Magnetospirillum Gryphiswaldense Strain MSR-1 Research Articles

Electroactivity of the magnetotactic bacteria Magnetospirillum magneticum AMB-1 and Magnetospirillum gryphiswaldense MSR-1

Magnetotactic bacteria reside in sediments and stratified water columns. They are named after their ability to synthesize internal magnetic particles that allow them to align and swim along the Earth’s magnetic field lines. Here, we show that two magnetotactic species, Magnetospirillum magneticum strain AMB-1 and Magnetospirillum gryphiswaldense strain MSR-1, are electroactive. Both M. magneticum and M. gryphiswaldense were able to generate current in microbial fuel cells with maximum power densities of 27 and 11 µW/m2, respectively. In the presence of the electron shuttle resazurin both species were able to reduce the crystalline iron oxide hematite (Fe2O3). In addition, M. magneticum could reduce poorly crystalline iron oxide (FeOOH). Our study adds M. magneticum and M. gryphiswaldense to the growing list of known electroactive bacteria, and implies that electroactivity might be common for bacteria within the Magnetospirillum genus.

Effects of culture conditions on denitrification activity by Magnetospirillum gryphiswaldense strain MSR-1

The application of denitrification function of Magnetospirillum gryphiswaldense strain MSR-1 has not received attention. Here, we studied the effects of different culture conditions, including carbon (C) and nitrogen (N) sources, C/N ratio, iron source species and addition, initial pH, and temperature on the growth, magnetic production, and denitrification activity and application potential of MSR-1 under anoxic and nutrient environments similar to that of urban domestic sewage. Sodium lactate remains the best carbon source for denitrification by MSR-1, although acetic acid is also promising. Nitrate is the best nitrogen source, where addition and the ratio to nitrite should be >3 mM and 1, respectively. At C/N between 3 and 7, initial pH between 6.5 and 7.5, and culture temperature from 25 to 35 °C, nitrate content (84 mg N/L) decreased to the lowest value in 24 h, concomitant with the lowest residual nitrite. A maximum nitrate removal rate of 88.22 % was observed in 16 h by the optimization of response surface methodology with C/N = 5.13, initial pH = 6.83, and culture temperature = 30.3 °C. MSR-1 effectively removed nitrate, with the advantage of a low C/N demand; furthermore, optimal pH and temperature for denitrification are similar to those used in the sewage biological treatment system. Here, the consistency between optimal denitrification conditions of MSR-1 and actual wastewater conditions was considered for the first time, demonstrating the application potential of MSR-1 for wastewater denitrification and providing a theoretical basis for the application of the strain in wastewater treatment.

Progress in affinity ligand-functionalized bacterial magnetosome nanoparticles for bio-immunomagnetic separation of HBsAg protein

Efficient Bio-immunomagnetic separation (BIMS) of recombinant hepatitis B surface antigen (rHBsAg) with high binding capacity was studied using affinity ligand immobilized bacterial magnetosome nanoparticles (Magnetospirillum gryphiswaldense strain MSR-1 bacteria) as an immunomagnetic sorbent. Our results showed immunomagnetic adsorption, acted by affinity interactions with the immobilized monoclonal antibody, offered higher antigen adsorption and desorption capacities as compared with the commercially available immunoaffinity sorbents. Four different ligand densities of the Hep-1 monoclonal antibody were examined during covalent immobilization on Pyridyl Disulfide-functionalized magnetosome nanoparticles for HBsAg immunomagnetic separation. The average of adsorption capacity was measured as 3 mg/ml in optimized immunomagnetic sorbent (1.056 mg rHBsAg/ml immunomagneticsorbent/5.5 mg of total purified protein) and 5mg/ml in immunoaffinity sorbent (0.876 mg rHBsAg/ml immunosorbent/5.5 mg total purified protein during 8 runs. Immunomagnetic sorbent demonstrated ligand leakage levels below 3 ng Mab/Ag rHBsAg during 12 consecutive cycles of immunomagnetic separation (IMS). The results suggest that an immunomagnetic sorbent with a lower ligand density (LD = 3 mg Mab/ml matrix) could be the best substitute for the immunosorbent used in affinity purification of r-HBsAg there are significant differences in the ligand density (98.59% (p-value = 0.0182)), adsorption capacity (97.051% (p-value = 0.01834)), desorption capacity (96.06% (p-value = 0.036)) and recovery (98.97% (p-value = 0.0231)). This study indicates that the immunosorbent approach reduces the cost of purification of Hep-1 protein up to 50% as compared with 5 mg Mab/ml immunoaffinity sorbent, which is currently used in large-scale production. As well, these results demonstrate that bacterial magnetosome nanoparticles (BMs) represent a promising alternative product for the economical and efficient immobilization of proteins and the immunomagnetic separation of Biomolecules, promoting innovation in downstream processing.

Novel Protein Mg2046 Regulates Magnetosome Synthesis in Magnetospirillum gryphiswaldense MSR-1 by Modulating a Proper Redox Status.

Magnetotactic bacteria (MTB) are a large, polyphyletic group of aquatic microorganisms capable of absorbing large amounts of iron and synthesizing intercellular nano-scaled nanoparticles termed magnetosomes. In our previous transcriptomic studies, we discovered that a novel gene (MGMSRv2_2046, termed as mg2046) in Magnetospirillum gryphiswaldense strain MSR-1 was significantly up-regulated during the period of magnetosome synthesis. In the present study, we constructed a MSR-1 mutant strain with deletion of mg2046 (termed Δmg2046) in order to evaluate the role of this gene in cell physiological status and magnetosome formation process. In comparison with wild-type MSR-1, Δmg2046 showed similar cell growth, but much lower cell magnetic response, smaller number and size of magnetosomes, and reduced iron absorption ability. mg2046 deletion evidently disrupted iron uptake, and redox equilibrium, and strongly inhibited transcription of dissimilatory denitrification pathway genes. Our experimental findings, taken together with results of gene homology analysis, indicate that Mg2046 acts as a positive regulator in MSR-1 under microaerobic conditions, responding to hypoxia signals and participating in regulation of oxygen metabolism, in part as a co-regulator of dissimilatory denitrification pathway with oxygen sensor MgFnr (MGMSRv2_2946, termed as Mg2946). Mg2046 is clearly involved in coupled regulation of cellular oxygen, iron and nitrogen metabolism under micro-aerobic or anaerobic conditions. Our findings help explain how MSR-1 cells initiate dissimilatory denitrification pathway and overcome energy deficiency under microaerobic conditions, and have broader implications regarding bacterial survival and energy metabolism strategies under hypoxia.

A Novel Highly Efficient Device for Growing Micro-Aerophilic Microorganisms.

This work describes a novel, simple and cost-effective culture system, named the Micro-Oxygenated Culture Device (MOCD), designed to grow microorganisms under particularly challenging oxygenation conditions. Two microaerophilic magnetotactic bacteria, a freshwater Magnetospirillum gryphiswaldense strain MSR-1 and a marine Magnetospira sp. strain QH-2, were used as biological models to prove the efficiency of the MOCD and to evaluate its specifications. Using the MOCD, growth rates of MSR-1 and QH-2 increased by four and twofold, respectively, when compared to traditional growing techniques using simple bottles. Oxystat-bioreactors have been typically used and specifically designed to control low dissolved oxygen concentrations, however, the MOCD, which is far less sophisticated was proven to be as efficient for both MSR-1 and QH-2 cultures with regard to growth rate, and even better for MSR-1 when looking at cell yield (70% increase). The MOCD enables a wide range of oxygenation conditions to be studied, including different O2-gradients. This makes it an innovative and ingenious culture device that opens up new parameters for growing microaerobic microorganisms.

Effects of Environmental Conditions on High-Yield Magnetosome Production by Magnetospirillum gryphiswaldense MSR-1

Background:Magnetotactic bacteria are a heterogeneous group of Gram-negative prokaryote cells that produce linear chains of magnetic particles called magnetosomes, intracellular organelles composed of magnetic iron particles. Many important applications have been defined for magnetic nanoparticles in biotechnology, such as cell separation applications, as well as acting as carriers of enzymes, antibodies, or anti-cancer drugs. Since the bacterial growth is difficult and the yield of magnetosome production is low, the application of magnetosome has not been developed on a commercial scale.Methods:Magnetospirillum gryphiswaldense strain MSR-1 was used in a modified current culture medium supplemented by different concentrations of oxygen, iron, carbon, and nitrogen, to increase the yield of magnetosomes.Results:Our improved MSR-1 culture medium increased magnetosome yield, magnetosome number per bacterial cell, magnetic response, and bacterial cell growth yield significantly. The yield of magnetosome increased approximately four times. The optimized culture medium containing 25 mM of Na-pyruvate, 40 mM of NaNO3, 200 µM of ferrous sulfate, and 5-10 ppm of dissolved oxygen (DO) resulted in 186.67 mg of magnetosome per liter of culture medium. The iron uptake increased significantly, and the magnetic response of the bacteria to the magnetic field was higher than threefold as compared to the previously reported procedures.Conclusion:This technique not only decreases the cultivation time but also reduces the production cost. In this modified method, the iron and DO are the major factors affecting the production of magnetosome by M. gryphiswaldense strain MSR-1. However, refining this technique will enable a further yield of magnetosome and cell density.

An Investigation of the Sensing Capabilities of Magnetotactic Bacteria.

We investigate the sensing capabilities of magnetotactic bacteria (Magnetospirillum gryphiswaldense strain MSR1) to MCF-7 breast cancer cells. Cancer cells are allowed to grow inside a capillary tube with depth of 200 $\mu \mathrm {m}$ and motion of magnetotactic bacteria is investigated under the influence of oxygen gradient and geomagnetic field. The influence of cancer cells is modeled to predict the oxygen gradient within the capillary tube in three-dimensional space. Our experimental motion analysis and count of motile magnetotactic bacteria indicate that they migrate towards less-oxygenated regions within the vicinity of cancer cells. Bands of magnetotactic bacteria with average concentration of 18.8±2.0% are observed in close proximity to MCF-7 cells $(h = 20~ \mu \mathrm {m})$, whereas the concentration at proximity of $190~ \mu \mathrm {m}$ is 5.0 ± 6.8%.

Flow cytometry as a rapid analytical tool to determine physiological responses to changing O2 and iron concentration by Magnetospirillum gryphiswaldense strain MSR-1

Magnetotactic bacteria (MTB) are a diverse group of bacteria that synthesise magnetosomes, magnetic membrane-bound nanoparticles that have a variety of diagnostic, clinical and biotechnological applications. We present the development of rapid methods using flow cytometry to characterize several aspects of the physiology of the commonly-used MTB Magnetospirillum gryphiswaldense MSR-1. Flow cytometry is an optical technique that rapidly measures characteristics of individual bacteria within a culture, thereby allowing determination of population heterogeneity and also permitting direct analysis of bacteria. Scatter measurements were used to measure and compare bacterial size, shape and morphology. Membrane permeability and polarization were measured using the dyes propidium iodide and bis-(1,3-dibutylbarbituric acid) trimethine oxonol to determine the viability and ‘health’ of bacteria. Dyes were also used to determine changes in concentration of intracellular free iron and polyhydroxylakanoate (PHA), a bacterial energy storage polymer. These tools were then used to characterize the responses of MTB to different O2 concentrations and iron-sufficient or iron-limited growth. Rapid analysis of MTB physiology will allow development of bioprocesses for the production of magnetosomes, and will increase understanding of this fascinating and useful group of bacteria.

Magnetic nanoparticles from Magnetospirillum gryphiswaldense increase the efficacy of thermotherapy in a model of colon carcinoma.

Magnetic nanoparticles (MNPs) are capable of generate heating power under the influence of alternating magnetic fields (AMF); this behaviour recently opened new scenarios for advanced biomedical applications, mainly as new promising tumor therapies. In this paper we have tested magnetic nanoparticles called magnetosomes (MNs): a class of MNPs naturally produced by magnetotactic bacteria. We extracted MNs from Magnetospirillum gryphiswaldense strain MSR-1 and tested the interaction with cellular elements and anti-neoplastic activity both in vitro and in vivo, with the aim of developing new therapeutic approaches for neoplastic diseases. In vitro experiments performed on Human Colon Carcinoma HT-29 cell cultures demonstrated a strong uptake of MNs with no evident signs of cytotoxicity and revealed three phases in the interaction: adherence, transport and accumulation in Golgi vesicles. In vivo studies were performed on subcutaneous tumors in mice; in this model MNs are administered by direct injection in the tumor volume, then a protocol consisting of three exposures to an AMF rated at 187 kHz and 23kA/m is carried out on alternate days, over a week. Tumors were monitored by Magnetic Resonance Imaging (MRI) to obtain information about MNs distribution and possible tissue modifications induced by hyperthermia. Histological analysis showed fibrous and necrotic areas close to MNs injection sites in mice subjected to a complete thermotherapy protocol. These results, although concerning a specific tumor model, could be useful to further investigate the feasibility and efficacy of protocols based on MFH. Magnetic nanoparticles naturally produced and extracted from bacteria seem to be promising candidates for theranostic applications in cancer therapy.

Manganese incorporation into the magnetosome magnetite: magnetic signature of doping

The biomineralization of magnetotactic bacterial magnetite nanoparticles is a topic of intense research due to the particles' narrow size distribution and magnetic properties. Incorporation of foreign metal ions into the crystal matrix of magnetotactic bacterial magnetite has been previously examined by a number of investigators. In this study, cells of a magnetotactic bacterium, Magnetospirillum gryphiswaldense strain MSR-1 were grown in the presence of manganese, ruthe- nium, zinc and vanadium, of which only manganese was incorporated within the magnetosome magnetite crystals. We demonstrate that the magnetic properties of magnetite crystals of magnetotactic bacteria can be significantly altered by the incorporation of metal ions, other than iron, in the crystal structure. The Verwey transition serves as a unique marker to probe the incorporation of the dopant within the magnetosome: manganese incorporation into the magnetite nanocrystals is signaled by a suppression oftheVerwey transition,aswell as by changes in the crystallinestructureand chemicalcomposition ofmagnetosome magnetite.

Surface expression of protein A on magnetosomes and capture of pathogenic bacteria by magnetosome/antibody complexes.

Magnetosomes are membrane-enclosed magnetite nanocrystals synthesized by magnetotactic bacteria (MTB). They display chemical purity, narrow size ranges, and species-specific crystal morphologies. Specific transmembrane proteins are sorted to the magnetosome membrane (MM). MamC is the most abundant MM protein of Magnetospirillum gryphiswaldense strain MSR-1. MamF is the second most abundant MM protein of MSR-1 and forms stable oligomers. We expressed staphylococcal protein A (SPA), an immunoglobulin-binding protein from the cell wall of Staphylococcus aureus, on MSR-1 magnetosomes by fusion with MamC or MamF. The resulting recombinant magnetosomes were capable of self-assembly with the Fc region of mammalian antibodies (Abs) and were therefore useful for functionalization of magnetosomes. Recombinant plasmids pBBR-mamC-spa and pBBR-mamF-spa were constructed by fusing spa (the gene that encodes SPA) with mamC and mamF, respectively. Recombinant magnetosomes with surface expression of SPA were generated by introduction of these fusion genes into wild-type MSR-1 or a mamF mutant strain. Studies with a Zeta Potential Analyzer showed that the recombinant magnetosomes had hydrated radii significantly smaller than those of WT magnetosomes and zeta potentials less than −30 mV, indicating that the magnetosome colloids were relatively stable. Observed conjugation efficiencies were as high as 71.24 μg Ab per mg recombinant magnetosomes, and the conjugated Abs retained most of their activity. Numbers of Vibrio parahaemolyticus (a common pathogenic bacterium in seafood) captured by recombinant magnetosome/Ab complexes were measured by real-time fluorescence-based quantitative PCR. One mg of complex was capable of capturing as many as 1.74 × 107 Vibrio cells. The surface expression system described here will be useful for design of functionalized magnetosomes from MSR-1 and other MTB.

Structure prediction of magnetosome-associated proteins

Magnetotactic bacteria (MTB) are Gram-negative bacteria that can navigate along geomagnetic fields. This ability is a result of a unique intracellular organelle, the magnetosome. These organelles are composed of membrane-enclosed magnetite (Fe3O4) or greigite (Fe3S4) crystals ordered into chains along the cell. Magnetosome formation, assembly, and magnetic nano-crystal biomineralization are controlled by magnetosome-associated proteins (MAPs). Most MAP-encoding genes are located in a conserved genomic region – the magnetosome island (MAI). The MAI appears to be conserved in all MTB that were analyzed so far, although the MAI size and organization differs between species. It was shown that MAI deletion leads to a non-magnetic phenotype, further highlighting its important role in magnetosome formation. Today, about 28 proteins are known to be involved in magnetosome formation, but the structures and functions of most MAPs are unknown. To reveal the structure–function relationship of MAPs we used bioinformatics tools in order to build homology models as a way to understand their possible role in magnetosome formation. Here we present a predicted 3D structural models’ overview for all known Magnetospirillum gryphiswaldense strain MSR-1 MAPs.

Magnetite Biomineralization in Magnetospirillum gryphiswaldense: Time-Resolved Magnetic and Structural Studies

Magnetotactic bacteria biosynthesize magnetite nanoparticles of high structural and chemical purity that allow them to orientate in the geomagnetic field. In this work we have followed the process of biomineralization of these magnetite nanoparticles. We have performed a time-resolved study on magnetotactic bacteria Magnetospirillum gryphiswaldense strain MSR-1. From the combination of magnetic and structural studies by means of Fe K-edge X-ray absorption near edge structure (XANES) and high-resolution transmission electron microscopy we have identified and quantified two phases of Fe (ferrihydrite and magnetite) involved in the biomineralization process, confirming the role of ferrihydrite as the source of Fe ions for magnetite biomineralization in M. gryphiswaldense. We have distinguished two steps in the biomineralization process: the first, in which Fe is accumulated in the form of ferrihydrite, and the second, in which the magnetite is rapidly biomineralized from ferrihydrite. Finally, the XANES analysis suggests that the origin of the ferrihydrite could be at bacterial ferritin cores, characterized by a poorly crystalline structure and high phosphorus content.

Bioengineered bioluminescent magnetotactic bacteria as a powerful tool for chip-based whole-cell biosensors

This paper describes the generation of genetically engineered bioluminescent magnetotactic bacteria (BL-MTB) and their integration into a microfluidic analytical device to create a portable toxicity detection system. Magnetospirillum gryphiswaldense strain MSR-1 was bioengineered to constitutively express a red-emitting click beetle luciferase whose bioluminescent signal is directly proportional to bacterial viability. The magnetic properties of these bacteria have been exploited as "natural actuators" to transfer the cells in the chip from the reaction to the detection area, optimizing the chip's analytical performance. A robust and cost-effective biosensor for the evaluation of sample toxicity, named MAGNETOX, based on lens-free contact imaging detection, has been developed. A microfluidic chip has been fabricated using multilayered black and transparent polydimethyl siloxane (PDMS) in which BL-MTB are incubated for 30 min with the sample, then moved by microfluidics, trapped, and concentrated in detection chambers by an array of neodymium-iron-boron magnets. The chip is placed in contact with a cooled CCD via a fiber optic taper to perform quantitative bioluminescence imaging after addition of luciferin substrate. A model toxic compound (dimethyl sulfoxide, DMSO) and a bile acid (taurochenodeoxycholic acid, TCDCA) were used to investigate the analytical performance of the MAGNETOX. Incubation with DMSO and TCDCA drastically reduces the bioluminescent signal in a dose-related manner. The generation of bacteria that are both magnetic and bioluminescent combines the advantages of easy 2D cell handling with ultra sensitive detection, offering undoubted potential to develop cell-based biosensors integrated into microfluidic chips.

A key time point for cell growth and magnetosome synthesis of Magnetospirillum gryphiswaldense based on real-time analysis of physiological factors

Pure culture of magnetotactic bacteria with high magnetosome yield has been achieved for only a few strains. The major obstacles involve the nutritional requirements and culture conditions of the cells. To increase cell density and magnetosome production, it is necessary to elucidate the physiological characteristics of a particular strain during cell growth and develop an appropriate artificial control strategy. Large-scale culture of Magnetospirillum gryphiswaldense strain MSR-1 was successfully performed for 48 h in a 42-L autofermentor, and several key physiological parameters were measured in real time. Maximal values of cell density (OD565) (19.4) and cell yield (dry weight) (4.76 g/L) were attained at 40 h. The key time point for cell growth and magnetosome formation was found to be 18–20 h. At this point, cells entered the log phase of growth, the maximal values of Cmag (1.78), iron content (0.47%), and magnetosome number (26 ± 3 per cell) were observed, superoxide dismutase (SOD) activity began to decrease more rapidly, ATP content dropped to an extremely low level (0.17 fmol), and reducing power (NADH/NAD+ ratio) began to increase very rapidly. Excessive levels of dissolved oxygen (≥20 ppb) and lactic acid in the medium caused notable cytotoxic effects after 20 h. Artificial control measures for fermentation must be based on realistic cell physiological conditions. At the key time point (18–20 h), cell density is high and magnetosomes have matured. The process of magnetosome synthesis involves a high consumption of ATP and reducing power, and the cells require replenishment of nutrients prior to the 18–20 h time point. Culture conditions that effectively minimize dissolved oxygen accumulation, lactic acid content, and reducing power at this point will enhance magnetosome yield without obvious inhibition of cell growth.

FeoB2 Functions in Magnetosome Formation and Oxidative Stress Protection in Magnetospirillum gryphiswaldense Strain MSR-1

Magnetotactic bacteria (MTB) synthesize unique organelles, the magnetosomes, which are intracellular nanometer-sized, membrane-enveloped magnetite. The biomineralization of magnetosomes involves the uptake of large amounts of iron. However, the iron metabolism of MTB is not well understood. The genome of the magnetotactic bacterium Magnetospirillum gryphiswaldense strain MSR-1 contains two ferrous iron transport genes, feoB1 and feoB2. The FeoB1 protein was reported to be responsible mainly for the transport of ferrous iron and to play an accessory role in magnetosome formation. To determine the role of feoB2, we constructed an feoB2 deletion mutant (MSR-1 ΔfeoB2) and an feoB1 feoB2 double deletion mutant (MSR-1 NfeoB). The single feoB2 mutation did not affect magnetite crystal biomineralization. MSR-1 NfeoB had a significantly lower average magnetosome number per cell (∼65%) than MSR-1 ΔfeoB1, indicating that FeoB2 plays a role in magnetosome formation when the feoB1 gene is deleted. Our findings showed that FeoB1 has a greater ferrous iron transport ability than FeoB2 and revealed the differential roles of FeoB1 and FeoB2 in MSR-1 iron metabolism. Interestingly, compared to the wild type, the feoB mutants showed increased sensitivity to oxidative stress and lower activities of the enzymes superoxide dismutase and catalase, indicating that the FeoB proteins help protect bacterial cells from oxidative stress.

Fluorescence imaging and targeted distribution of bacterial magnetic particles in nude mice

Bacterial magnetic particles (BMPs) are of interest as potential carriers of bioactive macromolecules, drugs, or liposomes. In this study, a high-pressure homogenizer was used to disrupt Magnetospirillum gryphiswaldense strain MSR-1 cells, and BMPs were purified. BMPs were labeled with fluorescence reagent 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocianin perchlorate (DiI) and injected into the tail vein of BALB/c nude mice. Distribution of fluorescence signals of DiI-BMPs in vivo was examined using a whole-body fluorescence imaging system. The result showed that fluorescence signals were detected in liver, stomach, intestine, lungs, and spleen. However, transmission electron microscopy of ultrathin sections indicated that BMPs were mainly present in liver and lungs, but not in the other organs. BMPs could be useful as carriers for targeted drug therapy of diseases of the liver or lung.

Fur in Magnetospirillum gryphiswaldense influences magnetosomes formation and directly regulates the genes involved in iron and oxygen metabolism.

Magnetospirillum gryphiswaldense strain MSR-1 has the unique capability of taking up large amounts of iron and synthesizing magnetosomes (intracellular magnetic particles composed of Fe3O4). The unusual high iron content of MSR-1 makes it a useful model for studying biological mechanisms of iron uptake and homeostasis. The ferric uptake regulator (Fur) protein plays a key role in maintaining iron homeostasis in many bacteria. We identified and characterized a fur-homologous gene (MGR_1314) in MSR-1. MGR_1314 was able to complement a fur mutant of E. coli in iron-responsive manner in vivo. We constructed a fur mutant strain of MSR-1. In comparison to wild-type MSR-1, the mutant strain had lower magnetosome formation, and was more sensitive to hydrogen peroxide and streptonigrin, indicating higher intracellular free iron content. Quantitative real-time RT-PCR and chromatin immunoprecipitation analyses indicated that Fur protein directly regulates expression of several key genes involved in iron transport and oxygen metabolism, in addition it also functions in magnetosome formation in M. gryphiswaldense.

Large-scale production of magnetosomes by chemostat culture of Magnetospirillum gryphiswaldense at high cell density

BackgroundMagnetotactic bacteria have long intrigued researchers because they synthesize intracellular nano-scale (40-100 nm) magnetic particles composed of Fe3O4, termed magnetosomes. Current research focuses on the molecular mechanisms of bacterial magnetosome formation and its practical applications in biotechnology and medicine. Practical applications of magnetosomes are based on their ferrimagnetism, nanoscale size, narrow size distribution, dispersal ability, and membrane-bound structure. However, the applications of magnetosomes have not yet been developed commercially, mainly because magnetotactic bacteria are difficult to cultivate and consistent, high yields of magnetosomes have not yet been achieved.ResultsWe report a chemostat culture technique based on pH-stat feeding that yields a high cell density of Magnetospirillum gryphiswaldense strain MSR-1 in an auto-fermentor. In a large-scale fermentor, the magnetosome yield was significantly increased by adjusting the stirring rate and airflow which regulates the level of dissolved oxygen (DO). Low concentration of sodium lactate (2.3 mmol l-1) in the culture medium resulted in more rapid cell growth and higher magnetosome yield than high concentration of lactate (20 mmol l-1). The optical density of M. gryphiswaldense cells reached 12 OD565 nm after 36 hr culture in a 42 L fermentor. Magnetosome yield and productivity were 83.23 ± 5.36 mg l-1 (dry weight) and 55.49 mg l-1 day-1, respectively, which were 1.99 and 3.32 times higher than the corresponding values in our previous study.ConclusionsCompared to previously reported methods, our culture technique with the MSR-1 strain significantly increased cell density, cell yield, and magnetosome yield in a shorter time window and thus reduced the cost of production. The cell density and magnetosome yield reported here are the highest so far achieved with a magnetotactic bacteria. Refinement of this technique will enable further increase of cell density and magnetosome yield.

MamO and mamE genes are essential for magnetosome crystal biomineralization in Magnetospirillum gryphiswaldense MSR-1

Four non-magnetic mutants of Magnetospirillum gryphiswaldense strain MSR-1 were isolated by transposon mutagenesis and found to contain interruption of either the mamO or mamE gene within the mamAB operon. Studies indicated that mamO and mamE genes are essential for magnetosome crystal biomineralization in MSR-1. This is the first report of a single gene ( mamO or mamE) whose mutation affects crystal biomineralization in MSR-1.