Xin-Xin Qian

Jia Liu

Nicolas Menguy

Jinhua Li

François Alberto

Zhaojie Teng

Tian Xiao

Wenyan Zhang
email: zhangwy@qdio.ac.cn

Long-Fei Wu
email: wu@imm.cnrs.fr

Long - Fei

Identification of novel species of marine magnetotactic bacteria affiliated with Nitrospirae phylum

Magnetotactic bacteria (MTB) are a group of Gram-negative bacteria characterized by synthesizing magnetosomes and swimming along geomagnetic field lines. Phylogenetically, they belong to different taxonomic lineages including Proteobacteria, Nitrospirae, Omnitrophica, Latescibacteria, and Planctomycetes phyla on the phylogenetic tree. To date, six Nitrospirae MTB phylotypes have been identified from freshwater or low salinity environments and described in the literature. Here we report the identification of two Nitrospirae MTB phylotypes collected, for the first time, from the marine environment. Both have a spherical morphology with a cell size of ~ 5 µM and similar motility but are different colors (black-brown and ivory-white) under the optic microscope. They synthesized bullet-shaped iron-oxide magnetosomes that were arranged in multiple bundles of chains.

Moreover, the cytoplasm of the black-brown Nitrospirae MTB contained sulfur inclusions that conferred on cells a rough, granular appearance. Phylogenetic analysis based on their 16S rRNA gene sequences revealed that they are two novel species and cluster with the previously reported MTB affiliated with the phylum Nitrospirae, thus extending the distribution of Nitrospirae MTB from freshwater to the marine environment.

Originality-Significance Statement

Magnetotactic bacteria (MTB) consist of a phylogenetic, physiological and morphologic heterogeneous group of gram-negative microbes. They are distributed worldwide at the oxic-anoxic interface in chemically stratified water columns or sediments. To date, despite Nitrospirae being one of the major phyla of MTB, they have only been found in freshwater or low-salinity environments. Here we report the identification of two novel species of Nitrospirae MTB, from the marine habitat for the first time. They have a spherical morphology and are relatively big in size approximately 5 x 3 μm. One contains abundant intracellular sulfur inclusions. This study shows the great diversity of Nitrospirae phylum and extends the distribution of Nitrospirae MTB into marine habitats.

Introduction

Magnetotactic bacteria (MTB) are a group of Gram-negative bacteria and exhibit the unique capacity of aligning and swimming along the geomagnetic field lines, a behavior referred to as magnetotaxis [START_REF] Uebe | Magnetosome biogenesis in magnetotactic bacteria[END_REF]. This capacity is based on the intracellular biomineralization of magnetite or greigite magnetic nanocrystal chains that give MTB cells a net magnetic moment dipole. MTB present various morphotypes including cocci, spirilla, rod-shaped, vibrio, and more complex multicellular aggregates, and have been found in aquatic environments from freshwater to marine ecosystems [START_REF] Blakemore | Magnetotactic bacteria[END_REF][START_REF] Bazylinski | Magnetosome formation in prokaryotes[END_REF]Lefèvre and Bazylinski, 2013). Phylogenetically, magnetotactic bacteria are members of several classes of the Proteobacteria phylum including the Alpha-, Gamma-, Delta-, Zeta-, candidate Lambda-, candidate Eta-classes, the Nitrospirae phylum, the candidate Omnitrophica phylum, the candidate Latescibacteria phylum and the Planctomycetes phylum (Lin et al., 2017a;[START_REF] Lin | Genomic expansion of magnetotactic bacteria reveals an early common origin of magnetotaxis with lineage-specific evolution[END_REF].

Compared with the Proteobacteria phylum, few MTB have been found to be phylogenetically affiliated with the phylum Nitrospirae and none of them have been cultured. This is consistent with the general difficulty in cultivating MTB due to their factitious physiological requirements [START_REF] Bazylinski | Magnetosome formation in prokaryotes[END_REF]. The first to be discovered and the most studied was Candidatus Magnetobacterium bavaricum [START_REF] Spring | Dominating Role of an Unusual Magnetotactic Bacterium in the Microaerobic Zone of a Freshwater Sediment[END_REF]. Ca.

Magnetobacterium bavaricum is a large, rod-shaped bacterium ranging from 8 to 10 μm in length, and contains 600-1000 bullet-shaped magnetite magnetosomes in 3-5 bundles of multiple chains [START_REF] Jogler | Cultivation-independent characterization of 'Candidatus Magnetobacterium bavaricum' via ultrastructural, geochemical, ecological and metagenomic methods[END_REF][START_REF] Jogler | Conservation of proteobacterial magnetosome genes and structures in an uncultivated member of the deep-branching Nitrospira phylum[END_REF]. Canditatus Magnetobacterium casensis shares similar morphology and 98.2% of the sequence identity of the 16S rRNA gene with Ca. Magnetobacterium bavaricum. Analysis of the draft genome sequence of Ca.

Magnetobacterium casensis reveals an autotrophic lifestyle. Pathways involved in denitrification, sulfur oxidation and sulfate reduction have also been predicted, which indicates its considerable capacity to adapt to variable environments [START_REF] Lin | Genomic insights into the uncultured genus 'Candidatus Magnetobacterium' in the phylum Nitrospirae[END_REF].

The third identified Nitrospirae MTB was a small rod-shaped bacterium collected from sediment of Waller See, in Germany and named MHB-1 [START_REF] Flies | Combined Approach for Characterization of Uncultivated Magnetotactic Bacteria from Various Aquatic Environments[END_REF]. This organism contains a single bundle of multiple chains of magnetosomes with bullet-shaped magnetite crystals. The fourth reported Nitrospirae MTB is a moderately thermophilic magnetotactic bacterium, HSMV-1, which remained viable and motile in samples kept between 25 and 62°C for several months [START_REF] Lefèvre | Moderately Thermophilic Magnetotactic Bacteria from Hot Springs in Nevada[END_REF]. HSMV-1 cells are small vibrios and biomineralize a single chain of bullet-shaped magnetosomes. Two Nitrospirae MTB phylotypes named LO-1 and MWB-1 were identified from southern Nevada, USA and Beijing, China, respectively [START_REF] Lefevre | Culture-independent characterization of a novel, uncultivated magnetotactic member of the Nitrospirae phylum[END_REF]Lin et al., 2012a). The two species have 6% phylogenetic divergence and possess a similar relatively big (~ 3 x 2.5μm) spherical morphology with bullet-shaped magnetosomes and 4-7 bundles of magnetosome chains parallel to the long axis of cells. Interestingly, the six described Nitrospirae MTB phylotypes have distinct cell appearance and different magnetosome arrangement including single chain, multiple chains with single bundle or multiple bundles. The obvious common feature is their bullet-shape magnetite magnetosomes. Another common property of the reported Nitrospirae MTB is that they dwell in low salinity habitats (< 3 ppt) [START_REF] Spring | Dominating Role of an Unusual Magnetotactic Bacterium in the Microaerobic Zone of a Freshwater Sediment[END_REF][START_REF] Flies | Combined Approach for Characterization of Uncultivated Magnetotactic Bacteria from Various Aquatic Environments[END_REF][START_REF] Lefevre | Culture-independent characterization of a novel, uncultivated magnetotactic member of the Nitrospirae phylum[END_REF]Lin et al., 2012a;Lin et al., 2012b). Therefore, it has been proposed that salinity might influence the distribution of MTB affiliated with Nitrospirae phylum (Lin et al., 2012b).

Here, we identified two novel Nitrospirae MTBs species and expand the distribution of Nitrospirae MTB to marine ecosystems.

Results and discussion

Morphology and swimming behaviour of black-brown cocci and ivory-white cocci

Differential interference contrast (DIC) microscopic examination showed the morphology of black-brown giant cocci (BGC) (Fig. 1 A, black arrows), ivory-white giant cocci (WGC) (Fig. 1 A, white arrows) and small cocci of MTB collected from the sediments in Six-Fours les Plages. BGC have a rough surface appearance with an average length of 5.39 ± 0.72 μm and an average width of 4.04 ± 0.33 μm (n=62). WGC have a smoother cell surface and a similar cell size with an average length of 5.21 ± 0.66 μm and an average width of 3.99 ± 0.30 μm (n=62) (B in Figure 1). During sample preparation for DNA amplification and sequencing, we observed that BGC cells began to disintegrate 5 min following their transfer from seawater to PBS buffer, and small granules were released from the cells (C1 in Figure 1). In contrast, WGC cells shrunk and became smaller in PBS buffer (C2 in Figure 1). Further analysis showed that approximately 50 % of the BGC and WGC cells remained motile and accumulated at the north edge of the cell suspension droplets where the salinity was decreased from 35 to 16 ppt by dilution with deionized water. The number of cells at the edge of the droplets decreased as the dilution increased, and the BGC and WGC cells almost disappeared completely from the droplets at 13 ppt salinity. This observation suggests a sensitivity of the marine cells to the freshwater conditions. BGC and WGC also exhibited different fluorescent properties. When excited with green light (510-550 nm), BGC emitted red fluorescence while no auto-fluorescence signals were observed in WGC under these conditions (D2 in Figure1).

BGC emitted stronger yellow auto-fluorescence than WGC under blue light (450-480 nm) excitation (D3 in Figure1). Similar intensity of green light was emitted by both BGC and WGC when excited by violet light (400-410 nm) (D4 in Figure1). WGC emitted stronger blue fluorescence under ultraviolet light (330-385 nm) compared to BGC (D5 in Figure 1). Illumination at these wavelengths had no impact on the motility of these MTB cells. Lack of culture samples prevents us from performing detailed spectrophotometry analysis to characterize the nature of the fluorescent molecules.

Most of the BGC and WGC exhibited north-seeking magnetotaxis behavior, i.e. swimming along the applied magnetic field lines, whereas 10% of cells were found to be south-seeking. Cells demonstrated a dynamic motility in the hanging drops for approximately 30 min then began to slow down gradually and stopped swimming at the end of one hour. The average swim velocities of BGC and WGC cells were 126.3 ± 45.0 μm•s -1 (n=52) and 148.9 ± 28.0 μm•s -1 (n=54), respectively (E in Figure 1). The corresponding maximal swim speeds of BGC (225.4 μm•s -1 ) and WGC (188.6 μm•s -1 ) were slower than the closest freshwater relative MTB MWB-1 (260 μm•s -1 ).

Phylogenetic analysis

In the environmental samples analyzed, BGC cells were the dominant MTB while WGC were approximately 4-fold less abundant as estimated by direct counting of the two morphotypes in droplets (Experimental Procedures and Supporting Information Figure S1).

We collected 8 WGC cells using the magnetic micro-sorting approach, and performed Whole Genome Amplification to get sufficient template DNA for further 16S rRNA gene amplification and cloning. We obtained 24 sequences of 16S rRNA genes that shared >99.3% identity between each other and thus formed one operational taxonomic unit (OTU-1, GenBank number MK203828). In parallel, we obtained 40 full-length 16S rRNA gene sequences of MTB prepared by a routine two-step magnetic collection (see Experimental Procedures). Among them 31 sequences share more than 97.1% identity and form another operational taxonomic unit (OTU-2, GenBank number MK073023). The nine other 16S rRNA sequences consisted of 6 OTUs that were all affiliated with Alphaproteobacteria. The high abundance of OTU-2 sequences detected is correlated with the dominant occurrence of BGC in this sample (Fig. 1). A Blast search in NCBI indicated that the two OTUs sharing 92% identity form a cluster that is affiliated with the phylum Nitrospirae (Fig. 2). They possess less than 97% identity with all 16S rRNA genes in the GenBank. The most closely related phylotypes are uncultured Nitrospirae MTBs clone MWB-1 (93% with OTU-1, 92% with OTU-2) and Candidatus Magnetoovum mohavensis LO-1 (94% with OTU-1, 92% with OTU-2). Therefore, the two OTUs represent a novel species of Nitrospirae.

Being one of the deep-branching MTB group, Nitrospirae MTB plays an important role when analyzing the origin and evolution of magnetosome biomineralization and magnetotaxis.

Lin et al. suggested that the magnetosome biomineralization and magnetotaxis was present before the separation of the Nitrospirae and Proteobacteria phyla, which was between ~3.38 and 3.21 Ga (Lin et al., 2017b). However, it remains unclear whether the acquisition of magnetosome island (MAI), that encodes essential proteins for magnetosome biosynthesis in the genome, occurred before or after the divergence between freshwater and marine Nitrospirae MTB. It is equally possible that the spherical cells of Nitrospirae phylum got MAI by horizontal gene transfer (HGT) then adapted into two different habitats or that they diverged into different habitats then acquired MAI separately. Future genomic comparison studies should provide answers to elucidate the evolutionary mechanism.

Corroboration of authenticity of giant cocci by fluorescence in situ hybridization

The BGC authenticity of OTU-2 was inferred from the fact that BGC is the most abundant morphotype in the analyzed samples. To corroborate this inference we designed a specific probe based on the conserved OTU-2 sequence and performed fluorescence in situ hybridization (FISH) analysis. As shown in Figure 3 A2, the universal bacterial probe hybridized with all the MTB, while the specific probe hybridized only with black-brown giant cocci. The WGC cells only hybridized with the bacteria universal probe and were used as a negative control. Therefore, the OTU-2 really corresponds to the abundant BGC morphotype.

We also confirmed the authenticity of the OTU-1 to the scarce WGC. A probe specific to the conserved sequences of OTU-1 was designed. We found that the specific probe hybridized only with the WGC morphotype while the universal probe hybridized with all bacterial cells including BGC that served as the negative control (Fig. 3,B1). After FISH identification, the same cover-slips were observed under scanning electron microscopy (SEM). As shown in B3 of Fig. 3, more than 8 bundles of multiple magnetosome chains aligned parallel to the long axis and close to the outer surface in the WGC cell. Correlative FISH-SEM analysis not only allowed identification of the OTU but also revealed characteristics of the magnetosome chains.

Magnetosome and element composition analyses

After corroborating their authenticities, we analyzed the magnetosome chains and element composition in BGC and WGC. Under DIC microscopy BGC appeared to contain brown granules inside the cells while WGC had a smooth appearance (Fig. 4A). Observations using High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) revealed that the BGC cells were filled with electron dense granules rich in sulfur (Fig. 4B, top cell with white inclusions, and 4C in red color) in addition to the magnetosomes chains (Fig. 4C green color). Further energy-dispersive X-ray spectroscopy (XEDS) analysis confirmed that the granules were rich in sulfur, the most abundant element in BGC cells but barely detectable in WGC (Fig. 4, D2 versus D1). The magnetosomes in WGC were arranged in bundles of multiple chains as revealed by FISH-SEM analysis. We attempted to identify the magnetosome crystals inside of BGC and WGC cells by electron diffraction or high-resolution electron microscopy without success due to the thickness of the cells. Although we identified isolated bullet-shaped crystals as magnetites on grids, but it is uncertain if they were from BGC or WGC because environmental samples containing other MTBs were used. We performed STEM-XEDS elemental map (Fig. 4C) and XEDS spectrum analyses (Fig. 4B, E1, E2, F1 and F2), and unambiguously showed that the crystals in BGC and WGC cells are iron-oxide minerals, which is consistent with the finding of only magnetite minerals in Nitrospirae MTB magnetosomes in the literature.

One essential difference between BGC and WGC is that BGC contain numerous sulfur-rich inclusions. Intracellular sulfur globules were also detected in Nitrospirae magnetotactic bacteria Ca. Magnetobacterium bavaricum, LO-1, MWB-1 and MYR-1 [START_REF] Jogler | Cultivation-independent characterization of 'Candidatus Magnetobacterium bavaricum' via ultrastructural, geochemical, ecological and metagenomic methods[END_REF][START_REF] Li | Biomineralization, crystallography and magnetic properties of bullet-shaped magnetite magnetosomes in giant rod magnetotactic bacteria[END_REF][START_REF] Lefevre | Culture-independent characterization of a novel, uncultivated magnetotactic member of the Nitrospirae phylum[END_REF]Lin et al., 2012a;[START_REF] Li | Crystal growth of bullet-shaped magnetite in magnetotactic bacteria of the Nitrospirae phylum[END_REF], and those in the Ca. Magnetobacterium bavarium are present in the form of cyclo-octa sulfur (S8) [START_REF] Eder | Sub-micrometer-scale mapping of magnetite crystals and sulfur globules in magnetotactic bacteria using confocal Raman micro-spectrometry[END_REF]. Intracellular deposition of sulfur may serve as an electron donor or an electron acceptor to provide energy for bacterial growth [START_REF] Cox | Organization and Elemental Analysis of P-, S-, and Fe-rich Inclusions in a Population of Freshwater Magnetococci[END_REF]. We observed that BGC became the dominant population during storage under laboratory conditions and were able to survive for more than ten months while maintaining vivacious magnetotactic motility. Therefore, it is plausible that the metabolism of intracellular sulfur-rich inclusions might contribute to the long-term survival of BGC under laboratory conditions.

In conclusion, we reported the discovery of two novel marine species of Nitrospirae MTB and their morphology and structural differences. Future genomic analysis would be helpful to understand the evolution and adaptation mechanisms of Nitrospirae magnetotactic bacteria to freshwater and marine environments.

Experimental Procedures

Sediment collection and MTB enrichment

Sediment samples were collected from Six-Four les Plages (43°07'N, 5°79'E) in the Mediterranean Sea, Southern France. The site was characterized by a salinity of ~35 ppt, pH of 7.5 and a temperature of 19°C at the time of sampling. Collected sediments were quickly transferred to 1000 ml plastic bottles, covered with approximately 600 ml of in situ seawater, transported to the laboratory, and incubated at room temperature without disturbance.

Black-brown giant cocci cells (BGC) in the sediment were magnetically enriched using the race-track method [START_REF] Wolfe | A 'capillary racetrack' method for isolation of magnetotactic bacteria[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]. The 16S rRNA genes were directly amplified from the collections. White giant cocci cells (WGC) were further collected by magnetic micro-sorting using a Transfer Man ONM-2D micromanipulator and an IM-9B CellTram Oil manual hydraulic pressure-control system installed on an IX51 microscope equipped with a 40 × LD objective [START_REF] Zhang | Characterization and phylogenetic identification of a species of spherical multicellular magnetotactic prokaryotes that produces both magnetite and greigite crystals[END_REF]. WGC cells were directly transferred to 3μl of PBS of the REPLI-g Single Cell Kit (QIAGEN). The PBS-suspended sample was frozen and thawed repeatedly, and the whole genome amplification (WGA) reaction was carried out using the REPLI-g Single Cell Kit [START_REF] Chen | A novel species of ellipsoidal multicellular magnetotactic prokaryotes from Lake Yuehu in China[END_REF]. The 16S rRNA gene sequences were obtained as previously described by Chen et al. [START_REF] Chen | A novel species of ellipsoidal multicellular magnetotactic prokaryotes from Lake Yuehu in China[END_REF].

Phylogenetic analysis

The 16S rDNA sequences were aligned using CLUSTAL W multiple alignment software.

Sequence identities were calculated with the BIOEDIT software. The phylogenetic tree was constructed via the neighbor-joining method using MEGA VERSION [START_REF] Kumar | MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets[END_REF], and boot-strap values were calculated from a 1000 replicates.

Figure legends Figure 1 .

 legends1 Figure legends

  (A) Differential interference contrast (DIC) micrograph shows magnetotaxis and accumulation of BGC (indicated by black arrows) and WGC (white arrows) at the north edge in a droplet. The big black arrow B indicates the direction of the magnetic field. (B) DIC micrograph shows the rough surface of BGC and smooth surface of WGC. (C) DIC micrograph shows both BGC and WGC under low salinity conditions. (C1) Cell inclusions released from BGC. (C2) Shrunken shapes of WGC. (D) DIC micrograph (D1) and fluorescence micrograph (D1-D5) shows auto-fluorescence of BGC and WGC excited by light at different wavelengths. (D1) BGC and WGC under white light. (D2) BGC emitted red fluorescence while WGC had no fluorescence emission when exposed to green light. (D3) BGC emitted stronger yellow fluorescence than WGC under blue light. (D4) BGC and WGC emitted similar green fluorescence under violet light. (D5) WGC emitted stronger blue fluorescence than BGC under ultraviolet light. (E) Swim tracks and velocities of WGC (n=54) and BGC (n=52). The scale bars: 5μm in A, C, D. 10μm in B and E.

Figure 2 .

 2 Figure 2. Phylogenetic tree of 16S rRNA gene sequences from Nitrospirae MTB.

Figure 3 .

 3 Figure 3. FISH identification of magnetotactic giant cocci.

Figure 4 .

 4 Figure 4. Element composition of BGC and WGC

Acknowledgments

We are grateful to H. Zhang and F.X. Wang for their assistance in the coordinated FISH-SEM experiments. X.X. Qian is grateful for her fellowship provided by the Chinese Scholarship Council. This work was supported by grants 41776131 and U1706208 from NSFC, grants 2016YFC0302502_02 and 2018YFC0309904 from National Key R&D Program of China, a grant XDB06010203 from the Strategic Priority Research Program, by a grant from the Excellence Initiative of Aix-Marseille University -A*Midex, a French "Investissements d'Avenir" programme, and grant from CNRS for LIA-MagMC. The FISH-SEM experiments were performed at the IGGCAS, Beijing, China and were supported by a NSFC grant 41522402.

Optical microscopy

The morphologies and motilities of the enriched MTB cells were examined under DIC microscopy (Olympus BX51 equipped with a DP71 camera; Olympus) using the hanging-drop method in an applied magnetic field. The ratio between BGC and WGC was estimated by counting the two morphotypes of cells at the edge of the droplets as shown in Supporting Information Figure S1. To analyze the sensitivity to the osmolality the collected cell suspensions were diluted with deionized water before observing their magnetotactic behavior in the droplets. The trajectories of the cells were obtained using the dark-field mode with an increased exposure time. To assess auto-fluorescence, fluorescence mirror units were used to expose the cells to green light (510-550 nm), blue light (450-480 nm), violet light (400-410 nm) and UV light (330-385 nm) with the corresponding filter sets of Olympus.

FISH analyses

BGC-specific probe (5'-ATACTTAATCTTCCGCAGACC-3') and WGC-specific probes (5'-GGTTCCACACCGAAGCATT-3') were designed according to the conserved 16S rRNA gene sequences of OTU-2 (GenBank number MK073023) and OTU-1 (GenBank number MK203828), respectively and evaluated for their specificity by using the online probe evaluation tools Ribosomal Database Project (RDP) https://rdp.cme.msu.edu/probematch/search.jsp [START_REF] Cole | The Ribosomal Database Project: improved alignments and new tools for rRNA analysis[END_REF][START_REF] Cole | Ribosomal Database Project: data and tools for high throughput rRNA analysis[END_REF]. The BGC-and WGC-specific probes were synthesized and fluorescently labeled with the hydrophilic sulfoindocyanine dye Cy3 at the 5' end. The universal bacterial probe EUB338

(5'-GCTGCCTCCCGTAGGAGT-3') was fluorescently labeled with fluorescein phosphoramidite 200 FAM at the 5' end and used as a positive control for FISH (Amann et al.,

Correlative FISH-SEM analyses

The FISH and correlative FISH-SEM analyses were carried out as described by Li et al. [START_REF] Li | Single-Cell Resolution of Uncultured Magnetotactic Bacteria via Fluorescence-Coupled Electron Microscopy[END_REF]. The same FISH coverslips of WGC were treated for SEM observations using a Zeiss Ultra-55 field-emission gun SEM (Carl Zeiss) operating at 5 kV [START_REF] Li | Single-Cell Resolution of Uncultured Magnetotactic Bacteria via Fluorescence-Coupled Electron Microscopy[END_REF].

HAADF-STEM elemental composition analysis

Magnetosome biomineralization was investigated using a JEM-2100F microscope (JEOLLtd) operating at 200 kV equipped with a field emission gun, an ultra-high resolution (UHR) pole piece and a Gatan energy filter (GIF2001) system (Gatan, Inc.). HAADF-STEM was used for Z-contrast imaging. Chemical compositional analysis was performed by EDXS elemental mapping in the HAADF-STEM mode. By using this method, nanometer-scale spatial distribution of most elements can be determined, and the relative abundance of each element at a certain location can be semi-quantitatively calculated [START_REF] Li | Single-Cell Resolution of Uncultured Magnetotactic Bacteria via Fluorescence-Coupled Electron Microscopy[END_REF].

Conflict of interest

The authors declare that they have no conflict of interest


Xin-Xin Qian

Claire-Lise Santini

Artemis Kosta

Nicolas Menguy

Hugo Le Guenno

Wenyan Zhang

Jinhua Li

Yi-Ran Chen

Jia Liu

François Alberto

Leon Espinosa

Tian Xiao

Long-Fei Wu
email: wu@imm.cnrs.fr

Long - Fei

Juxtaposed membranes underpin cellular adhesion and display unilateral cell division of multicellular magnetotactic prokaryotes

Multicellular magnetotactic prokaryotes (MMPs) exhibit peculiar coordination of swimming along geomagnetic field lines. Approximate 40-80 cells assemble, with a helical geometry or axisymmetry, into spherical or ellipsoidal MMPs, respectively. To contribute to a comprehensive understanding of bacterial multicellularity here we took multiple microscopic approaches to study the diversity, assembly, reproduction and motility of ellipsoidal MMPs.

Using correlative fluorescence in situ hybridization and scanning electron microscopy analysis, we found an unexpected diversity in populations of ellipsoidal MMPs in the Mediterranean Sea. The high-pressure freezing/freeze substitution fixation technique allowed us to show, for the first time, that cells adhere via juxtaposed membranes and are held together by a rimming lattice. Fluorescence confocal microscopy and ultra-thin section images revealed not only the one-layer hollow three-dimensional architecture, but also periphery-core unilateral constriction of constituent cells and unidirectional binary fission of the ellipsoidal MMPs. This finding suggests the evolution toward MMPs multicellularity via the mechanism of incomplete separation of offspring. Remarkably, thousands of flagellar at the periphery surface of cells underpin the coordinated swimming of MMPs in response to mechanical, chemical, magnetic and optical stimuli, including a magnetotactic photokinesis behavior. Together these results unveil the unique structure and function property of ellipsoidal MMPs.

Introduction

Multicellularity evolved independently and repeatedly through two major mechanisms, incomplete separation of daughter cells after division and motile cell aggregation [START_REF] Bonner | The origins of multicellularity[END_REF]. Morphological development results in the unbranched filaments with differentiated cells of cyanobacteria, the branched syncytial filaments of actinomycetes, and the fruiting bodies of myxobacteria [START_REF] Claessen | Bacterial solutions to multicellularity: a tale of biofilms, filaments and fruiting bodies[END_REF]. These multicellular bacteria reproduce, besides binary cell fission, by sporulation or fragmentation of filaments to form propagules.

The major modes of multicellular motility appear as the motile filaments of cyanobacteria and the social foraging for food by swarming myxobacteria [START_REF] Claessen | Bacterial solutions to multicellularity: a tale of biofilms, filaments and fruiting bodies[END_REF]. Other types of multicellular prokaryotes with distinct features of multicellularity remain poorly investigated.

During the evolution various organisms have developed magnetoreception capacity to exploit geomagnetic field as an orientation cue and maps while migrating or homing [START_REF] Johnsen | The physics and neurobiology of magnetoreception[END_REF]. Several hypotheses have been proposed to account for the mechanism of the magnetoreception. Increasing evidence supports a potential compass function of magnetite particles in magnetotactic bacteria (MTB). MTB consist of a heterogeneous group of Gram-negative bacteria that are capable of producing single domain magnetite (Fe 3 O 4 ) or greigite (Fe 3 S 4 ) crystals in intracellular organelles termed magnetosomes [START_REF] Bazylinski | Magnetosome formation in prokaryotes[END_REF]. The magnetosomes enable bacteria to align to and swim along geomagnetic field lines, a behavior is referred to as magnetotaxis. The hypothetic function of magnetotaxis is to simplify a three-dimensional search to a one-dimensional movement for cells to efficiently find and maintain at the preferable oxic-anoxic transition zone in chemically stratified sediments [START_REF] Bazylinski | Magnetosome formation in prokaryotes[END_REF]. Besides the most abundant unicellular MTB, a group of multicellular magnetotactic prokaryotes (MMPs) has attracted particularly our interest because they have opted both multicellular and magnetotactic properties during their evolution.

Multicellular magnetotactic prokaryotes were first discovered in the Rodrigo de Freitas lagoon in Brazil by [START_REF] Farina | Ultrastructure of a magnetotactic microorganism[END_REF]. Typically, 15-45 bacterial cells arrange with a helical geometry in a spherical or mulberry-like multicellular entity [START_REF] Keim | Structure, behavior, ecology and diversity of multicellular magnetotactic prokaryotes[END_REF]. This type of MMPs has been observed worldwide [START_REF] Rodgers | Intercellular structure in a many-celled magnetotactic prokaryote[END_REF][START_REF] Simmons | Unexpected diversity in populations of the manycelled magnetotactic prokaryote[END_REF][START_REF] Winklhofer | Magnetic optimization in a multicellular magnetotactic organism[END_REF][START_REF] Wenter | Ultrastructure, tactic behaviour and potential for sulfate reduction of a novel multicellular magnetotactic prokaryote from North Sea sediments[END_REF][START_REF] Shapiro | Multicellular photo-magnetotactic bacteria[END_REF][START_REF] Zhou | Adaptation of spherical multicellular magnetotactic prokaryotes to the geochemically variable habitat of an intertidal zone[END_REF]Zhang et al., 2014a). Besides the spherical MMPs (sMMPs), we have identified another morphotype, the ellipsoidal or pineapple-like MMPs (eMMPs) in the Mediterranean Sea [START_REF] Lefèvre | Characterization of mediterranean magnetotactic bacteria[END_REF][START_REF] Chen | Novel species and expanded distribution of ellipsoidal multicellular magnetotactic prokaryotes[END_REF], the China Sea, and the Pacific Ocean [START_REF] Zhou | A novel genus of multicellular magnetotactic prokaryotes from the Yellow Sea[END_REF][START_REF] Chen | A novel species of ellipsoidal multicellular magnetotactic prokaryotes from Lake Yuehu in China[END_REF][START_REF] Du | Temporal distributions and environmental adaptations of two types of multicellular magnetotactic prokaryote in the sediments of Lake Yuehu, China[END_REF][START_REF] Dong | The detection of magnetotactic bacteria in deep sea sediments from the east Pacific Manganese Nodule Province[END_REF][START_REF] Teng | Diversity and Characterization of Multicellular Magnetotactic Prokaryotes From Coral Reef Habitats of the Paracel Islands, South China Sea[END_REF].

Approximately 60 cells aggregate into four to six interlacing circles to form a ~ 5-10 µM ellipsoidal entity. Both morphotypes synthesize magnetite or greigite, or both, magnetosomes.

The common features for the two morphotype MMPs are that the periphery surface of their cells is covered by thousands of flagella whilst the inner surface surrounds a core lumen.

Both display two kinds of typical swimming behaviors of MMPs when inspected in droplets.

North-seeking MMPs swim and accumulate at the north edge of droplets. Randomly, they escape from the north edge and swim southward with acceleration. At variable distances, MMPs decelerate, stop the motility, and swim with acceleration northward back to the north edge [START_REF] Rodgers | Intercellular structure in a many-celled magnetotactic prokaryote[END_REF][START_REF] Simmons | Spatiotemporal distribution of marine magnetotactic bacteria in a seasonally stratified coastal salt pond[END_REF][START_REF] Zhou | A novel genus of multicellular magnetotactic prokaryotes from the Yellow Sea[END_REF][START_REF] Zhou | Adaptation of spherical multicellular magnetotactic prokaryotes to the geochemically variable habitat of an intertidal zone[END_REF]Zhang et al., 2014a;[START_REF] Chen | A novel species of ellipsoidal multicellular magnetotactic prokaryotes from Lake Yuehu in China[END_REF][START_REF] Chen | Novel species and expanded distribution of ellipsoidal multicellular magnetotactic prokaryotes[END_REF]. South-seeking MMPs exhibit a similar escape motion, but in the opposite direction (Lins de Barros et al., 1990;[START_REF] Lins | Imaging faces of shadowed magnetite (Fe(3)O(4)) crystals from magnetotactic bacteria with energy-filtering transmission electron microscopy[END_REF]Keim et al., 2004a;[START_REF] Greenberg | Observation of magnetoreceptive behavior in a multicellular magnetotactic prokaryote in higher than geomagnetic fields[END_REF]. This behavior is referred to as escape motility or ping-pong motion. In addition, illumination with blue, violet, or UV light triggers the MMPs to swim away from the droplet edges opposite to the magnetotaxis direction, which is referred to as a photophobic response or is sometimes miscalled negative phototaxis [START_REF] Shapiro | Multicellular photo-magnetotactic bacteria[END_REF][START_REF] Zhou | A novel genus of multicellular magnetotactic prokaryotes from the Yellow Sea[END_REF][START_REF] Zhou | Adaptation of spherical multicellular magnetotactic prokaryotes to the geochemically variable habitat of an intertidal zone[END_REF]Zhang et al., 2014a;[START_REF] Chen | A novel species of ellipsoidal multicellular magnetotactic prokaryotes from Lake Yuehu in China[END_REF][START_REF] Chen | Novel species and expanded distribution of ellipsoidal multicellular magnetotactic prokaryotes[END_REF] To gain more insight into the bacterial multicellularity, we performed multiple microscopy analyses of ellipsoidal MMPs. The results obtained contributed to better understanding about diversity, architecture, reproduction and motility of this peculiar multicellular prokaryote model organism.

Results

Diversity of multicellular magnetotactic prokaryotes

In general, single phylotype of MMPs was identified at a given sampling site [START_REF] Abreu | Candidatus Magnetoglobus multicellularis', a multicellular, magnetotactic prokaryote from a hypersaline environment[END_REF][START_REF] Wenter | Ultrastructure, tactic behaviour and potential for sulfate reduction of a novel multicellular magnetotactic prokaryote from North Sea sediments[END_REF][START_REF] Zhou | A novel genus of multicellular magnetotactic prokaryotes from the Yellow Sea[END_REF][START_REF] Zhou | Adaptation of spherical multicellular magnetotactic prokaryotes to the geochemically variable habitat of an intertidal zone[END_REF][START_REF] Kolinko | Single-cell genomics reveals potential for magnetite and greigite biomineralization in an uncultivated multicellular magnetotactic prokaryote[END_REF]Zhang et al., 2014a;[START_REF] Chen | A novel species of ellipsoidal multicellular magnetotactic prokaryotes from Lake Yuehu in China[END_REF][START_REF] Chen | Novel species and expanded distribution of ellipsoidal multicellular magnetotactic prokaryotes[END_REF]. An exception has been reported by Simmons et al. [START_REF] Simmons | Unexpected diversity in populations of the manycelled magnetotactic prokaryote[END_REF], they found five phylotypes of spherical MMPs in samples collected from Little Sippewissette salt marsh (Falmouth, MA, USA). The low natural abundance and similar morphology of MMPs prevented analysis of their diversity.

Recently, [START_REF] Li | Singlecellresolution of uncultured magnetotactic bacteria via fluorescence-coupled electron microscopy[END_REF] applied a coordinated FISH-SEM analysis procedure to efficiently identify the phylotypes of magnetotactic bacteria with simultaneous characterization of their magnetic crystals. Here, we used this procedure to assess the diversity of ellipsoidal MMPs of the Mediterranean Sea (Fig. 1A). We collected magnetotactic bacteria (MTB) by racetrack magnetic enrichment and obtained five 16S rRNA gene sequences that potentially belonged to MMPs. One of them (SF-1) was identical to the previously identified ellipsoidal MMPs collected from this site [START_REF] Chen | Novel species and expanded distribution of ellipsoidal multicellular magnetotactic prokaryotes[END_REF], and the others shared higher than 90% identity with SF-1. Using correlative FISH-SEM analysis, we confirmed the authenticity of the ellipsoidal MMPs SF-2 to SF-5 and showed speciesdependent cell morphology and magnetosome arrangement in the MMPs (Fig. 1B, C, D, andE). These results unambiguously show the diversity of ellipsoidal MMPs at this sampling site.

In parallel, we also used magnetic micromanipulation to sort MMPs collected from Lake Yuehu and Six-Fours-les-Plages (see Experimental Procedures), and amplified and sequenced their 16S rRNA genes as previously reported [START_REF] Kolinko | Single-cell genomics reveals potential for magnetite and greigite biomineralization in an uncultivated multicellular magnetotactic prokaryote[END_REF]Zhang et al., 2014a;[START_REF] Chen | A novel species of ellipsoidal multicellular magnetotactic prokaryotes from Lake Yuehu in China[END_REF][START_REF] Chen | Novel species and expanded distribution of ellipsoidal multicellular magnetotactic prokaryotes[END_REF]. In total, we obtained 11 new 16S rRNA gene sequences, identifying five species belonging to three genera of spherical MMPs and six species belonging to three genera of ellipsoidal MMPs (Fig. 2A), using a 97% and 95% sequence identity as the threshold for classifying species and genera, respectively [START_REF] Tindall | Notes on the characterization of prokaryote strains for taxonomic purposes[END_REF]. Our study has therefore extended the total number of identified spherical and ellipsoidal MMPs to eleven species belonging to six genera and nine species belonging to six genera, respectively. They formed branches of a MMP clade that were affiliated with Deltaproteobacteria but were distinguished from the multicellular myxobacteria (Fig. 2B).

The worldwide distribution and the great diversity of species justify the consideration of MMPs as a distinct taxonomic group of multicellular prokaryotes.

Cellular arrangement within ellipsoidal MMPs

Previously we have reported the architecture of ellipsoidal MMPs using fluorescence microscopy, transmission electron microscope (TEM), scan electron microscope (SEM), and focused ion beam SEM (FIB-SEM) [START_REF] Zhou | A novel genus of multicellular magnetotactic prokaryotes from the Yellow Sea[END_REF][START_REF] Chen | A novel species of ellipsoidal multicellular magnetotactic prokaryotes from Lake Yuehu in China[END_REF][START_REF] Chen | Novel species and expanded distribution of ellipsoidal multicellular magnetotactic prokaryotes[END_REF][START_REF] Leao | Ultrastructure of ellipsoidal magnetotactic multicellular prokaryotes depicts their complex assemblage and cellular polarity in the context of magnetotaxis[END_REF]. Approximately 40-80 cells arrange in interlacing circles with a pineapplelike ellipsoidal morphology. Partly because of the MMP size, the resolution that can be achieved by these microscopy techniques is limited. In addition, knowledge about overall three-dimensional arrangement of cells in ellipsoidal MMPs needs to be improved. HPF/FS fixation is an optimal method to preserve the structures present in the living cell up to a thickness of 200 µm. Here, we used HPF/FS, for the first time, in combination with TEM and fluorescence confocal microscopy to study the ultrastructure of MMPs. As previously reported [START_REF] Leao | Ultrastructure of ellipsoidal magnetotactic multicellular prokaryotes depicts their complex assemblage and cellular polarity in the context of magnetotaxis[END_REF] but with more detail, we observed the conspicuous peripherycore architecture (Fig. 3A, B, C, E andG), an axisymmetrical arrangement of cells along the eMMP longitudinal axis (Fig. 3A), a "open space" at one end (Fig. 3A, top) and a core lumen in the center of ellipsoidal MMPs (Fig. 3G andK). We also found at surface of the MMPs, flagella (Fig. 3A andI), and, in the cytoplasm, magnetite magnetosomes (Fig. 3A,B,D,F,G and H), lipid granules, (Fig. 3A, D, G and I), and periphery bars at the external side of the cells (green arrows in Fig. 3J and SI, Fig. S2).

Besides these structures, ultrathin sections of HPF/FS samples unveiled an unreported electron dense dashed line surrounded the outermost surface (Fig. 3A, G, H, and J, yellow arrows). The dashes were approximately 11.6 x 20.7 nm (average of 193 points from two MMPs) and aligned with an interval distance of 10.3 nm (average of 183 points from two MMPs) along the perimeter of a matrix outside the outer membrane of the MMPs (Fig. 3G, H, andJ). The dashed lines on two successive ultrathin sections arranged in a similar pattern (SI, Fig. S1). Therefore, it is likely that the regular distributed dashes are a cut-view of a lattice that holds the cells together (Fig. 3G, H, andJ). Underneath the matrix layer, there are the well-preserved three-layer structures of the outer membrane, peptidoglycan, and the inner membrane (Fig. 3G, H, I and J). The high quality of preservation unveiled unambiguously that juxtaposed membranes tightly connect cells together to assemble into a multicellular entity (Fig. 3D and G, red asterisks). Close to the periphery side of the inner membranes, a fence-like structure (f) was observed in some cells (cell 4 in Fig. 3G andJ), which recalls the structure of photosynthetic membrane lamellae in prokaryotic cells. The laser confocal analysis and reconstitution of ultrathin section images clearly revealed that ellipsoidal MMPs consist of only one layer of cells that are all connected to the core lumen (Fig. 3C,E, SI Movie 1). Taken together, juxtaposed membranes adhere cells together surrounding the core lumen to achieve a one-layer hollow structure that is held by a well-patterned lattice at the surface of ellipsoidal MMPs.

Reproduction of ellipsoidal MMPs

Ellipsoidal MMPs consist of cells that are arranged axisymmetrically along the long body axis and radially on the short axis dissection. We investigated how they reproduce to maintain the inherent periphery-core architecture and a magnetic polarity. Ultrathin sections of HPF/FS-or chemically fixed MMPs revealed that all dividing cells exhibit a unilateral indentation of the cell wall from the periphery towards the core lumen (Fig. 4B andC, red asterisks and yellow triangles). The high-quality preservation of the membrane structures by HPF/FS exposes the process of constituent cell division. As shown on two successive ultrathin slices of an HPF/FS-fixed eMMP (Fig. 4D andE1), outer membranes bend inward from the peripheral surface and the two sides then join together, doubling the thickness, and invaginate into the cytoplasm. The constriction furrow penetrates unilaterally into the cytoplasm without initiating indentation of the membranes at the core lumen side, even when the constriction furrows into three-fifths of the depth of the cytoplasm (Fig. 4D, E1, E2, and E3). We analyzed ultrathin sections with the core lumen of eleven MMPs and found approximately 50% of cells having a constriction furrow. In all cases, we observed the indentation occurring unilaterally only from the periphery toward the lumen. The inward bending and doubling thickness of the outer membrane at the onset of cytokinesis displayed the same structure as the outer membrane between two neighboring cells (Fig. 4D andE compared to F, red asterisks). As shown in Fig. 4D, the outer membranes connect the cells together. Therefore, the juxtaposed membrane implies a characteristic adhesion of MMP cells. Moreover, the surface lattice and matrix seem to prevent the cells from separation from the aggregates after the division. These observations strongly suggest the evolution of MMP toward multicellularity via the mechanism of incomplete separation of offspring [START_REF] Bonner | The origins of multicellularity[END_REF].

Further fluorescence confocal microscopy inspection of cells shows membranes that separated neighboring cells on the surface stack images (Fig. 4G1, yellow arrows). However, on the deeper stack images in some cases, the membranes disappeared progressively first at the center, leading to 'H-shaped' cells (Fig. 4G2 to G4), and then at the opposite corners of the dividing cells (SI Movie S2). These results are consistent with the hypothesis that unilateral constriction of cell envelope occurs from the periphery toward the core lumen parallel to the long axis of MMPs. Moreover, the "H-shaped" cells that were previously observed using FIB-SEM microscopy reflect a stage of cell division instead of a permanent morphology of MMP cells. Notably the HPF/FS slice in Fig. 4H shows the split of an ellipsoidal multicellular magnetotactic prokaryote, which corresponds to one state of the division process revealed by confocal microscopy (Fig. 5I). A collection of confocal analyses corroborated the hypothetical unidirectional split of the eMMP entity using fluorescence staining to visualize the membrane (Fig. 4I, SI Movie S3) or intracellular lipid granules (Fig. 4J, SI Movie S4).

Bounce motion, axial magnetotaxis, and magnetotactic-photokinesis of ellipsoidal MMPs

Multicellular magnetotactic prokaryotes possessed multiple magnetosome chains arranged along their long axis (Fig. 5A) and were covered by flagella (Fig. 5B). The HPF/FS thin section images showed that the flagella have a diameter of approximately 22 nm with an interval distance of 68 nm (Fig. 3I). If the flagella are separated by equal longitudinal and latitudinal distances, we can assume a flagella density of 1 flagellum per 8100 nm squares (123 flagella per µm square), which could lead to approximately ten thousand flagella on a 4

x 8 µm MMP. It is similar to the 147 flagella motors per µm square estimated for spherical MMPs [START_REF] Silva | Flagellar apparatus of south-seeking many-celled magnetotactic prokaryotes[END_REF]. The multiple bundles of magnetosomes and thousands of flagella underpin the canonical magnetotactic behavior of MMPs. Previous studies of MMPs swimming in droplets have shown the typical escape and photophobic motilities. To gain deeper insight into these behaviors we further analyzed MMPs swimming in microfluidic channels (see Experimental Procedures) to mitigate the air-liquid-solid border effect, and irregular light transmission that occurs in droplets.

The swim velocity of ellipsoidal MMPs collected from environmental samples when compared one to another, displayed up to a ten-fold variation and the velocities decreased progressively as the observation time increased. We observed eMMP swimming northward with an average velocity of 34.2 ± 14.7 µm/s (n=12). When they performed escape or pingpong motion eMMPs swam southward at 20.0 ± 9.3 µm/s (n=14) with instantaneous acceleration ranging from 87.6 to 6.3 µm/s 2 , which was followed by a return northward swimming at of 13.5 ± 6.1 µm/s (n=10). Despite the substantial variation of velocity among individuals the pattern of each type of swimming behavior was very well conserved and examination of instantaneous velocity change of a given multicellular magnetotactic prokaryote in response to an environmental stimulus is meaningful. We observed backward motility of eMMPs accumulated at north side in microchannels (Fig. 5C, SI Movie 5).

Notably the backward motion in microchannels shares a similar pattern with the escape or ping-pong motion of MMPs in droplets; eMMP swim to and accumulate at the north edge in droplets or in microchannels, where some of them randomly make a backward swim with acceleration. The mechanism involved in eMMPs sensing of microchannel walls might be different from that of sensing the surface/border of the droplets although both the wall and the border prevent MMPs from further northward swim. We also observed a conspicuous backward motion when MMPs encountered obstacles (Fig. 5D, SI Movie 6). The channel walls and obstacles blocked the forward north-seeking motility and triggered the MMPs to swim backwards in order to circumvent the obstacles. Hence, we propose that the two behaviors are considered a bounce motion. Moreover, at times MMPs randomly changed their swimming direction southward and subsequently changed back to a north-seeking swim (Fig. 5E, SI Movie 7), which is typical behavior of axial magnetotaxis [START_REF] Bazylinski | Magnetosome formation in prokaryotes[END_REF]. The stochastic backward motion may play a similar physiological function to the tumbling of Escherichia coli that allows bacteria to randomly explore the favorable direction in which to go. It is noteworthy that the backward swims in bounce motion and axial magnetotaxis started with the highest acceleration (red arrows and 'ac' in Fig. 5D and5E) and had higher instantaneous velocity than the forward swim (red curves versus blue curves).

To characterize the photosensitive magnetotaxis of MMPs in microchannels, we applied two light-sources, i.e. a transmission light for observation and imaging and a UV (385 nm, 203 µW/cm 2 ) epi-illumination on a defined area for analyzing the photo-effect on swimming behavior. The swimming of MMPs was maintained within the illumination spots via application of an alternate uniform magnetic field (10 Gs, 0.5 Hz) in order to periodically reverse the swimming direction of MMPs before they left the illumination area. As shown in Fig. 5F1, an eMMP initially swam northward at 100 µm/s in the UV spot (green track). After the reversal of the field direction, the MMP immediately reversed its swimming direction to keep the north-seeking swim (Fig. 5F1 the U-turn of the green dot track, 3F2 the gap of green track at frame 40 and the change of velocity from positive to negative). Approximately 0.2 seconds later, the MMP suddenly changed its swimming direction to southward (red arrow and "r") with an acceleration (ac) that doubled the velocity, reaching approximately 200 µm/s (Fig. 5F1, SI Movie 8 and Movie 9). Therefore, the illumination resulted in not only the photophobic response (SI Movie 10), but also in photokinesis of the magnetotaxis, i.e. a change to the direction and velocity of motility.

Discussion

Our structure and behavior analyses unveil hallmark features of multicellularity that distinguishes MMPs from other extensively studied multicellular bacteria. Both myxobacteria and MMPs are affiliated with Deltaproteobacteria (Fig. 2B). Lying on the boundary between uni-and multicellular organisms, myxobacteria grow and divide as proper unicellular Gramnegative bacteria. However, they feed socially via collective predation. Moreover, under starvation conditions, hundreds of thousands of cells form compact and symmetric structures, i.e. fruiting bodies that confer on myxobacteria selective advantages for spore dispersion and cooperative-feeding [START_REF] Kaiser | Building a multicellular organism[END_REF]. On the contrary, MMPs are patterned, permanent multicellular entities. They are constructed via tight cell membrane juxtaposition and an outer matrix envelope. The spherical and ellipsoidal morphotypes of MMPs display different assembly symmetries. The structures of the well-studied spherical MMPs consist of 15-40 cells that are arranged with a helical symmetry into a mulberry-like morphology [START_REF] Farina | Ultrastructure of a magnetotactic microorganism[END_REF][START_REF] Rodgers | Intercellular structure in a many-celled magnetotactic prokaryote[END_REF][START_REF] Farina | Magnetic iron-sulphur crystals from a magnetotactic microorganism[END_REF]Lins de Barros et al., 1990;[START_REF] Mann | Magnetotactic bacteria: microbiology, biomineralization, palaeomagnetism and biotechnology[END_REF][START_REF] Rodgers | Intercellular structure in a many-celled magnetotactic prokaryote[END_REF][START_REF] Lins | Imaging faces of shadowed magnetite (Fe(3)O(4)) crystals from magnetotactic bacteria with energy-filtering transmission electron microscopy[END_REF]Keim et al., 2004b). Although the short-axis dissection of ellipsoidal MMPs exhibits radial symmetry like spherical MMPs, but only the ellipsoidal MMP cells axisymmetrically assemble into several rows to form a pineapple-like morphology. At present no unicellular stage has been observed for MMPs ((Keim et al., 2004a;Keim et al., 2004b;[START_REF] Abreu | Cell viability in magnetotactic multicellular prokaryotes[END_REF][START_REF] Zhou | A novel genus of multicellular magnetotactic prokaryotes from the Yellow Sea[END_REF][START_REF] Abreu | Deciphering unusual uncultured magnetotactic multicellular prokaryotes through genomics[END_REF]Zhang et al., 2014a;[START_REF] Chen | A novel species of ellipsoidal multicellular magnetotactic prokaryotes from Lake Yuehu in China[END_REF][START_REF] Chen | Novel species and expanded distribution of ellipsoidal multicellular magnetotactic prokaryotes[END_REF], and FISH in this study). Our phylogenetic analysis, based on 16S rRNA gene sequences, revealed that the MMP cluster is located distantly to myxobacteria and other unicellular magnetotactic bacteria (Fig. 2B).

Interestingly, the genes involved in magnetosome biosynthesis are well conserved among uni-and multicellular magnetotactic bacteria from the Deltaproteobacteria class [START_REF] Leao | Ultrastructure of ellipsoidal magnetotactic multicellular prokaryotes depicts their complex assemblage and cellular polarity in the context of magnetotaxis[END_REF]. This result cannot distinguish between acquisitions by nonmagnetic multicellular microbes of magnetosome coding genes via horizontal gene transfer from evolution of MMPs from unicellular magnetotactic Deltaproteobacteria. Future genomic phylogenetic analyses should shed light on the evolution mechanism of MMPs. The data presented here support the hypothesis that myxobacteria and MMPs gained their multicellularity in separate events.

Coordinated swimming behavior is a fundamental feature that emerged during the evolution of multicellularity. The paradigm of bacterial chemotaxis is underpinned by intracellular diffusion of phosphorylated proteins and their binding to flagellar motors to steer the swimming behavior in response to environmental stimuli [START_REF] Wadhams | Making sense of it all: Bacterial chemotaxis[END_REF]).

MMPs exhibit a highly complex motion. In addition to the escape swim and photophobic response, here we showed the bounce motion, axial magnetotaxis, and magneto-photokinesis behavior of ellipsoidal MMPs. While unicellular magnetotactic bacteria display aero-magnetotaxis and photophobic behavior [START_REF] Frankel | Magneto-aerotaxis in marine coccoid bacteria[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], magnetophotokinesis has been observed only for multicellular magnetotactic prokaryotes. MMPs dwell in intertidal sediments as deep as 30 cm and undergo seasonal, and probably also daily, vertical movement in response to nutrient distribution changes [START_REF] Liu | Seasonal changes in the vertical distribution of two types of multicellular magnetotactic prokaryotes in the sediment of Lake Yuehu, China[END_REF]. Shapiro et al. have suggested that photophobic behavior enables MMPs to optimize their location to adapt to circadian variation of chemical gradients and light intensity [START_REF] Shapiro | Multicellular photo-magnetotactic bacteria[END_REF]. Indeed genes involved in controlling circadian rhythm were found in the genomes of spherical [START_REF] Abreu | Deciphering unusual uncultured magnetotactic multicellular prokaryotes through genomics[END_REF] and ellipsoidal MMP strain SF-1 (SI Table S1). The bounce motility and photokinesis capacity may be selective strategies for MMPs to move to favorable places in sediments. Moreover, MMPs swim faster than most unicellular bacteria and have large sizes, which imply an advantage in mitigating predation risk.

Bacterial photo-sensing might rely directly on dedicated photoreceptor systems or indirectly on the products of photosynthesis or other illumination by-products, i.e. reactive oxygen species, ATP, change of intracellular redox or force proton motif. The fence-like structure (Fig. 3J), which looks like photosynthetic membrane lamellae, could be an appropriate candidate for accommodating photoreceptors involved in MMPs photo-sensing. Multicellular magnetotactic prokaryotes displayed a helical trajectory of swimming and were illuminated with UV-light perpendicularly to the translation direction. They changed the magnetotaxis direction and velocity suddenly within the illumination area. Therefore, the magneto-photokinesis is unlikely to be a result of the detection of an intracellular spatial light gradient.

The sudden change of swim direction under constant illumination would suggest a cumulating effect of periodical exposure of photoreceptive structures to UV-light or production of harmful by-products. Therefore, multicellular magnetotactic prokaryotes reversed their swimming direction to escape from the deleterious light. It remains an enigma how thousands of flagella of 60-80 cells coordinate their rotations to propel the helical translation of MMPs. Therefore, the mechanism that governs the change of magnetotaxis direction is inexplicable. Both HPF/FS section and confocal data revealed that the core lumen connects all cells in a MMP, and the lumen thus might play a crucial role in the communication required to regulate the operation of flagella at the surface.

Reproduction of MMPs shows some particularities compared to other multicellular prokaryotes. Until now, no single-cell stage has been observed in the life cycle of MMPs (Keim et al., 2004a;Keim et al., 2004b;[START_REF] Abreu | Cell viability in magnetotactic multicellular prokaryotes[END_REF][START_REF] Abreu | Candidatus Magnetoglobus multicellularis', a multicellular, magnetotactic prokaryote from a hypersaline environment[END_REF][START_REF] Abreu | Deciphering unusual uncultured magnetotactic multicellular prokaryotes through genomics[END_REF]. Keim et al. have proposed that spherical MMPs reproduce by two major steps: division of individual cells via invaginations of the cell membrane at the external side, and "8"-shaped torsion fission of MMPs into two equal spheres (Keim et al., 2004b). Here, the highly preserved structure of ultrathin sections unambiguously reveals the unilateral constriction of MMP cells (Fig. 4). It has been reported recently that nematode symbiont rodshaped Gammaproteobacteria initiate division by membrane invagination only at the proximal pole that is attached to the host cuticle [START_REF] Pende | Hostpolarized cell growth in animal symbionts[END_REF]. The fission progresses by membrane invagination at the distal end and the two constriction furrows penetrate longitudinally towards the center until there is a complete division. Similarly, electron cryotomography analysis of unicellular bacteria reveals that some dividing cells from eight bacterial species initiate indentations of the cell envelope on only one side of the cells at the early-constriction state [START_REF] Yao | Short FtsZ filaments can drive asymmetric cell envelope constriction at the onset of bacterial cytokinesis[END_REF]. Subsequently, all mid-constriction cells exhibit constriction on both sides of the cell body, and the division furrows converge into a neck-like structure in the late-constriction cells, which indicates bidirectional division of these cells.

Cyanelles are primitive photosynthetic organelles of protists Cyanophora paradoxa. During their reproduction a shallow furrow starts asymmetrically on one side of the division plane, resulting in kidney-shaped cyanelles. Subsequently the furrow extends, covers, and deepens around the entire circumference of the division plane, leading to dumbbell-shaped cyanelles and a complete division [START_REF] Chang | Developmental regulation of oligosialylation in zebrafish[END_REF]. The MMP cells are distinguished from other asymmetrically constricted unicellular cells by the apparent absence of bilateral indentations of the membrane even at late stages of the division. The periphery-core architecture of MMPs might prevent the symmetric bilateral constriction. The periphery bars, which are located underneath the outer curved membrane and close to the magnetosome chains in MMP cells (Fig. 3J, SI Appendix, Fig. S2), might play a role in determining the onset of unilateral cell constriction. Interestingly, the unicellular magnetotactic bacterium Magnetospirillum gryphiswaldense reproduces via asymmetric, wedge-like constriction of the cell wall at the division site, which leads to snapping of the magnetosome chain into halves in the two offspring with minimal magnetostatic attraction [START_REF] Katzmann | Magnetosome chains are recruited to cellular division sites and split by asymmetric septation[END_REF]. Unilateral indentation of the MMP cell wall seems to be a similar mechanism of the segregation of magnetosome chains in the multiple daughter cells that remain tightly connected after division.

The lack of MMP cultures prevents detailed study of the mechanisms of cell and entity divisions. Whole-genome sequencing and analysis might circumvent this obstacle. Moreover, it is crucial to develop a dedicated microscope device in order to determine precisely the wavelength spectra that trigger photokinesis and to perform real-time inspection of the MMP lifecycle in microchannels. Using multicellular magnetotactic prokaryotes as a novel model of multicellular organisms could provide a different way of thinking about the evolution of multicellularity. The periphery-core architecture restricts the mechanisms that govern cell division, cell-cell adhesion and communication, swimming coordination, and distinguishes MMPs from the filamentous cyanobacteria and actinomycetes.

Experimental Procedures

Phylogenetic analysis

Sediment samples were collected from Six-Fours-les-Plages (43°07'N, 5°79'E) of the Mediterranean Sea, or Lake Yuehu (37°21'N, 122°34'E) of the China Sea. Magnetotactic bacteria were magnetically enriched using the race-track method [START_REF] Wolfe | A "capillary racetrack" method for isolation of magnetotactic bacteria[END_REF]. The 16S rRNA genes were directly amplified from the collected samples and sequenced, or after further collection of MMPs by magnetic manipulation microsorting, and then amplified and sequenced as previously reported [START_REF] Chen | A novel species of ellipsoidal multicellular magnetotactic prokaryotes from Lake Yuehu in China[END_REF]. The ellipsoidal MMPs were sorted using a TransferMan ONM-2D micromanipulator and an IM-9B CellTram Oil manual hydraulic pressure-control system installed on an IX51 microscope equipped with a 40 × LD objective (all from Olympus). One droplet (20 μl) containing magnetically collected MMPs and several droplets of 20 μl filter-sterilized (0.2 μm pore size) sample water were placed onto a glass slide. The ellipsoidal MMPs were transferred using micromanipulator from magnetic collections successively to the droplets of filter-sterilized sample water. In the transferred washing steps, the MMPs were aspirated and expulsed by the glass capillary (at least 10 times) for removal of contaminants and extracellular DNA [START_REF] Chen | A novel species of ellipsoidal multicellular magnetotactic prokaryotes from Lake Yuehu in China[END_REF].

Finally, two to ten purified MMPs were directly transferred to one reaction tube containing 3 μl of PBS, which was in REPLI-g Single Cell Kit (cat # 150343; QIAGEN, German). The PBS-suspended sample was frozen and thawed repeatedly, and the WGA reaction was carried out using the REPLI-g Single Cell Kit. Only the samples containing single homogenous 16S rRNA sequence were used in this study.

Characterization of swimming behavior

Swimming behaviors of MMPs were analyzed either in droplets with the optical microscopes Olympus BX51 or Nikon TiE [START_REF] Chen | A novel species of ellipsoidal multicellular magnetotactic prokaryotes from Lake Yuehu in China[END_REF], or in microfluidic channels with a Magnetodrome device (2D and 3D µ-Slide Chemotaxis channels, Ibidi, GmbH, Germany; or home-made channels as described in (Zhang et al., 2014b)). To characterize photokinesis behavior, we modified the Zeiss Axio Observer A1 microscope on the Magnetodrome device (Zhang et al., 2014b) by replacing the HBO 100 light source with Zeiss Colibri 7. A UV light (385 nm) at 203 µW/cm 2 was used to trigger photophobic and photokinesis behavior. The swimming of MMPs was maintained within the UV spot by application of an alternate uniform magnetic field (10 Gs, 0.5 Hz) in order to periodically reverse the swimming direction of MMP before they left the illumination area. The motility was analyzed using MTrackJ Plugins of ImageJ [START_REF] Meijering | Methods for cell and particle tracking[END_REF].

Microscopy analyses

Routine optical microscopy observation was performed with Zeiss Axiostar Plus, Zeiss Axio Vert 200M, and Olympus BX51. To perform laser confocal analysis, MMPs were fixed with 1% paraformaldehyde for 1.5 hours at room temperature, stained with 15 µg/ml Nil red (for lipids), 7.5 µg/ml FM4-64 (for membranes), and 2µg/ml DAPI (for chromosomal DNA) and observed with the Olympus FV1000 microscope with laser 405 nm excitation and 425-475 nm emission for DAPI and 543 nm excitation and 555-655 nm emission for Nil Red and FM4-64. Images were collected at a series of focal levels at 0.17 µm intervals. Threedimensional image reconstitution was performed using ImageJ and Amira software.

Correlative FISH-SEM analyses were carried out on coverslips as described by [START_REF] Li | Singlecellresolution of uncultured magnetotactic bacteria via fluorescence-coupled electron microscopy[END_REF]. Briefly, we designed four probes that specifically target to the 16S rRNA gene sequences of ellipsoidal MMPs (SI Appendix, Table S2) and evaluated their specificity using the online probe evaluation tool Ribosomal Database Project [START_REF] Cole | The Ribosomal Database Project: improved alignments and new tools for rRNA analysis[END_REF][START_REF] Cole | Ribosomal Database Project: data and tools for high throughput rRNA analysis[END_REF]. The MMP-specific probes were synthesized and fluorescently labeled with the hydrophilic sulfoindocyanine dye Cy3 at the 5' end. The universal bacterial probe EUB338 (Table S1) was fluorescently labeled with fluorescein phosphoramidite 200 FAM at the 5' end and used for FISH as a positive-control probe for bacteria [START_REF] Amann | Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology[END_REF]. FISH observation was performed with a Nikon TiE microscope by following the protocols reported by [START_REF] Simmons | Unexpected diversity in populations of the manycelled magnetotactic prokaryote[END_REF] and [START_REF] Li | Singlecellresolution of uncultured magnetotactic bacteria via fluorescence-coupled electron microscopy[END_REF]. The same coverslips were treated for SEM observation using a Zeiss Ultra-55 field-emission gun SEM (Carl Zeiss, Germany) operating at 5 kV [START_REF] Li | Singlecellresolution of uncultured magnetotactic bacteria via fluorescence-coupled electron microscopy[END_REF]. General SEM morphology analyses of MMPs were carried out using a KVKV-2800B microscope as described by Chen et al. [START_REF] Chen | A novel species of ellipsoidal multicellular magnetotactic prokaryotes from Lake Yuehu in China[END_REF].

Transmission electron microscopy

Magnetotactic bacteria were pelleted, high-pressure frozen, freeze substituted, embedded in Epon resin (Medium Grade), as reported in [START_REF] Mcdonald | Rapid embedding methods into epoxy and LR White resins for morphological and immunological analysis of cryofixed biological specimens[END_REF]. Because only low amount of MMPs could be collected from fresh environmental samples each time, cells of marine unicellular photobacterium cultures were added during HPF/FS preparation. Ultrathin sections (60-90 nm) were cut with an ultracryomicrotome (EM UC7 Leica) and stained with uranyl acetate and lead citrate [START_REF] Reynolds | The use of lead citrate at high pH as an electron-opaque stain in electron microscopy[END_REF]. The samples were analyzed using a Tecnai 200KV electron microscope (FEI) and digital acquisitions were made using a numeric camera (Eagle, FEI). Alternatively, the grids were analyzed using STEM-HAADF for Zcontrast imaging, using a JEM-2100F microscope (JEOLLtd., Tokyo, Japan) operating at 200 kV equipped with a field emission gun, an ultra-high resolution pole piece and a Gatan energy filter (GIF2001) system (Gatan, Inc., Pleasanto, 242 CA, USA) to determine element composition. High resolution TEM images were analyzes with Fast Fourier Transform (FFT) procedure using ImageJ 1.52o software (http://imagej.nih.gov/ij/). grants from CNRS for LIA-MagMC. The FISH-SEM experiments were performed at the 457 IGGCAS, Beijing, China and were supported by a NSFC grant 41522402. 458 (green track) changing its swimming direction in response to the reversal of the alternate uniform magnetic field (10 Gs, 0.5 Hz). After 0.2 second it abruptly reversed its swimming direction to south-seeking (arrow "r" and red track) with acceleration (ac) and continued a south-seeking motion out of the UV spot. The movies were recorded at 60 frames per second (fps) for D and E and 30 fps for F.

Figure 1 .

 1 Figure 1. Identification of Mediterranean MMPs by correlative microscopy.

Figure 2 .

 2 Figure 2. Diversity and phylogenetic tree of MMPs.

Figure 3 .

 3 Figure 3. Ultrastructure of ellipsoidal MMPs.

Figure 4 .

 4 Figure 4. Division of constituent cells and reproduction of MMPs.

Fig

  Fig. 3G. (E2) and (E3) are magnified images of (E1) to show the end (yellow triangles) of the

Figure 5 .

 5 Figure 5. Bounce motion and photokinesis of ellipsoidal MMPs.

Acknowledgments

We thank A. Bernadac and Y. Pan for critical reading of the manuscript, A. Bernadac for suggestions for sample preparation and N. Petersen for Magnetodrome assistance. We are grateful to H. Zhang and F.X. Wang for their assistance in the coordinated FISH-SEM experiments. This work was supported by a funding from the Excellence Initiative of Aix-Marseille University -A*Midex, a French "Investissements d'Avenir" programme, by grants 41330962 and 41506147 from NSFC, grant 2018YFC0309904 from National Key R&D Program of China, grant XDB06010203 from the Strategic Priority Research Program and