Elodie C T Descamps

Caroline L Monteil

Nicolas Menguy

Nicolas Ginet

David Pignol

Dennis A Bazylinski

Christopher T Lefèvre
email: christopher.lefevre@cea.fr

a magnetotactic bacterium from a brackish desert spring able to biomineralize greigite and magnetite, that represents a novel lineage in the Desulfobacteraceae

Keywords: sp nov., Desulfamplus magnetovallimortis gen. nov., sp. nov Desulfamplus magnetovallimortis, BW-1, magnetotactic bacteria, greigite, magnetite, magnetotaxis, magnetosome, Deltaproteobacteria, sulfate-reducing bacteria

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Desulfamplus magnetovallimortis gen. nov.

Introduction

Magnetotactic bacteria represent a phylogenetically, ecologically and metabolically diverse group of prokaryotes able to biomineralize single-magnetic-domain nanocrystals of magnetite (an iron oxide (Fe(II)Fe(III) 2 O 4 )) or greigite (an iron sulfide (Fe(II)Fe(III) 2 S 4 )) in specific organelles [START_REF] Bazylinski | Magnetosome formation in prokaryotes[END_REF]. These organelles, called magnetosomes, are aligned in one or more chains within the cytoplasm and cause cells to actively swim along the Earth's magnetic field lines [START_REF] Frankel | Magnetoaerotaxis in marine coccoid bacteria[END_REF]. This behavior, termed magnetotaxis, makes the search for optimal concentrations of certain nutrients and redox conditions within water columns and sediments more efficient by simplifying a three-dimensional search to a linear search [START_REF] Frankel | Magnetoaerotaxis in marine coccoid bacteria[END_REF].

Although magnetotactic bacteria were discovered 40 years ago [START_REF] Blakemore | Magnetotactic Bacteria[END_REF], relatively few strains have been cultivated in pure culture. Officially described and cultured magnetotactic species are all magnetite-producers and belong to the genera Magnetospirillum [START_REF] Maratea | Aquaspirillum magnetotacticum sp nov, a magnetic spirillum[END_REF][START_REF] Matsunaga | Magnetite formation by a magnetic bacterium capable of growing aerobically[END_REF][START_REF] Schleifer | The genus Magnetospirillum gen. nov. description of Magnetospirillum gryphiswaldense sp. nov. and transfer of Aquaspirillum magnetotacticum to Magnetospirillum magnetotacticum comb[END_REF], Desulfovibrio [START_REF] Sakaguchi | Desulfovibrio magneticus sp nov., a novel sulfate-reducing bacterium that produces intracellular single-domain-sized magnetite particles[END_REF], Magnetovibrio [START_REF] Bazylinski | Magnetovibrio blakemorei, gen. nov. sp. nov., a new magnetotactic bacterium (Alphaproteobacteria: Rhodospirillaceae) isolated from a salt marsh[END_REF], Magnetococcus [START_REF] Bazylinski | Magnetococcus marinus gen. nov., sp. nov., a marine, magnetotactic bacterium that represents a novel lineage (Magnetococcaceae fam. nov.; Magnetococcales ord. nov.) at the base of the Alphaproteobacteria[END_REF] and Magnetospira [START_REF] Williams | Magnetospira thiophila gen. nov., sp. nov., a marine magnetotactic bacterium that represents a novel lineage within the Rhodospirillaceae (Alphaproteobacteria)[END_REF]. The candidate genus name of Magnetoglobus has also been officially proposed for a magnetotactic multicellular prokaryote [START_REF] Abreu | Candidatus Magnetoglobus multicellularis", a multicellular, magnetotactic prokaryote from a hypersaline environment[END_REF].

Greigite-producing magnetotactic bacteria were first described in 1983 [START_REF] Farina | Ultrastructure of a magnetotactic bacterium[END_REF] but the mineral composition of the crystals in the magnetosomes of these organisms was only identified in 1990 [START_REF] Farina | Magnetic iron-sulfur crystals from a magnetotactic microorganism[END_REF][START_REF] Mann | Biomineralization of ferrimagnetic greigite (Fe3S4) and iron pyrite (FeS2) in a magnetotactic bacterium[END_REF]. Until recently, they were thought to be confined to marine habitats [START_REF] Simmons | Spatiotemporal distribution of marine magnetotactic bacteria in a seasonally stratified coastal salt pond[END_REF]. There are two known morphological types of greigite-producers: a group of morphologically unique, multicellular magnetotactic prokaryotes (MMPs), and a group of large rod-shaped bacteria [46]. While a good deal of information has been acquired regarding the MMPs from culture-independent, environmental [START_REF] Abreu | Greigite magnetosome membrane ultrastructure in "Candidatus Magnetoglobus multicellularis[END_REF][START_REF] Bazylinski | Magnetotactic Bacteria[END_REF][START_REF] Simmons | Unexpected diversity in populations of the many-celled magnetotactic prokaryote[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] Zhang | Characterization and phylogenetic identification of a species of spherical multicellular magnetotactic prokaryotes that produces both magnetite and greigite crystals[END_REF] and genomics studies [START_REF] Abreu | Common ancestry of iron oxide-and iron-sulfide-based biomineralization in magnetotactic bacteria[END_REF][START_REF] Abreu | Deciphering unusual uncultured magnetotactic multicellular prokaryotes through genomics[END_REF][START_REF] Kolinko | Single-cell genomics reveals potential for magnetite and greigite biomineralization in an uncultivated multicellular magnetotactic prokaryote[END_REF], their isolation in axenic culture has not yet been successful, although Candidatus Magnetoglobus multicellularis was co-cultured in a medium where cells reduced sulfate and used succinate as an electron donor [START_REF] Abreu | Deciphering unusual uncultured magnetotactic multicellular prokaryotes through genomics[END_REF]. The MMP group represents an assemblage of obligately multicellular organisms that consist of about 10-60 genetically identical cells and is phylogenetically affiliated with the Desulfobacteraceae family within the Deltaproteobacteria class of the Proteobacteria phylum [START_REF] Chen | Novel species and expanded distribution of ellipsoidal multicellular magnetotactic prokaryotes[END_REF][START_REF] Delong | Multiple evolutionary origins of magnetotaxis in bacteria[END_REF][START_REF] Simmons | Unexpected diversity in populations of the many-celled magnetotactic prokaryote[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] Zhang | Characterization and phylogenetic identification of a species of spherical multicellular magnetotactic prokaryotes that produces both magnetite and greigite crystals[END_REF][START_REF] Zhou | A novel genus of multicellular magnetotactic prokaryotes from the Yellow Sea[END_REF]. The large rod-shaped greigite and/or magnetite-producing bacteria have been studied for several decades using culture-independent techniques from environmental samples [START_REF] Bazylinski | Controlled biomineralization of magnetite (Fe3O4) and greigite (Fe3S4)[END_REF][START_REF] Bazylinski | Fe3O4 and Fe3S4 in a bacterium[END_REF][START_REF] Heywood | Structure, morphology and growth of biogenic greigite[END_REF][START_REF] Simmons | Population dynamics of marine magnetotactic bacteria in a meromictic salt pond described with qPCR[END_REF][START_REF] Simmons | Spatiotemporal distribution of marine magnetotactic bacteria in a seasonally stratified coastal salt pond[END_REF] but have only been phylogenetically identified [START_REF] Lefèvre | A cultured greigite-producing magnetotactic bacterium in a novel group of sulfate-reducing bacteria[END_REF][START_REF] Wang | High diversity of magnetotactic deltaproteobacteria in a freshwater niche[END_REF] and cultured [START_REF] Lefèvre | A cultured greigite-producing magnetotactic bacterium in a novel group of sulfate-reducing bacteria[END_REF] recently. The greigite-producing, rod-shaped bacteria also belong to the Desulfobacteraceae family [START_REF] Lefèvre | A cultured greigite-producing magnetotactic bacterium in a novel group of sulfate-reducing bacteria[END_REF].

The first isolation and cultivation of a large, rod-shaped greigite-producing magnetotactic bacterium, referred to as Candidatus Desulfamplus magnetomortis (renamed here Desulfamplus magnetovallimortis) strain BW-1, was recently described [START_REF] Lefèvre | A cultured greigite-producing magnetotactic bacterium in a novel group of sulfate-reducing bacteria[END_REF]. In silico analyses of this organism revealed the presence of two different magnetosome gene clusters. Based on comparative genomics, one magnetosome gene cluster was proposed to be responsible for greigite biomineralization and the other for magnetite synthesis [START_REF] Lefèvre | A cultured greigite-producing magnetotactic bacterium in a novel group of sulfate-reducing bacteria[END_REF][START_REF] Lefèvre | Comparative genomic analysis of magnetotactic bacteria from the Deltaproteobacteria provides new insights into magnetite and greigite magnetosome genes required for magnetotaxis[END_REF]. The presence of two similar clusters was also identified in the MMP Ca. Magnetomorum strain HK-1 collected from samples from the North Sea, Germany, supporting the ability of this bacterium to synthesize magnetite and greigite magnetosomes. Magnetosome genes involved in greigite formation were first described in Ca. Magnetoglobus multicellularis [START_REF] Abreu | Common ancestry of iron oxide-and iron-sulfide-based biomineralization in magnetotactic bacteria[END_REF]. Finally, greigite magnetosome genes were also identified in a draft genome of an uncultivated bacterium belonging to the candidate phylum Latescibacteria [START_REF] Lin | A putative greigite-type magnetosome gene cluster from the candidate phylum Latescibacteria[END_REF]. Although there is no evidence that this bacterium is able to produce greigite, it is possible that greigite biomineralization extends to this phylum as well as the Deltaproteobacteria.

Desulfamplus magnetovallimortis strain BW-1 T (Bad Water-1) is the subject of the current study. This magnetotactic bacterium was isolated from a brackish spring at Badwater Basin, Death Valley National Park, California, USA [START_REF] Lefèvre | A cultured greigite-producing magnetotactic bacterium in a novel group of sulfate-reducing bacteria[END_REF]. Using cultivation experiments and genomic approaches, here we fully characterize and name this strain that represents the first greigite-producing magnetotactic bacterium isolated in axenic culture.

Materials and methods

Sample collection, enrichment and purification

Strain BW-1 T was isolated from mud and water collected from the brackish spring located at Badwater Basin in Death Valley, California, USA [START_REF] Lefèvre | A cultured greigite-producing magnetotactic bacterium in a novel group of sulfate-reducing bacteria[END_REF]. For isolation of strain BW-1, cells of magnetotactic bacteria were first magnetically concentrated from water samples using the magnetic capillary racetrack technique [START_REF] Wolfe | A "capillary racetrack" method for isolation of magnetotactic bacteria[END_REF] and then inoculated into a modified semisolid, oxygen concentration gradient ([O 2 ]-gradient) medium based on a growth medium originally designed for Desulfobacterium vacuolatum. The medium consisted of an artificial seawater (ASW) base diluted to the approximate salinity of the water sample, containing (per liter): NaCl, 20 g; MgCl 2 •6H 2 O, 3 g; Na 2 SO 4 , 3 g; KCl, 0.5 g; and CaCl 2 •2H 2 O, 0.2 g. To this was added (per liter) the following, in order, prior to autoclaving: 50 µL of 0.2% (wt/vol) aqueous resazurin, 5 mL of modified Wolfe's mineral elixir [START_REF] Frankel | Magnetoaerotaxis in marine coccoid bacteria[END_REF], 0.3 g of NH 4 Cl, 2.4 g of HEPES, 2 g of Casamino acids and 2 g of Agar Noble (both from Difco). The pH of the medium was then adjusted to 7.0 prior to autoclaving. Once the medium cooled to about 45°C, the following solutions were added (per liter), in order, from stock solutions (except for the cysteine, which was made fresh and filter sterilized directly into the medium): 0.5 mL of vitamin solution [START_REF] Frankel | Magnetoaerotaxis in marine coccoid bacteria[END_REF]; 1.8 mL of 0.5 M KHPO 4 /KH 2 PO 4 buffer, pH 7.0; 3 mL of 0.01 M FeCl 2 dissolved in 0.02 N HCl; and L-cysteine to give a final concentration of 0.4 g/L. The medium (10 mL) was dispensed into sterile, 15 x 125 mm screw-capped test tubes. All cultures were incubated at 28°C and, after approximately one week, an aggregate of cells formed in the anaerobic zone of the medium. The large rod-shaped cells were not magnetotactic in this medium and it was only when they were transferred in a specific liquid medium that cells became magnetotactic. An axenic culture of strain BW-1 T was obtained after three successive rounds of dilution to extinction in this latter medium. Purity of the cultures was determined using light microscopy and by amplification and sequencing of the 16S rRNA gene.

Media and culture conditions

For magnetosome formation, cells of strain BW-1 T were grown in BWM medium in 250 mL Schott bottles containing 100 mL of liquid anaerobic medium closed by a cap and a rubber stopper. The formula of the medium was (per liter): 20 g NaCl, 3 g MgCl 2 •6H 2 O, 3 g Na 2 SO 4 , 0.2 g CaCl 2 •2H 2 O and 0.5 g KCl. To a liter of the basal medium, 5 ml modified Wolfe's mineral elixir [START_REF] Frankel | Magnetoaerotaxis in marine coccoid bacteria[END_REF], 50 µL 1% aqueous resazurin, 0.3 g NH 4 Cl, 2.4 g 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 1 g fumaric acid were added and the pH adjusted to 7.5 with 10 M NaOH. The medium was then autoclaved. After autoclaving, 0.5 mL of an anaerobic stock solution of vitamins [START_REF] Frankel | Magnetoaerotaxis in marine coccoid bacteria[END_REF]; 1.8 mL of 0.5 M KHPO 4 buffer (pH 7.0); 2 mL or 10 mL of 10 mM FeCL 2 •4H 2 O (in 0.02 M HCl) and 0.4 g of freshly made filtered sterilized cysteine were added to the medium. The medium was then bubbled with O 2 -free N 2 for 30 min. The medium was inoculated with 5 mL of a culture in exponential growth phase and incubated at 22°C. Electron donors and acceptors were tested in this growth medium. The following compounds were tested as terminal electron acceptors (sodium salts, when appropriate): nitrate (2 mM), nitrite (2 mM), fumarate (20 mM), trimethylamine oxide (TMAO; 15 mM), dimethylsulfoxide (DMSO; 15 mM), sulfur (5 mM), sulfite (5 mM), thiosulfate (5 mM), tetrationate (5 mM), dithionite (5 mM) and N 2 O (1 atm in the headspace). Except for the tubes containing nitrous oxide (N 2 O), all others were flushed with O 2 -free N 2 . To test for heterotrophic growth, potential carbon sources were added to tubes to give a final concentration of 0.1% (wt/vol or vol/vol) using the BWM liquid medium minus fumaric acid. The following carbon sources were tested (sodium salts for acids, L-enantiomers were used for amino acids, D-enantiomers for sugars): alanine, aspartic acid, glutamic acid, glutamine, glycine, isoleucine, leucine, proline, serine, valine, butanol, glucose, casamino acids, yeast extract, butyrate, malate, pyruvate, acetate, fumarate, gluconic acid, lactic acid and succinate. To test for autotrophic growth, fumaric acid was omitted from the medium, 2.8 mL of a solution of 0.8 M NaHCO 3 (autoclaved dry; sterile water added after autoclaving to make the fresh stock solution) was added to the medium which was then bubbled with H 2 for 30 min. If growth was observed, the culture was transferred twice further in the same medium in triplicate. Fumaric acid was used as the positive control and sterile H 2 O was used as the negative control. Tubes were incubated for at least 3 weeks at 28°C.

Whole genome sequencing and annotation

Whole genome sequencing of Desulfamplus magnetovallimortis strain BW-1 was previously described [START_REF] Lefèvre | Monophyletic origin of magnetotaxis and the first magnetosomes[END_REF]. The draft genome sequence partially annotated was submitted to the European Nucleotide Archive and carries the accession number PRJEB14757. The genome was assembled using the MicroScope platform resulting in 108 scaffolds and 346 contigs for a total length of 6,784,998 bases. Automatic and manual sequence annotations were performed using the MicroScope platform from the Genoscope [START_REF] Vallenet | MicroScope--an integrated microbial resource for the curation and comparative analysis of genomic and metabolic data[END_REF]. There are 11.28% of Nosferatu Repeated Regions (excluding undetermined bases). CDS length is about 942.13 bp with an intergenic length of 210 bp. A total of 6,137 CDS are present in the genome.

Phylogenetic analysis

Evolutionary relationships between Desulfamplus magnetovallimortis BW-1 T and its relatives were investigated at both 16S rRNA sequence and whole genome resolutions. proteins. Core amino acids sequences were aligned independently using MAFFT 7 [START_REF] Katoh | MAFFT multiple sequence alignment software version 7: improvements in performance and usability[END_REF].

Alignments were trimmed to remove positions composed of more than 95% gaps and concatenated into a single alignment of 23,813 amino acids positions among which

Chemotaxonomic analyzes

For fatty acid and respiratory quinones analyses, sulfate-reducing cells of strain BW-1 T , grown in liquid medium with fumarate as electron donor were harvested during late exponential phase. Fatty acid analysis and respiratory quinones was carried out by the Identification Service of the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Braunschweig, Germany. Fatty acids were extracted, purified, methylated and quantified by gas chromatography using the standard Microbial

Identification System [START_REF] Kämpfer | Numerical analysis of fatty acid patterns of coryneform bacteria and related taxa[END_REF][START_REF] Kuykendall | Fatty Acids, Antibiotic Resistance, and Deoxyribonucleic Acid Homology Groups of Bradyrhizobium japonicum[END_REF][START_REF] Miller | Single derivatization method for routine analysis of bacterial whole-cell fatty acid methyl esters, including hydroxy acids[END_REF]. Respiratory quinones were analyzed by thin layer chromatography using the standard Microbial Identification System [START_REF] Tindall | A Comparative Study of the Lipid Composition of Halobacterium saccharovorum from Various Sources[END_REF][START_REF] Tindall | Lipid composition of Halobacterium lacusprofundi[END_REF].

Electron microscopy

Transmission electron microscopy (TEM) of whole cells was done using a Tecnai (FEI Company, Hillsboro OR) model G2 Biotwin electron microscope using an accelerated voltage of 100 kV. High resolution transmission microscopy (HRTEM) was carried out on a Jeol 2100F microscope. This machine, operating at 200 kV, is equipped with a Schottky emission gun, an ultra-high-resolution pole piece, and an ultrathin window JEOL X-ray Energy Dispersive Spectrometer. HRTEM images were obtained with a Gatan US 4000 CCD camera.

A drop of culture containing cells of strain BW-1 T was deposited onto TEM copper grids coated with a carbon film, which were then washed and dried in air. The grids were stained with 1% uranyl acetate in particular to observe flagella.

Results and discussion

Cell morphology

Light microscopy and TEM observations show that cells of strain BW-1 T are relatively large, rod-shaped and possess a single polar flagellum that measures 3-4 times the cell's body length (Fig. 1A). Although the majority of the cells are not motile in the lag phase, most of the cells in the exponential and stationary phases are motile. Cells have a smooth motility in the presence or absence of a magnetic field with an approximate speed of 30 µm.s -1 . Cultured cells synthesize a large electron-dense inclusion of unknown composition approximately 500 nm in diameter (Fig. 1B) that were not observed in cells collected from the environment. Specific chemical elements in this inclusion were not detected using energy-dispersive X-ray spectroscopy (Fig. 1C). This inclusion was more commonly observed in cells in stationary or early exponential growth phases.

Growth characteristics

Cells growing in BWM medium appeared to be more motile and contained more magnetosomes than other growth media tested (see Magnetosomes section). To increase the growth rate, yeast extract was added (0.2 g/L) to BWM medium and the cultures incubated at 28°C. Under such conditions, cells were still motile but did not show any magnetic response under the light microscope and only biomineralized weakly crystallized particles as observed by TEM. The minimum doubling time during exponential growth phase in modified BWM medium was about 6-8 hours. Physiological as well as morphological properties of strain BW-1 T are listed in Table 1 and compared to closely related species.

Table 1. Selected characteristics that differentiate strain BW-1 T from its closest relatives.

NR, Not reported; +, good growth; (+), poor growth; -, no growth. Reference strains: 1, Desulfobacterium autotrophicum (data from [START_REF] Strittmatter | Genome sequence of Desulfobacterium autotrophicum HRM2, a marine sulfate reducer oxidizing organic carbon completely to carbon dioxide[END_REF] and [START_REF] Brysch | Lithoautotrophic growth of sulfate-reducing bacteria, and description of Desulfobacterium autotrophicum gen. nov., sp[END_REF]); 2, Desulfobacula toluolica (data from [START_REF] Kuever | Reclassification of Desulfobacterium phenolicum as Desulfobacula phenolica comb. nov. and description of strain SaxT as Desulfotignum balticum gen. nov., sp[END_REF]); 3, Desulfotignum balticum (data from [START_REF] Kuever | Reclassification of Desulfobacterium phenolicum as Desulfobacula phenolica comb. nov. and description of strain SaxT as Desulfotignum balticum gen. nov., sp[END_REF]); 4, Desulfospira joergensenii (data from [START_REF] Finster | Desulfospira joergensenii, gen. nov., sp. nov., a new Sulfate-reducing Bacterium Isolated from Marine Surface Sediment[END_REF]); 5, Desulfoconvexum algidum (data from [START_REF] Könneke | Desulfoconvexum algidum gen. nov., sp. nov., a psychrophilic sulfate-reducing bacterium isolated from a permanently cold marine sediment[END_REF]); Desulfobacter postgatei (data from [START_REF] Widdel | Studies on dissimilatory sulfate-reducing bacteria that decompose fatty acids. I. Isolation of new sulfate-reducing bacteria enriched with acetate from saline environments. Description of Desulfobacter postgatei gen. nov., sp[END_REF]). [START_REF] Lefèvre | Growth of magnetotactic sulfate-reducing bacteria in oxygen concentration gradient medium[END_REF], we tested BW-1 T for this ability in an [O 2 ]-gradient medium where 264 fumaric acid was added as the electron donor. In presence of sulfate, cells of strain BW-265 1 T only grew as aggregates below the oxic-anoxic interface. In absence of sulfate, no 266 growth was observed showing their inability to use O 2 as electron acceptor. Strain BW-1 T 267 represents, to date, the only cultured magnetotactic bacterium unable to grow 268 microaerobically raising questions on the role(s) of magnetosomes in this bacterium. It is 269 possible that the conditions for aerobic growth of BW-1 T remain to be elucidated or that 270 this ability was lost due to long term transfer of the culture under strictly anaerobic 271 conditions. Moreover, as it was reported for Desulfobacterium autotrophicum [START_REF] Strittmatter | Genome sequence of Desulfobacterium autotrophicum HRM2, a marine sulfate reducer oxidizing organic carbon completely to carbon dioxide[END_REF], strain 272 BW-1 T also has in its genome cydAB genes coding for a cytochrome d ubiquinol oxidase 273 that could enable strain BW-1 T to respire oxygen at low concentrations [START_REF] Green | The nucleotide sequence of the cyd locus encoding the two subunits of the cytochrome d terminal oxidase complex of Escherichia coli[END_REF][START_REF] Lemos | The "strict" anaerobe Desulfovibrio gigas contains a membrane-bound oxygen-reducing respiratory chain[END_REF].

Characteristic

Additionally, strain BW-1 T possesses a catalase-peroxidase (KatG), a selenocysteinecontaining peroxidase, rubrerythrin (Rbr) and a rubredoxin oxidoreductases (Rbo) for oxygen detoxification.

Glutamate, glutamine, casamino acids, yeast extract, fumarate, lactate, malate, pyruvate and succinate supported growth and are thus potential carbon sources for BW-1 T (Table 1).

In BWM medium, cultures in exponential to late-exponential growth phase can be stored for several weeks at 4°C before having to be transferred into fresh medium. For long term storage, concentrated cells remained viable for several years in BWM medium supplemented with 10% (final concentration) dimethylsulfoxide (DMSO) at -80°C.

Magnetosome biomineralization

Individual cells of strain BW-1 T grown in BWM medium contained one or two magnetosome chains composed of magnetite and/or greigite (Fig. 2A and2B). Magnetite crystals produced by BW-1 T were consistently bullet-shaped, whereas greigite crystals were pleomorphic. When a cell biomineralizes magnetite and greigite magnetosomes, both types of particles appear to be in the same chain likely sharing the same MamK filament. We previously showed that the composition of magnetosome mineral crystals is correlated with the concentration of hydrogen sulfide produced during growth (sulfatereduction) and affected by external environmental conditions, for example, redox potential [START_REF] Bazylinski | Controlled biomineralization of magnetite (Fe3O4) and greigite (Fe3S4)[END_REF][START_REF] Lefèvre | A cultured greigite-producing magnetotactic bacterium in a novel group of sulfate-reducing bacteria[END_REF]. Indeed, when sulfide accumulates (> 0.3 mM) in the growth medium, most magnetosomes contain greigite [START_REF] Lefèvre | A cultured greigite-producing magnetotactic bacterium in a novel group of sulfate-reducing bacteria[END_REF]. Since the initial description of strain BW-1 T [START_REF] Kuever | Reclassification of Desulfobacterium phenolicum as Desulfobacula phenolica comb. nov. and description of strain SaxT as Desulfotignum balticum gen. nov., sp[END_REF], we have improved in vitro magnetosome formation using BWM medium.

A relatively high concentration of iron in the medium (> 100 µM) appeared to be favorable for magnetite rather than greigite formation. It seems that magnetite production requires a high concentration of iron and a relatively high redox potential/low sulfide concentration. Under such conditions, hydrogen sulfide, produced during growth and sulfate reduction, is trapped as FeS by the relatively high concentration of iron in the growth medium. Conversely, greigite formation seems favored at lower iron concentrations (< 20 µM) and low redox potential/high sulfide concentration. Under these conditions, most of the hydrogen sulfide produced during cell growth is not trapped and reduces the redox potential of the growth medium. Genes involved in magnetosome formation were previously described in detail [START_REF] Lefèvre | Comparative genomic analysis of magnetotactic bacteria from the Deltaproteobacteria provides new insights into magnetite and greigite magnetosome genes required for magnetotaxis[END_REF] and will not be further discussed here. We have grown strain BW-1 T consecutively since 2010 under these anaerobic media and cells were always able to produce magnetosomes. Non-magnetotactic mutant were never obtained in our growth conditions as it was reported for other magnetotactic species. Indeed, the loss of magnetosome formation and, thus, magnetotaxis by some cultivated MTB occurs relatively easily in culture following the loss of magnetosome genes [START_REF] Dubbels | Evidence for a copper-dependent iron transport system in the marine, magnetotactic bacterium strain MV-1[END_REF][START_REF] Schübbe | Characterization of a spontaneous nonmagnetic mutant of Magnetospirillum gryphiswaldense reveals a large deletion comprising a putative magnetosome island[END_REF]. The absence of integrase and/or transposase genes in close proximity of the magnetosome gene clusters [START_REF] Lefèvre | Comparative genomic analysis of magnetotactic bacteria from the Deltaproteobacteria provides new insights into magnetite and greigite magnetosome genes required for magnetotaxis[END_REF] indicates that this region is relatively stable in the genome of BW-1 T and could explain why the magnetotactic phenotype was never lost for this strain.

Phylogeny of strain BW-1 T

Phylogenomics analyses confirmed the placement of strain BW-1 T in the order Desulfobacterales, more specifically in the family Desulfobacteraceae (Fig. 3A). The three closest 16S rRNA gene sequences from type strains are those of Desulfobacterium vacuolatum (93.3%), Desulfobacterium autotrophicum (92.0%) and Desulfobacula toluolica (89.4). The closest genetically related species to BW-1 T , for which a whole genome was sequenced and available, is Desulfobacterium autotrophicum HRM2 (=DSM 3382 T ). Amino acid sequences from coding DNA sequences between the two species are 58.15% identical based on the KostasLab two-way average amino acid identity (AAI) calculator (http://enve-omics.ce.gatech.edu/aai/) (Fig. 4) [START_REF] Rodriguez-R | Bypassing cultivation to identify bacterial species: culture-independent genomic approaches identify credibly distinct clusters, avoid cultivation bias, and provide true insights into microbial species[END_REF]. Such a genetic divergence between two genomes is generally found among bacterial strains belonging to different genera [START_REF] Rodriguez-R | Bypassing cultivation to identify bacterial species: culture-independent genomic approaches identify credibly distinct clusters, avoid cultivation bias, and provide true insights into microbial species[END_REF]. A 16S rRNA based tree containing BW-1 T and relatives confirmed that the most recent ancestor of BW-1 T is shared with Desulfobacterium species (Fig. 3B). Together with this phylogenetic evidence, ecological and phenotypic features of BW-1 T compared to those of its most closely related species support our contention that BW-1 T is the first representative of a new genus we name here as Desulfamplus. In addition, among other compared characteristics, no Desulfobacterium spp. were shown to be magnetotactic and magnetosome genes are not present in the genome of Desulfobacterium autotrophicum HRM2, the only genome sequenced and available from members of this genus [START_REF] Strittmatter | Genome sequence of Desulfobacterium autotrophicum HRM2, a marine sulfate reducer oxidizing organic carbon completely to carbon dioxide[END_REF]. Moreover, only 26-31% of their genome sequences are shared (Fig. 4).

The genus Desulfamplus likely contains several species since other magnetotactic bacteria closely related to BW-1 T have been described from natural environments [START_REF] Lefèvre | A cultured greigite-producing magnetotactic bacterium in a novel group of sulfate-reducing bacteria[END_REF][START_REF] Simmons | Spatiotemporal distribution of marine magnetotactic bacteria in a seasonally stratified coastal salt pond[END_REF][START_REF] Wang | High diversity of magnetotactic deltaproteobacteria in a freshwater niche[END_REF]. This clade appears to consist of at least two groups that could potentially be divided into two separate genera based on 16S rRNA gene sequence divergence [START_REF] Lefèvre | Ecology, diversity, and evolution of magnetotactic bacteria[END_REF][START_REF] Lefèvre | A cultured greigite-producing magnetotactic bacterium in a novel group of sulfate-reducing bacteria[END_REF].

New cultures and new genomes sequenced from those magnetotactic relatives to BW-1 T are clearly necessary to determine the number of different genera these groups represent and how widespread this group of sulfate-reducing bacteria is. AAI values were estimated with the web-based KostasLab AAI calculator using reciprocal best hits (http://enve-omics.ce.gatech.edu/aai/) for each pair of genomes. The radius of each light grey circle is proportional to the AAI value. All data were organized according to the phylogeny given in Figure 3A.

Some additional metabolic features and details

We previously showed evidence that strain BW-1 T relies on sulfate-reduction for growth [START_REF] Lefèvre | A cultured greigite-producing magnetotactic bacterium in a novel group of sulfate-reducing bacteria[END_REF]. The genome of strain BW-1 T contains several sets of genes that encode the major metabolic components for respiration and reduction of sulfate. We identified a number of these genes that encode key enzymes of this pathway including two sulfate Almost all cultured magnetotactic bacteria tested possess the capability of N 2 fixation [START_REF] Bazylinski | Magnetotactic Bacteria[END_REF] and strain BW-1 T appears to be no exception as its genome contains a cluster of nif genes (nifHEKBDJ) demonstrating it clearly has the potential to perform this environmentally important process.

Chemotaxonomy

The cellular fatty acids of strain BW-1 T were C are generally the major fatty acids (Table 2).

Table 2. Major cellular fatty acids of Desulfamplus magnetovallimortis strain BW-1 T and some phylogenetically related species.

Strains: Desulfamplus magnetovallimortis gen. nov., sp. nov. BW-1 T ; Desulfobacterium autotrophicum strain HRM2 (data from [START_REF] Kuever | Reclassification of Desulfobacterium phenolicum as Desulfobacula phenolica comb. nov. and description of strain SaxT as Desulfotignum balticum gen. nov., sp[END_REF]); Desulfotignum balticum strain Sax T [START_REF] Kuever | Reclassification of Desulfobacterium phenolicum as Desulfobacula phenolica comb. nov. and description of strain SaxT as Desulfotignum balticum gen. nov., sp[END_REF]; Desulfospira joergensenii strain B331 T [START_REF] Finster | Desulfospira joergensenii, gen. nov., sp. nov., a new Sulfate-reducing Bacterium Isolated from Marine Surface Sediment[END_REF]; Desulfobacter postgatei strain DSM 2034 T [START_REF] Kuever | Reclassification of Desulfobacterium phenolicum as Desulfobacula phenolica comb. nov. and description of strain SaxT as Desulfotignum balticum gen. nov., sp[END_REF]; Desulfobacula toluolica strain DSM 7467 T [START_REF] Kuever | Reclassification of Desulfobacterium phenolicum as Desulfobacula phenolica comb. nov. and description of strain SaxT as Desulfotignum balticum gen. nov., sp[END_REF].

First

  , genomes of several Deltaproteobacteria type strains were downloaded from the public database NCBI (updated up to March 2016) and uploaded into the MicroScope platform. These included at least one representative of each order (Desulfobacterales, and Desulfovibrionales) with the exception of the Desulfobacterales and Desulfovibrionales, for which several genomes were included in the dataset. A core genome (i.e., genes detected in all genomes compared, for which protein sequences shared > 50% identity over > 80% of their length), was defined with the Microscope comparative genomics tool and categorized into several MicroScope gene families (MICFAM). Among the 131 MICFAM shared by the 24 genomes, 70 were represented by a unique sequence per genome and 32 out of those coded for ribosomal

Fig. 1 .

 1 Fig. 1. Transmission electron microscope (TEM) images of whole cells of strain BW-1 T showing (A) the single polar flagellum and (B) the large inclusion of unknown composition and function. (C) TEM and scanning-transmission electron microscope (STEM) images showing a large inclusion analyzed with energy-dispersive X-ray spectroscopy. Elemental spectrum of the inclusion (left; black star inside the inclusion) compared to that of background (right; white star outside the inclusion) showing that the inclusion is not composed by specific elements. White arrows indicate magnetosomes. Scale bars represent 200 nm.

Fig. 2 .

 2 Fig. 2. Transmission electron microscope (TEM) images of cells of strain BW-1 T . (A) Cell grown in BWM medium containing 100 µM iron producing only magnetite magnetosomes, (B) high-resolution TEM (HRTEM) image of a magnetite crystal and corresponding selected area diffraction (SAED) of crystal viewed along the [110] zone axis (C). (D) Cell grown in BWM medium containing 20 µM iron, cell produces only greigite magnetosomes, (E) HRTEM image of greigite crystal and corresponding SAED of crystal viewed along the [110] zone axis (F).

Fig. 3 .

 3 Fig. 3. Phylogenetic position of Desulfamplus magnetovallimortis strain BW-1 T within the Deltaproteobacteria class. (A) Unrooted ML phylogeny showing the evolutionary relationships between Desulfamplus magneotovallimortis strain BW-1 T with 23 sequenced genomes of Deltaproteobacteria type

Fig. 4 .

 4 Fig.4. Core genome sizes and percentages average amino acid identity (AAI) between Desulfamplus magnetovallimortis BW-1 T and its closest relatives. Core genes are defined by MaGe as genes detected in both genomes, for which coding protein sequences shared a minimum of 50% identity over a minimum of 80% of their length. The radius of each dark grey circle is proportional to the number of detected genes.

  adenylyltransferases (MTBBW1_1630032 and MTBBW1_1740012), a set of adenylylsulfate reductases (aprAB) (MTBBW1_350011 and MTBBW1_350012), ), adenosyl phosphosulfate and quinoneinteracting membrane-bound oxidoreductase (qmoABC) (MTBBW1_350014, MTBBW1_350015 and MTBBW1_350016).

16 : 1

 161 ω7c (25.2% of the total fatty acids), C 16:0 (23.3%), C 17:1 ω6c (13.1%), C 15:0 (9.7%), C17:0 (4.9%), C 15:1 ω6c (4.6%), C 18:1 ω7c (4.4%), C 17:1 ω8c (3.5%), C 14:0 (3.2%), C 18:1 ω9c (1.2%). Strain BW-1 T has a fatty acid profile typical of members of the Desulfobacteraceae, where C 16:0 and C 16:1

  Only few genomes of members of the Desulfobacteraceae family are currently available. Thus, a phylogenetic tree based on the 16S rRNA gene sequences was built to determine the relatedness of Desulfamplus magnetovallimortis BW-1 T within the existing diversity of Desulfobacteraceae species already described. For this purpose, 43 sequences of cultured type strains > 1,200 bp available in the Ribosomal Database Project (https://rdp.cme.msu.edu) were downloaded, aligned and trimmed as described above. A ML tree was also built following the same procedure than described above but under a GAMMA model of rate heterogeneity, estimating a Generalized Time Reversible (GTR) model for nucleotide substitution and an estimate of the proportion of invariable sites.

18,161 were polymorphic. A maximum-likelihood (ML) tree was built with RAxML 8.2.6

[START_REF] Stamatakis | RAxML version 8: a tool for phylogenetic analysis and postanalysis of large phylogenies[END_REF]

under the GAMMA model of rate heterogeneity using empirical AA frequencies and the LG substitution model (selected with the Bayesian criterion). A total of 200 bootstrap replicates automatically determined by the MRE-based bootstopping criterion were conducted under the rapid bootstrapping algorithm, among which 100 were sampled to generate proportional support values.

416 Description of Desulfamplus gen. nov. 417

  95%). This result is consistent with the respiratory quinones usually observed 409 in species of the Desulfocateraceae family, where the menaquinone MK-7 is generally 410 found as a major respiratory quinone. 411 412 Based on the data presented here, strain BW-1 T is considered to represent a novel 413 species of a new genus, for which the name Desulfamplus magnetovallimortis gen. nov., 414 sp. nov. is proposed. 415

	406
	Fatty acid	BW-1 T	Desulfobacterium	Desulfotignum	Desulfospira	Desulfobacter	Desulfobacula
			autotrophicum	balticum	joergensenii	postgatei	toluolica
	C 14:0	3.2	2.8	3.7	13.9	15.4	8.3
	iso-C 15:0	-	-	-	1.8	0.7	-
	C 15:1 ω9c	-	6.5	0.8	-	-	-
	C 15:0	9.7	5.1	0.2	1.3	3.1	1.6
	3-OH C 14:0	2.2	-	-	1.6	-	-
	C 16:1 ω7c	25.2	-	-	-	-	-
	C 16:1 ω9c	-	30.7	8.5	38.9	10.0	19.7
	C 16:0	23.3	13.2	24.3	28.4	20.9	31.2
	C 16:0 10-	-	7.4	14.0	-	11.2	17.6
	methyl
	C 17:1 ω11c	-	10.5	-	-	-	-
	C 17:1 ω8c	3.5	-	-	-	-	-
	C 17:1 ω6c	13.1	-	-	-	-	-
	C 17:0 cyclo	-	-	19.5	2.4	31.5	2.6
	C 17:0	4.9	2.9	0.4	0.6	2.3	0.7
	C 16:0 3-OH	-	0.5	-	2.3	-	-
	C 18:1 ω13c	-	-	-	-	-	-
	C 18:1 ω11c	-	2.7	7.4	5.3	1.2	6.0
	C 18:1 ω9c	1.2	-	3.7	-	-	-
	C 18:1 ω7c	4.4	-	-	-	-	-
	C 18:0	0.4	1.1	9.8	0.7	0.5	3.1
	iso-C 19:0	-	-	1.7	-	-	2.5
	C 19:0 cyclo	-	-	1.1	-	-	-
	407
	408	Respiratory quinones analysis of strain BW-1 T shows the presence of MK-6 (5%)
		and MK-7 (

Acknowledgements

This work was supported by a project from the Deutsche Forschungsgemeinschaft and the French National Research Agency (DFG-ARN: GROMA) and the ANR MEFISTO. D.A.B. is supported by U.S. National Science Foundation grant EAR-1423939.

Desulfamplus (De.sul.fo.am'plus. N.L. prep. de from; N.L. pref. sulfo-used for N.L. sulfuratis sulfate; L.adj. amplus, -a, -um, large; N.L.n. masc. a large sulfatereducer).

Cells are Gram-negative, relatively large rods and motile by mean of a single polar flagellum. They are strict anaerobes, using sulfate as terminal electron acceptor that is reduced to sulfide. The two main cellular fatty acids are C 16:1 ω7c and C 16:0 while the major respiratory quinone is the menaquinone MK-7. The genus Desulfamplus belongs to the Deltaproteobacteria class in the Proteobacteria phylum with its closest relative species belonging to genus Desulfobacterium. The type species is Desulfamplus magnetovallimortis BW-1 T . The Taxonumber is GA00026.

Description of Desulfamplus magnetovallimortis sp. nov.

Desulfamplus magnetovallimortis (ma.gne.to.val.li.mor'tis; ; L.adj.of greek origin magnes magnetic; L.n.fem. valles, -is valley; L.n.fem. mors, mortis death; magnetovallimortis referring to a magnetic bacterium isolated from the Death Valley in California, USA, where the type strain was isolated from).

Species description is as for genus, with the following additional characteristics.

Cells are, on average, 2 µm long and 1 µm wide. Capable of chemoorganoheterotrophic growth, using fumarate, lactate, pyruvate, malate and succinate as electron donors and carbon sources. Sulfate and thiosulfate are used as terminal electron acceptors. Addition of at least 10 g NaCl L -1 is necessary for growth; optimum NaCl concentration for growth is 15-25 g L -1 . Temperature requirements: T min , 10°C; T opt , 28°C; T max , 35 °C. The pH range for growth is 6.5-7.5; optimum 7. The G-C content of the genome is 41 mol%. The major cellular fatty acids are C 16:1 ω7c, C 16:0 , C 17:1 ω6c, C 15:0 , C17:0, C 15:1 ω6c, C 18:1 ω7c, C 17:1 ω8c and C 14:0 . The type strain, BW-1 T (=JCM 18010 T =DSM 103535 T ) was isolated from a brackish spring in Death Valley National Park (California, USA). The Taxonumber is TA00086.