Discovery of antibacterial biogenic magnetosome nanoparticles from Providencia sp. MTBPRB-1: Screening, purification and characterization.

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.

Exploring the host range for genetic transfer of magnetic organelle biosynthesis.

Magnetosomes produced by magnetotactic bacteria have great potential for application in biotechnology and medicine due to their unique physicochemical properties and high biocompatibility. Attempts to transfer the genes for magnetosome biosynthesis into non-magnetic organisms have had mixed results. Here we report on a systematic study to identify key components needed for magnetosome biosynthesis after gene transfer. We transfer magnetosome genes to 25 proteobacterial hosts, generating seven new magnetosome-producing strains. We characterize the recombinant magnetosomes produced by these strains and demonstrate that denitrification and anaerobic photosynthesis are linked to the ability to synthesize magnetosomes upon the gene transfer. In addition, we show that the number of magnetosomes synthesized by a foreign host negatively correlates with the guanine-cytosine content difference between the host and the gene donor. Our findings have profound implications for the generation of magnetized living cells and the potential for transgenic biogenic magnetic nanoparticle production.

Phylogenetics and biomineralization of a novel magnetotactic Gammaproteobacterium from a freshwater lake in Beijing, China.

Magnetotactic bacteria (MTB) have the remarkable capability of producing intracellularly membrane-enveloped magnetic nanocrystals (i.e. magnetosomes) and swimming along geomagnetic field lines. Despite more than 50 years of research, bacterial diversity and magnetosome biomineralization within MTB are relatively less known in the Gammaproteobacteria class than other groups. This is incompatible with the status of Gammaproteobacteria as the most diverse class of gram-negative bacteria with a number of ecologically important bacteria. Here, we identify a novel MTB strain YYHR-1 affiliated with the Gammaproteobacteria class of the Pseudomonadota phylum from a freshwater lake. In YYHR-1, most magnetosome crystals are organized into a long chain aligned along the cell long axis; unusually, a few small superparamagnetic crystals are located at the side of the chain, off the main chain axis. Micromagnetic simulations indicate that magnetostatic interactions among adjacent crystals within a chain reduce the Gibbs energy to enhance chain stability. Genomic analysis suggests that duplication of magnetosome gene clusters may result in off-chain magnetosomes formation. By integrating available genomic data from Gammaproteobacteria, the phylogenetic position of MTB in this class is reassigned here. Our new findings expand knowledge about MTB diversity and magnetosome biomineralization, and deepen understanding of the phylogenetics of the Gammaproteobacteria.

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.

Intracellular transformation and disposal mechanisms of magnetosomes in macrophages and cancer cells.

Magnetosomes are magnetite nanoparticles biosynthesized by magnetotactic bacteria. Given their potential clinical applications for the diagnosis and treatment of cancer, it is essential to understand what becomes of them once they are within the body. With this aim, here we have followed the intracellular long-term fate of magnetosomes in two cell types: cancer cells (A549 cell line), because they are the actual target for the therapeutic activity of the magnetosomes, and macrophages (RAW 264.7 cell line), because of their role at capturing foreign agents. It is shown that cells dispose of magnetosomes using three mechanisms: splitting them into daughter cells, excreting them to the surrounding environment, and degrading them yielding less or non-magnetic iron products. A deeper insight into the degradation mechanisms by means of time-resolved X-ray absorption near-edge structure (XANES) spectroscopy has allowed us to follow the intracellular biotransformation of magnetosomes by identifying and quantifying the iron species occurring during the process. In both cell types there is a first oxidation of magnetite to maghemite and then, earlier in macrophages than in cancer cells, ferrihydrite starts to appear. Given that ferrihydrite is the iron mineral phase stored in the cores of ferritin proteins, this suggests that cells use the iron released from the degradation of magnetosomes to load ferritin. Comparison of both cellular types evidences that macrophages are more efficient at disposing of magnetosomes than cancer cells, attributed to their role in degrading external debris and in iron homeostasis.

Recent advances in studies on magnetosome-associated proteins composing the bacterial geomagnetic sensor organelle.

Magnetotactic bacteria (MTB) generate a membrane-enclosed subcellular compartment called magnetosome, which contains a biomineralized magnetite or greigite crystal, an inner membrane-derived lipid bilayer membrane, and a set of specifically targeted associated proteins. Magnetosomes are formed by a group of magnetosome-associated proteins encoded in a genomic region called magnetosome island. Magnetosomes are then arranged in a linear chain-like positioning, and the resulting magnetic dipole of the chain functions as a geomagnetic sensor for magneto-aerotaxis motility. Recent metagenomic analyses of environmental specimens shed light on the sizable phylogenetical diversity of uncultured MTB at the phylum level. These findings have led to a better understanding of the diversity and conservation of magnetosome-associated proteins. This review provides an overview of magnetosomes and magnetosome-associated proteins and introduces recent topics about this fascinating magnetic bacterial organelle.

Influence of protozoan grazing on magnetotactic bacteria on intracellular and extracellular iron content.

Magnetotactic bacteria (MTB) ubiquitously inhabit the oxic-anoxic interface or anaerobic areas of aquatic environments. MTB biomineralize magnetite or greigite crystals and synthesize an organelle known as magnetosome. This intrinsic ability of MTB allows them to accumulate iron to levels 100-1000 times higher than those in non-magnetotactic bacteria (non-MTB). Therefore, MTB considerably contributes to the global iron cycle as primary iron suppliers in the aquatic environmental food chain. However, to the best of our knowledge, there have been no reports describing the effects of trophic interactions between MTB and their protist grazers on the iron distributions in MTB grazers and the extracellular milieu. Herein, we evaluated the effects of MTB grazing using a model species of protist (Tetrahymena pyriformis) and a model species of MTB (Magnetospirillum magneticum AMB-1). MTB-fed T. pyriformis exhibited a magnetic response and contained magnetite crystals in their vacuoles. Fluorescence imaging using a ferrous ion-specific fluorescent dye revealed that the cellular ferrous ion content was five times higher in MTB-fed T. pyriformis than in non-MTB grazers. Moreover, soluble iron concentrations in the spent media increased with time during MTB predation. This study provides experimental evidence to delineate the importance of trophic interactions of MTB on iron distributions.

Large-Scale Cultivation of Magnetotactic Bacteria and the Optimism for Sustainable and Cheap Approaches in Nanotechnology.

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.

Tuning the Magnetic Response of Magnetospirillum magneticum by Changing the Culture Medium: A Straightforward Approach to Improve Their Hyperthermia Efficiency.

Magnetotactic bacteria Magnetospirillum magneticum AMB-1 have been cultured using three different media: magnetic spirillum growth medium with Wolfe's mineral solution (MSGM + W), magnetic spirillum growth medium without Wolfe's mineral solution (MSGM - W), and flask standard medium (FSM). The influence of the culture medium on the structural, morphological, and magnetic characteristics of the magnetosome chains biosynthesized by these bacteria has been investigated by using transmission electron microscopy, X-ray absorption spectroscopy, and X-ray magnetic circular dichroism. All bacteria exhibit similar average size for magnetosomes, 40-45 nm, but FSM bacteria present slightly longer subchains. In MSGM + W bacteria, Co2+ ions present in the medium substitute Fe2+ ions in octahedral positions with a total Co doping around 4-5%. In addition, the magnetic response of these bacteria has been thoroughly studied as functions of both the temperature and the applied magnetic field. While MSGM - W and FSM bacteria exhibit similar magnetic behavior, in the case of MSGM + W, the incorporation of the Co ions affects the magnetic response, in particular suppressing the Verwey (∼105 K) and low temperature (∼40 K) transitions and increasing the coercivity and remanence. Moreover, simulations based on a Stoner-Wolhfarth model have allowed us to reproduce the experimentally obtained magnetization versus magnetic field loops, revealing clear changes in different anisotropy contributions for these bacteria depending on the employed culture medium. Finally, we have related how these magnetic changes affect their heating efficiency by using AC magnetometric measurements. The obtained AC hysteresis loops, measured with an AC magnetic field amplitude of up to 90 mT and a frequency, f, of 149 kHz, reveal the influence of the culture medium on the heating properties of these bacteria: below 35 mT, MSGM - W bacteria are the best heating mediators, but above 60 mT, FSM and MSGM + W bacteria give the best heating results, reaching a maximum heating efficiency or specific absorption rate (SAR) of SAR/f ≈ 12 W g-1 kHz-1.

Magnetosome-inspired synthesis of soft ferrimagnetic nanoparticles for magnetic tumor targeting.

Magnetic targeting is one of the most promising approaches for improving the targeting efficiency by which magnetic drug carriers are directed using external magnetic fields to reach their targets. As a natural magnetic nanoparticle (MNP) of biological origin, the magnetosome is a special "organelle" formed by biomineralization in magnetotactic bacteria (MTB) and is essential for MTB magnetic navigation to respond to geomagnetic fields. The magnetic targeting of magnetosomes, however, can be hindered by the aggregation and precipitation of magnetosomes in water and biological fluid environments due to the strong magnetic attraction between particles. In this study, we constructed a magnetosome-like nanoreactor by introducing MTB Mms6 protein into a reverse micelle system. MNPs synthesized by thermal decomposition exhibit the same crystal morphology and magnetism (high saturation magnetization and low coercivity) as natural magnetosomes but have a smaller particle size. The DSPE-mPEG-coated magnetosome-like MNPs exhibit good monodispersion, penetrating the lesion area of a tumor mouse model to achieve magnetic enrichment by an order of magnitude more than in the control groups, demonstrating great prospects for biomedical magnetic targeting applications.

Periplasmic Bacterial Biomineralization of Copper Sulfide Nanoparticles.

Metal sulfides are a common group of extracellular bacterial biominerals. However, only a few cases of intracellular biomineralization are reported in this group, mostly limited to greigite (Fe3 S4 ) in magnetotactic bacteria. Here, a previously unknown periplasmic biomineralization of copper sulfide produced by the magnetotactic bacterium Desulfamplus magnetovallimortis strain BW-1, a species known to mineralize greigite (Fe3 S4 ) and magnetite (Fe3 O4 ) in the cytoplasm is reported. BW-1 produces hundreds of spherical nanoparticles, composed of 1-2 nm substructures of a poorly crystalline hexagonal copper sulfide structure that remains in a thermodynamically unstable state. The particles appear to be surrounded by an organic matrix as found from staining and electron microscopy inspection. Differential proteomics suggests that periplasmic proteins, such as a DegP-like protein and a heavy metal-binding protein, could be involved in this biomineralization process. The unexpected periplasmic formation of copper sulfide nanoparticles in BW-1 reveals previously unknown possibilities for intracellular biomineralization that involves intriguing biological control and holds promise for biological metal recovery in times of copper shortage.

In vitro magnetosome remineralization for silver-magnetite hybrid magnetosome biosynthesis and used for healing of the infected wound.

BACKGROUND: Magnetosomes (BMPs) are organelles of magnetotactic bacteria (MTB) that are responsible for mineralizing iron to form magnetite. In addition, BMP is an ideal biomaterial that is widely used in bio- and nano-technological applications, such as drug delivery, tumor detection and therapy, and immunodetection. The use of BMPs to create multifunctional nanocomposites would further expand the range of their applications. RESULTS: In this study, we firstly demonstrate that the extracted BMP can remineralize in vitro when it is exposed to AgNO3 solution, the silver ions (Ag+) were transported into the BMP biomembrane (MM) and mineralized into a silver crystal on one crystal plane of Fe3O4. Resulting in the rapid synthesis of an Ag-Fe3O4 hybrid BMP (BMP-Ag). The synergy between the biomembrane, Fe3O4 crystal, and unmineralized iron enabled the remineralization of BMPs at an Ag+ concentration ≥ 1.0 mg mL-1. The BMP-Ag displayed good biocompatibility and antibacterial activity. At a concentration of 2.0 mg/mL, the BMP-Ag and biomembrane removed Ag-Fe3O4 NPs inhibited the growth of gram-negative and gram-positive bacteria. Thus using BMP-Ag as a wound dressing can effectively enhance the contraction of infected wounds. CONCLUSIONS: This study represents the first successful attempt to remineralize organelles ex vivo, realizing the biosynthesis of hybrid BMP and providing an important advancement in the synthesis technology of multifunctional biological nanocomposites.

Biogeochemical fingerprinting of magnetotactic bacterial magnetite.

Biominerals are important archives of the presence of life and environmental processes in the geological record. However, ascribing a clear biogenic nature to minerals with nanometer-sized dimensions has proven challenging. Identifying hallmark features of biologically controlled mineralization is particularly important for the case of magnetite crystals, resembling those produced by magnetotactic bacteria (MTB), which have been used as evidence of early prokaryotic life on Earth and in meteorites. We show here that magnetite produced by MTB displays a clear coupled C-N signal that is absent in abiogenic and/or biomimetic (protein-mediated) nanometer-sized magnetite. We attribute the presence of this signal to intracrystalline organic components associated with proteins involved in magnetosome formation by MTB. These results demonstrate that we can assign a biogenic origin to nanometer-sized magnetite crystals, and potentially other biominerals of similar dimensions, using unique geochemical signatures directly measured at the nanoscale. This finding is significant for searching for the earliest presence of life in the Earth's geological record and prokaryotic life on other planets.

Magnetic Particle Imaging of Magnetotactic Bacteria as Living Contrast Agents Is Improved by Altering Magnetosome Arrangement.

Superparamagnetic iron oxide nanoparticles (SPIONs) can be used as imaging agents to differentiate between normal and diseased tissue or track cell movement. Magnetic particle imaging (MPI) detects the magnetic properties of SPIONs, providing quantitative and sensitive image data. MPI performance depends on the size, structure, and composition of nanoparticles. Magnetotactic bacteria produce magnetosomes with properties similar to those of synthetic nanoparticles, and these can be modified by mutating biosynthetic genes. The use of Magnetospirillum gryphiswaldense, MSR-1 with a mamJ deletion, containing clustered magnetosomes instead of typical linear chains, resulted in improved MPI signal and resolution. Bioluminescent MSR-1 with the mamJ deletion were administered into tumor-bearing and healthy mice. In vivo bioluminescence imaging revealed the viability of MSR-1, and MPI detected signals in livers and tumors. The development of living contrast agents offers opportunities for imaging and therapy with multimodality imaging guiding development of these agents by tracking the location, viability, and resulting biological effects.

Identification of sulfate-reducing magnetotactic bacteria via a group-specific 16S rDNA primer and correlative fluorescence and electron microscopy: Strategy for culture-independent study.

Magnetotactic bacteria (MTB) biomineralize intracellular magnetic nanocrystals and swim along geomagnetic field lines. While few axenic MTB cultures exist, living cells can be separated magnetically from natural environments for analysis. The bacterial universal 27F/1492R primer pair has been used widely to amplify nearly full-length 16S rRNA genes and to provide phylogenetic portraits of MTB communities. However, incomplete coverage and amplification biases inevitably prevent detection of some phylogenetically specific or non-abundant MTB. Here, we propose a new formulation of the upstream 390F primer that we combined with the downstream 1492R primer to specifically amplify 1100-bp 16S rRNA gene sequences of sulfate-reducing MTB in freshwater sediments from Lake Weiyanghu, Xi'an, northwestern China. With correlative fluorescence in situ hybridization and scanning/transmission electron microscopy, three novel MTB strains (WYHR-2, WYHR-3 and WYHR-4) from the Desulfobacterota phylum were identified phylogenetically and structurally at the single-cell level. Strain WYHR-2 produces bullet-shaped magnetosome magnetite, while the other two strains produce both cubic/prismatic greigite and bullet-shaped magnetite. Our results expand knowledge of bacterial diversity and magnetosome biomineralization of sulfate-reducing MTB. We also propose a general strategy for identifying and characterizing uncultured MTB from natural environments.

Potential and whole-genome sequence-based mechanism of elongated-prismatic magnetite magnetosome formation in Acidithiobacillus ferrooxidans BYM.

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 â„ƒ. 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.

A novel colorimetric technique for estimating iron in magnetosomes of magnetotactic bacteria based on linear regression.

Magnetotactic bacteria (MTB) use iron from their habitat to create magnetosomes, a unique organelle required for magnetotaxis. Due to a lack of cost-effective assay methods for estimating iron in magnetosomes, research on MTB and iron-rich magnetosomes is limited. A systemized assay was established in this study to quantify iron in MTB using ferric citrate colorimetric estimation. With a statistically significant R2 value of 0.9935, the iron concentration range and wavelength for iron estimation were optimized using linear regression. This colorimetric approach and the inductively coupled plasma optical emission spectrometry (ICP-OES) exhibited an excellent correlation R2 value of 0.961 in the validatory correlative study of the iron concentration in the isolated magnetotactic bacterial strains. In large-scale screening studies, this less-expensive strategy could be advantageous.

Defining Local Chemical Conditions in Magnetosomes of Magnetotactic Bacteria.

Defining chemical properties of intracellular organelles is necessary to determine their function(s) as well as understand and mimic the reactions they host. However, the small size of bacterial and archaeal microorganisms often prevents defining local intracellular chemical conditions in a similar way to what has been established for eukaryotic organelles. This work proposes to use magnetite (Fe3O4) nanocrystals contained in magnetosome organelles of magnetotactic bacteria as reporters of elemental composition, pH, and redox potential of a hypothetical environment at the site of formation of intracellular magnetite. This methodology requires combining recent single-cell mass spectrometry measurements together with elemental composition of magnetite in trace and minor elements. It enables a quantitative characterization of chemical disequilibria of 30 chemical elements between the intracellular and external media of magnetotactic bacteria, revealing strong transfers of elements with active influx or efflux processes that translate into elemental accumulation (Mo, Se, and Sn) or depletion (Sr and Bi) in the bacterial internal medium of up to seven orders of magnitude relative to the extracellular medium. Using this concept, we show that chemical conditions in magnetosomes are compatible with a pH of 7.5-9.5 and a redox potential of -0.25 to -0.6 V.

Magnetotactic bacteria: concepts, conundrums, and insights from a novel in situ approach using digital holographic microscopy (DHM).

Magnetotactic bacteria (MTB) are a diverse group of highly motile Gram-negative microorganisms with the common ability to orient along magnetic field lines, a behavior known as magnetotaxis. Ubiquitous in aquatic sediment environments, MTB are often microaerophilic and abundant at the oxic/anoxic interface. Magnetic field sensing is accomplished using intracellular, membrane-encased, iron-containing minerals known as magnetosomes. The chemistry, morphology and arrangement of magnetosomes differs substantially among different MTB. Although magnetic field sensing mechanisms, genetic bases and protein functions have been elucidated in select model organisms such as the Magnetospirillum strains and Desulfovibrio RS-1, not all findings are applicable to diverse clades of MTB. As the number of identified species has increased, it has become evident that many of the characteristics and mechanisms once presumed to be prototypical of MTB are in fact not universal. Here we present a general overview of the current state of MTB research for readers outside of the realm of prokaryotic research, focusing on recent discoveries, knowledge gaps and future directions. In addition, we report new insights acquired using holographic technology to observe and quantify microbial responses in magnetic fields that are earth-strength or weaker, providing a new ecophysiological approach to in situ MTB research.

Magnetotactic advantage in stable sediment by long-term observations of magnetotactic bacteria in Earth's field, zero field and alternating field.

Magnetotactic bacteria (MTB) rely on magnetotaxis to effectively reach their preferred living habitats, whereas experimental investigation of magnetotactic advantage in stable sediment is currently lacking. We studied two wild type MTB (cocci and rod-shaped M. bavaricum) in sedimentary environment under exposure to geomagnetic field in the laboratory, zero field and an alternating field whose polarity was switched every 24 hours. The mean concentration of M. bavaricum dropped by ~50% during 6 months in zero field, with no clear temporal trend suggesting an extinction. Cell numbers recovered to initial values within ~1.5 months after the Earth's field was reset. Cocci displayed a larger temporal variability with no evident population changes in zero field. The alternating field experiment produced a moderate decrease of M. bavaricum concentrations and nearby extinction of cocci, confirming the active role of magnetotaxis in sediment and might point to a different magnetotactic mechanism for M. bavaricum which possibly benefited them to survive field reversals in geological periods. Our findings provide a first quantification of magnetotaxis advantage in sedimentary environment.