly inhibited the growth of Staphylococcus sp., Pseudomonas aeruginosa, and Klebsiella pneumoniae. Magnetosomes were found to contain cuboidal iron crystals with an average size of 42 nm measured by particle size analysis and scanning electron microscope analysis. The energy dispersive X-ray examination revealed that Fe and O were present in the extracted magnetosomes. The extracted magnetosome nanoparticles displayed maximum absorption at 260 nm in the UV-Vis spectrum. The distinct magnetite peak in the Fourier transform infrared (FTIR) spectroscopy spectra was observed at 574.75 cm−1. More research is needed into the intriguing prospect of biogenic magnetosome nanoparticles for antibacterial applications. [ABSTRACT FROM AUTHOR]

An integrated scalable platform for fermentative production and downstream processing of bacterial magnetosome products is advanced. Long magnetosome chains, high cellular magnetism, and low numbers of polyhydroxyalkanoate granules were obtained during the exponential growth phase of a two-stage continuous high cell density fermentation of M. gryphiswaldense MSR-1. Centrifugally concentrated 20% (w/v) suspensions of exponential phase cells were disrupted with high efficiency (--92%) in a single pass through a Constant Systems Cell Disruptor operated at 10 kpsi, releasing --75% of the cellular iron. Magnetosomes were recovered in partially purified form from crude whole cell disruptates by rotor-stator high-gradient magnetic separation. Further purification/polishing was achieved by magnetically enhanced density separation in an aqueous micellar two-phase system (a new technique developed in this work as a low-cost alternative to sucrose gradient ultracentrifugation). The unoptimised 4-step process delivered highly purified magnetosomes (ca. 50 and 80-fold with respect to polyhydroxyalkanoate and protein) in > 50% yield, with no evidence of crystal coat damage, acceptable reduction (--35%) in median magnetosome chain length, and magnetic properties (pot-bellied hysteresis loop, coercivity = 9.8 mT, 'squareness' = 0.32) expected of isolated magnetosome chains. Though demonstrated in batch mode, the platform displays potential for end-to-end continuous manufacture of future magnetosome-based products. [ABSTRACT FROM AUTHOR]

Magnetotactic bacteria are microorganisms that produce intracellular magnetic nanoparticles organized in chains, conferring a magnetic moment to the bacterial body that allows it to swim following the geomagnetic field lines. Magnetotactic bacteria usually display two swimming polarities in environmental samples: the South-seeking (SS) polarity and the North-seeking (NS) polarity, characterized by the bacteria swimming antiparallel or parallel to the magnetic field lines, respectively. It has been observed that in the presence of inhomogeneous magnetic fields, NS magnetotactic bacteria can change their swimming polarity to SS or vice versa. The present study analyzes populations of NS cocci obtained from SS cocci isolated in the presence of a magnet. The aim was to study differences in the swimming characteristics and magnetic moment among both populations of cocci. For that, trajectories were recorded and the velocity and angle among the velocity and the applied magnetic field were calculated. In addition, micrographs from both SS and NS cocci were obtained and their magnetosomes were measured to analyze their length, width, aspect ratio and magnetic moment, to finally obtain the magnetic moment for each coccus. The results showed the following properties of NS relative to SS cocci: higher velocities, narrow bacterial magnetic moment distribution, higher dispersion in the distribution of angles among the velocity and the applied magnetic field and lower magnetic field sensibility. Those differences cannot be explained by the simple change in magnetic polarity of the magnetosome chain and can be related to the existence of an active magnetoreceptive process in magnetotactic bacteria. [ABSTRACT FROM AUTHOR]

Magnetosomes are biogenic magnetic nanoparticles with unique material properties, such as high crystallinity and strong magnetization as well as uniform and tunable shapes and sizes. In particular, the ability to genetically modify their enveloping membrane could render isolated magnetosomes as versatile tools for a variety of potential applications in the biomedical field. However, the biocompatibility of the bacterially produced particles has so far not been fully been assessed. In this study, different biocompatibility and toxicity factors of magnetosomes isolated from the model organism Magnetospirillum gryphiswaldense were investigated and tested with THP-1, Jurkat cells, and primary human-derived material such as peripheral blood mononuclear cells (PBMCs). The obtained data demonstrate generally good biocompatibility for all tested magnetosome concentrations as well as high blood compatibility. Only slight but not significant complement activation was observed, with no indications for plasma coagulation or hemolysis. However, the presence of lipopolysaccharides originating from the Gram-negative cell wall of M. gryphiswaldense caused activation of PBMCs expressed as significant cytokine release. Thus, this endotoxicity represents an issue for possible in vivo applications, which needs to be addressed in future studies. Nevertheless, the results show that, overall, magnetosomes have high potential for different (bio)-medical applications and are already well-suited for the fields of in vitro diagnostics. [ABSTRACT FROM AUTHOR]

Liquid marbles are widely known for their potential biomedical applications, especially due to their versatility and ease of preparation. In the present work, we prepared liquid marbles with various cores composed of water, agar-based hydrogels, magnetic fluids, or non-aqueous substances. As a coating material, we used biocompatible particles of plant origin, such as turmeric grains and Lycopodium pollen. Additionally, we provided marbles with magnetic properties by incorporating either magnetosomes or iron oxide nanoparticles as a powder or by injecting another magnetic fluid. Structures obtained in this way were stable and susceptible to manipulation by an external magnetic field. The properties of the magnetic components of our marbles were verified using electron paramagnetic resonance (EPR) spectroscopy and vibrating sample magnetometry (VSM). Our approach to encapsulation of active substances such as antibiotics within a protective hydrogel core opens up new perspectives for the delivery of hydrophobic payloads to the inherently hydrophilic biological environment. Additionally, hydrogel marbles enriched with magnetic materials showed promise as biocompatible heating agents under alternating magnetic fields. A significant innovation of our research was also the fabrication of composite structures in which the gel-like core was surrounded without mixing by a magnetic fluid covered on the outside by the particle shell. Our liquid marbles, especially those with a hydrogel core and magnetic content, due to the ease of preparation and favorable properties, have great potential for biomedical use. The fact that we were able to simultaneously produce, functionalize (by filling with predefined cargo), and manipulate (by means of an external magnetic field) several marbles also seems to be important from an application point of view. [ABSTRACT FROM AUTHOR]

Aims Magnetotactic bacteria (MTB) can use their unique intracellular magnetosome organelles to swim along the Earth's magnetic field. They play important roles in the biogeochemical cycles of iron and sulfur. Previous studies have shown that the applied magnetic fields could affect the magnetosome formation and antioxidant defense systems in MTB. However, the molecular mechanisms by which magnetic fields affect MTB cells remain unclear. We aim to better understand the dark at 28°C–29°C for 20 h, as shownthe interactions between magnetic fields and cells, and the mechanism of MTB adaptation to magnetic field at molecular levels. Methods and results We performed microbiological, transcriptomic, and genetic experiments to analyze the effects of a weak static magnetic field (SMF) exposure on the cell growth and magnetosome formation in the MTB strain Magnetospirillum magneticum AMB-1. The results showed that a 1.5 mT SMF significantly promoted the cell growth but reduced magnetosome formation in AMB-1, compared to the geomagnetic field. Transcriptomic analysis revealed decreased expression of genes primarily involved in the sulfate reduction pathway. Consistently, knockout mutant lacking adenylyl-sulfate kinase CysC did no more react to the SMF and the differences in growth and C mag disappeared. Together with experimental findings of increased reactive oxidative species in the SMF-treated wild-type strain, we proposed that cysC , as a key gene, can participate in the cell growth and mineralization in AMB-1 by SMF regulation. Conclusions This study suggests that the magnetic field exposure can trigger a bacterial oxidative stress response involved in AMB-1 growth and magnetosome mineralization by regulating the sulfur metabolism pathway. CysC may serve as a pivotal enzyme in mediating sulfur metabolism to synchronize the impact of SMF on both growth and magnetization of AMB-1. [ABSTRACT FROM AUTHOR]

Magnetotactic bacteria (MTB) are a phylogenetically diverse group of bacteria remarkable for their ability to biomineralize magnetite (Fe3O4) or greigite (Fe3S4) in organelles called magnetosomes. The majority of genes required for magnetosome formation are encoded by a magnetosome gene island (MAI). Most previous genetic studies of MTB have focused on the MAI, using screens to identify key MAI genes or targeted genetics to isolate specific genes and their function in one specific growth condition. This is the first study that has taken an unbiased approach to look at many different growth conditions to reveal key genes both inside and outside the MAI. Here, we conducted random barcoded transposon mutagenesis (RB-TnSeq) in Magnetospirillum magneticum AMB-1. We generated a library of 184,710 unique strains in a wild-type background, generating ∼34 mutant strains for each gene. RB-TnSeq also allowed us to determine the essential gene set of AMB-1 under standard laboratory growth conditions. To pinpoint novel genes that are important for magnetosome formation, we subjected the library to magnetic selection screens under varied growth conditions. We compared biomineralization under standard growth conditions to biomineralization under high-iron and anaerobic conditions, respectively. Strains with transposon insertions in the MAI gene mamT had an exacerbated biomineralization defect under both high-iron and anaerobic conditions compared to standard conditions, adding to our knowledge of the role of MamT in magnetosome formation. Mutants in an ex-MAI gene, amb4151, are more magnetic than wild-type cells under anaerobic conditions. All three of these phenotypes were validated by creating a markerless deletion strain of the gene and evaluating with TEM imaging. Overall, our results indicate that growth conditions affect which genes are required for biomineralization and that some MAI genes may have more nuanced functions than was previously understood. IMPORTANCE Magnetotactic bacteria (MTB) are a group of bacteria that can form nano-sized crystals of magnetic minerals. MTB are likely an important part of their ecosystems, because they can account for up to a third of the microbial biomass in an aquatic habitat and consume large amounts of iron, potentially impacting the iron cycle. The ecology of MTB is relatively understudied; however, the cell biology and genetics of MTB have been studied for decades. Here, we leverage genetic studies of MTB to inform environmental studies. We expand the genetic toolset for studying MTB in the lab and identify novel genes, or functions of genes, that have an impact on biomineralization.

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.

Magnetotactic bacteria are a diverse group of microorganisms that use intracellular chains of ferrimagnetic nanocrystals, produced within magnetosome organelles, to align and navigate along the geomagnetic field. Several conserved genes for magnetosome formation have been described, but the mechanisms leading to distinct species-specific magnetosome chain configurations remain unclear. Here, we show that the fragmented nature of magnetosome chains in Magnetospirillum magneticum AMB-1 is controlled by genes mcaA and mcaB. McaA recognizes the positive curvature of the inner cell membrane, while McaB localizes to magnetosomes. Along with the MamK actin-like cytoskeleton, McaA and McaB create space for addition of new magnetosomes in between pre-existing magnetosomes. Phylogenetic analyses suggest that McaA and McaB homologs are widespread among magnetotactic bacteria and may represent an ancient strategy for magnetosome positioning.

The combined passive and active targeting of tumoral tissue remains an active and relevant cancer research field. Here, we exploit the properties of two highly magnetic nanomaterials, magnetosomes and ultramagnetic liposomes, in order to magnetically target prostate adenocarcinoma tumors, implanted orthotopically or subcutaneously, to take into account the role of tumor vascularization in the targeting efficiency. Analysis of organ biodistribution in vivo revealed that, for all conditions, both nanomaterials accumulate mostly in the liver and spleen, with an overall low tumor retention. However, both nanomaterials were more readily identified in orthotopic tumors, reflecting their higher tumor vascularization. Additionally, a 2- and 3-fold increase in nanomaterial accumulation was achieved with magnetic targeting. In summary, ultramagnetic nanomaterials show promise mostly in the targeting of highly-vascularized orthotopic murine tumor models. [ABSTRACT FROM AUTHOR]

BackgroundMagnetic nanoparticles such as magnetosomes modified with antibodies allow a high probability of their interaction with targets of interest. Magnetosomes biomineralized by magnetotactic bacteria are in homogeneous nanoscale size and have crystallographic structure, and high thermal and colloidal stability. Camelidae derived nanobodies (Nbs) are small in size, thermal stable, highly water soluble, easy to produce, and fusible with magnetosomes. We aimed to functionalize Nb-magnetosomes for the analysis of the insecticide fipronil.ResultsThree recombinant magnetotactic bacteria (CF, CF+ , and CFFF) biomineralizing magnetosomes with different abundance of Nbs displayed on the surface were constructed. Compared to magnetosomes from the wild type Magnetospirillum gryphiswaldense MSR-1, all of the Nb-magnetosomes biosynthesized by strains CF, CF+ , and CFFF showed a detectable level of binding capability to fipronil-horseradish peroxidase (H2-HRP), but none of them recognized free fipronil. The Nb-magnetosomes from CFFF were oxidized with H2O2 or a glutathione mixture consisting of reduced glutathione and oxidized glutathione in vitro and their binding affinity to H2-HRP was decreased, whereas that to free fipronil was enhanced. The magnetosomes treated with the glutathione mixture were employed to develop an enzyme-linked immunosorbent assay for the detection of fipronil in water samples, with average recoveries in a range of 78-101%.ConclusionsThe economical and environmental-friendly Nb-magnetosomes biomineralized by the bacterial strain MSR-1 can be potentially applied to nanobody-based immunoassays for the detection of fipronil or nanobody-based assays in general.

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.

BackgroundThe discovery of membrane-enclosed, metabolically functional organelles in Bacteria has transformed our understanding of the subcellular complexity of prokaryotic cells. Biomineralization of magnetic nanoparticles within magnetosomes by magnetotactic bacteria (MTB) is a fascinating example of prokaryotic organelles. Magnetosomes, as nano-sized magnetic sensors in MTB, facilitate cell navigation along the local geomagnetic field, a behaviour referred to as magnetotaxis or microbial magnetoreception. Recent discovery of novel MTB outside the traditionally recognized taxonomic lineages suggests that MTB diversity across the domain Bacteria are considerably underestimated, which limits understanding of the taxonomic distribution and evolutionary origin of magnetosome organelle biogenesis.ResultsHere, we perform the most comprehensive metagenomic analysis available of MTB communities and reconstruct metagenome-assembled MTB genomes from diverse ecosystems. Discovery of MTB in acidic peatland soils suggests widespread MTB occurrence in waterlogged soils in addition to subaqueous sediments and water bodies. A total of 168 MTB draft genomes have been reconstructed, which represent nearly a 3-fold increase over the number currently available and more than double the known MTB species at the genome level. Phylogenomic analysis reveals that these genomes belong to 13 Bacterial phyla, six of which were previously not known to include MTB. These findings indicate a much wider taxonomic distribution of magnetosome organelle biogenesis across the domain Bacteria than previously thought. Comparative genome analysis reveals a vast diversity of magnetosome gene clusters involved in magnetosomal biogenesis in terms of gene content and synteny residing in distinct taxonomic lineages. Phylogenetic analyses of core magnetosome proteins in this largest available and taxonomically diverse dataset support an unexpectedly early evolutionary origin of magnetosome biomineralization, likely ancestral to the origin of the domain Bacteria.ConclusionsThese findings expand the taxonomic and phylogenetic diversity of MTB across the domain Bacteria and shed new light on the origin and evolution of microbial magnetoreception. Potential biogenesis of the magnetosome organelle in the close descendants of the last bacterial common ancestor has important implications for our understanding of the evolutionary history of bacterial cellular complexity and emphasizes the biological significance of the magnetosome organelle. Video Abstract.

Magnetotactic bacteria (MTB) are aquatic microorganisms that are able to biomineralize membrane-enclosed magnetic nanoparticles called magnetosomes. Inside the MTB, magnetosomes are arranged in a chain that allows MTB to align and navigate along the Earth's magnetic field. When isolated from the MTB, magnetosomes display a number of potential applications for targeted cancer therapies, such as magnetic hyperthermia, localized drug delivery, or tumor monitoring. The characteristics and properties of magnetosomes for these applications exceed in several aspects those of synthetic magnetic nanoparticles. Likewise, the whole MTB can also be considered as promising agents for cancer treatment, taking advantage of their self-propulsion capability provided by their flagella and the guidance capabilities ensured by their magnetosome chain. Indeed, MTB are envisaged as nanobiots that can be guided and manipulated by external magnetic fields and are naturally attracted toward hypoxic areas, such as the tumor regions, while retaining the therapeutic and imaging capacities of the isolated magnetosomes. Moreover, unlike most of the bacteria currently tested in clinical trials for cancer therapy, MTB are not pathogenic but could be engineered to deliver and/or express specific cytotoxic molecules. In this article, we will review the progress and perspectives of this emerging research field and will discuss the main challenges to overcome before the use of MTB can be successfully applied in the clinic. [ABSTRACT FROM AUTHOR]

At currently, approximately 70 species of magnetotactic bacteria have been identified; thus, there is an urgent need to identify more magnetotactic bacteria from diverse environmental sources with potential applications in industry and biotechnology. To the best of our knowledge, this is the first magnetotactic bacterial strain discovered in Pakistan. The first magnetotactic bacteria, Magnetospirillum moscoviense MS-24, was isolated from Banjosa Lake (Rawalakot), Pakistan, in the current investigation. Magnetospirillum moscoviense MS-24 was screened using the Racetrack method. The Magnetospirillum moscoviense MS-24 were physically characterised using Atomic Force Microscopy, High-Resolution Scanning Electron Microscopy, and Transmission Electron Microscopy. The current study used microscopy to illustrate the shape of bacteria and to find a very obvious chain of magnetosomes within the bacterial cell. The Magnetospirillum moscoviense MS-24 measured about 4 ± 0.04 µm in length and 600 ± 0.02 nm in diameter. The microfluidic chip experiments were also used to detect magnetotaxis behaviour in bacteria. [ABSTRACT FROM AUTHOR]

Magnetosome-producing microorganisms can sense and move toward the redox gradient and have been extensively studied in terrestrial and shallow marine sediment environments. However, given the difficulty of sampling, magnetotactic bacteria (MTB) are poorly explored in deep-sea hydrothermal fields. In this study, a deep-sea hydrothermal vent chimney from the Southern Mariana Trough was collected using a remotely operated submersible. The mineralogical and geochemical characterization of the vent chimney sample showed an internal iron redox gradient. Additionally, the electron microscopy of particles collected by magnetic separation from the chimney sample revealed MTB cells with bullet-shaped magnetosomes, and there were minor occurrences of cuboctahedral and hexagonal prismatic magnetosomes. Genome-resolved metagenomic analysis was performed to identify microorganisms that formed magnetosomes. A metagenome-assembled genome (MAG) affiliated with Nitrospinae had magnetosome genes such as mamA, mamI, mamM, mamP, and mamQ. Furthermore, a diagnostic feature of MTB genomes, such as magnetosome gene clusters (MGCs), including mamA, mamP, and mamQ, was also confirmed in the Nitrospinae-affiliated MAG. Two lines of evidence support the occurrence of MTB in a deep-sea, inactive hydrothermal vent environment. [ABSTRACT FROM AUTHOR]

For the potential in vitro/in vivo applications of magnetic iron oxide nanoparticles, their stability in different physiological fluids has to be ensured. This important prerequisite includes the preservation of the particles' stability during the envisaged application and, consequently, their invariance with respect to the transfer from storage conditions to cell culture media or even bodily fluids. Here, we investigate the colloidal stabilities of commercial nanoparticles with different coatings as a model system for biogenic iron oxide nanoparticles (magnetosomes) isolated from magnetotactic bacteria. We demonstrate that the stability can be evaluated and quantified by determining the intensity-weighted average of the particle sizes (Z-value) obtained from dynamic light scattering experiments as a simple quality criterion, which can also be used as an indicator for protein corona formation. [ABSTRACT FROM AUTHOR]

Magnetotactic bacteria (MTB) are ubiquitous aquatic microorganisms that mineralize dissolved iron into intracellular magnetic crystals. After cell death, these crystals are trapped into sediments that remove iron from the soluble pool. MTB may significantly impact the iron biogeochemical cycle, especially in the ocean where dissolved iron limits nitrogen fixation and primary productivity. A thorough assessment of their impact has been hampered by a lack of methodology to measure the amount of, and variability in, their intracellular iron content. We quantified the iron mass contained in single MTB cells of Magnetospirillum magneticum strain AMB-1 using a time-resolved inductively coupled plasma-mass spectrometry methodology. Bacterial iron content depends on the external iron concentration, and reaches a maximum value of ~10-6 ng of iron per cell. From these results, we calculated the flux of dissolved iron incorporation into environmental MTB populations and conclude that MTB may mineralize a significant fraction of dissolved iron into crystals.

Many species of bacteria can manufacture materials on a finer scale than those that are synthetically made. These products are often produced within intracellular compartments that bear many hallmarks of eukaryotic organelles. One unique and elegant group of organisms is at the forefront of studies into the mechanisms of organelle formation and biomineralization. Magnetotactic bacteria (MTB) produce organelles called magnetosomes that contain nanocrystals of magnetic material, and understanding the molecular mechanisms behind magnetosome formation and biomineralization is a rich area of study. In this Review, we focus on the genetics behind the formation of magnetosomes and biomineralization. We cover the history of genetic discoveries in MTB and key insights that have been found in recent years and provide a perspective on the future of genetic studies in MTB.

Bacterial magnetic particles (BMPs) are an attractive carrier material for immunoassays because of their nanoscale size, dispersal ability, and membrane-bound structure. Antitetrabromobisphenol-A (TBBPA) nanobodies (Nbs) in the form of monovalence (Nb1), bivalence (Nb2), and trivalence (Nb3) were biotinylated and immobilized onto streptavidin (SA)-derivatized BMPs to construct the complexes of BMP-SA-Biotin-Nb1, -Nb2, and -Nb3, respectively. An increasing order of binding capability of BMP-SA-Biotin-Nb1, -Nb2, and -Nb3 to TBBPA was observed. These complexes showed high resilience to temperature (90 °C), methanol (100%), high pH (12), and strong ionic strength (1.37 M NaCl). A BMP-SA-Biotin-Nb3-based enzyme linked immunosorbent assay (ELISA) for TBBPA dissolved in methanol was developed, showing a half-maximum inhibition concentration (IC50) of 0.42 ng mL-1. TBBPA residues in landfill leachate, sewage, and sludge samples determined by this assay were in a range of -1, -1, and -1 (dw), respectively, correlating well with the results by liquid chromatography tandem mass spectrometry. The BMP-SA-Biotin-Nb3 was reusable at least three times without significant loss of the binding capability. The BMP-SA-Biotin-Nb3-based ELISA, with a total assay time of less than 30 min, is promising for the rapid monitoring of TBBPA in the environment.

Abstract Background One of the most complex prokaryotic organelles are magnetosomes, which are formed by magnetotactic bacteria as sensors for navigation in the Earth’s magnetic field. In the alphaproteobacterium Magnetospirillum gryphiswaldense magnetosomes consist of chains of magnetite crystals (Fe3O4) that under microoxic to anoxic conditions are biomineralized within membrane vesicles. To form such an intricate structure, the transcription of > 30 specific structural genes clustered within the genomic magnetosome island (MAI) has to be coordinated with the expression of an as-yet unknown number of auxiliary genes encoding several generic metabolic functions. However, their global regulation and transcriptional organization in response to anoxic conditions most favorable for magnetite biomineralization are still unclear. Results Here, we compared transcriptional profiles of anaerobically grown magnetosome forming cells with those in which magnetosome biosynthesis has been suppressed by aerobic condition. Using whole transcriptome shotgun sequencing, we found that transcription of about 300 of the > 4300 genes was significantly enhanced during magnetosome formation. About 40 of the top upregulated genes are directly or indirectly linked to aerobic and anaerobic respiration (denitrification) or unknown functions. The mam and mms gene clusters, specifically controlling magnetosome biosynthesis, were highly transcribed, but constitutively expressed irrespective of the growth condition. By Cappable-sequencing, we show that the transcriptional complexity of both the MAI and the entire genome decreased under anaerobic conditions optimal for magnetosome formation. In addition, predominant promoter structures were highly similar to sigma factor σ70 dependent promoters in other Alphaproteobacteria. Conclusions Our transcriptome-wide analysis revealed that magnetite biomineralization relies on a complex interplay between generic metabolic processes such as aerobic and anaerobic respiration, cellular redox control, and the biosynthesis of specific magnetosome structures. In addition, we provide insights into global regulatory features that have remained uncharacterized in the widely studied model organism M. gryphiswaldense, including a comprehensive dataset of newly annotated transcription start sites and genome-wide operon detection as a community resource (GEO Series accession number GSE197098).

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. [ABSTRACT FROM AUTHOR]

Melanoma is the most aggressive and metastasis-prone form of skin cancer. Conventional therapies include chemotherapeutic agents, either as small molecules or carried by FDA-approved nanostructures. However, systemic toxicity and side effects still remain as major drawbacks. With the advancement of nanomedicine, new delivery strategies emerge at a regular pace, aiming to overcome these challenges. Stimulus-responsive drug delivery systems might considerably reduce systemic toxicity and side-effects by limiting drug release to the affected area. Herein, we report the development of paclitaxel-loaded lipid-coated manganese ferrite magnetic nanoparticles (PTX-LMNP) as magnetosomes synthetic analogs, envisaging the combined chemo-magnetic hyperthermia treatment of melanoma. PTX-LMNP physicochemical properties were verified, including their shape, size, crystallinity, FTIR spectrum, magnetization profile, and temperature profile under magnetic hyperthermia (MHT). Their diffusion in porcine ear skin (a model for human skin) was investigated after intradermal administration via fluorescence microscopy. Cumulative PTX release kinetics under different temperatures, either preceded or not by MHT, were assessed. Intrinsic cytotoxicity against B16F10 cells was determined via neutral red uptake assay after 48 h of incubation (long-term assay), as well as B16F10 cells viability after 1 h of incubation (short-term assay), followed by MHT. PTX-LMNP-mediated MHT triggers PTX release, allowing its thermal-modulated local delivery to diseased sites, within short timeframes. Moreover, half-maximal PTX inhibitory concentration (IC50) could be significantly reduced relatively to free PTX (142,500×) and Taxol® (340×). Therefore, the dual chemo-MHT therapy mediated by intratumorally injected PTX-LMNP stands out as a promising alternative to efficiently deliver PTX to melanoma cells, consequently reducing systemic side effects commonly associated with conventional chemotherapies. [ABSTRACT FROM AUTHOR]

Liquid marbles are widely known for their potential biomedical applications, especially due to their versatility and ease of preparation. In the present work, we prepared liquid marbles with various cores composed of water, agar-based hydrogels, magnetic fluids, or non-aqueous substances. As a coating material, we used biocompatible particles of plant origin, such as turmeric grains and Lycopodium pollen. Additionally, we provided marbles with magnetic properties by incorporating either magnetosomes or iron oxide nanoparticles as a powder or by injecting another magnetic fluid. Structures obtained in this way were stable and susceptible to manipulation by an external magnetic field. The properties of the magnetic components of our marbles were verified using electron paramagnetic resonance (EPR) spectroscopy and vibrating sample magnetometry (VSM). Our approach to encapsulation of active substances such as antibiotics within a protective hydrogel core opens up new perspectives for the delivery of hydrophobic payloads to the inherently hydrophilic biological environment. Additionally, hydrogel marbles enriched with magnetic materials showed promise as biocompatible heating agents under alternating magnetic fields. A significant innovation of our research was also the fabrication of composite structures in which the gel-like core was surrounded without mixing by a magnetic fluid covered on the outside by the particle shell. Our liquid marbles, especially those with a hydrogel core and magnetic content, due to the ease of preparation and favorable properties, have great potential for biomedical use. The fact that we were able to simultaneously produce, functionalize (by filling with predefined cargo), and manipulate (by means of an external magnetic field) several marbles also seems to be important from an application point of view.

Spin wave logic circuits using quantum oscillations of spins (magnons) as carriers of information have been proposed for next generation computing with reduced energy demands and the benefit of easy parallelization. Current realizations of magnonic devices have micrometer sized patterns. Here we demonstrate the feasibility of biogenic nanoparticle chains as the first step to truly nanoscale magnonics at room temperature. Our measurements on magnetosome chains (ca 12 magnetite crystals with 35 nm particle size each), combined with micromagnetic simulations, show that the topology of the magnon bands, namely anisotropy, band deformation, and band gaps are determined by local arrangement and orientation of particles, which in turn depends on the genotype of the bacteria. Our biomagnonic approach offers the exciting prospect of genetically engineering magnonic quantum states in nanoconfined geometries. By connecting mutants of magnetotactic bacteria with different arrangements of magnetite crystals, novel architectures for magnonic computing may be (self-) assembled.

We report the fabrication of highly pure magnetosomes that are synthesized by magnetotactic bacteria (MTB) using pharmaceutically compatible growth media, i.e., without compounds of animal origin (yeast extracts), carcinogenic, mutagenic, or toxic for reproduction (CMR) products, and other heavy metals than iron. To enable magnetosome medical applications, these growth media are reduced and amended compared with media commonly used to grow these bacteria. Furthermore, magnetosomes are made non-pyrogenic by being extracted from these micro-organisms and heated above 400 °C to remove and denature bacterial organic material and produce inorganic magnetosome minerals. To be stabilized, these minerals are further coated with citric acid to yield M-CA, leading to fully reconstructed chains of magnetosomes. The heating properties and anti-tumor activity of highly pure M-CA are then studied by bringing M-CA into contact with PC3-Luc tumor cells and by exposing such assembly to an alternating magnetic field (AMF) of 42 mT and 195 kHz during 30 min. While in the absence of AMF, M-CA are observed to be non-cytotoxic, they result in a 35% decrease in cell viability following AMF application. The treatment efficacy can be associated with a specific absorption rate (SAR) value of M-CA, which is relatively high in cellular environment, i.e., SARcell = 253 ± 11 W/gFe, while being lower than the M-CA SAR value measured in water, i.e., SARwater = 1025 ± 194 W/gFe, highlighting that a reduction in the Brownian contribution to the SAR value in cellular environment does not prevent efficient tumor cell destruction with these nanoparticles. Key points: • Highly pure magnetosomes were produced in pharmaceutically compatible growth media • Non-pyrogenic and stable magnetosomes were prepared for human injection • Magnetosomes efficiently destroyed prostate tumor cells in magnetic hyperthermia [ABSTRACT FROM AUTHOR]

Magnetic parameters of magnetosomes inside the bacteria of MSR-1, LBB-42, AMB-1, SP-1, BB-1, and SO-1 strains of the genus Magnetospirillum fixed by 5% formalin in the nutrient medium were estimated by measurements of the nonlinear longitudinal response to a weak ac magnetic field (NLR-M2) at room temperature. For the BB-1, MSR-1, and AMB-1 strains, the measurements of the electron magnetic resonance (EMR) spectra with the special X-band spectrometer for wide-line registration were also carried out. To trace the evolution of the magnetic state of the magnetosomes during the long-term storage, freshly prepared samples ("new") and samples after a year of storage at 4 °C ("old") were studied. The assessment of the state of the bacteria ensemble in the medium after the long-term storage was carried out for one typical strain (BB-1) using atomic force microscopy. The stable single-domain state of magnetic centers in the magnetosomes indicating their proximity to a superparamagnetic (SPM) regime was found at the scan frequency 0.02 Hz of the steady magnetic field. This allowed a semi-quantitative analysis of M2 data to be carried out with the formalism based on the numerical solution of the kinetic Fokker–Planck equation for SPM particles. Processing the NLR-M2 data demonstrated the presence of two kinds of magnetosomes in both the "new" and "old" samples: (i) those with the large magnetic moment (the "heavy", monodisperse mode) and (ii) those with the comparatively small magnetic moment (the "light", highly dispersed mode). The EMR spectra were formed mostly by the "heavy" fraction for both samples. The presence of two peaks in the spectra evidenced the presence of conventional uniaxial magnetic anisotropy in the magnetosomes. The appearance of one or two additional peaks in the spectra in the "old" fraction of some strains implied their instability at the long-term storage, even when fixed by formalin and sealed in the nitrogen atmosphere. [ABSTRACT FROM AUTHOR]

Magnetotactic bacteria (MTB), a diverse group of marine and freshwater microorganisms, have attracted the scientific community's attention since their discovery. These bacteria biomineralize ferrimagnetic nanocrystals, the magnetosomes, or biological magnetic nanoparticles (BMNs), in a single or multiple chain(s) within the cell. As a result, cells experience an optimized magnetic dipolar moment responsible for a passive alignment along the lines of the geomagnetic field. Advances in MTB cultivation and BMN isolation have contributed to the expansion of the biotechnological potential of MTB in recent decades. Several studies with mass-cultured MTB expanded the possibilities of using purified nanocrystals and whole cells in nano- and biotechnology. Freshwater MTB were primarily investigated in scaling up processes for the production of BMNs. However, marine MTB have the potential to overcome freshwater species applications due to the putative high efficiency of their BMNs in capturing molecules. Regarding the use of MTB or BMNs in different approaches, the application of BMNs in biomedicine remains the focus of most studies, but their application is not restricted to this field. In recent years, environment monitoring and recovery, engineering applications, wastewater treatment, and industrial processes have benefited from MTB-based biotechnologies. This review explores the advances in MTB large-scale cultivation and the consequent development of innovative tools or processes. [ABSTRACT FROM AUTHOR]

Shijiao Ma,1 Chenchen Gu,1 Junjie Xu,1 Jinxin He,2 Shuli Li,1 Haolan Zheng,1 Bo Pang,1 Ying Wen,1 Qiaojun Fang,3 Weiquan Liu,1 Jiesheng Tian1 1State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, 100193, People’s Republic of China; 2College of Veterinary Medicine, Shanxi Agriculture University, Taigu, Shanxi, 030801, People’s Republic of China; 3CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing, 100190, People’s Republic of ChinaCorrespondence: Weiquan Liu; Jiesheng Tian, Tel/Fax +8610-62732676 ; +8610-62731440, Email weiquan8@cau.edu.cn; tianhome@cau.edu.cnPurpose: Nanoparticles (NPs) decorated with functional ligands are promising candidates for cancer diagnosis and treatment. However, numerous studies have shown that chemically coupled targeting moieties on NPs lose their targeting capability in the biological milieu because they are shielded or covered by a “protein corona”. Herein, we construct a functional magnetosome that recognizes and targets cancer cells even in the presence of protein corona.Methods: Magnetosomes (BMPs) were extracted from magnetotactic bacteria, M. gryphiswaldense (MSR-1), and decorated with trastuzumab (TZ) via affibody (RA) and glutaraldehyde (GA). The engineered BMPs are referred to as BMP-RA-TZ and BMP-GA-TZ. Their capacities to combine HER2 were detected by ELISA, the quantity of plasma corona proteins was analyzed using LC-MS. The efficiencies of targeting SK-BR-3 were demonstrated by confocal laser scanning microscopy and flow cytometry.Results: Both engineered BMPs contain up to ∼ 0.2 mg TZ per mg of BMP, while the quantity of HER2 binding to BMP-RA-TZ is three times higher than that binding to BMP-GA-TZ. After incubation with normal human plasma or IgG-supplemented plasma, GA-TZ-containing BMPs have larger hydrated radii and more surface proteins in comparison with RA-TZ-containing BMPs. The TZ-containing BMPs all can be targeted to and internalized in the HER2-overexpressing breast cancer cell line SK-BR-3; however, their targeting efficiencies vary considerably: 50– 75% for RA-TZ-containing BMPs and 9– 19% for GA-TZ-containing BMPs. BMPs were incubated with plasma (100%) and cancer cells to simulate human in vivo environment. In this milieu, BMP-RA-TZ uptake efficiency of SK-BR-3 reaches nearly 80% (slightly lower than for direct interaction with BMP-RA-TZ), whereas the BMP-GA-TZ uptake efficiency is < 17%.Conclusion: Application of the RA scaffold promotes and orients the arrangement of targeting ligands and reduces the shielding effect of corona proteins. This strategy improves the targeting capability and drug delivery of NP in a simulated in vivo milieu.Keywords: affibody, protein corona, magnetosomes, human epidermal growth factor receptor 2, HER2

Magnetosomes are complex bacterial organelles that serve as model systems for studying bacterial cell biology, biomineralization, and global iron cycling. Magnetosome biogenesis is primarily studied in two closely related Alphaproteobacteria of the genus Magnetospirillum that form cubooctahedral-shaped magnetite crystals within a lipid membrane. However, chemically and structurally distinct magnetic particles have been found in physiologically and phylogenetically diverse bacteria. Due to a lack of molecular genetic tools, the mechanistic diversity of magnetosome formation remains poorly understood. Desulfovibrio magneticus RS-1 is an anaerobic sulfate-reducing deltaproteobacterium that forms bullet-shaped magnetite crystals. A recent forward genetic screen identified 10 genes in the conserved magnetosome gene island of D. magneticus that are essential for its magnetic phenotype. However, this screen likely missed mutants with defects in crystal size, shape, and arrangement. Reverse genetics to target the remaining putative magnetosome genes using standard genetic methods of suicide vector integration have not been feasible due to the low transconjugation efficiency. Here, we present a reverse genetic method for targeted mutagenesis in D. magneticus using a replicative plasmid. To test this method, we generated a mutant resistant to 5-fluorouracil by making a markerless deletion of the upp gene that encodes uracil phosphoribosyltransferase. We also used this method for targeted marker exchange mutagenesis by replacing kupM, a gene identified in our previous screen as a magnetosome formation factor, with a streptomycin resistance cassette. Overall, our results show that targeted mutagenesis using a replicative plasmid is effective in D. magneticus and may also be applied to other genetically recalcitrant bacteria.IMPORTANCE Magnetotactic bacteria (MTB) are a group of organisms that form intracellular nanometer-scale magnetic crystals though a complex process involving lipid and protein scaffolds. These magnetic crystals and their lipid membranes, termed magnetosomes, are model systems for studying bacterial cell biology and biomineralization and are potential platforms for biotechnological applications. Due to a lack of genetic tools and unculturable representatives, the mechanisms of magnetosome formation in phylogenetically deeply branching MTB remain unknown. These MTB contain elongated bullet-/tooth-shaped magnetite and greigite crystals that likely form in a manner distinct from that of the cubooctahedral-shaped magnetite crystals of the genetically tractable MTB within the Alphaproteobacteria Here, we present a method for genome editing in Desulfovibrio magneticus RS-1, a cultured representative of the deeply branching MTB of the class Deltaproteobacteria This marks a crucial step in developing D. magneticus as a model for studying diverse mechanisms of magnetic particle formation by MTB.

Magnetic nanoparticles (MNPs) are useful for many biomedical applications, but it is challenging to synthetically produce them in large numbers with uniform properties and surface functionalization. Magnetotactic bacteria (MTB) produce magnetosomes with homogenous sizes, shapes, and magnetic properties. Consequently, there is interest in using MTB as biological factories for MNP production. Nonetheless, MTB can only be grown to low yields, and wild-type strains produce low numbers of MNPs/bacterium. There are also limited technologies to facilitate the selection of MTB with different magnetic contents, such as MTB with compromised and enhanced biomineralization ability. Here, we describe a magnetic microfluidic platform combined with transient cold/alkaline treatment to temporarily reduce the rapid flagellar motion of MTB without compromising their long-term proliferation and biomineralization ability for separating MTB on the basis of their magnetic contents. This strategy enables live MTB to be enriched, which, to the best of our knowledge, has not been achieved with another previously described magnetic microfluidic device that makes use of ferrofluid and heat. Our device also facilitates the high-throughput (25,000 cells/min) separation of wild-type Magnetospirillum gryphiswaldense (MSR-1) from nonmagnetic ΔmamAB MSR-1 mutants with a sensitivity of up to 80% and isolation purity of up to 95%, as confirmed with a gold-standard fluorescent-activated cell sorter (FACS) technique. This offers a 25-fold higher throughput than other previously described magnetic microfluidic platforms (1,000 cells/min). The device can also be used to isolate Magnetospirillum magneticum (AMB-1) mutants with different ranges of magnetosome numbers with efficiencies close to theoretical estimates. We believe this technology will facilitate the magnetic characterization of genetically engineered MTB for a variety of applications, including using MTB for large-scale, controlled MNP production.IMPORTANCE Our magnetic microfluidic technology can greatly facilitate biological applications with magnetotactic bacteria, from selection and screening to analysis. This technology will be of interest to microbiologists, chemists, and bioengineers who are interested in the biomineralization and selection of magnetotactic bacteria (MTB) for applications such as directed evolution and magnetogenetics.

Magnetosomes (MAGs) extracted from magnetotactic bacteria are well-defined membrane-enveloped single-domain magnetic nanoparticles. Due to their superior magnetic and structural properties, MAGs constitute potential materials that can be manipulated via genetic and chemical engineering for use in biomedical and biotechnological applications. However, the long-term effects exerted by MAGs on cells are of concern in the context of in vivo applications. Meanwhile, it remains relatively unclear which mechanisms are employed by cells to process and degrade MAGs. Hence, a better understanding of MAGs' degradation and fundamental signal modulations occurring throughout this process is essential. In the current study, we investigated the potential actions of MAGs on endothelial cells over a 10-day period. MAGs were retained in cells and found to gradually gather in the lysosome-like vesicles. Meanwhile, iron-ion release was observed. Proteomics further revealed a potential cellular mechanism underlying MAGs degradation, in which a group of proteins associated with vesicle biogenesis, and lysosomal enzymes, which participate in protein hydrolysis and lipid degradation, were rapidly upregulated. Moreover, the released iron triggered the regulation of the iron metabolic profiles. However, given that the levels of cell oxidative damage were relatively stable, the released iron ions were handled by iron metabolic profiles and incorporated into normal metabolic routes. These results provide insights into the cell response to MAGs degradation that may improve their in vivo applications. [ABSTRACT FROM AUTHOR]

The synthesis of magnetosomes in magnetotactic bacteria (MTB) represents probably one of Earth's most ancient forms of biomineralization. The evolution of magnetosomes and the origin of magnetotaxis date back to the Archean Eon, 4.4–2.5 Ga ago. Magnetosomes consist of fine magnetite nanocrystals coated with a lipidic envelope. Their findings in eukaryotic cells and animals support the evolutionary success of otherwise energetically very demanding biocrystallization. Moreover, the conservation of magnetite biomineralization genes in all domains of life has been proposed very recently. Therefore, it is not surprising that magnetosomes have attracted attention from various scientific fields, including mineralogy, microbiology, biochemistry, biophysics, and bioengineering. Here, we review the most recent iron flow findings that lead to magnetite nanocrystals' biomineralization in MTB. We emphasize the historical milestones that formed the evolution of magnetosomes and magnetotaxis functionality. Finally, we discuss the usability of these unique structures in biomedical, biotechnological, environmental, and nutritional applications. [ABSTRACT FROM AUTHOR]

Background: One of the most complex prokaryotic organelles are magnetosomes, which are formed by magnetotactic bacteria as sensors for navigation in the Earth's magnetic field. In the alphaproteobacterium Magnetospirillum gryphiswaldense magnetosomes consist of chains of magnetite crystals (Fe3O4) that under microoxic to anoxic conditions are biomineralized within membrane vesicles. To form such an intricate structure, the transcription of > 30 specific structural genes clustered within the genomic magnetosome island (MAI) has to be coordinated with the expression of an as-yet unknown number of auxiliary genes encoding several generic metabolic functions. However, their global regulation and transcriptional organization in response to anoxic conditions most favorable for magnetite biomineralization are still unclear. Results: Here, we compared transcriptional profiles of anaerobically grown magnetosome forming cells with those in which magnetosome biosynthesis has been suppressed by aerobic condition. Using whole transcriptome shotgun sequencing, we found that transcription of about 300 of the > 4300 genes was significantly enhanced during magnetosome formation. About 40 of the top upregulated genes are directly or indirectly linked to aerobic and anaerobic respiration (denitrification) or unknown functions. The mam and mms gene clusters, specifically controlling magnetosome biosynthesis, were highly transcribed, but constitutively expressed irrespective of the growth condition. By Cappable-sequencing, we show that the transcriptional complexity of both the MAI and the entire genome decreased under anaerobic conditions optimal for magnetosome formation. In addition, predominant promoter structures were highly similar to sigma factor σ70 dependent promoters in other Alphaproteobacteria. Conclusions: Our transcriptome-wide analysis revealed that magnetite biomineralization relies on a complex interplay between generic metabolic processes such as aerobic and anaerobic respiration, cellular redox control, and the biosynthesis of specific magnetosome structures. In addition, we provide insights into global regulatory features that have remained uncharacterized in the widely studied model organism M. gryphiswaldense, including a comprehensive dataset of newly annotated transcription start sites and genome-wide operon detection as a community resource (GEO Series accession number GSE197098). [ABSTRACT FROM AUTHOR]

Magnetic nanocarriers have attracted attention in translational oncology due to their ability to be employed both for tumor diagnostics and therapy. This review summarizes data on applications of synthetic and biogenic magnetic nanoparticles (MNPs) in oncological theranostics and related areas. The basics of both types of MNPs including synthesis approaches, structure, and physicochemical properties are discussed. The properties of synthetic MNPs and biogenic MNPs are compared with regard to their antitumor therapeutic efficiency, diagnostic potential, biocompatibility, and cellular toxicity. The comparative analysis demonstrates that both synthetic and biogenic MNPs could be efficiently used for cancer theranostics, including biosensorics and drug delivery. At the same time, reduced toxicity of biogenic particles was noted, which makes them advantageous for in vivo applications, such as drug delivery, or MRI imaging of tumors. Adaptability to surface modification based on natural biochemical processes is also noted, as well as good compatibility with tumor cells and proliferation in them. Advances in the bionanotechnology field should lead to the implementation of MNPs in clinical trials. [ABSTRACT FROM AUTHOR]

A number of materials are studied in the field of magnetic hyperthermia. In general, the most promising ones appear to be iron oxide particle nanosystems. This is also indicated in some clinical trial studies where iron-based oxides were used. On the other hand, the type of material itself provides a number of variations on how to tune hyperthermia indicators. In this paper, magnetite nanoparticles in various forms were analyzed. The nanoparticles differed in the core size as well as in the form of their arrangement. The arrangement was determined by the nature of the surfactant. The individual particles were covered chemically by dextran; in the case of chain-like particles, they were encapsulated naturally in a lipid bilayer. It was shown that in the case of chain-like nanoparticles, except for relaxation, a contribution from magnetic hysteresis to the heating process also appears. The influence of the chosen methodology of magnetic field generation was also analyzed. In addition, the influence of the chosen methodology of magnetic field generation was analyzed. The application of a rotating magnetic field was shown to be more efficient in generating heat than the application of an alternating magnetic field. However, the degree of efficiency depended on the arrangement of the magnetite nanoparticles. The difference in the efficiency of the rotating magnetic field versus the alternating magnetic field was much more pronounced for individual nanoparticles (in the form of a magnetic fluid) than for systems containing chain nanoparticles (magnetosomes and a mix of magnetic fluid with magnetosomes in a ratio 1:1). [ABSTRACT FROM AUTHOR]

Iron is a crucial element integral to various fundamental biological molecular mechanisms, including magnetosome biogenesis in magnetotactic bacteria (MTB). Magnetosomes are formed through the internalization and biomineralization of iron into magnetite crystals. However, the interconnected mechanisms by which MTB uptake and regulate intracellular iron for magnetosome biomineralization remain poorly understood, particularly at the single-cell level. To gain insights we employed a holistic multiscale approach, i . e ., from elemental iron species to bacterial populations, to elucidate the interplay between iron uptake dynamics and magnetosome formation in Magnetospirillum gryphiswaldense MSR-1 under near-native conditions. We combined a correlative microscopy approach integrating light and X-ray tomography with analytical techniques, such as flow cytometry and inductively coupled plasma spectroscopy, to evaluate the effects of iron and oxygen availability on cellular growth, magnetosome biogenesis, and intracellular iron pool in MSR-1. Our results revealed that increased iron availability under microaerobic conditions significantly promoted the formation of longer magnetosome chains and increased intracellular iron uptake, with a saturation point at 300 μM iron citrate. Beyond this threshold, additional iron did not further extend the magnetosome chain length or increase total intracellular iron levels. Moreover, our work reveals (i) a direct correlation between the labile Fe 2+ pool size and magnetosome content, with higher intracellular iron concentrations correlating with increased magnetosome production, and (ii) the existence of an intracellular iron pool, distinct from magnetite, persisting during all stages of biomineralization. This study offers insights into iron dynamics in magnetosome biomineralization at a single-cell level, potentially enhancing the industrial biomanufacturing of magnetosomes.

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.

For the potential in vitro/in vivo applications of magnetic iron oxide nanoparticles, their stability in different physiological fluids has to be ensured. This important prerequisite includes the preservation of the particles’ stability during the envisaged application and, consequently, their invariance with respect to the transfer from storage conditions to cell culture media or even bodily fluids. Here, we investigate the colloidal stabilities of commercial nanoparticles with different coatings as a model system for biogenic iron oxide nanoparticles (magnetosomes) isolated from magnetotactic bacteria. We demonstrate that the stability can be evaluated and quantified by determining the intensity-weighted average of the particle sizes (Z-value) obtained from dynamic light scattering experiments as a simple quality criterion, which can also be used as an indicator for protein corona formation.

A magnetosome-producing bacterium Acidithiobacillus ferrooxidans BYM (At. ferrooxidans BYM) was isolated and magnetically screened. The magnetosome yield from 0.5896 to 13.1291 mg/g was achieved under different aeration rates, ferrous sulfate, ammonium sulfate, and gluconic acid concentrations at 30 ℃. TEM observed 6–9 magnetosomes in size of 20–80 nm irregularly dispersed in a cell. STEM-EDXS and HRTEM-FFT implied that the elongated-prismatic magnetite magnetosomes with {110} crystal faces grown along the [111] direction. Whole-genome sequencing and annotation of BYM showed that 3.2 Mb chromosome and 47.11 kb plasmid coexisted, and 322 genes associated with iron metabolism were discovered. Ten genes shared high similarity with magnetosome genes were predicted, providing sufficient evidence for the magnetosome-producing potential of BYM. Accordingly, we first proposed a hypothetic model of magnetosome formation including vesicle formation, iron uptake and mineralization, and magnetite crystal maturation in At. ferrooxidans. These indicated that At. ferrooxidans BYM would be used as a commercial magnetosome-producing microorganism. [ABSTRACT FROM AUTHOR]

Microbes that synthesize minerals, a process known as microbial biomineralization, contributed substantially to the evolution of current planetary environments through numerous important geochemical processes. Despite its geological significance, the origin and evolution of microbial biomineralization remain poorly understood. Through combined metagenomic and phylogenetic analyses of deep-branching magnetotactic bacteria from the Nitrospirae phylum, and using a Bayesian molecular clock-dating method, we show here that the gene cluster responsible for biomineralization of magnetosomes, and the arrangement of magnetosome chain(s) within cells, both originated before or near the Archean divergence between the Nitrospirae and Proteobacteria This phylogenetic divergence occurred well before the Great Oxygenation Event. Magnetotaxis likely evolved due to environmental pressures conferring an evolutionary advantage to navigation via the geomagnetic field. Earth's dynamo must therefore have been sufficiently strong to sustain microbial magnetotaxis in the Archean, suggesting that magnetotaxis coevolved with the geodynamo over geological time.

Magnetotactic bacteria possess cellular compartments called magnetosomes that sense magnetic fields. Alignment of magnetosomes in the bacterial cell is necessary for their function, and this is achieved through anchoring of magnetosomes to filaments composed of the protein MamK. MamK is an actin homolog that polymerizes upon ATP binding. Here, we report the structure of the MamK filament at ∼6.5 Å, obtained by cryo-Electron Microscopy. This structure confirms our previously reported double-stranded, nonstaggered architecture, and reveals the molecular basis for filament formation. While MamK is closest in sequence to the bacterial actin MreB, the longitudinal contacts along each MamK strand most closely resemble those of eukaryotic actin. In contrast, the cross-strand interface, with a surprisingly limited set of contacts, is novel among actin homologs and gives rise to the nonstaggered architecture.