Jinhua Li
email: lijinhua@mail.iggcas.ac.cn

Nicolas Menguy

Eric Leroy

Andrew P Roberts

Peiyu Liu

Yongxin Pan

Biomineralization and magnetism of uncultured magnetotactic coccus strain THC-1 with non-chained magnetosomal magnetic nanoparticles

Keywords: Magnetotactic bacteria, Non-chain magnetosome, Biomineralization, Magnetic properties, THC-1, Magnetofossil identification

Key Points

• Magnetotactic bacteria (MTB) produce chain-organized, single-domain magnetite with distinctive magnetic properties.

• We reported unusual arrangement and magnetic properties of non-chained magnetite in uncultured magnetotactic coccus THC-1.

• Combing magnetic and electron microscopic analyses are necessary to identify magnetofossils from the geological record.

records (magnetofossils) are ideal magnetic carriers and are used to reconstruct paleomagnetic and paleoenvironmental information. Here, we studied the biomineralization and magnetic properties of magnetosomal magnetite produced by uncultured magnetotactic coccus strain THC-1 by combining transmission electron microscope (TEM) and magnetic approaches. High-resolution TEM and TEM tomography observations reveal that THC-1 produces elongated hexagonal prismatic magnetite that is elongated along the [111] crystallographic direction. Most of the magnetite within THC-1 cells has roughly uniform crystal orientations, but is dispersed without obvious chain assembly. Whole-cell THC-1 samples have pot-bellied hysteresis loops and a Verwey transition temperature of ~112 K. In contrast to MTB cells with magnetosome chain(s), THC-1 cells have a teardrop first-order reversal curve distribution that is indicative of

Plain Language Summary

Magnetotactic bacteria (MTB) are intriguing because they produce magnetic nanocrystals of magnetite (Fe3O4) or greigite (Fe3S4) (magnetosomes) within their cells and form magnetic nanofossils (magnetofossils) within geological records. The identification of magnetofosssils for paleoenvironmental and paleomagnetic purposes is heavily based on our knowledge from some modern MTB which often organize magnetosomes into chain(s).

We studied here the biomineralization and magnetism of uncultured magnetotactic coccus strain THC-1 in which magnetosomal magnetite particles are dispersed without an obvious chain assembly. In contrast to whole-cell MTB samples with magnetosome chain(s), THC-1 has distinctive magnetic properties such as teardrop FORC distribution and enhanced Verwey transition behavior. Together with previous studies, our results indicate that MTB magnetite has distinctive magnetic properties but not necessarily unique compared to other magnetite types. This indicates that combining bulk magnetic measurements and electron microscopic observations remains necessary to identify magnetofossils from the geological record.

Introduction

Magnetotactic bacteria (MTB) are morphologically and phylogenetically diverse prokaryotes that biomineralize iron minerals and swim along geomagnetic field lines, a behavior known as magnetotaxis [START_REF] Bazylinski | Magnetosome formation in prokaryotes[END_REF]. Since their independent discovery by Bellini in the 1960s [START_REF] Bellini | On a unique behavior of freshwater bacteria[END_REF] and Blakemore in the 1970s [START_REF] Blakemore | Magnetotactic bacteria[END_REF], MTB have attracted extensive interest from biology to geology (e.g., [START_REF] Amor | Ironbiomineralizing organelle in magnetotactic bacteria: function, synthesis and preservation in ancient rock samples[END_REF][START_REF] Bazylinski | Magnetosome formation in prokaryotes[END_REF][START_REF] Kopp | The identification and biogeochemical interpretation of fossil magnetotactic bacteria[END_REF], and even in materials science and biomedical nanotechnology (e.g., Faivre & Schüler, 2008;[START_REF] Prozorov | Novel magnetic nanomaterials inspired by magnetotactic bacteria: topical review[END_REF].

MTB have been studied widely for several reasons in geological applications. First, MTB provide an ideal model system for studying biologically controlled mineralization (BCM) because they produce intracellular, membrane-enveloped nanocrystals of magnetite (Fe3O4) or greigite (Fe3S4) within magnetosomes that are generally arranged into chain structures (e.g., [START_REF] Bazylinski | Biologically controlled mineralization in prokaryotes[END_REF][START_REF] Uebe | Magnetosome biogenesis in magnetotactic bacteria[END_REF]. Second, MTB have also been studied widely to understand how organisms sense geomagnetic fields and how magnetotaxis or the magnetoreception system evolves within prokaryotes and eukaryotes over geological time (e.g., [START_REF] Kirschvink | Magnetite-based magnetoreception[END_REF][START_REF] Leão | Magnetosome magnetite biomineralization in a flagellated protist: evidence for an early evolutionary origin for magnetoreception in eukaryotes[END_REF][START_REF] Lin | On the origin of microbial magnetoreception[END_REF][START_REF] Monteil | Magnetoreception in Microorganisms[END_REF][START_REF] Pósfai | Magnetic nanocrystals in organisms[END_REF]. Third, MTB are important functional microbiota in driving biogeochemical cycling of elements that are essential for life (e.g., C, N, S, P, and Fe) due to the fact that they are distributed widely within the oxic-anoxic interface (OAI) of freshwater, brackish, and marine sediments and chemically stratified water columns (e.g., Lefèvre & Bazylinski, 2013;[START_REF] Lin | Diversity and ecology of and biomineralization by magnetotactic bacteria[END_REF], as indicated by the general presence of sulfur globules or phosphate inclusions in addition to magnetosomes (e.g., Bazylinski et al., 2004;[START_REF] Cox | Organization and elemental analysis of P-, S-, and Fe-rich inclusions in a population of freshwater magnetococci[END_REF][START_REF] Li | Crystal growth of bullet-shaped magnetite in magnetotactic bacteria of the Nitrospirae phylum[END_REF][START_REF] Li | Phylogenetic and structural identification of a novel magnetotactic Deltaproteobacterium strain WYHR-1 from a freshwater lake[END_REF][START_REF] Rivas-Lamelo | Magnetotactic bacteria as a new model for P sequestration in the ferruginous Lake Pavin[END_REF][START_REF] Schulz-Vogt | Effect of large magnetotactic bacteria with polyphosphate inclusions on the phosphate profile of the suboxic zone in the Black Sea[END_REF]. Finally, the remains of MTB within sediments or sedimentary rocks (i.e., magnetofossils) are ideal carriers of paleomagnetic signals because of their magnetic stable single domain (SD) properties and of potentially important paleoenvironmental information because of their unique crystal morphologies and chain assembly (e.g., [START_REF] Chang | Coupled microbial bloom and oxygenation decline recorded by magnetofossils during the Palaeocene-Eocene Thermal Maximum[END_REF][START_REF] He | Magnetofossil abundance and diversity as paleoenvironmental proxies: a case study from southwest Iberian margin sediments[END_REF][START_REF] Hesse | Evidence for bacterial paleoecological origin of mineral magnetic cycles in oxic and sub-oxic Tasman Sea sediments[END_REF][START_REF] Kirschvink | Ultrafine-grained magnetite in deep-sea sediments: possible bacterial magnetofossils[END_REF][START_REF] Kopp | The identification and biogeochemical interpretation of fossil magnetotactic bacteria[END_REF][START_REF] Larrasoaña | Paleomagnetic and paleoenvironmental implications of magnetofossil occurrences in late Miocene marine sediments from the Guadalquivir Basin, SW Spain[END_REF][START_REF] Petersen | Fossil bacterial magnetite in deep-sea sediments from the South Atlantic Ocean[END_REF][START_REF] Roberts | Searching for single domain magnetite in the "pseudo-single-domain" sedimentary haystack: implications of biogenic magnetite preservation for sediment magnetism and relative paleointensity determinations[END_REF][START_REF] Tarduno | Biogeochemical remanent magnetization in pelagic sediments of the western equatorial Pacific Ocean[END_REF][START_REF] Yamazaki | Organic carbon flux controls the morphology of magnetofossils in marine sediments[END_REF].

To study the biomineralization and magnetism of magnetic particles produced by modern MTB can provide mineralogical and physical evidence to better understand the process and mechanisms of BCM and magnetotaxis in the MTB system (e.g., [START_REF] Li | The link between biomineralization and fossilization of bacteria: Insights from field and experimental studies[END_REF][START_REF] Pósfai | Magnetic nanocrystals in organisms[END_REF]. Such studies are also crucial for developing effective criteria or approaches to identify magnetofossils from sediments or rocks (e.g., [START_REF] Amor | Ironbiomineralizing organelle in magnetotactic bacteria: function, synthesis and preservation in ancient rock samples[END_REF][START_REF] Jimenez-Lopez | Magnetite as a prokaryotic biomarker: a review[END_REF][START_REF] Kopp | The identification and biogeochemical interpretation of fossil magnetotactic bacteria[END_REF][START_REF] Roberts | Magnetic properties of pelagic marine carbonates[END_REF]. Despite discovery of huge MTB diversity over the past four decades [START_REF] Kolinko | Singlecell genomics of uncultivated deep-branching magnetotactic bacteria reveals a conserved set of magnetosome genes[END_REF]Lefèvre & Bazylinski, 2013;[START_REF] Lin | Genomic expansion of magnetotactic bacteria reveals an early common origin of magnetotaxis with lineage-specific evolution[END_REF][START_REF] Liu | Speciesspecific magnetosome biomineralization as a foundation for environmental interpretation of ancient magnetofossils[END_REF], in-depth studies of the biomineralization and magnetism of magnetosomal magnetic particles have only been carried out for several bacterial strains grown in axenic culture (e.g., [START_REF] Chariaou | Anisotropy of bullet-shaped magnetite nanoparticles in the magnetotactic bacteria Desulfovibrio magneticus sp. strain RS-1[END_REF][START_REF] Ding | Deletion of the ftsZ-like gene results in the production of superparamagnetic magnetite magnetosomes in Magnetospirillum gryphiswaldense[END_REF][START_REF] Gehring | The detection of magnetotactic bacteria and magnetofossils by means of magnetic anisotropy[END_REF][START_REF] Kobayashi | Experimental observation of magnetosome chain collapse in magnetotactic bacteria: sedimentological, paleomagnetic, and evolutionary implications[END_REF][START_REF] Kopp | Chains, clumps, and strings: magnetofossil taphonomy with ferromagnetic resonance spectroscopy[END_REF][START_REF] Li | A strong angular dependence of magnetic properties of magnetosome chains: Implications for rock magnetism and paleomagnetism[END_REF][START_REF] Li | Magnetite magnetosome and fragmental chain formation of Magnetospirillum magneticum AMB-1: transmission electron microscopy and magnetic observations[END_REF]Li et al., 2012;[START_REF] Moskowitz | Rock magnetic criteria for the detection of biogenic magnetite[END_REF][START_REF] Moskowitz | A comparison of magnetite particles produced anaerobically by magnetotactic and dissimilatory iron-reducing bacteria[END_REF][START_REF] Moskowitz | Magnetic properties of magnetotactic bacteria[END_REF][START_REF] Pósfai | Properties of intracellular magnetite crystals produced by Desulfovibrio magneticus strain RS-1[END_REF], or for a few uncultured but phylogenetically identified strains that were separated directly from laboratory microcosms of natural sediments [START_REF] Li | Biomineralization, crystallography and magnetic properties of bullet-shaped magnetite magnetosomes in giant rod magnetotactic bacteria[END_REF]Lin & Pan, 2009;Pan, Petersen, Winklhofer, et al., 2005). Based on morphological, crystallographic, magnetic, and chemical evidence, criteria or approaches have been proposed for identifying magnetofossils from sediments or rocks (e.g., [START_REF] Amor | Chemical signature of magnetotactic bacteria[END_REF][START_REF] Arató | Crystal-size and shape distributions of magnetite from uncultured magnetotactic bacteria as a potential biomarker[END_REF][START_REF] Chang | Discrimination of biogenic and detrital magnetite through a double Verwey transition temperature[END_REF][START_REF] Egli | Characterization of individual rock magnetic components by analysis of remanence curves. 3. Bacterial magnetite and natural processes in lakes[END_REF][START_REF] Egli | Detection of noninteracting single domain particles using first-order reversal curve diagrams[END_REF][START_REF] Gehring | S-band ferromagnetic resonance spectroscopy and the detection of magnetofossils[END_REF][START_REF] Gehring | The detection of magnetotactic bacteria and magnetofossils by means of magnetic anisotropy[END_REF][START_REF] Kind | Combined use of magnetometry and spectroscopy for identifying magnetofossils in sediments[END_REF]Li et al., 2012;[START_REF] Moskowitz | Rock magnetic criteria for the detection of biogenic magnetite[END_REF][START_REF] Thomas-Keprta | Elongated prismatic magnetite crystals in ALH84001 carbonate globules: potential Martian magnetofossils[END_REF]. Although widely used, some of these criteria or approaches need to be reassessed because the crystal morphologies and arrangements of magnetosomal magnetic particles within modern MTB are diverse (e.g., [START_REF] Li | The link between biomineralization and fossilization of bacteria: Insights from field and experimental studies[END_REF][START_REF] Li | Bulletshaped magnetite biomineralization within a magnetotactic Deltaproteobacterium: implications for magnetofossil identification[END_REF][START_REF] Liu | Speciesspecific magnetosome biomineralization as a foundation for environmental interpretation of ancient magnetofossils[END_REF][START_REF] Pósfai | Phylogenetic significance of composition and crystal morphology of magnetosome minerals[END_REF], and spatial arrangements and/or chemical compositions of magnetofossils within the geological record may be altered during burial (e.g., [START_REF] Kopp | The identification and biogeochemical interpretation of fossil magnetotactic bacteria[END_REF][START_REF] Li | The link between biomineralization and fossilization of bacteria: Insights from field and experimental studies[END_REF]Li et al., 2012;[START_REF] Roberts | Magnetic properties of pelagic marine carbonates[END_REF][START_REF] Rodelli | Diagenetic fate of biogenic soft and hard magnetite in chemically stratified sedimentary environments of Mamanguá Ría, Brazil[END_REF][START_REF] Tarduno | Biogeochemical remanent magnetization in pelagic sediments of the western equatorial Pacific Ocean[END_REF]. Documenting the diversity, biomineralization, and magnetism of magnetosomal magnetic nanoparticles within modern MTB is, therefore, crucial for understanding the magnetofossil record.

Here, by combining advanced transmission electron microscope (TEM) and rock magnetic approaches, we studied systematically the biomineralization and magnetism of uncultured magnetotactic coccus strain THC-1 in which magnetosomal magnetite particles are not arranged into a chain structure but are dispersed. Strain THC-1 was recently discovered in sediments from Tanghe River, Qinghuangdao city, China [START_REF] Liu | Speciesspecific magnetosome biomineralization as a foundation for environmental interpretation of ancient magnetofossils[END_REF].

Phylogenetically, it is affiliated with the Etaproteobacteria class of the Proteobacteria phylum, with ~99% sequence similarity in 16S rRNA genes to Magnetococcus clone CF22 from a freshwater habitat in Northern Germany [START_REF] Flies | Combined approach for characterization of uncultivated magnetotactic bacteria from various aquatic environments[END_REF]. We compare here the crystal morphologies and spatial arrangements, and the corresponding magnetic properties of magnetosomal magnetite from strain THC-1 and other diverse modern MTB and discuss the potential for precise magnetofossil identification from ancient sediments or rocks.

Materials and methods

Sample preparation

Water and sediment samples were collected from Tanghe River, Qinhuangdao city, eastern China. Tanghe River flows through the city into the Bohai Sea. The sampling site is located in the urban river section (39°55′43″N, 119°33′13″E) in a freshwater environment with salinity of 0.31 ppt, pH of 7.99, and 22°C temperature during sampling (September 2015). Surface sediments were collected near the river bank at water depths of ~3-5 m and transferred to 1-l plastic bottles with ~2:1 sediment:water ratio. The bottles were shipped to the laboratory and stored at room temperature in dim light to set up microcosms. MTB in the microcosms were routinely checked with the hanging-drop technique using an Olympus microscope BX51 equipped with phase-contrast fluorescence and a DP70 digital camera system (Olympus Corp., Tokyo, Japan). The microcosms that contained dominant THC-1 were selected for cell collection using a homemade magnetic separation apparatus. To enrich the sample for subsequent microscopic and magnetic measurements, ~500 ml of slurry from the microcosms was used to magnetically separate the THC-1 cells. The collected THC-1 cells were washed three times with Milli-Q water, resuspended in ~500 µl of Milli-Q water, and then prepared for microscopic and magnetic experiments. About 10 µl of the concentrated THC-1 suspension was deposited onto carbon-coated copper grids for TEM observations. The rest of the THC-1 cells were loaded into a non-magnetic gelatin capsule by centrifugation (10000 rpm), and dried at room temperature for 24 h within an anaerobic glove box for magnetic measurements. TEM and magnetic samples were stored under pure nitrogen at -20°C in a refrigerator prior to measurements.

Transmission electron microscopy

Conventional TEM observations were performed on a JEM2100 microscope (JEOL Ltd., Tokyo, Japan) operating at 200 kV at the Institute of Geology and Geophysics, Chinese Academy of Sciences (IGGCAS, Beijing, China). High-resolution TEM (HRTEM) and scanning TEM (STEM) observations in high-angle annular dark field (HAADF) mode were carried out on a JEM-2100F microscope (JEOL Ltd., Tokyo, Japan) at 200 kV at the Institut de Minéralogie, de Physique des Matériaux et de Cosmochimie (IMPMC, Paris, France). Microchemical analyses were made by energy dispersive X-ray spectrometry (EDX) elemental mapping in HAADF-STEM mode. Crystal lengths (along the long axis) and widths (perpendicular to the long axis) were measured from TEM images. Crystal habits of magnetosomal magnetite particles were determined by a combination of Fourier analyses of HRTEM images for individual particles and crystallographic investigations, as previously described (Faivre et al., 2008;[START_REF] Li | Crystal growth of bullet-shaped magnetite in magnetotactic bacteria of the Nitrospirae phylum[END_REF]. Idealized shapes were modeled using the KrystalShaper software package. HAADF-STEM tomography was carried out on a FEI Tecnai F20 microscope at 200 kV at the Institut de Chimie et des Matériaux de Paris Est (ICPME, Thiais, France). The tomographic tilt series was acquired at 1° tilt increments to a maximum tilt angular range of ±70° using the DigitalMicrographTM (Gatan) STEM tomography module. Tomography alignment, reconstruction, and visualization were performed using the Gatan 3D Reconstruction and Visualization software.

Rock magnetic measurements

Low-temperature magnetic measurements were made with a Quantum Design Magnetic Property Measurement System (MPMS XP-5) with 5.0 × 10 -10 Am 2 sensitivity and a residual magnetic field less than 0.2 mT. Zero-field-cooled (ZFC) and field-cooled (FC) curves were obtained by cooling the sample from 300 to 10 K in zero magnetic field and in a 2.5-T field, respectively, followed by imparting a saturation isothermal remanent magnetization (SIRM) to the sample in a 2.5-T field at 10 K (SIRM10K_2.5T), and then by measuring SIRM10K_2.5T during warming back to 300 K in zero field. The thermal dependence of SIRM, obtained by saturating the sample in a 2.5-T field at 300 K (SIRM300K_2.5T), was measured in zero field during a cooling-warming cycle (300 → 10 → 300 K). The Verwey transition temperature (Tv) is the temperature at which the first derivative of the FC curve reaches a maximum value. The δ-ratio is defined by δFC/δZFC, where δFC and δZFC are calculated as δ = (M80K -M150K)/M80K, where M80K and M150K are the remanences measured at 80 and 150 K, respectively [START_REF] Moskowitz | Rock magnetic criteria for the detection of biogenic magnetite[END_REF].

Room-temperature magnetic experiments were carried out using an alternating gradient magnetometer (AGM) Micromag 2900 (Princeton Measurement Corporation, United States) with 1.0 × 10 -11 Am 2 sensitivity. A hysteresis loop was measured in a 1 T maximum field with 200 ms averaging time. The saturation magnetization (Ms), saturation remanence (Mrs), and coercivity (Bc), were determined after applying a high-field (0.7-1 T) slope correction for high-field linear contributions. Static IRM and DC demagnetization curves were measured to 0.5 T in 0.5 mT increments on a demagnetized sample. The coercivity of remanence (Bcr) was determined from the DC demagnetization curve. To simplify comparison of remanence results and to calculate the R-value of the Wohlfarth-Cisowski test [START_REF] Cisowski | Interacting vs. non-interacting single domain behavior in natural and synthetic samples[END_REF], the IRM acquisition curve was normalized to the SIRM, and the backfield demagnetization curve was rescaled as ½(1+IRM(-H)/SIRM).

First-order reversal curve (FORC) measurements (Pike et al., 1999;[START_REF] Roberts | First-order reversal curve diagrams: a new tool for characterizing the magnetic properties of natural samples[END_REF] were also made at room temperature using the Micromag 2900 AGM. One hundred FORCs were measured using a 500-mT saturating field and 200-ms averaging time. The FORC diagram was calculated using the FORCinel v3.06 software (Harrison and Feinberg, 2008). The horizontal (Bc) and vertical (Bi) axes indicate the microcoercivity and interaction field distribution for SD particles, respectively (Pike et al., 1999;[START_REF] Roberts | First-order reversal curve diagrams: a new tool for characterizing the magnetic properties of natural samples[END_REF]. The characteristic FORC coercivity (Bc,CFORC) and peak FORC coercivity (Bc,PFORC) are given by the coercivity value where the Bc distribution reaches its maximum on the marginal coercivity distribution and the horizontal profile across Bi = 0 mT, respectively [START_REF] Egli | Theoretical aspects of dipolar interactions and their appearance in firstorder reversal curves of thermally activated single-domain particles[END_REF]Pike et al., 1999;[START_REF] Winklhofer | Extracting the intrinsic switching field distribution in perpendicular media: a comparative analysis[END_REF]. The half-width interaction field (Bi,1/2) is defined as the interaction field value where the broadest Bi distribution reduces to half of its maximum [START_REF] Egli | Detection of noninteracting single domain particles using first-order reversal curve diagrams[END_REF]Li et al., 2012;Pike et al., 1999).

Results

Morphological and chemical features of THC-1

TEM observations confirm that our sample contains dominantly THC-1 cells (>~90%) (Fig. 1B). As previously described [START_REF] Liu | Speciesspecific magnetosome biomineralization as a foundation for environmental interpretation of ancient magnetofossils[END_REF], THC-1 cells are coccoid in shape with 2.5 ± 0.2 µm average diameter (n = 54). They contain two flagella bundles, with on average 35 ± 15 magnetite particles per cell (n = 54) (Fig. 1A-C). The magnetite particles are elongated prisms with 70.6 ± 8.6 nm average length and 42.3 ± 5.2 nm average width (n = 551). They are dispersed and not organized into linear chains (Fig. 1D,1E). As expected, both the crystal length and width distributions are skewed negatively, with nearly constant width/length ratio (Fig. 1F-H). This indicates that magnetite particles within THC-1 grew homothetically within the magnetosome membrane, which is typical for crystal growth of prismatic and cubo-octahedral magnetosomal magnetite [START_REF] Isambert | Transmission electron microscopy study of magnetites in a freshwater population of magnetotactic bacteria[END_REF][START_REF] Jandacka | Size distributions of nanoparticles from magnetotactic bacteria as signatures of biologically controlled mineralization[END_REF][START_REF] Li | Magnetite magnetosome and fragmental chain formation of Magnetospirillum magneticum AMB-1: transmission electron microscopy and magnetic observations[END_REF][START_REF] Li | Single-cell resolution of uncultured magnetotactic bacteria via fluorescence-coupled electron microscopy[END_REF][START_REF] Zhang | Magnetotactic coccus strain SHHC-1 affiliated to Alphaproteobacteria forms octahedral magnetite magnetosomes[END_REF].

Further TEM observations and EDX analyses in HAADF-STEM mode reveal diverse inclusions within THC-1 cells (Figs. 1, 2, S1, S2). Besides magnetite particles, three kinds of intracellular inclusions are recognized from morphological and chemical features. These include sulfur globules, which are electron-dense granules with sizes that generally range from ~200 to ~1000 nm (Fig. 1C, Fig. S1). Calcium-rich polyphosphate inclusions have irregular shapes and sizes generally between ~200 and ~500 nm (Fig. 1D, Fig. S2); some have spherical shapes with sizes generally ranging from ~ 500 to ~1000 nm. In contrast to the sulfur globules and polyphosphate inclusions, they are electron-transparent and obviously lack cellular matter (e.g., C, N, O, P, and S) (Figs. 2,S1,S2). This indicates that they are vacuoles that may contain inorganic matter rather than lipids or sugars (Li et al., 2020b). Notably, both magnetosomes and polyphosphate inclusions appear to occur in all THC-1 cells (Figs. S1, S2), while sulfur globules and vacuoles are not always present (Figs.

1B, 1C, S2). Some THC-1 cells contain all four inclusion types (Fig. 2). Sulfur globules and/or polyphosphate inclusions are found widely in MTB along with magnetosomes (e.g., Bazylinski et al., 2004;[START_REF] Cox | Organization and elemental analysis of P-, S-, and Fe-rich inclusions in a population of freshwater magnetococci[END_REF][START_REF] Li | Crystal growth of bullet-shaped magnetite in magnetotactic bacteria of the Nitrospirae phylum[END_REF][START_REF] Li | Phylogenetic and structural identification of a novel magnetotactic Deltaproteobacterium strain WYHR-1 from a freshwater lake[END_REF][START_REF] Rivas-Lamelo | Magnetotactic bacteria as a new model for P sequestration in the ferruginous Lake Pavin[END_REF][START_REF] Schulz-Vogt | Effect of large magnetotactic bacteria with polyphosphate inclusions on the phosphate profile of the suboxic zone in the Black Sea[END_REF], which indicates that MTB may serve as an efficient bacterial shuttle for C, N, S, and Fe across aquatic OAI environments and, therefore, contribute significantly to their global biogeochemical cycling [START_REF] Li | Magnetotaxis as an adaptation to enable bacterial shuttling of microbial sulfur and sulfur cycling across aquatic oxic-anoxic interfaces[END_REF][START_REF] Rivas-Lamelo | Magnetotactic bacteria as a new model for P sequestration in the ferruginous Lake Pavin[END_REF][START_REF] Schulz-Vogt | Effect of large magnetotactic bacteria with polyphosphate inclusions on the phosphate profile of the suboxic zone in the Black Sea[END_REF]. EDX spectra extracted from the carbon film covering the TEM grid (green dashed circle in Fig. 2A), cell wall (blue dashed circle in Fig. 2A), vacuole (red dashed circle in Fig. 2A), sulfur globule (cyan dashed circle in Fig. 2A), polyphosphate inclusion (yellow dashed circle in Fig. 2B), and magnetite particles (purple dashed circle in Fig. 2B). Notes, the Si and Cu signals on the EDX spectral originates from the TEM grid.

Spatial arrangement and crystal habits of magnetosomal magnetite in THC-1

The special arrangement of magnetosomal magnetite particles within THC-1 cells and their morphology were investigated by HAADF-STEM tomography (Fig. 3). Threedimensional visualization demonstrates that magnetite in THC-1 occurs as elongated hexagonal prisms that are terminated by two large crystal faces at the bottom and top, with angles truncated by several small crystal faces. In contrast to linear-chain or linear-bundle assembling of magnetosome chains within most MTB (e.g., [START_REF] Kobayashi | Experimental observation of magnetosome chain collapse in magnetotactic bacteria: sedimentological, paleomagnetic, and evolutionary implications[END_REF]Li et al., 2015;[START_REF] Li | Bulletshaped magnetite biomineralization within a magnetotactic Deltaproteobacterium: implications for magnetofossil identification[END_REF], most magnetite particles within THC-1 are dispersed without an obvious chain assembly. Few particles appear to be clustered, possibly due to cell shrinkage during sample preparation. A dispersed arrangement of most particles and a clustered arrangement for some particles coexist within THC-1 cells. Despite their spatial arrangement, the particles are not oriented randomly within the cell; most particles have roughly uniform orientations (Fig. 3F, Movie S1). This compromise particle assembly would produce a net magnetic dipole moment for THC-1 cells, so that they also behave in a magnetic field like MTB with magnetosome chains (Lefèvre, Abreu, et al., 2011).

Magnetic properties of THC-1 magnetosomal magnetite

The remanence in ZFC/FC-SIRM10K_2.5T warming and SIRM300K_2.5T cooling-warming cycling curves for THC-1 cells decrease significantly at ~112 K (Tv) (Fig. 5A,5B). This indicates the Verwey transition of magnetite. δFC, δZFC, and δ-ratio values are 0.425, 0.304, and 1.397, respectively. δFC and δZFC values are significantly higher and the δ-ratio is lower than for whole-cell MTB samples in which magnetosomal magnetite is generally organized into closely-packed chain(s) [START_REF] Li | Magnetite magnetosome and fragmental chain formation of Magnetospirillum magneticum AMB-1: transmission electron microscopy and magnetic observations[END_REF][START_REF] Li | Biomineralization, crystallography and magnetic properties of bullet-shaped magnetite magnetosomes in giant rod magnetotactic bacteria[END_REF]Li et al., 2012;Lin & Pan, 2009;[START_REF] Moskowitz | Rock magnetic criteria for the detection of biogenic magnetite[END_REF]Pan, Petersen, Winklhofer, et al., 2005;[START_REF] Wang | Characterizing and optimizing magnetosome production of Magnetospirillum sp. XM-1 isolated from Xi'an City Moat, China[END_REF].

In addition, the cooling-warming cycling SIRM300K_2.5T curve is nearly reversible with a small remanence loss (~3.6%) after cycling (Fig. 5B). A room-temperature hysteresis loop for THC-1 cells is pot-bellied with hysteresis parameters Bc, Bcr, Bcr/Bc, and Mrs/Ms of 36.3 mT, 44.9 mT, 1.24, and 0.49, respectively (Fig. 5C). Normalized IRM acquisition and demagnetization curves are not symmetric, with a gradual remanence increase mainly between ~10 and 90 mT, while the latter has a gradual remanence decrease from ~0 to ~80 mT (Fig. 5D). The curves cross at R = 0.37 in the Wohlfarth-Cisowski test [START_REF] Cisowski | Interacting vs. non-interacting single domain behavior in natural and synthetic samples[END_REF], which is lower than the expected value of 0.5 for non-interacting SD particles. This indicates moderate inter-particle magnetostatic interactions within THC-1 cells (Li et al., 2012).

The magnetic domain state, magnetostatic interactions, and coercivity distribution of magnetosomal magnetite within THC-1 cells were further assessed with a FORC diagram (Pike et al., 2009;[START_REF] Roberts | First-order reversal curve diagrams: a new tool for characterizing the magnetic properties of natural samples[END_REF], which contains features related to the spatial arrangement of magnetite particles within THC-1 cells (Fig. 6). First, the FORC distribution has a single peak coercivity (Bc,PFORC = 52.6 mT) along the horizontal axis, which is typical of SD behavior (Fig. 6A). Both the horizontal profile at Bi = 0 mT and the marginal coercivity distribution are asymmetric and skewed negatively. Compared to the former, the latter is shifted obviously to lower coercivities (Bc,CFORC = 43.0 mT) (Fig. 6B).

Second, the FORC distribution has a teardrop shape, which is consistent with TEM observations that indicate that most magnetite particles are dispersed (i.e., non-interacting) and some are clustered (interacting). The FORC distribution has maximum vertical width at Bc = ~38 mT, with Bi values that extend to ~20 mT. The FORC distribution also has a central ridge that is concentrated over a relatively narrow area along the horizontal axis (Bi < ±4 mT). The characteristic interaction field Bi,1/2 is 2.9 mT at Bc = 38 mT and 1.3 mT at Bc = 58 mT, which indicates that most magnetite particles within THC-1 cells interact weakly. The FORC results are consistent with HAADF-STEM tomography observations (Fig. 3) and of teardrop and central ridge structures in experimental and simulated FORC distributions for collapsed (i.e., magnetically interacting) magnetosome chains [START_REF] Harrison | FORCulator: a micromagnetic tool for simulating firstorder reversal curve diagrams[END_REF]Li et al., 2012). Third, the FORC diagram has a negative response in the lower left-hand region, which is centered about (Bc, Bi) = (0, -40) mT. Such negative regions are observed for whole-cell MTB samples [START_REF] Ding | Deletion of the ftsZ-like gene results in the production of superparamagnetic magnetite magnetosomes in Magnetospirillum gryphiswaldense[END_REF][START_REF] Li | Magnetite magnetosome and fragmental chain formation of Magnetospirillum magneticum AMB-1: transmission electron microscopy and magnetic observations[END_REF]Li et al., 2010;Lin & Pan, 2009;Pan, Petersen, Winklhofer, et al., 2005;[START_REF] Zhu | Isolation and characterization of a marine magnetotactic spirillum axenic culture QH-2 from an intertidal zone of the China Sea[END_REF], which are due to randomly oriented, SD particles with uniaxial anisotropy [START_REF] Egli | Detection of noninteracting single domain particles using first-order reversal curve diagrams[END_REF][START_REF] Muxworthy | Influence of magnetostatic interactions on first-order-reversal-curve (FORC) diagrams: a micromagnetic approach[END_REF][START_REF] Newell | A high-precision model of first-order reversal curve (FORC) functions for single-domain ferromagnets with uniaxial anisotropy[END_REF]. This is consistent with the dimension distributions for magnetite particles within THC-1 (Fig. 1F-H), which fall within the SD size range in the domain state diagram of [START_REF] Muxworthy | Critical superparamagnetic/single-domain grain sizes in interacting magnetite particles: implications for magnetosome crystals[END_REF].

In addition to the conventional FORC characteristics for SD particles, two small negative regions are distributed symmetrically on both sides of the FORC distribution at Bc = ~70 mT. These regions are likely to be due to single vortex behavior, as reported by [START_REF] Pike | An investigation of magnetic reversal in submicron-scale Co dots using first order reversal curve diagrams[END_REF]. Given that the magnetite particle size and length distributions for THC-1 (Fig. 1F-H) fall entirely within the SD size range, and that THC-1 cells only contain such particles, it is not expected that this behavior is due to particles in the single vortex size range, which for the width/length distributions shown in Fig. 1H will have lengths close to 200 nm [START_REF] Muxworthy | Critical superparamagnetic/single-domain grain sizes in interacting magnetite particles: implications for magnetosome crystals[END_REF]. Instead, it is likely that vortex superstructures are being produced by flux closure among magnetostatically interacting SD particles (e.g., [START_REF] Harrison | Direct imaging of nanoscale magnetic interactions in minerals[END_REF] ), and even in other Bacterial lineages [START_REF] Kolinko | Singlecell genomics of uncultivated deep-branching magnetotactic bacteria reveals a conserved set of magnetosome genes[END_REF]Lefèvre & Bazylinski, 2013;[START_REF] Lin | Genomic expansion of magnetotactic bacteria reveals an early common origin of magnetotaxis with lineage-specific evolution[END_REF][START_REF] Liu | Speciesspecific magnetosome biomineralization as a foundation for environmental interpretation of ancient magnetofossils[END_REF]. In agreement with bacterial diversity, biomineralization within MTB is highly diverse in terms of the number, size, morphology, and spatial assembly of magnetite crystals [START_REF] Li | Bulletshaped magnetite biomineralization within a magnetotactic Deltaproteobacterium: implications for magnetofossil identification[END_REF][START_REF] Liu | Speciesspecific magnetosome biomineralization as a foundation for environmental interpretation of ancient magnetofossils[END_REF][START_REF] Pósfai | Phylogenetic significance of composition and crystal morphology of magnetosome minerals[END_REF]). An intrinsic relationship has been found between magnetite morphology and MTB phylogenetic affiliations [START_REF] Li | Bulletshaped magnetite biomineralization within a magnetotactic Deltaproteobacterium: implications for magnetofossil identification[END_REF][START_REF] Pósfai | Phylogenetic significance of composition and crystal morphology of magnetosome minerals[END_REF]. For example, MTB in the Nitrospirae phylum generally produce kinked, elongated, bullet-shaped magnetite with final crystal elongation along the [001] (e.g., Candidatus

Magnetotactic casensis strain MYR-1) [START_REF] Li | Crystal growth of bullet-shaped magnetite in magnetotactic bacteria of the Nitrospirae phylum[END_REF] or [110] directions (e.g., Candidatus

Thermomagnetovibrio paiutensis strain HSMV-1) [START_REF] Lefèvre | Morphological features of elongated-anisotropic magnetosome crystals in magnetotactic bacteria of the Nitrospirae phylum and the Deltaproteobacteria class[END_REF].

Magnetotactic Deltaproteobacteria form bullet-shaped magnetite, but with straight particles elongated along the [001] direction, with generally one flat {001} face (e.g.,

Candidatus

Magnetcampylobacter weiyangensis strain WYHR-1) ( [START_REF] Li | Bulletshaped magnetite biomineralization within a magnetotactic Deltaproteobacterium: implications for magnetofossil identification[END_REF] or a double-triangle shape at its bottom (e.g., strains RS-1 and AV-1) [START_REF] Lefèvre | Morphological features of elongated-anisotropic magnetosome crystals in magnetotactic bacteria of the Nitrospirae phylum and the Deltaproteobacteria class[END_REF][START_REF] Pósfai | Properties of intracellular magnetite crystals produced by Desulfovibrio magneticus strain RS-1[END_REF]. In contrast, magnetotactic Alphaproteobacteria, Etaproteobacteria, and Gammaproteobacteria generally produce magnetite with symmetric morphologies, including octahedral {111} forms (e.g., Magnetofaba australis strain IT-1 and uncultured magnetotactic coccus strain SHHC-1) [START_REF] Araujo | Combined genomic and structural analyses of a cultured magnetotactic bacterium reveals its niche adaptation to a dynamic environment[END_REF][START_REF] Zhang | Magnetotactic coccus strain SHHC-1 affiliated to Alphaproteobacteria forms octahedral magnetite magnetosomes[END_REF], cubo-octahedral ({111}+{100} forms) (e.g., Magnetotactic spirillum strains MS-1, MMS-1, MSR-1, and AMB-1) (Faivre et al., 2008;[START_REF] Li | A strong angular dependence of magnetic properties of magnetosome chains: Implications for rock magnetism and paleomagnetism[END_REF][START_REF] Mann | Structure, morphology and crystal growth of bacterial magnetite[END_REF]Meldrum et al., 1993b), and elongated hexagonal prisms with {111}+{110} forms (e.g.,

Magnetovibrio blakemorei strain MV-1) [START_REF] Sparks | Structure and morphology of magnetite anaerobically-produced by a marine magnetotactic bacterium and a dissimilatory iron-reducing bacterium[END_REF] or {111}+{110}+{100}

forms (e.g., Magnetococcus marinus strain MC-1, magnetotactic Gammaproteobacteria strain SHHR-1) [START_REF] Li | Single-cell resolution of uncultured magnetotactic bacteria via fluorescence-coupled electron microscopy[END_REF]Meldrum et al., 1993a). Consistent with previous observations, this study demonstrates that magnetotactic Alphaproteobacteria strain THC-1 biomineralizes elongated hexagonal magnetite with six {110} faces forming prism sides two {111} faces terminating the prism at both ends, and six {111}, {110}, and {100} faces truncating the twelve prism angles (Fig. 4). This crystal habit appears to be common for prismatic magnetosomal magnetite [START_REF] Li | Single-cell resolution of uncultured magnetotactic bacteria via fluorescence-coupled electron microscopy[END_REF]Meldrum et al., 1993a;[START_REF] Pósfai | Phylogenetic significance of composition and crystal morphology of magnetosome minerals[END_REF]. However, morphological differences in particle size, elongation (aspect ratio), and crystal face volume are observed clearly within elongated prismatic magnetite produced by different MTB species/strains [START_REF] Li | Single-cell resolution of uncultured magnetotactic bacteria via fluorescence-coupled electron microscopy[END_REF]Meldrum et al., 1993aMeldrum et al., , 1993b;;[START_REF] Pósfai | Phylogenetic significance of composition and crystal morphology of magnetosome minerals[END_REF]this study). This further confirms that magnetite biomineralization within magnetosomes is genetically controlled and, therefore, species/strain specific [START_REF] Li | Bulletshaped magnetite biomineralization within a magnetotactic Deltaproteobacterium: implications for magnetofossil identification[END_REF][START_REF] Liu | Speciesspecific magnetosome biomineralization as a foundation for environmental interpretation of ancient magnetofossils[END_REF][START_REF] Pósfai | Phylogenetic significance of composition and crystal morphology of magnetosome minerals[END_REF].

HAADF-STEM tomography results demonstrate that magnetosomal magnetite within THC-1 cells is dispersed without obvious chain structures. This situation contrasts with that for some uncultured MTB in which magnetosomal magnetite is arranged in aggregated clusters or in clumped chains (e.g., [START_REF] Cox | Organization and elemental analysis of P-, S-, and Fe-rich inclusions in a population of freshwater magnetococci[END_REF]Lin & Pan, 2009;[START_REF] Liu | Speciesspecific magnetosome biomineralization as a foundation for environmental interpretation of ancient magnetofossils[END_REF][START_REF] Moench | Bilophococcus magnetotacticus gen. nov. sp. nov., a motile, magnetic coccus[END_REF]. In these cases, the cellular magnetic dipole moment cannot be maximized to the sum of all magnetic particles in each cell, as is the case for the linear chain(s) that occur within most MTB (e.g., [START_REF] Dunin-Borkowski | Magnetic microstructure of magnetotactic bacteria by electron holography[END_REF][START_REF] Hanzlik | Pulsed-field-remanence measurements on individual magnetotactic bacteria[END_REF][START_REF] Li | A strong angular dependence of magnetic properties of magnetosome chains: Implications for rock magnetism and paleomagnetism[END_REF][START_REF] Li | Crystal growth of bullet-shaped magnetite in magnetotactic bacteria of the Nitrospirae phylum[END_REF][START_REF] Li | Biomineralization, crystallography and magnetic properties of bullet-shaped magnetite magnetosomes in giant rod magnetotactic bacteria[END_REF]. This partially explains why MTB without linear chains generally produce more magnetic particles to produce a sufficient cellular magnetic dipole moment for magnetotaxis [START_REF] Liu | Speciesspecific magnetosome biomineralization as a foundation for environmental interpretation of ancient magnetofossils[END_REF]. The process and molecular mechanism that produce the spatial arrangement of non-chain magnetic particles are not yet clear because pure cultures of these MTB are not available and genomic information is sparse.

However, we show that dispersed magnetic particles occur within wild-type MTB and have magnetic properties that are distinct from those of chain-organized particles. For example, the whole-cell THC-1 sample has a teardrop FORC distribution, R < 0.5 in the Wohlfarth-Cisowski test, with relatively high δFC and δZFC, and low δ-ratio values (Fig. 5). This indicates moderate to weak inter-particle interactions (i.e., negative magnetostatic interactions) within THC-1 cells, which contrasts with generally strong intra-chain interactions (i.e., positive magnetostatic interactions) for magnetosome chains within other MTB (e.g., [START_REF] Li | A strong angular dependence of magnetic properties of magnetosome chains: Implications for rock magnetism and paleomagnetism[END_REF][START_REF] Li | Biomineralization, crystallography and magnetic properties of bullet-shaped magnetite magnetosomes in giant rod magnetotactic bacteria[END_REF]Li et al., 2012;[START_REF] Moskowitz | Rock magnetic criteria for the detection of biogenic magnetite[END_REF][START_REF] Moskowitz | Magnetic properties of magnetotactic bacteria[END_REF]Pan, Petersen, Winklhofer, et al., 2005;[START_REF] Wang | Characterizing and optimizing magnetosome production of Magnetospirillum sp. XM-1 isolated from Xi'an City Moat, China[END_REF]. Despite lacking intra-chain interactions, the observed particle elongations will produce significant uniaxial anisotropy for individual magnetite particles within THC-1 [START_REF] Körnig | Magnetite crystal orientation in magnetosome chains[END_REF][START_REF] Moreno | The role of faceting and elongation on the magnetic anisotropy of magnetite Fe3O4 nanocrystals[END_REF], which results in a pot-bellied hysteresis loop with Mrs/Ms close to 0.5, as expected for non-interacting or weakly interacting uniaxial SD particles [START_REF] Stoner | A mechanism of magnetic hysteresis in heterogeneous alloys[END_REF]. This particle shape also causes a bifurcation between FC and ZFC curves below Tv, with the FC remanence much greater than the ZFC remanence [START_REF] Carter-Stiglitz | Low-temperature remanence in stable single domain magnetite[END_REF].

Magnetism of MTB magnetite and implications for magnetofossil identification

BCM provides MTB magnetite with distinctive mineralogical, crystallographic, chemical, and magnetic features that are used to identify magnetofossils from sediments or sedimentary rocks (e.g., [START_REF] Amor | Ironbiomineralizing organelle in magnetotactic bacteria: function, synthesis and preservation in ancient rock samples[END_REF][START_REF] Jimenez-Lopez | Magnetite as a prokaryotic biomarker: a review[END_REF][START_REF] Kopp | The identification and biogeochemical interpretation of fossil magnetotactic bacteria[END_REF][START_REF] Li | The link between biomineralization and fossilization of bacteria: Insights from field and experimental studies[END_REF][START_REF] Moskowitz | Rock magnetic criteria for the detection of biogenic magnetite[END_REF]. Based on the fact that most MTB magnetite has narrow size distributions, stable SD sizes, chain assembly, and relatively low Tv, rock magnetic approaches have been proposed to assess the presence of magnetofossils within geological records. These include the ratio of anhysteretic remanent magnetization (ARM) to IRM [START_REF] Petersen | Fossil bacterial magnetite in deep-sea sediments from the South Atlantic Ocean[END_REF][START_REF] Egli | Characterization of individual rock magnetic components by analysis of remanence curves. 3. Bacterial magnetite and natural processes in lakes[END_REF], decomposition of IRM acquisition curves [START_REF] Egli | Characterization of individual rock magnetic components by analysis of remanence curves. 3. Bacterial magnetite and natural processes in lakes[END_REF]Pan, Petersen, Davila, et al., 2005), normalized vector difference sum (VDS) demagnetization curves [START_REF] Heslop | Quantifying magnetite magnetofossil contributions to sedimentary magnetizations[END_REF], FORC measurements [START_REF] Egli | Detection of noninteracting single domain particles using first-order reversal curve diagrams[END_REF][START_REF] Heslop | Characterizing magnetofossils from firstorder reversal curve (FORC) central ridge signatures[END_REF][START_REF] Kind | Combined use of magnetometry and spectroscopy for identifying magnetofossils in sediments[END_REF][START_REF] Roberts | Searching for single domain magnetite in the "pseudo-single-domain" sedimentary haystack: implications of biogenic magnetite preservation for sediment magnetism and relative paleointensity determinations[END_REF][START_REF] Roberts | Domain state diagnosis in rock magnetism: evaluation of potential alternatives to the day diagram[END_REF], ferromagnetic resonance (FMR) spectra [START_REF] Gehring | S-band ferromagnetic resonance spectroscopy and the detection of magnetofossils[END_REF][START_REF] Gehring | The detection of magnetotactic bacteria and magnetofossils by means of magnetic anisotropy[END_REF][START_REF] Kind | Combined use of magnetometry and spectroscopy for identifying magnetofossils in sediments[END_REF][START_REF] Kopp | Chains, clumps, and strings: magnetofossil taphonomy with ferromagnetic resonance spectroscopy[END_REF][START_REF] Weiss | Ferromagnetic resonance and low-temperature magnetic tests for biogenic magnetite[END_REF], the Moskowitz test (δFC/δZFC) [START_REF] Moskowitz | Rock magnetic criteria for the detection of biogenic magnetite[END_REF], the δ-plot (δFC/δZFC vs. δZFC) (Li et al., 2012), and double Verwey transition temperatures [START_REF] Chang | Discrimination of biogenic and detrital magnetite through a double Verwey transition temperature[END_REF]. Despite being used widely, limitations and uncertainties remain for these bulk magnetic methods especially for quantitative magnetofossil identification because magnetofossils have diverse crystal morphologies, particle sizes, and spatial arrangements, or occur in mixtures with magnetic minerals with non-MTB origins (e.g., [START_REF] Kopp | The identification and biogeochemical interpretation of fossil magnetotactic bacteria[END_REF][START_REF] Li | The link between biomineralization and fossilization of bacteria: Insights from field and experimental studies[END_REF]Li et al., 2012;[START_REF] Roberts | Magnetic mineral diagenesis[END_REF][START_REF] Roberts | Searching for single domain magnetite in the "pseudo-single-domain" sedimentary haystack: implications of biogenic magnetite preservation for sediment magnetism and relative paleointensity determinations[END_REF].

On the one hand, the bulk magnetic properties of MTB magnetite are related closely to the morphology and spatial arrangement of magnetic particles produced by different MTB groups or species/strains or even the same species/strains under different environmental growth conditions (Fig. 7). As expected, most unoriented whole-cell MTB magnetite samples have magnetic properties typical of noninteracting or weakly interacting uniaxial SD particles because of their chain assembly within cells, i.e., Mrs/Ms values are close to 0.5 (e.g., Li et al., 2012;[START_REF] Moskowitz | Magnetic properties of magnetotactic bacteria[END_REF]Pan, Petersen, Winklhofer, et al., 2005). However, studies are revealing increasingly that the coercivity of MTB magnetite varies among groups or species/strains (Fig. 7A,B). They can range from Bc = 16.9 mT and Bcr = 18.9 mT for strain MSR-1 with cubo-octahedral magnetite [START_REF] Katzmann | Analysis of magnetosome chains in magnetotactic bacteria by magnetic measurements and automated image analysis of electron micrographs[END_REF] to Bc = 30.5 mT and Bcr = 37.4 mT for strain AMB-1 with elongated cubo-octahedral magnetite (Li et al., 2012) to Bc = 36.3 mT and Bcr = 44.9 mT for strain THC-1 with elongated prismatic magnetite (this study), and to Bc = 54.5 mT and Bcr = 61 mT for strain MYR-1 with bullet-shaped magnetite [START_REF] Li | Biomineralization, crystallography and magnetic properties of bullet-shaped magnetite magnetosomes in giant rod magnetotactic bacteria[END_REF]. Values of Bc, Bcr, Bcr/Bc and Mrs/Ms, can also be affected significantly by the degree of chain alignment, chain collapse, and particle aggregation (Fig. 7A-C) [START_REF] Kobayashi | Experimental observation of magnetosome chain collapse in magnetotactic bacteria: sedimentological, paleomagnetic, and evolutionary implications[END_REF][START_REF] Kopp | Chains, clumps, and strings: magnetofossil taphonomy with ferromagnetic resonance spectroscopy[END_REF][START_REF] Li | A strong angular dependence of magnetic properties of magnetosome chains: Implications for rock magnetism and paleomagnetism[END_REF][START_REF] Li | Biomineralization, crystallography and magnetic properties of bullet-shaped magnetite magnetosomes in giant rod magnetotactic bacteria[END_REF]Li et al., 2012;[START_REF] Moskowitz | Magnetic properties of magnetotactic bacteria[END_REF]. Measurements parallel to magnetosome chains produce higher Bc and Mrs/Ms values, and lower Bcr and Bcr/Bc values, while perpendicular measurements yield lower Bc and Mrs/Ms values, and higher Bcr and

Bcr/Bc values [START_REF] Li | A strong angular dependence of magnetic properties of magnetosome chains: Implications for rock magnetism and paleomagnetism[END_REF][START_REF] Li | Biomineralization, crystallography and magnetic properties of bullet-shaped magnetite magnetosomes in giant rod magnetotactic bacteria[END_REF]. Chain collapse and particle aggregation, which often happens during magnetofossil burial, can reduce the coercivity (Bc, Bcr) and Mrs/Ms of MTB magnetite due to increasing inter-particle interactions and decreasing intrachain interactions [START_REF] Kobayashi | Experimental observation of magnetosome chain collapse in magnetotactic bacteria: sedimentological, paleomagnetic, and evolutionary implications[END_REF][START_REF] Kopp | Chains, clumps, and strings: magnetofossil taphonomy with ferromagnetic resonance spectroscopy[END_REF]Li et al., 2012;[START_REF] Moskowitz | Magnetic properties of magnetotactic bacteria[END_REF]. Environmental factors such as oxygen concentration, iron source, and medium composition can strongly affect the particle number, grain size, and even stoichiometry of biogenic magnetite particles, which results in obvious coercivity changes even for the same MTB species/strain [START_REF] Ding | Deletion of the ftsZ-like gene results in the production of superparamagnetic magnetite magnetosomes in Magnetospirillum gryphiswaldense[END_REF][START_REF] Katzmann | Analysis of magnetosome chains in magnetotactic bacteria by magnetic measurements and automated image analysis of electron micrographs[END_REF][START_REF] Li | Controlled cobalt doping in the spinel structure of magnetosome magnetite: new evidences from element-and site-specific X-ray magnetic circular dichroism analyses[END_REF]Li & Pan, 2012;[START_REF] Staniland | Controlled cobalt doping of magnetosomes in vivo[END_REF].

As mentioned above, compared to stoichiometric magnetite with a Verwey transition at ~122 K, MTB magnetite has obviously lower Tv values (Fig. 7D). Tv generally ranges from 112 K to 98 K for MTB magnetite among different bacterial species/strains [START_REF] Chang | Discrimination of biogenic and detrital magnetite through a double Verwey transition temperature[END_REF][START_REF] Ding | Deletion of the ftsZ-like gene results in the production of superparamagnetic magnetite magnetosomes in Magnetospirillum gryphiswaldense[END_REF][START_REF] Li | Biomineralization, crystallography and magnetic properties of bullet-shaped magnetite magnetosomes in giant rod magnetotactic bacteria[END_REF]Lin & Pan, 2009;[START_REF] Wang | Characterizing and optimizing magnetosome production of Magnetospirillum sp. XM-1 isolated from Xi'an City Moat, China[END_REF][START_REF] Zhu | Isolation and characterization of a marine magnetotactic spirillum axenic culture QH-2 from an intertidal zone of the China Sea[END_REF], and even down to ~85 K [START_REF] Pósfai | Properties of intracellular magnetite crystals produced by Desulfovibrio magneticus strain RS-1[END_REF]. Chain alignment, chain collapse, and particle aggregation appear to not affect Tv [START_REF] Kopp | Chains, clumps, and strings: magnetofossil taphonomy with ferromagnetic resonance spectroscopy[END_REF][START_REF] Li | A strong angular dependence of magnetic properties of magnetosome chains: Implications for rock magnetism and paleomagnetism[END_REF]Li et al., 2012). However, crystal orientation and spatial arrangement can influence significantly Verwey transition behavior in thermal demagnetization curves of a lowtemperature SIRM [START_REF] Li | A strong angular dependence of magnetic properties of magnetosome chains: Implications for rock magnetism and paleomagnetism[END_REF][START_REF] Li | Biomineralization, crystallography and magnetic properties of bullet-shaped magnetite magnetosomes in giant rod magnetotactic bacteria[END_REF]Li et al., 2012;[START_REF] Moskowitz | Rock magnetic criteria for the detection of biogenic magnetite[END_REF][START_REF] Pósfai | Properties of intracellular magnetite crystals produced by Desulfovibrio magneticus strain RS-1[END_REF]. For example, the Verwey transition for bullet-shaped magnetite is smeared in both FC-and ZFC-SIRM10K_2.5T warming curves and is even absent in cooling-warming cycling curves of SIRM300K_2.5T because individual particles are elongated and oriented along the [001] crystal direction (i.e., the magnetocrystalline hard axis for magnetite), which results in low δFC and δZFC values [START_REF] Li | Biomineralization, crystallography and magnetic properties of bullet-shaped magnetite magnetosomes in giant rod magnetotactic bacteria[END_REF][START_REF] Pósfai | Properties of intracellular magnetite crystals produced by Desulfovibrio magneticus strain RS-1[END_REF]. For MTB magnetite with cubo-octahedral and prismatic morphologies that are generally elongated and oriented along the [111] direction (i.e., the magnetocrystalline easy axis for magnetite), the Verwey transition becomes more obvious with higher δFC and δZFC values than those for bullet-shaped MTB magnetite (e.g., [START_REF] Ding | Deletion of the ftsZ-like gene results in the production of superparamagnetic magnetite magnetosomes in Magnetospirillum gryphiswaldense[END_REF][START_REF] Li | Magnetite magnetosome and fragmental chain formation of Magnetospirillum magneticum AMB-1: transmission electron microscopy and magnetic observations[END_REF]Li et al., 2012;Lin & Pan, 2009;[START_REF] Moskowitz | Rock magnetic criteria for the detection of biogenic magnetite[END_REF][START_REF] Wang | Characterizing and optimizing magnetosome production of Magnetospirillum sp. XM-1 isolated from Xi'an City Moat, China[END_REF][START_REF] Zhu | Isolation and characterization of a marine magnetotactic spirillum axenic culture QH-2 from an intertidal zone of the China Sea[END_REF]this study). Compared to whole-cell MTB magnetite samples, isolated particles have increasing δFC and δZFC values and lower δFC/δZFC values due to the decreasing uniaxial anisotropy of magnetosome chain(s) or increasing inter-particle magnetostatic interactions or both [START_REF] Kobayashi | Experimental observation of magnetosome chain collapse in magnetotactic bacteria: sedimentological, paleomagnetic, and evolutionary implications[END_REF][START_REF] Kopp | Chains, clumps, and strings: magnetofossil taphonomy with ferromagnetic resonance spectroscopy[END_REF]Li et al., 2012;[START_REF] Moskowitz | Rock magnetic criteria for the detection of biogenic magnetite[END_REF].

Consistent with early observations by [START_REF] Moskowitz | Rock magnetic criteria for the detection of biogenic magnetite[END_REF], most measurements of intact MTB cells have δFC/δZFC > 2.0, i.e., meeting the so-called Moskowitz test. However, some MTB magnetite do have δFC/δZFC < 2.0 due to loosely packed chain structures (e.g., [START_REF] Ding | Deletion of the ftsZ-like gene results in the production of superparamagnetic magnetite magnetosomes in Magnetospirillum gryphiswaldense[END_REF][START_REF] Zhu | Isolation and characterization of a marine magnetotactic spirillum axenic culture QH-2 from an intertidal zone of the China Sea[END_REF] or dispersed assembly within cells (this study). In addition, the Tv and other magnetic behavior of MTB magnetite can be modified significantly by environmental conditions in which bacteria are growing [START_REF] Li | Controlled cobalt doping in the spinel structure of magnetosome magnetite: new evidences from element-and site-specific X-ray magnetic circular dichroism analyses[END_REF]Li & Pan, 2012;[START_REF] Staniland | Controlled cobalt doping of magnetosomes in vivo[END_REF].

On the other hand, magnetosomal magnetite has distinctive but not unique properties compared to non-MTB magnetite. Some experiments indicate that iron-reducing and ironoxidizing bacteria can produce extracellular SD magnetite under special conditions (e.g., high-temperature or long-term cultures), although they generally form superparamagnetic (SP) magnetite under normal conditions [START_REF] Miot | Formation of single domain magnetite by Green Rust oxidation promoted by microbial anaerobic nitrate-dependent iron oxidation[END_REF][START_REF] Moskowitz | A comparison of magnetite particles produced anaerobically by magnetotactic and dissimilatory iron-reducing bacteria[END_REF][START_REF] Vali | Formation of tabular single-domain magnetite induced by Geobacter metallireducens GS-15[END_REF][START_REF] Zhang | Formation of singledomain magnetite by a thermophilic bacterium[END_REF]. SD magnetite with equidimensional cubo-octahedral or elongated prismatic morphologies can be produced by chemical synthesis based on solution-phase reactions or thermal decomposition (e.g., [START_REF] Baumgartner | Formation of magnetite nanoparticles at low temperature: from superparamagnetic to stable single domain particles[END_REF][START_REF] Golden | A simple inorganic process for formation of carbonates, magnetite, and sulfides in Martian meteroite ALH84001[END_REF][START_REF] Jimenez-Lopez | Magnetite as a prokaryotic biomarker: a review[END_REF]. Such non-MTB SD magnetite can produce comparable magnetic properties (e.g., coercivity, Mrs/Ms) to those for MTB magnetite (Fig. 7A-C) [START_REF] Dunlop | Coercive forces and coercivity spectra of submicron magnetites[END_REF][START_REF] Dunlop | Magnetite: behavior near the single-domain threshold[END_REF][START_REF] Levi | Properties of single-domain, pseudo-single-domain, and multidomain magnetite[END_REF][START_REF] Miot | Formation of single domain magnetite by Green Rust oxidation promoted by microbial anaerobic nitrate-dependent iron oxidation[END_REF][START_REF] Özdemir | A preliminary magnetic study of soil samples from west-central Minnesota[END_REF][START_REF] Vali | Formation of tabular single-domain magnetite induced by Geobacter metallireducens GS-15[END_REF]. This indicates that magnetic approaches based only on coercivity or hysteresis properties cannot identify magnetofossils precisely (e.g., [START_REF] Roberts | A Critical Appraisal of the "Day" Diagram[END_REF].

Magnetic methods that can effectively indicate the uniaxial SD behavior of magnetosome chain(s) have proven to be most useful for identifying magnetofossils within sediments, such as FORC, FMR, and low-temperature magnetic measurements (e.g., [START_REF] Chang | Discrimination of biogenic and detrital magnetite through a double Verwey transition temperature[END_REF][START_REF] Egli | Detection of noninteracting single domain particles using first-order reversal curve diagrams[END_REF][START_REF] Kind | Combined use of magnetometry and spectroscopy for identifying magnetofossils in sediments[END_REF][START_REF] Kopp | The identification and biogeochemical interpretation of fossil magnetotactic bacteria[END_REF][START_REF] Li | The link between biomineralization and fossilization of bacteria: Insights from field and experimental studies[END_REF][START_REF] Roberts | Domain state diagnosis in rock magnetism: evaluation of potential alternatives to the day diagram[END_REF][START_REF] Weiss | Ferromagnetic resonance and low-temperature magnetic tests for biogenic magnetite[END_REF]. As shown in Fig. 7D-F, compared to chemically synthesized stoichiometric magnetite, MTB magnetite has relatively low Tv, δFC and δZFC values, and higher δFC/δZFC values. On the δ-plot (Li et al., 2012), data for most MTB magnetite are distributed within a region with δZFC = 0.36 and δFC/δZFC = 1.2 [START_REF] Ding | Deletion of the ftsZ-like gene results in the production of superparamagnetic magnetite magnetosomes in Magnetospirillum gryphiswaldense[END_REF][START_REF] Li | A strong angular dependence of magnetic properties of magnetosome chains: Implications for rock magnetism and paleomagnetism[END_REF][START_REF] Li | Controlled cobalt doping in the spinel structure of magnetosome magnetite: new evidences from element-and site-specific X-ray magnetic circular dichroism analyses[END_REF]Li & Pan, 2012;Li et al., 2010;Li et al., 2012;Lin & Pan, 2009;[START_REF] Moskowitz | Rock magnetic criteria for the detection of biogenic magnetite[END_REF]Pan, Petersen, Winklhofer, et al., 2005;[START_REF] Wang | Characterizing and optimizing magnetosome production of Magnetospirillum sp. XM-1 isolated from Xi'an City Moat, China[END_REF][START_REF] Zhu | Isolation and characterization of a marine magnetotactic spirillum axenic culture QH-2 from an intertidal zone of the China Sea[END_REF] this study), while data for oxidized MTB magnetite and chemically synthesized magnetite fall outside this region [START_REF] Moskowitz | Rock magnetic criteria for the detection of biogenic magnetite[END_REF] (Fig. 7F). Data for natural sediment samples can fall either within or outside this region depending on magnetofossil concentration and preservation [START_REF] Housen | Depth distribution of magnetofossils in nearsurface sediments from the Blake/Bahama Outer Ridge, western North Atlantic Ocean, determined by low-temperature magnetism[END_REF][START_REF] Liu | Insolation driven biomagnetic response to the Holocene Warm Period in semi-arid East Asia[END_REF]Pan, Petersen, Davila, et al., 2005). Extracellular magnetite produced by iron-reducing bacteria, when they produce elongated SD particles, can have comparable magnetic properties to those of MTB magnetite, so they are not easily distinguished from isolated magnetosomal magnetite using bulk magnetic measurements [START_REF] Vali | Formation of tabular single-domain magnetite induced by Geobacter metallireducens GS-15[END_REF][START_REF] Weiss | Ferromagnetic resonance and low-temperature magnetic tests for biogenic magnetite[END_REF]. et al., 1977), (D) Verwey transition (Tv), (E) δ-ratio [START_REF] Moskowitz | Rock magnetic criteria for the detection of biogenic magnetite[END_REF], and (F) δ-plot (Li et al., 2012). Room-temperature magnetic parameters (Bc, Bcr, Bcr/Bc, and Mrs/Ms) of magnetite samples from MTB are from [START_REF] Moskowitz | A comparison of magnetite particles produced anaerobically by magnetotactic and dissimilatory iron-reducing bacteria[END_REF] (whole-cell MV-1), [START_REF] Moskowitz | Rock magnetic criteria for the detection of biogenic magnetite[END_REF] this study (whole-cell THC-1).

Conclusions

We have investigated chemical, morphological, and magnetic features of magnetosomal magnetite within uncultured magnetotactic coccus strain THC-1 using a combination of TEM and rock magnetic approaches. We demonstrate that strain THC-1 forms sulfur-rich globules, Ca-rich polyphosphate inclusions, and vacuoles in addition to magnetosomal magnetite. Most magnetite particles in THC-1 cells are dispersed without an obvious chain assembly. Mature magnetite crystals have an elongated prismatic morphology that can be modelled as a hexagonal prism with six {110} side faces, terminated by two {111} faces at both ends, and truncated by six {111}, six {110}, and six {100} faces at the prism angles.

The studied whole-cell THC-1 sample has a pot-bellied hysteresis loop with Bc = 36.3 mT, Bcr = 44.9 mT, and Mrs/Ms = 0.49. Its Tv value is ~112 K. In contrast to whole-cell MTB samples with magnetosome chain(s), THC-1 has a teardrop FORC distribution that indicates moderate inter-particle interactions. Due to the absence of magnetosome chain(s),

THC-1 has relatively high δFC (0.425) and δZFC (0.304), and low δFC/δZFC value (1.397).

Together with previous studies, our results indicate that some MTB species/strains do form magnetite particles without linear chain configurations. Despite producing typical SD particles, MTB magnetite has diverse magnetic properties that are related closely to crystal morphology and spatial arrangement within different MTB groups or species/strains, or in the same species/strain under different environmental conditions. Furthermore, some non-MTB magnetite has magnetic properties that are comparable to those for MTB magnetite.

This indicates that combining bulk magnetic measurements and microscopic observations (e.g., TEM) remains necessary to identify magnetofossils from the geological record.

Figure 1 .

 1 Figure 1. Morphological features of THC-1 cells. (A) Optical microscope image of living

Figure 2 .

 2 Figure 2. HAADF-STEM EDX elemental mapping of a THC-1 cell. (A-I) HAADF-STEM

  HRTEM observations of nine representative particles reveal that the side faces of prisms are {110} faces, bottom and top faces are {111} faces, and truncated faces at the prism angles are {111}, {110}, and {001} faces (Figs.4, S3). Together with 3D HAADF-STEM tomography and 2D HRTEM observations, the ideal crystal habit of THC-1 magnetosomal magnetite can be modeled as an elongated hexagonal prism in which the cylinder has six {110} faces, the bottom and top ends are terminated by two {111} faces, and the twelve angles at both ends are truncated by six {111}, six {110}, and six {001} faces. Elongation is along the [111] crystal direction of magnetite (Fig.4).

Figure 3 .

 3 Figure 3. Spatial arrangement and 3D morphology of THC-1 magnetosomal magnetite

Figure 4 .

 4 Figure 4. (A-D) HRTEM analyses of four representative THC-1 magnetosomal magnetite

  , as demonstrated by the FORC simulations of Harrison et al. (2019).

Figure 5 .

 5 Figure 5. Magnetic properties of THC-1 magnetosomal magnetite (whole-cell sample). (A)

Figure 6 .

 6 Figure 6. (A) FORC diagram for THC-1 magnetosomal magnetite (whole-cell sample). (B)

Figure 7 .

 7 Figure 7. Data distributions and comparison of magnetic properties for magnetite with

  (whole-cell and isolated MS-1 magnetite), Lin & Pan (2009) (whole-cell YDC-1), Zhu et al. (2010) (whole-cell QH-2), Li et al. (2010) (unoriented and oriented whole-cell MYR-1), Li et al. (2012) (whole-cell and isolated AMB-1 magnetite), Li & Pan (2012) (whole-cell AMB-1 grown under different oxygen conditions), Katzmann et al. (2013) (whole-cell MSR-1), Li, Ge et al. (2013) (Oriented whole-cell AMB-1), Wang et al. (2015) (whole-cell XM-1), Li et al. (2016) (whole-cell AMB-1 grown with different cobalt concentrations), and this study (whole-cell THC-1). Room-temperature magnetic parameters for extracellular magnetite from iron-reducing bacteria are from Moskowitz et al. (1989) (GS-1) and Vali et al. (2004) (GS-15, nontraditional 668-h culture), and iron-oxidizing bacteria are from Miot et al. (2014) (BoFeN1). Room-temperature magnetic parameters of chemically synthesized magnetite are from Dunlop (1972) (acicular magnetite; 200 nm), Levi & Merrill (1978) (acicular magnetite; 350 nm), Özdemir & Banerjee (1982) (equidimensional magnetite; 25, 120, 190, and 995 nm) and Dunlop (1986) (cubic magnetite; 37, 76, 100, and 220 nm). Room-temperature magnetic parameters of natural MTB or magnetofossil-rich samples are from Pan et al. (2005b) (surface sediments, Lake Chiemsee, Germany), Liu et al. (2015) (DL670 and DL750), and Roberts et al. (2011) (ODP Hole 738B). Low-temperature magnetic parameters (Tv, δFC, and δZFC) for magnetite from MTB are from Moskowtiz et al. (1993) (whole-cells of MS-1, MV-1, MV-2, and isolated magnetite MS-1 particles), Pan, Petersen, Winklhofer, et al. (2005a) (uncultured MTB, P2, and P3 whole-cells), Ding et al. (2010) (whole-cell MSR-1), Zhu et al. (2010) (whole-cell QH-2), Lin & Pan (2009) (whole-cell YDC-1), Li et al. (2010) (whole-cell MYR-1), Li et al. (2012) (whole-cell and isolated AMB-1 magnetite), Li, Ge et al. (2013) (oriented whole-cell AMB-1), Li & Pan (2012) (whole-cell AMB-1 grown under different oxygen conditions), Li et al. (2016) (whole-cell AMB-1 grown with different cobalt conditions), Wang et al. (2015) (whole-cell XM-1),

Acknowledgments

This study was supported financially by the National Natural Science Foundation of China (grants no. 41920104009, 41890843, and 41621004), The Senior User Project of RVKEXUE2019GZ06 (Center for Ocean Mega-Science, Chinese Academy of Sciences), and the Australian Research Council (grant DP160100805). We thank Mrs. Fuxian Wang and Mr. Heng Zhang for assistance with field sampling and sample preparation.

Supplementary Movie S1. HAADF-STEM tomography results for magnetosomal magnetite particles within a single cell that demonstrate the dispersed assembly of magnetite particles within THC-1 magnetosomes.