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

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

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.

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

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

Swimming polarity inversion in uncultured magnetotactic cocci.

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.

In situ magnetic identification of giant, needle-shaped magnetofossils in Paleocene-Eocene Thermal Maximum sediments.

Near-shore marine sediments deposited during the Paleocene-Eocene Thermal Maximum at Wilson Lake, NJ, contain abundant conventional and giant magnetofossils. We find that giant, needle-shaped magnetofossils from Wilson Lake produce distinct magnetic signatures in low-noise, high-resolution first-order reversal curve (FORC) measurements. These magnetic measurements on bulk sediment samples identify the presence of giant, needle-shaped magnetofossils. Our results are supported by micromagnetic simulations of giant needle morphologies measured from transmission electron micrographs of magnetic extracts from Wilson Lake sediments. These simulations underscore the single-domain characteristics and the large magnetic coercivity associated with the extreme crystal elongation of giant needles. Giant magnetofossils have so far only been identified in sediments deposited during global hyperthermal events and therefore may serve as magnetic biomarkers of environmental disturbances. Our results show that FORC measurements are a nondestructive method for identifying giant magnetofossil assemblages in bulk sediments, which will help test their ecology and significance with respect to environmental change.

Purification, Characterization, and Assessment of Anticancer Activity of Iron Oxide Nanoparticles Biosynthesized by Novel Thermophilic Bacillus tequilensis ASFS1 .

Magnetic nanoparticles (MNPs), particularly iron oxide nanoparticles (IONPs), are a fascinating group of nanoparticles that have been considerably investigated for biomedical applications because of their superparamagnetic properties, biodegradable nature, and biocompatibility. A novel Gram-positive moderately thermophilic bacterial strain, namely Bacillus tequilensis ASFS.1, was isolated and identified. This strain is capable of producing superparamagnetic Fe3O4 nanoparticles and exhibiting magnetotaxis behavior. This strain swimming behavior was investigated under static and dynamic environments, where it behaved very much similar to the magnetotaxis in magnetotactic bacteria. This study is the first report of a bacterium from the Bacillaceae family that has the potential to intracellular biosynthesis of IONPs. MNPs were separated by a magnetic and reproducible method which was designed for the first time for this study. In addition, UV-visible spectrophotometer, Fourier-transform infrared spectroscopy, vibrating sample magnetometer, field emission scanning electron microscopy (FESEM), X-ray diffraction, and thermal gravimetric analysis were utilized to characterize the bio-fabricated magnetite nanoparticles. Analysis of the particle size distribution pattern of the biogenic MNPs by FESEM imaging revealed the size range of 10-100 nm with the size range of 10-40 nm MNPs being the most frequent particles. VSM analysis demonstrated that biogenic MNPs displayed superparamagnetic properties with a high saturation magnetization value of 184 emu/g. After 24 h treatment of 3T3, U87, A549, MCF-7, and HT-29 cell lines with the biogenic MNPs, IC50 values were measured to be 339, 641, 582, 149, and 184 µg mL-1, respectively. This study presents the novel strain ASFS.1 capable of magnetotaxis by the aid of its magnetite nanoparticles and paving information on isolation, characterization, and in vitro cytotoxicity of its MNPs. The MNPs showed promising potential for biomedical applications, obviously subject to additional studies.

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.

Non-pyrogenic highly pure magnetosomes for efficient hyperthermia treatment of prostate cancer.

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.

External magnetic field have significant effects on diversity of magnetotactic bacteria in sediments from Yangtze River, Chagan Lake and Zhalong Wetland in China.

Magnetotactic bacteria (MTB) can rapidly relocate to optimal habitats by magnetotaxis, and play an important role in iron biogeochemical cycling. This study aimed to evaluate the contribution of the external magnetostatic field to the diversity of MTB in freshwater sediments from Yangtze River (Changjiang River, CJ), Chagan Lake (CGH) and Zhalong Wetland (ZL). The magnetic field intensity was tightly associated with the community richness of MTB in CJ, whereas it was closely related to the diversity of MTB in CGH and ZL (p < 0.05), elucidating a significant variation in the community composition of MTB. Magnetic exposure time appeared more significant correlation with community richness than diversity for MTB in CJ and CGH (p < 0.05), while an opposite relationship existed in ZL (p < 0.01). Herbaspirillum (93.81-96.48 %) dominated in the sediments of these surfacewatesr regardless of waterbody types, while it shifted to Magnetospirillum in ZL under 100 Gs magnetic field. The network connectivity and stability of MTB deteriorate with the increase of magnetic field intensity. Functional analysis showed that the Two-component system and ABC transporter system of MTB obviously responded to magnetic field intensity and exposure time. Our findings will pave the way to understanding the response mechanism of MTB community in freshwater sediments to the external magnetostatic field.

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.

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.

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.

Fundidesulfovibrio magnetotacticus sp. nov., a sulphate-reducing magnetotactic bacterium, isolated from sediments and freshwater of a pond.

A sulphate-reducing magnetotactic bacterium, designated strain FSS-1T, was isolated from sediments and freshwater of Suwa Pond located in Hidaka, Saitama, Japan. Strain FSS-1T was a motile, Gram-negative and curved rod-shaped bacterium that synthesizes bullet-shaped magnetite (Fe3O4) nanoparticles in each cell. Strain FSS-1T was able to grow in the range of pH 6.5-8.0 (optimum, pH 7.0), 22-34 °C (optimum, 28 °C) and with 0-8.0 g l-1 NaCl (optimum, 0-2.0 g l-1 NaCl). Strain FSS-1T grew well in the presence of 50 µM ferric quinate as an iron source. The major fatty acids were anteiso-C15 : 0, iso-C15 : 0 and anteiso-C17 : 0. The major menaquinone was MK-7 (H2). Strain FSS-1T contained desulfoviridin, cytochrome c 3 and catalase, but did not contain oxidase. Strain FSS-1T used fumarate, lactate, pyruvate, malate, formate/acetate, succinate, tartrate, ethanol, 1-propanol, peptone, soytone and yeast extract as electron donors, while the strain used sulphate, thiosulphate and fumarate as electron acceptors. Fumarate was fermented in the absence of electron acceptors. Analysis of the 16S rRNA gene sequence showed that strain FSS-1T is a member of the genus Fundidesulfovibrio. The gene sequence showed 96.7, 95.0, 92.0, 91.2 and 91.4% similarities to the most closely related members of the genera Fundidesulfovibrio putealis B7-43T, Fundidesulfovibrio butyratiphilus BSYT, Desulfolutivibrio sulfoxidireducens DSM 107105T, Desulfolutivibrio sulfodismutans ThAc01T and Solidesulfovibrio magneticus RS-1T, respectively. The DNA G+C content of strain FSS-1T was 67.5 mol%. The average nucleotide identity value between strain FSS-1T and F. putealis B7-43T was 80.7 %. Therefore, strain FSS-1T represents a novel species within the genus Fundidesulfovibrio, for which the name Fundidesulfovibrio magnetotacticus sp. nov. is proposed (=JCM 32405T=DSM 110007T).

Occurrence of south- and north-seeking multicellular magnetotactic prokaryotes in a coastal lagoon in the South Hemisphere.

Magnetotactic bacteria (MTB) response to the magnetic field can be classified into north-seeking (NS) and south-seeking (SS), which usually depends on their inhabiting site in the North and South Hemisphere, respectively. However, uncommon inverted polarity was observed on both hemispheres. Here, we studied magnetotactic multicellular prokaryotes (MMPs) from a coastal lagoon in Brazil collected in April and August 2014. MMPs from the first sampling period presented both magnetotactic behaviors, while MMPs collected in August/2014 were only SS. Phylogenetic analysis based on the 16S rRNA coding gene showed that these organisms belong to the Deltaproteobacteria class. The 16S rRNA gene sequences varied among MMPs regardless of the sampling period, and similarity values were not related to the type of magnetotactic response presented by the microorganisms. Therefore, differences in the magnetotactic behavior might result from the physiological state of MMPs, the availability of resources, or the instability of the chemical gradient in the environment. This is the first report of NS magnetotactic behavior on MMPs from the South Hemisphere.

Production and characterization of naturally occurring antibacterial magnetite nanoparticles from magnetotactic Bacillus sp. MTB17.

AIMS: This study envisaged the isolation and characterization of magnetite nanoparticles (MNPs) from magnetotactic bacteria (MTB) and the evaluation of their antibacterial efficacy. METHODS AND RESULTS: MNPs were extracted from 20 motile but morphologically different MTB, and they were subjected to antibacterial activity assay. These MNPs were found to be highly effective against Vibrio cholerae. MTB17 was considered as the potent MTB strain based on the antibacterial activity. The MNPs of MTB17 were isolated and validated by UV-Visible spectroscopy, particle size analysis, FTIR analysis, and PXRD. CONCLUSIONS: Isolation and characterization of ~85 nm MNPs from MTB is reported, and it is highly active against all the gram-positive and gram-negative strains tested. SIGNIFICANCE AND IMPACT OF THE STUDY: This study focuses on a novel use of biogenic magnetite MNPs as an antibacterial agent, which can be further explored using in vivo studies.

Continuous Production of Biogenic Magnetite Nanoparticles by the Marine Bacterium Magnetovibrio blakemorei Strain MV-1T with a Nitrous Oxide Injection Strategy.

Magnetotactic bacteria (MTB) produce magnetosomes, which are membrane-embedded magnetic nanoparticles. Despite their technological applicability, the production of magnetite magnetosomes depends on the cultivation of MTB, which results in low yields. Thus, strategies for the large-scale cultivation of MTB need to be improved. Here, we describe a new approach for bioreactor cultivation of Magnetovibrio blakemorei strain MV-1T. Firstly, a fed-batch with a supplementation of iron source and N2O injection in 24-h pulses was established. After 120 h of cultivation, the production of magnetite reached 24.5 mg∙L-1. The maximum productivity (16.8 mg∙L-1∙day-1) was reached between 48 and 72 h. However, the productivity and mean number of magnetosomes per cell decreased after 72 h. Therefore, continuous culture in the chemostat was established. In the continuous process, magnetite production and productivity were 27.1 mg∙L-1 and 22.7 mg∙L-1∙day-1, respectively, at 120 h. This new approach prevented a decrease in magnetite production in comparison to the fed-batch strategy.

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

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

Magnetotactic bacteria in vertical sediments of volcanic lakes in NE China appear Alphaproteobacteria dominated distribution regardless of waterbody types.

Magnetotactic bacteria (MTB) distribute widely in sediment habitats and play critical roles in iron cycling. Here, the vertical distribution of morphology and phylogenetic diversity of MTB in sediments (0-15 cm) of three lakes (open waterbody, Bailonghu, BL; semi-enclosed waterbody, Yaoquanhu, YQ; enclosed waterbody, Yueyapao, YY) in Wudalianchi volcanic field (China) were investigated. TEM showed the appearance of coccoid, rod-shaped, oval-shaped, and arc-shaped MTB. With the increase of BL sediment depth, the number of rod-shaped and spherical MTB decreased and increased, respectively. High-throughput sequencing indicated that Alphaproteobacterial MTB dominantly thrived in these lakes regardless of waterbody types. In BL and YY, the dominant genus was Magnetospirillum (44.99-70.80%) which showed a peak in the middle layer. In YQ, the genus Magnetospira was dominant in the upper (52.36%) and middle (66.56%) layer and Magnetococcus (69.63%) existed dominantly in the bottom layer. The vertical distribution of MTB in sediments of these lakes decreased first and then increased. Functional analysis showed that ABC transporter and two-component system of MTB changed significantly with the sediment depth. RDA indicated that the distribution of Magnetospirillum was positively associated with sulfide, pH, and TC. These findings will expand our knowledge of the vertical distribution of MTB in volcanic lakes.

The cation diffusion facilitator protein MamM's cytoplasmic domain exhibits metal-type dependent binding modes and discriminates against Mn2.

Cation diffusion facilitator (CDF) proteins are a conserved family of divalent transition metal cation transporters. CDF proteins are usually composed of two domains: the transmembrane domain, in which the metal cations are transported through, and a regulatory cytoplasmic C-terminal domain (CTD). Each CDF protein transports either one specific metal or multiple metals from the cytoplasm, and it is not known whether the CTD takes an active regulatory role in metal recognition and discrimination during cation transport. Here, the model CDF protein MamM, an iron transporter from magnetotactic bacteria, was used to probe the role of the CTD in metal recognition and selectivity. Using a combination of biophysical and structural approaches, the binding of different metals to MamM CTD was characterized. Results reveal that different metals bind distinctively to MamM CTD in terms of their binding sites, thermodynamics, and binding-dependent conformations, both in crystal form and in solution, which suggests a varying level of functional discrimination between CDF domains. Furthermore, these results provide the first direct evidence that CDF CTDs play a role in metal selectivity. We demonstrate that MamM's CTD can discriminate against Mn2+, supporting its postulated role in preventing magnetite formation poisoning in magnetotactic bacteria via Mn2+ incorporation.

A Novel Magnetotactic Alphaproteobacterium Producing Intracellular Magnetite and Calcium-Bearing Minerals.

Magnetotactic bacteria (MTB) are prokaryotes that form intracellular magnetite (Fe3O4) or greigite (Fe3S4) nanocrystals with tailored sizes, often in chain configurations. Such magnetic particles are each surrounded by a lipid bilayer membrane, called a magnetosome, and provide a model system for studying the formation and function of specialized internal structures in prokaryotes. Using fluorescence-coupled scanning electron microscopy, we identified a novel magnetotactic spirillum, XQGS-1, from freshwater Xingqinggong Lake, Xi'an City, Shaanxi Province, China. Phylogenetic analyses based on 16S rRNA gene sequences indicate that strain XQGS-1 represents a novel genus of the Alphaproteobacteria class in the Proteobacteria phylum. Transmission electron microscopy analyses reveal that strain XQGS-1 forms on average 17 ± 3 magnetite magnetosome particles with an ideal truncated octahedral morphology, with an average length and width of 88.3 ± 11.7 nm and 83.3 ± 11.0 nm, respectively. They are tightly organized into a single chain along the cell long axis close to the concave side of the cell. Intrachain magnetic interactions likely result in these large equidimensional magnetite crystals behaving as magnetically stable single-domain particles that enable bacterial magnetotaxis. Combined structural and chemical analyses demonstrate that XQGS-1 cells also biomineralize intracellular amorphous calcium phosphate (2 to 3 granules per cell; 90.5- ± 19.3-nm average size) and weakly crystalline calcium carbonate (2 to 3 granules per cell; 100.4- ± 21.4-nm average size) in addition to magnetite. Our results expand the taxonomic diversity of MTB and provide evidence for intracellular calcium phosphate biomineralization in MTB. IMPORTANCE Biomineralization is a widespread process in eukaryotes that form shells, teeth, or bones. It also occurs commonly in prokaryotes, resulting in more than 60 known minerals formed by different bacteria under wide-ranging conditions. Among them, magnetotactic bacteria (MTB) are remarkable because they might represent the earliest organisms that biomineralize intracellular magnetic iron minerals (i.e., magnetite [Fe3O4] or greigite [Fe3S4]). Here, we report a novel magnetotactic spirillum (XQGS-1) that is phylogenetically affiliated with the Alphaproteobacteria class. In addition to magnetite crystals, XQGS-1 cells form intracellular submicrometer calcium carbonate and calcium phosphate granules. This finding supports the view that MTB are also an important microbial group for intracellular calcium carbonate and calcium phosphate biomineralization.