*Magnetovirga frankeli* gen. Nov., sp. nov., a
magnetotactic bacterium isolated from the Salton Sea,
California, that represents a novel lineage in the
*Gammaprotoeobacteria*

Mila Sirinelli-Kojadinovic, Elsa C A Turrini, Emma Ropion, Béatrice Alonso, Marine Bergot, Emilie Gachon, Philippe Ortet, Paul E D Soto-Rodriguez, Caroline L Monteil, Christopher T. Lefèvre

► To cite this version:

Mila Sirinelli-Kojadinovic, Elsa C A Turrini, Emma Ropion, Béatrice Alonso, Marine Bergot, et al. *Magnetovirga frankeli* gen. Nov., sp. nov., a magnetotactic bacterium isolated from the Salton Sea, California, that represents a novel lineage in the *Gammaprotoeobacteria*. Systematic and Applied Microbiology, 2025, 48 (4), pp.126621. 10.1016/j.syapm.2025.126621. hal-05350599

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*Magnetovirga frankeli* gen. Nov., sp. nov., a magnetotactic bacterium isolated from the Salton Sea, California, that represents a novel lineage in the *Gammaproteobacteria*

Mila Sirinelli-Kojadinovic<sup>a</sup>, Elsa C.A. Turrini<sup>a</sup>, Emma Ropion<sup>a</sup>, Béatrice Alonso<sup>a</sup>, Marine Bergot<sup>a</sup>, Emilie Gachon<sup>a</sup>, Philippe Ortet<sup>a</sup>, Paul E.D. Soto-Rodriguez<sup>a,b</sup>, Caroline L. Monteil<sup>a</sup>, Christopher T. Lefevre<sup>a,*</sup>

ARTICLE INFO

Keywords:

Magnetotactic bacteria
Magnetotaxis
Chemolithoautotrophy
Magnetosome
Biomineralization
Gammaproteobacteria

ABSTRACT

A magnetotactic bacterium, designated strain SS-5<sup>T</sup>, was isolated from the Salton Sea, a highly saline lake in California, USA, and cultivated in axenic culture. The Gram-negative cells of strain SS-5<sup>T</sup> are relatively small and rod-shaped and possess a single polar flagellum (monotrichous). This strain is a magnetotactic bacterium producing magnetite nanocrystals aligned in one chain per cell. Strain SS-5<sup>T</sup> is a microaerophile that grows chemolithoautotrophically while reducing oxygen as a terminal electron acceptor. Optimal growth occurred at pH 7.0-7.3 and 28-36 °C with thiosulfate used as an electron donor and sodium bicarbonate as a carbon source. Based on its genome sequence, the G + C content is 61.6 %. Phylogenomic and phylogenetic analyses indicate that strain SS-5<sup>T</sup> belongs to the *Sedimenticolaceae* family within the *Gammaproteobacteria* class. Based on average amino acid identity, strain SS-5<sup>T</sup> can be considered as a novel species of a new genus, for which the name *Magnetovirga frankeli* is proposed. The type strain of *M. frankeli* is SS-5<sup>T</sup> (=DSM 116211<sup>T</sup> = JCM 39467<sup>T</sup>).

Introduction

Magnetotactic bacteria represent a phylogenetically, ecologically
and metabolically diverse group of prokaryotes able to biomineralize
nanocrystals of magnetite (Fe₃O₄) or greigite (Fe₃S₄) in specific organ-
elles, named magnetosomes (Uebe and Schüler, 2016). Magnetosomes
are aligned in one or more chains within the cytoplasm and cause cells to
actively swim along the Earth's magnetic field lines (Frankel and
Bazylinski, 2006). This behavior, termed magnetotaxis, makes the
search for optimal concentrations of certain nutrients and redox condi-
tions within water columns and sediments more efficient by simplifying
a three-dimensional search to a linear search (Frankel and Bazylinski,
2009).

Magnetotactic bacteria (MTB) were discovered almost 50 years ago. Using the Genome Taxonomy Database (GTDB) (Wan et al., 2024), they have been reported in 17 phyla of bacteria including, for the majority of described MTB, the *Pseudomonadota*, *Desulfobacterota*, *Nitrospirota* and *Omnitrophota* (Wan et al., 2024). Among the 14 strains of MTB isolated

in axenic culture and characterized so far, 9 are representatives of the
*Alphaproteobacteria* class and 2 are representatives of the *Candidatus*
Magnetococcia class of the *Pseudomonadota* phylum and 3 are repre-
sentatives of the *Desulfobacterota* phylum: two of the *Desulfovibrionia*
class and one of the *Desulfobacteria* class.

Magnetotactic *Gammaproteobacteria* have been mentioned for the first time in 2007 (Simmons et al., 2007) in an environmental study carried out on the seasonally stratified Salt Pond, Massachusetts, USA. Magnetotactic bacteria collected in the water column of this saline lake were various in morphology and the type of magnetosome crystals biomineralized. Among this diversity, it was reported the presence of a magnetotactic bacterium biomineralizing greigite that was considered to be affiliated to the *Gammaproteobacteria*. However, the examination of the phylogenetic relationship of this organism raised doubts to its affiliation with the *Gammaproteobacteria* (Lefèvre et al., 2012). Indeed, the large greigite-producing rods they described was most likely affiliated with the *Desulfobacterota* as its ultrastructure was very similar to *Desulfamplus magnetovallimortis* strain BW-1, a magnetotactic bacterium

<footnote><sup>a</sup> Aix-Marseille Université, CEA, CNRS, BIAM, UMR7265, Institute of Biosciences and Biotechnologies of Aix-Marseille, Saint-Paul-lez-Durance, France</footnote>

<footnote><sup>b</sup> Instituto de estudios avanzados IUDEA, Departamento de Física, Universidad de La Laguna C/Astrofísico Francisco Sánchez, s/n.E-38203 Tenerife, Spain</footnote>

<footnote>* Corresponding author.
E-mail address: christopher.lefevre@cea.fr (C.T. Lefevre).</footnote>
later characterized (Lefèvre et al., 2011; Descamps et al., 2017). It is
likely that a magnetotactic gammaproteobacterium was present in their
samples based on transmission electron microscopy images that showed
similarities to magnetotactic *Gammaproteobacteria* (e.g. Fig. 2C
(Simmons et al., 2007)) later described. However, it is only in 2012 that
magnetotactic *Gammaproteobacteria* were unequivocally described with
the culture of two bacteria: one tentatively assigned to the *Thiotrichales*
order, strain BW-2 isolated from a brackish pond in Death Valley, Cal-
ifornia, and another affiliated to the *Chromatiales*, strain SS-5 isolated
from the highly saline Salton Sea, California (Lefèvre et al., 2012). Since
this study, strain SS-5 has been used to decipher the different mecha-
nisms underlying magnetotactic behaviors along with other models in
culture (Lefèvre et al., 2014) and enabled to characterize a new micro-
bial division scheme (Lefèvre et al., 2015). The complete genome se-
quences of strains BW-2 and SS-5 have been released recently (Geurink
et al., 2020; Trubitsyn et al., 2021).

Since then, the presence of magnetotactic *Gammaproteobacteria* in
several aquatic environments across the world was reported, including
(i) a giant rod-shaped MTB isolated from a freshwater pond in Kana-
zawa, Japan (Taoka et al., 2014), (ii) a bacterium found in a marine
bivalve *Thyasira* cf. *gouldi* harvested from the fjord of Bonne Bay,
Newfoundland, Canada (Dufour et al., 2014), (iii) strain NS-1, a north-
seeking MTB, isolated from the Piripiri Lagoon, Rio de Janeiro state,
Brazil, located in the Southern Hemisphere (Leao et al., 2016), (iv) strain
SHHR-1 isolated from brackish sediments collected from the Shihe River
estuary in Qinhuangdao City, China, that is phylogenetically and
morphologically closely related to strain SS-5 (Li et al., 2017), (v) strain
MRC-13 isolated from the intertidal zone in the northern part of Yuehu
Lake, a shallow coastal lagoon located on the east side of Shandong
Peninsula, China (Du et al., 2017), (vi) strains ZSR-1 and FZSR-2 isolated
from a salt evaporation pool in Bohai Bay, at the Fuzhou saltern, Dalian
City, eastern China (Liu et al., 2022), (vii) strain YYHR-1 isolated from a
freshwater lake in Beijing, China (Zhang et al., 2023) and viii), strains
CCP3 and CCP4 that are calcium carbonate producing magnetotactic
bacteria isolated from the freshwater Lake Pavin (Mangin et al., 2025).

So far, four groups of magnetotactic *Gammaproteobacteria* have been
described, two in the *Chromatiales* order including one monophyletic
group containing strains SS-5, NS-1, SHHR-1, ZSR-1, FZSR-2 and the
symbiont of *Thyasira* cf. *gouldi*, and one group containing strain CCP4
from the *Chromatiaceae* family. The third group is represented by the
giant magnetotactic bacteria strains GRS-1 and CCP3 that belongs to the
order named CAIRSR01 without official name (Mangin et al., 2025). The
fourth group belongs to the *Thiotrichales* order and includes strains BW-
2, MRC-13 and YYHR-1. With the revision of the current classification
and bacterial nomenclature, these affiliations slightly changed over time
and now it includes the *Chromatiales* and a candidate order close to the
*Nitrosococcales* remaining to be described. Additional observation sug-
gest that these MTB could be even more diversified in *Gammaproteo-
bacteria* based on metagenomics studies (Lin et al., 2017a; Lin et al.,
2017b; Lin et al., 2020; Uzun et al., 2020). However, the phylogenetic
affiliation of these metagenomes remains unknown and there is no ev-
idence that these metagenomes belong to magnetotactic bacteria as the
presence of magnetosome genes in a genome was shown not to be a
proof of magnetotaxis capability (Dziuba et al., 2023). We can also
mention the report of other bacteria affiliated to the *Gammaproteobac-
teria* class tentatively described as magnetotactic including *Acid-
ithiobacillus ferrooxidans* (Liu et al., 2008; Yan et al., 2013, 2016; Zhang
et al., 2018; Zhao et al., 2022) or *Pseudomonas aeruginosa* (Jayaraman
et al., 2021; Khan et al., 2021). However, none of these studies allow for
the unambiguous identification of magnetosomes similar to those syn-
thetized by MTB nor they showed the presence of orthologous genes
than those involved in magnetosomes formation.

Despite the culture of the two magnetotactic *Gammaproteobacteria*
strains BW-2 and SS-5 in 2012, none have been characterized so far.
Here, we fully characterize and name *Magnetovirga frankeli* strain SS-5T
(Salton Sea-5) using cultivation experiments, microscopy and genomic

approaches. This magnetotactic bacterium isolated from the highly saline Salton Sea in California, USA (Lefèvre et al., 2012) represents the first magnetotactic gammaproteobacterium characterized and enrich the repertoire of type strains in the poorly known *Sedimenticolaceae* family in the *Chromatiales* order.

Materials and methods

Sample collection, enrichment and purification

Strain SS-5T was isolated from the Salton Sea, a highly saline endo-
rheic rift lake located directly on the San Andreas Fault and located
below sea level (~69 m) (Lefèvre et al., 2012). The site where the sample
containing strain SS-5T was taken was situated at the southeastern shore
of the Salton Sea, near the boat ramp at Red Hill Marina County Park
(33.20° N, 115.61° W). The salinity here was 52 ppt and samples were
collected from a reddish pool containing large numbers of halophilic
bacteria as determined by microscopic observation. One liter plastic
bottles were filled to about 0.2-0.3 of their volume with sediment, then
filled to their capacity with water that overlaid the sediment. Air bub-
bles were excluded. Once in the laboratory, samples were stored under
dim light at room temperature (~25 °C).

For isolation of strain SS-5T, cells of magnetotactic bacteria were first
magnetically concentrated from water samples using the magnetic
capillary racetrack technique (Wolfe et al., 1987) and then inoculated
into a semisolid, oxygen concentration gradient ([O₂]-gradient) me-
dium. The medium consisted of an artificial seawater (ASW) containing
(per liter): 37.8 g NaCl, 5.4 g MgCl₂·6H₂O, 5.4 g Na₂SO₄, 0.5 g
CaCl₂·2H₂O and 0.9 g KCl. To a liter of this mixture, 5 mL modified
Wolfe's mineral elixir (Wolin et al., 1963; Bazylinski et al., 2000), 0.2
mL 1 % aqueous resazurin and 0.3 g NH₄Cl was added and the pH
adjusted to 7.0. 2.0 g of Bacto-Agar (Difco Laboratories, Detroit, MI) was
then added after which the medium was autoclaved. After autoclaving,
the following was added as sterile stock solutions (per liter): 0.5 mL of
vitamin solution (Frankel et al., 1997); 2.8 mL of 0.5 M KHPO₄ buffer
pH 7.0; 3 mL of 4.0 % sodium thiosulfate (Na₂S₂O₃·5H₂O); 3 mL of 10
mM FeSO₄·7H₂O (in 0.02 N HCl); 10 mL of a filter-sterilized, freshly-
made 5.0 % sodium bicarbonate (NaHCO₃) solution; and 10 mL of a
filter-sterilized, freshly-made 4.0 % neutralized cysteine·HCl·2H₂O
solution. Screw-capped glass culture tubes were filled to ~80 % of their
volume with medium. The medium was allowed to sit at room temper-
ature for several hours to solidify and to allow the O₂ concentration
gradient to form as evidenced by the presence of a pink (oxidized) zone
near the surface and a colorless (reduced) zone in the lower part of the
tubes.

Axenic cultures were obtained by dilution to extinction three times in
succession in the semi-solid growth medium containing thiosulfate as
the electron donor. Purity of the cultures was determined using light
microscopy and 16S rRNA gene sequencing (Lefèvre et al., 2012).

Media and culture conditions

In standard conditions, SS-5T was grown in the medium used for its
isolation (described above) i.e. in an oxygen-gradient highly saline
medium in autotrophic conditions using thiosulfate as an electron donor
and NaHCO3 as a carbon source. A few modifications were however
made: the amount of agar was reduced to 1.2 g.L⁻¹ and sodium bicar-
bonate (NaHCO3) was added at a 15 mM final concentration before
autoclaving. In addition, the source of iron was modified to FeCl2 (30 µM
final concentration). Before dispensing the medium into sterile tubes at
~80 % of their volume and allowing for the medium to solidify and the
oxygen gradient to form, pH of the medium was re-adjusted to 7.2.
Bacterial cultures were inoculated at 1:100 to 1:200 dilutions from one
week-old pre-cultures and were grown at 28 °C in an incubator.

SS-5T standard growth conditions were used to investigate the effect
of different parameters on SS-5T growth with appropriate modifications.
Thus, to explore SS-5T temperature, pH and NaCl concentration tolerances, one of these parameters was independently modified.

In order to study the effects of different carbon and electron sources, thiosulfate and NaHCO₃ were omitted in the SS-5T standard medium and were replaced by one of the following molecules at 10 mM final concentrations: formate, acetate, propionate, lactate, malate, succinate, fumarate, pyruvate or ethanol. For the negative growth control, thiosulfate and NaHCO₃ were replaced by water. These different molecules were tested with O₂ as an electron acceptor using the standard protocol (i.e. the medium was allowed to sit at room temperature for several hours to solidify and to allow the O₂ concentration gradient to form) or using nitrate as an electron acceptor. For the later condition, NaNO₃ was added to the SS-5T standard medium at a 10 mM final concentration. After dispensing the medium in sterile tubes and inoculating the solidified medium, tubes were flushed for 20 min with N₂ to be in anaerobic growth conditions.

For all tested conditions, culture tubes were inoculated with 10 µL of bacteria collected from the aerotactic band of a semisolid standard oxygen gradient pre-culture. Growth was assessed after 7 days of incubation. Growth was considered as positive if a bacterial band equivalent to the one observed in the standard growth conditions (i.e. growth in an oxygen-gradient highly saline medium in autotrophic conditions using thiosulfate as an electron donor and NaHCO₃ as a carbon source) was observed and if the band was still observed after 3 transfers. Growth was considered as negative if no band was observed or if the band was very thin and corresponded to the residual bacteria remaining from the inoculum, similar to what could be observed in the negative control. All growth tests were done in three biological replicates.

### Movement and magnetotactic response analysis

A customized magnetic microscope equipped with a triaxial Helmholtz coil set and controller (C-SpinCoil-XYZ, Micro Magnetics Inc.) and an Andor Zyla 5.5 high-speed camera was used (Bennet et al., 2014). The 3D-axis Helmholtz coils can generate DC magnetic fields with a precision of 5 % of Earth's magnetic field (±2.5 µT). Using the setup, we programmed the switching of the magnetic field between -3.5 and +3.5 mT for the U-turn. For U-turn measurements, the magnetic field was fixed for 2 s before switching. The switching was repeated to collect at least three U-turns in the field of view. The trajectories and U-turn of 218 cells were extracted and smoothed by a tracking script written in Python and based on the Trackpy package algorithm. The data were smoothed using a Gaussian filter with the standard deviation for the Gaussian kernel set to 1. For both trajectory and U-turn measurements, a 20× objective (N.A. 0.45) was used. The mathematical relation to calculate the cells magnetic moment from the U-turn time were as previously described (Chevrier et al., 2023).

### Whole genome sequencing and annotation

Whole genome sequencing and assembly of Magnetovirga frankeli strain SS-5T was performed in a previous study (Trubitsyn et al., 2021). The closed SS-5T genome sequence of 3,729,439 bases was deposited in GenBank under the BioProject number PRJNA491989 and BioSample number SAMN10093509. Automatic and manual sequence annotations were performed using the MicroScope platform (Vallenet et al., 2019). The SS-5T genome has a GC content of 61.65 % on average and 11.52 % Nosferatu Repeated Regions (excluding undetermined bases). It comprises 3318 CDS with an average length of 908 bp intergenic length of 146.71 bp.

### Phylogenetic analysis

First, GTDB-Tk v2.1.1 (Chaumeil et al., 2022) was used to assign SS-5T to a taxonomic group based on the GTDB classification (Parks et al., 2022). Once the taxonomic group identified, all genomes of good quality

(i.e. > 90 % complete with <5 % redundancy according to completion values on GTDB) were downloaded from the Genbank database (Benson et al., 2012) in October 2024, and rapidly re-annotated with PROKKA v1.14.6 (Seemann, 2014) to homogenize coding sequence predictions. The sequences of the 120 (bac120) phylogenetically informative markers used in GTDB (Chaumeil et al., 2022), were then extracted using pyhmmer 0.8.0 (Larralde and Zeller, 2023). To limit the number of gaps due to incomplete assemblies, we selected only genomes in which more than 90 % of markers were detected, and eliminated markers detected in less than 90 % of genomes. Multiple sequence alignments (MSAs) were then performed with MAFFT v7.490 (Katoh and Standley, 2013), trimmed with BMGE v1.12 (Criscuolo and Gribaldo, 2010) for each marker and concatenated into a single alignment. Trees were constructed using the maximum likelihood method implemented in the IQ-TREE v2.2 software (Nguyen et al., 2015; Minh et al., 2020) and a substitution model was selected from the global alignment with Modelfinder (Kalyaanamoorthy et al., 2017) with the -MFP option. Branch robustness was estimated by the ultrafast bootstrap method implemented in IQ-TREE (1000 resamples) (Nguyen et al., 2015; Minh et al., 2020). Trees were drawn and edited using FigTree v1.4.4 (Rambaut, 2010) and Inkscape software (Inkscape Project, 2020, https://inkscape.org). The average amino-acid identity (AAI) and average nucleotide identity (ANI) values were also used for genus delineation (Konstantinidis and Tiedje, 2005; Rodriguez-R and Konstantinidis, 2014) using CompareM (https://github.com/donovan-h-parks/CompareM) and FastANI (https://github.com/ParBLiSS/FastANI) respectively.

### Biochemical assays

Catalase activity was determined by generation of O₂ gas bubbles when cell suspensions were added to aqueous 3 % H₂O₂ solution. Oxidase activity was tested with the N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride assay using oxidase test disks (Sigma-Aldrich). *Escherichia coli* was used as positive control.

### Chemotaxonomic analyzes

For fatty acid, respiratory quinone and polar lipid analyses, and in order to avoid agar contamination, strain SS-5T was grown in bottles with septum caps in a standard liquid medium containing no agar and flushed at 1 % O₂. Cultures were inoculated at a 1:50 dilution from semisolid standard aerotactic bands. After 2 weeks of growth at 28 °C without agitation, cultures were harvested through two centrifugations: a first one at 4000g during 20 min and at 20 °C and a second one at 16,500 g during 5 min at 20 °C. The pellets were conserved at -80 °C and were then lyophilized. Fatty acid, respiratory quinone and polar lipid analyses were carried out by the Identification Service of the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Braunschweig, Germany. Fatty acids were analyzed after conversion into fatty acid methyl esters (FAMEs) by saponification, methylation and extraction (Sasser, 1990), respiratory quinones were analyzed by HPLC using a reverse phase column recording absorption spectra (Vieira et al., 2021) and polar lipids were detected using spray reagents specific for defined functional groups (Bligh and Dyer, 1959; Tindall et al., 2007).

### Electron microscopy

Transmission electron microscopy (TEM) was done using a Tecnai (FEI Company, Hillsboro OR) model G² BioTWIN electron microscope using an accelerated voltage of 100 kV.

For the observation of whole cells of strain SS-5T, a 20-µL drop collected on the aerotactic band of a semisolid oxygen gradient culture was deposited onto TEM copper grids coated with a carbon/formvar film, which were then washed and dried in air.
Thin-sectioned cells of strain SS-5<sup>T</sup> were prepared from three aero-tactic bands collected in three different semisolid oxygen-gradient cultures, concentrated by centrifugation and fixed in a 0.25 % (w/v) glutaraldehyde solution in sodium cacodylate buffer (0.1 M, pH 7.4) prepared in a similar artificial seawater than that used for the culture medium. Fixed cells were kept at 4 °C for at least 24 h. Cells were then washed in this same buffer and post-fixed for 1 h with 1 % (w/v) of osmium tetroxide. Magnetic cells were then dehydrated with successive ethanol baths with increasing concentrations and finally embedded in a monomeric resin (Epon 812). All of the chemicals used for histological preparation were purchased from Electron Microscopy Sciences. Sections (100 nm thick and 3 mm long) were made with the ultramicrotome UC7RT (Leica Microsystems GmbH), deposited onto TEM copper grids and stained with uranyless solution (Electron Microscopy Sciences) for 10 min and Reynolds lead citrate 3 % (Electron Microscopy Sciences) for 3 min.

## Results and discussion

### Cell morphology and motility

Light microscopy and TEM observations showed that cells of strain SS-5<sup>T</sup> are rod-shaped (Figs. 1 and 2). Cells of strain SS-5<sup>T</sup> have a smooth motility with frequent back and forth event in the presence or absence of a magnetic field with a speed of 56 ± 3 µm.s<sup>-1</sup>. When observed using the hanging drop assay under the light microscope, the majority of the cells are attracted by the south pole of the applied magnetic field meaning that most of the cells are north-seeking (Fig. 1A). The magnetotactic mechanism of strain SS-5<sup>T</sup> has been described as unipolar directed towards the oxic zone (Lefèvre et al., 2014). Indeed, when cells are observed under a capillary with an oxygen gradient opposed to the magnetic field, the cells form an aerotactic band at the oxic-anoxic interface that will move towards the oxic part of the capillary if the magnetic field is switched.

The magnetic moment of cells of strain SS-5<sup>T</sup> were obtained by analyzing their U-turn motion which relates the radius and turning time of cells upon external magnetic field reversal (Esquivel et al., 1983). We obtained an average absolute value of the magnetic moment M = 1.67 ± 0.12 × 10<sup>-16</sup> A m<sup>2</sup>. Similar values have been obtained in other MTB (Dunin-Borkowski, 1998; Nadkarni et al., 2013) including the model strain Magnetospirillum gryphiswaldense MSR-1, i.e. 2.5 ± 0.5 × 10<sup>-16</sup> A m<sup>2</sup> (Pichel et al., 2018) or 7.7 × 10<sup>-16</sup> A m<sup>2</sup> (Zahn et al., 2017).

Cells of strain SS-5<sup>T</sup> possess a single polar flagellum that measures 3–4 times the cell's body length (Fig. 2A). They produce a single chain of elongated magnetite magnetosomes (Fig. 2); in some cases two chains

could be observed (Fig. 2C) (Lefèvre et al., 2012). Ultrathin sections of cells of strain SS-5<sup>T</sup> showed typical Gram-negative cell membrane and the vesicle surrounding the magnetite particles of the magnetosomes (Fig. 2D and E). Strain SS-5<sup>T</sup> produces sulfur inclusions that are refractive when observed under the light microscope (Fig. 1C) and delimited by a double membrane typical of cells of the Gammaproteobacteria class (Fig. 2F) (Methner et al., 2023). To finish, cells of strain SS-5<sup>T</sup> also produce polyphosphate inclusions, generally located at the cell poles that are electron dense when observed under the TEM (Fig. 2) (Lefèvre et al., 2012). Strain SS-5<sup>T</sup> has been shown to have a unique dividing scheme in order to conserve its magnetic orientation. Indeed, during cell division the new flagellum is synthetized at the septum (Lefèvre et al., 2015).

### Growth characteristics

Since SS-5<sup>T</sup> was isolated, it is routinely grown in an oxygen-gradient highly saline medium in chemolithoautotrophic conditions using thiosulfate as an electron donor and bicarbonate as a carbon source. Bacterial growth is observed as a band forming at the oxic-anoxic transition zone in the medium. Bacteria can grow at temperatures ranging from 13 °C to 36 °C, with an optimum between 28 °C and 36 °C and have an optimal growth for a pH between 7 and 7.3 (Table 1). No growth is observed for pH lower than 6 and higher than 8.5. SS-5 can grow with NaCl concentrations ranging from 0 to 6 % and shows an optimal growth for concentrations between 3.78 and 4 % (Table 1). SS-5<sup>T</sup> cannot grow in an oxygen-gradient medium in chemoorganoheterotrophic conditions. Indeed, as shown in Table 1, no growth is observed when acetate, ethanol, formate, fumarate, lactate, malate, pyruvate, propionate or succinate are used as electron donors and carbon sources (10 mM). In a previous study (Lefèvre et al., 2012), it was mentioned that cells of strain SS-5<sup>T</sup> showed low levels of growth on succinate. Unfortunately, we were not able to reproduce this observation.

In addition, SS-5<sup>T</sup> is unable to grow in anaerobic conditions using nitrate (10 mM) as an electron acceptor both in chemolithoautotrophic and in chemoorganoheterotrophic conditions. Indeed, as shown in Table 1, no denitrification was observed when using, thiosulfate (5 mM) as an electron donor and bicarbonate (15 mM) as a carbon source or when acetate, ethanol, formate, fumarate, lactate, malate, pyruvate, propionate or succinate were used as electron donors and carbon sources (10 mM).

We also assessed the potential capacity for oxidase and catalase activities by strain SS-5<sup>T</sup>, both tests were negative.

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Light microscope images showing the shape of strain SS-5<sup>T</sup>. A) North-seeking cells at the edge of a 'hanging drop' in a magnetic field. B and C) Cells observed between slide and coverslip with some cells showing refractive, intracellular, sulfur-rich globules (C).</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Transmission electron microscopy (TEM) images of cells of strain SS-5T. A) Whole cell showing the single polar flagellum at black arrows. B) Aggregate of cells deposited on the TEM grid directly from an aerotactic band showing the presence in the cell of a single chain of elongated magnetosomes. C) Cell with two magnetosome chains. D) Longitudinal ultrathin section of a SS-5T cell showing the external membrane typical of a Gram-negative bacterium and the magnetosome membrane; the frame represents the image in panel E) that shows in higher magnification the magnetosome membrane pointed with white arrows. F) Longitudinal ultrathin section of a cell with two visible polyphosphate globules (P) and one sulfur inclusion (S).</figcaption></figure>

## Chemotaxonomy

The cellular fatty acids of strain SS-5T were C₁₆:₁ w7c (34.5 % of the total fatty acids), C₁₆:₀ (25.4 %), C₁₈:₁ w7c (22.5 %), C₁₈:₀ (6.4 %), C₁₈:₁ w9c (1.4 %), C₁₀:₀ 3OH (1.1 %) and C₁₈:₁ w5c (1.0 %). We compared the cellular fatty acids composition of strain SS-5T with related species of the genera *Thiolapillus* and *Sedimenticola* (Table S1), overall the major fatty acids of these taxa appear to be C₁₆:₁ ω7c, C₁₆:₀ and C₁₈:₁ ω7c (Narasingarao and Häggblom, 2006; Nunoura et al., 2014; Flood et al., 2015; Slobodkina et al., 2023).

Respiratory quinones analysis of strain SS-5T showed only the presence of ubiquinone Q-8. Similar observation was made for S. *hydrogenitrophicus*, S. *thiotaurini* and T. *brandeum* with also small amount of Q-9 and Q-7 detected for S. *hydrogenitrophicus* and S. *thiotaurini*, respectively (Nunoura et al., 2014; Flood et al., 2015; Slobodkina et al., 2023).

Aminolipids were the dominant polar lipids (Fig. S1). This observation differs from polar lipids observed for S. *hydrogenitrophicus*, S. *thiotaurini* and T. *brandeum* for which dominant polar lipids are phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol (Nunoura et al., 2014; Flood et al., 2015; Slobodkina et al., 2023).

## Phylogeny of strain SS-5T

Sequence alignments of the SS-5T 16S rRNA gene sequence against the NCBI database using the BLASTN algorithm with default settings suggested that Magnetovirga frankeli represented a novel genus of the Sedimenticolaceae family within the order Chromatiales of the Gammaproteobacteria. This assignation was further confirmed by the GTDB-Tk v2.1.1 analysis (Chaumeil et al., 2022), the phylogenomic trees (Fig. 3A and Fig. S2) and AAI pairwise comparisons with other isolate genomes and MAGs (metagenome-assembled genomes) (Fig. 3B). In

GTDB, SS-5T belongs to a genus g_JADGBD01 without a valid name (i.e. listed in LPSN, https://lpsn.dsmz.de/) in the Sedimenticolaceae family described by Slobodkina et al. (Slobodkina et al., 2023) and validated by LPSN. This genus is represented by two species only: that represented by SS-5T and another one represented by the MAG nS315bin38 assembled from magnetically-sorted populations of sediments in China (Lin et al., 2020). Based on the relative evolutionary divergence (RED) calculation (Parks et al., 2018) and AAI percentage (estimated to 84.8 %), both bacteria belong to different species.

A phylogenomic tree with a high-quality representative species of each Sedimenticolaceae genus showed that the closest genera (i.e. represented by MAGs Modern_marine.mb.35, L181_Sed1 and RIFOX-YD12_FULL_61_37) are composed of uncultivated species whose genomes were assembled from MAGs (Fig. S2). Their AAI percentage values calculated with SS-5T are all close to the 65 % threshold recommended to define the genus boundary (Konstantinidis and Tiedje, 2005; Konstantinidis et al., 2017). This delineation is further supported by the RED calculation from GTDB-tk. The 16S rRNA gene sequence identity percentage and AAI values between SS-5T and the most closely related type strains are presented in Table 2. The comparison with the type strains of the different genera of the Sedimenticolaceae family revealed values that are lower than the threshold criterion for prokaryotic genus delineation proposed to be 45 %–65 % (AAI) and 92–95 % (16 s rRNA) (Konstantinidis et al., 2017). Calculated ANI values between SS-5T and the most closely related type strains were much lower (i.e. < 90 %) than the threshold of 95 % to define a species. Thus, our phylogenomic analyses identified the taxonomic position of strain SS-5T as a representative of a new genus and species in the family Sedimenticolaceae. (See Table 3.)

The Sedimenticolaceae family being represented by few type strains with their genome sequenced, we looked for 16S rRNA gene sequences that could be close to SS-5T in public NCBI and SILVA databases
<table><caption>**Table 1**

Comparative characteristics of strain SS-5<sup>T</sup> and the most closely related type strains of the *Sedimenticolaceae* family.</caption><thead><tr><th>Characteristics</th><th>Magnetovirga frankeli SS-5<sup>T</sup></th><th>Thiolapillus brandeum Hiromi1<sup>T</sup></th><th>Sedimenticola hydrogenitrophicus SB48<sup>T</sup></th><th>Sedimenticola selenatireducens AK4OH1<sup>T</sup></th><th>Sedimenticola thiotaurini SIP-G1<sup>T</sup></th></tr></thead><tbody><tr><td>Isolation source</td><td>Highly saline lake</td><td>Hydrothermal vent chimney</td><td>Terrestrial mud volcano</td><td>Estuarine sediments</td><td>Salt marsh sediments</td></tr><tr><td>Cell morphology</td><td>Rod</td><td>Rod</td><td>Rod</td><td>Rod</td><td>Rod</td></tr><tr><td>Flagellar apparatus</td><td>1 polar flagellum</td><td>Non-motile</td><td>1 polar flagellum</td><td>Motile in certain conditions</td><td>Motile in certain conditions</td></tr><tr><td><strong>Growth characteristics</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>pH optimum</td><td>7-7.3</td><td>6.5</td><td>7.5</td><td>nd</td><td>nd</td></tr><tr><td>Temperature optimum (°C)</td><td>28-36</td><td>40</td><td>30</td><td>25</td><td>35</td></tr><tr><td>NaCl optimum (% w/v)</td><td>3.78-4</td><td>3.0</td><td>1.5-2.0</td><td>2.3</td><td>3.5</td></tr><tr><td>Autotrophy</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td><strong>Electron acceptors</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>O<sub>2</sub></td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Nitrate</td><td>-</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td><strong>Electron donors</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Thiosulfate</td><td>+</td><td>+</td><td>+/-</td><td>+</td><td>+</td></tr><tr><td>H<sub>2</sub>S</td><td>+</td><td>nd</td><td>-</td><td>+</td><td>+</td></tr><tr><td>Acetate</td><td>-</td><td>nd</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Ethanol</td><td>-</td><td>nd</td><td>+</td><td>nd</td><td>nd</td></tr><tr><td>Formate</td><td>-</td><td>-</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Fumarate</td><td>-</td><td>-</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Lactate</td><td>-</td><td>nd</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Malate</td><td>-</td><td>nd</td><td>+</td><td>+</td><td>+</td></tr><tr><td>pyruvate</td><td>-</td><td>-</td><td>+</td><td>+</td><td>+</td></tr><tr><td>propionate</td><td>-</td><td>nd</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Succinate</td><td>-</td><td>-</td><td>+</td><td>+</td><td>+</td></tr><tr><td><strong>Biochemical characteristics</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Oxidase</td><td>-</td><td>nd</td><td>nd</td><td>+</td><td>+</td></tr><tr><td>Catalase</td><td>-</td><td>nd</td><td>nd</td><td>-</td><td>+</td></tr></tbody></table>

All growth characteristics tests obtained for SS-5<sup>T</sup> were performed in triplicates.

+, positive; -, negative; +/-, weak growth. nd, not determined.

**Note:** For *Thiolapillus brandeum* Hiromi1<sup>T</sup>, chemolithoheterotrophic growth was observed with formate, fumarate, pyruvate and succinate used as carbon sources coupled with sulfur oxidation.

*Thiolapillus brandeum* Hiromi1<sup>T</sup>, data from Nunoura et al. (2014).

*Sedimenticola hydrogenitrophicus* SB48<sup>T</sup>, data from Slobodkina et al. (2023).

*Sedimenticola selenatireducens* AK4OH1<sup>T</sup>, data from Narasingarao and Häggblom (2006).

*Sedimenticola thiotaurini* SIP-G1<sup>T</sup>, data from Flood et al. (2015).

<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Genome-based classification of SS-5<sup>T</sup>. (A) Maximum-likelihood tree showing the genetic relationships of SS-5<sup>T</sup> with representative isolate genomes of the *Sedimenticolaceae* family (order *Chromatiales*, class *Gammaproteobacteria*) based on the alignment of 120 conserved proteins and the substitution matrix LG + F + R5. Only one genome of good quality (i.e. > 90 % complete with <5 % redundancy according to CheckM v1.0.18 (Parks et al., 2015)) was used for each *Sedimenticolaceae* genus. “C.” stands for the candidatus state. Accession numbers are given for each genome. In GTDB, the candidatus species noted with a “*” represents another genus “Sedimenticola_A” to be named. The tree was rooted with representative members of the closest family of the *Chromatiales* order (i.e. *Chromatiaceae*, grey group). Branches length represents the number of substitutions per site. The circles plotted on the internal nodes represent the statistical support estimated with an ultrafast bootstrap method and 1000 replicates, which was considered satisfactory when the value was greater than 95 %. A version including MAGs representing all *Sedimenticolaceae* genera is given in Fig. S2. (B) Heatmap of AAI relatedness among the representative isolate genomes of the *Sedimenticolaceae* family. The symbols represent genomes of panel A. The grey scale represents the percentage of AAI.</figcaption></figure>

(November 2024). This search showed that most of the *Sedimenticolaceae* bacteria in collection are represented in the whole genome tree (Fig. 3A). Only two sequences of uncultured magnetotactic bacteria with an identity percentage greater than 94 were retrieved: the magnetotactic *Gammaproteobacteria* strains SHHR-1 (Li et al., 2017) and FZSR-2 (Liu et al., 2022) (Genbank accession numbers KX344069.1 and

MW466773.1, respectively). With a 16S rRNA gene sequence nearly identical (> 99.9 %), these two bacteria belong to the same species than SS-5<sup>T</sup>.
<caption>**Table 2**</caption>

16S rRNA gene sequence identity and AAI values (%) between strain SS-5T and the type strains of the *Magneticolaceae* family.

<table><thead><tr><th>Strain</th><th>16S rRNA</th><th>AAI</th></tr></thead><tbody><tr><td><i>Thiolapillus brandeum</i> Hiromi1T</td><td>91.52</td><td>61.87</td></tr><tr><td><i>Sedimenticola hydrogenitrophicus</i> SB48T</td><td>92.38</td><td>61.49</td></tr><tr><td><i>Sedimenticola selenatireducens</i> AK4OH1T</td><td>91.68</td><td>61.65</td></tr><tr><td><i>Sedimenticola thiotaurini</i> SIP-G1T</td><td>91.88</td><td>61.66</td></tr></tbody></table>

*Biogeography, abundance and ecology of bacteria related to strain SS-5T*

We investigated the biogeography of strain SS-5T closest species by analyzing the metadata (e.g. isolation source) associated with the most similar genomes and 16S rRNA gene sequences. According to the data available, *Magnetovirga* represents a cosmopolitan group of magnetotactic species only. Indeed, isolated bacteria or MAGs available for this group were obtained after a magnetic concentration. The other genome available is another putative magnetotactic bacterium nS315bin38 with a medium-quality genome that was isolated from sediments of the alkaline (pH 8.5) and saline (4.3 ppt) Qinghai Lake, in China (Lin et al., 2020). In addition, strains ZSR-1 and FZSR-2 were isolated from the magnetic concentration of sediments located in a salt evaporation pool in Bohai Bay, at the Fuzhou saltern, Dalian City, eastern China. The two 16S rRNA gene sequences most closely related to strain SS-5T were from uncultured MTB isolated from brackish sediments (~23.9 ppt, pH 7.5) of the Shihe River estuary in Qinhuangdao City, China (Li et al., 2017) and from a salt evaporation pool (salinity 38.4 ppt, pH 8.01) in Bohai Bay, at the Fuzhou saltern, Dalian City, eastern China (Liu et al., 2022) for strains SHHR-1 and FZSR-2, respectively.

The undescribed genera forming a monophyletic group with *Magnetovirga* are composed of uncultivated species only without magnetotactic representative. The high-quality MAGs representing these genera: Modern_marine_mb.35 (GCA_018812525.1), RIFOXYD12_FULL_61_37 (GCA_001801995.1) and L181_Sed1 (GCA_025656895.1) were obtained from a groundwater sample from a deep geological site excavated in crystalline bedrock of the Fennoscandian Shield in the Swedish Nuclear Fuel (57.264°N 16.3936°E), a groundwater sample from a Rifle well in Colorado, USA (39.537°N 107.783°W) and the gill of the mollusk *Bathyaustriella thionipta* harvested from sediment of the hydrothermal vent at the Macauley Cone, New Zealand (30.22°S 178.45°W), respectively.

Although strain SS-5T belongs to a new genus with only few uncultured representatives, it appears that bacteria from this genus and its closest relatives are widespread in the Northern Hemisphere in freshwater, brackish, marine and highly saline aquatic habitats and are free-living or symbiotic microorganisms.

*Metabolic genomic analysis*

*Magnetosome biomineralization*

Genes involved in magnetosome formation of strain SS-5T have

partially been described (Lefèvre and Wu, 2013), here we represent the whole magnetosome gene cluster and its specificity (Fig. 4).

Strain SS-5T has been consecutively grown since 2010 under microaerobic conditions and cells were always able to produce magnetosomes. Non-magnetotactic mutants 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 (Schübbe et al., 2003). The absence of tRNA, integrase and/or transposase genes in close proximity of the magnetosome gene cluster indicates that this region is relatively stable in the genome of SS-5T and could explain why the magnetotactic phenotype was never lost for this strain. Thus, in SS-5T genome, the magnetosome gene cluster is not arranged within a clearly delineated genomic island. Moreover, the magnetosome gene cluster is 63.7 % GC content compared with 61.6 % GC content of the genome.

The *mamAB* cluster in SS-5T genome is formed by 29,731 bp. The gene order of *mam* genes is generally conserved when compared to the *mamAB* operon of magnetotactic *Alphaproteobacteria*. The SS-5T genome lacks the putative cytoskeletal attachment protein MamJ. This protein appears to be specific to magnetotactic species of the *Magnetospirillum* genus.

In most MTB genomes, the cluster containing *mamC* is found apart from the main cluster that contains magnetosome genes. This is also the case for SS-5T where *mamC* forms a cluster with *mamCOP*. In SS-5T genome, the cluster containing *mamC* is 5072 bp long, it consists of a cluster *mamPOCD*. Two genes coding for hypothetical proteins with unknown function are localized between *mamC* and *mamD*.

In MTB genomes, two clusters of the *feoAB* operon, involved in iron transport, are generally found, one specific to the MTB (named *feoABm* in the magnetotactic *Alphaproteobacteria* and *feoBAm* in the magnetotactic *Desulfobacterota*) and another *feoAB* cluster that has the same origin as in other bacteria (Lefèvre et al., 2013). Interestingly, in strain SS-5T genome, the *feoABm* operon is absent. A *feoAB* operon is found in the genome, not attached to the *mamAB* operon. This operon might support both the iron transport to maintain iron homeostasis within the cell and magnetosome formation.

*Signal transduction and chemotaxis*

The regulation proteins (i.e. proteins that modulate the expression of specific genes or the activity of other proteins in response to environmental or cellular signals) present in the genome of strain SS-5T were predicted using our P2RP tool (Barakat et al., 2013) based on our P2CS (Prokaryotic two-Component Systems) (Ortet et al., 2015) and P2TF (Prokaryotic Transcription Factors) (Ortet et al., 2012) databases. Out of the 3318 proteins, 112 were identified as transcription factors (50 transcription factors, 14 one component systems, 15 response regulators, 11 sigma factors, and 22 others), and 113 were classified as belonging to two-component systems (TCSs) and chemotaxis genes, representing a total of 7 % of the proteome. This high number of genes encoding for signal transduction histidine kinases responsible for signal

<caption>**Table 3**</caption>

Genome characteristics according to GTDB of *Magnetovirga frankeli* strain SS-5T compared to the type strains of the *Magneticolaceae* family.

<table><thead><tr><th>Strain</th><th>Magnetovirga frankeli<br>SS-5T</th><th>Thiolapillus brandeum<br>Hiromi1T</th><th>Sedimenticola hydrogenitrophicus<br>SB48T</th><th>Sedimenticola selenatireducens AK4OH1T</th><th>Sedimenticola<br>thiotaurini<br>SIP-G1T</th></tr></thead><tbody><tr><td>GenBank ID</td><td>GCA_009497875.2</td><td>GCF_000828615.1</td><td>GCF_025175675.1</td><td>GCF_000428045.1</td><td>GCF_001007875.1</td></tr><tr><td>Genome size, Mbp</td><td>3.7</td><td>3.2</td><td>4.7</td><td>4.6</td><td>4.0</td></tr><tr><td>G + C DNA content, %</td><td>61.7</td><td>56.2</td><td>60.7</td><td>56.7</td><td>57.6</td></tr><tr><td>Genome completeness, %</td><td>99.1</td><td>100</td><td>100</td><td>100</td><td>100</td></tr><tr><td>Contamination, %</td><td>0.6</td><td>0.25</td><td>1.25</td><td>2.51</td><td>0.47</td></tr><tr><td>CDSs</td><td>3318</td><td>2962</td><td>4185</td><td>4246</td><td>3645</td></tr><tr><td>Complete rRNAs</td><td>2</td><td>1</td><td>2</td><td>1</td><td>2</td></tr><tr><td>tRNA (total)</td><td>52</td><td>38</td><td>50</td><td>48</td><td>50</td></tr><tr><td>No. contigs</td><td>1</td><td>3</td><td>132</td><td>41</td><td>2</td></tr></tbody></table>
<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Magnetosome gene cluster (MGC) of strain SS-5T. Each arrow represents a magnetosome gene previously described in the MTB model strain *Magnetospirillum gryphiswaldense* MSR-1 (Uebe and Schüler, 2016). Grey genes are genes of unknown function or not conserved in MTB. Checkerboards represent collapsed regions spacing two sets of magnetosome genes.</figcaption></figure>

detection (Ortet et al., 2012) is in line with others microaerophilic MTB. For instance, 107, 212 and 87 signal transduction histidine kinases were detected in the genomes of *Magnetospirillum magneticum* strain AMB-1, *Magnetospirillum magnetotacticum* strain MS-1 and *Magnetococcus marinus* strain MC-1, respectively (Schübbe et al., 2009).

As previously mentioned, strain SS-5 displays a strong aerotaxis under very low concentrations of oxygen (i.e. 3 ± 3 µM, (Lefèvre et al., 2014)) and appears to quickly locate and maintain a position at the oxic-anoxic interface. Similar to most gradient-loving bacteria, the genome of strain SS-5 T contains a relatively large number of known chemotaxis genes, suggesting an important role for these genes in the ecology of SS-5 T (e.g. locating and maintaining their optimal position at the oxic-anoxic interface). We detected 5 major chemotaxis operons called *cheOp1–5*. Two of them contains more than 9 genes directly associated to chemotaxis. *cheOp2* contains 3 genes encoding putative chemoreceptors (MCP), 1 *cheA*, 1 *cheY*, 2 *cheW*, 1 *cheB*, 1 *cheR* and 1 *cheD* genes. *cheOp3* has the same genetic composition and organization but with only 2 MCPs. The other three contain the genes essential for chemotaxis (i.e. *cheA*, *cheY*, *cheB*, *cheW*). We found 18 genes encoding MCPs, 7 of which are present in the 5 major operons. In addition, we identified multiple additional motility related genes outside of *cheOp1–5* operons. Previous studies on model magnetotactic strains also revealed the presence of multiple chemotaxis pathways. For instance in *Magnetospirillum gryphiswaldense* strain MSR-1, four *che* operons encoding chemotaxis-related proteins have been detected and studied (Pfeiffer et al., 2020).

With the hope to establish strain SS-5 T as a future model to study magnetosomes formation and magnetotactic mechanisms, we believe the study of the signal transduction and chemotaxis genes mentioned above will allow us to increase our understanding of the functioning of the magneto-aerotactic mechanisms in polar magnetotactic bacteria.

### Autotrophic growth

Strain SS-5T has been shown to grow chemolithoautotrophically using sulfide and thiosulfate as electron donors and sodium bicarbonate/CO2 as the major source of carbon. “Green-like” ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) genes were previously identified, indicating strain SS-5T has the genetic potential to employ the Calvin-Benson-Bassham cycle for autotrophy (Lefèvre et al., 2012).

### Nitrogen metabolism

Strain SS-5T is one of the few MTB that does not demonstrate nitrogenase activity (Lefèvre et al., 2012) and thus it is not surprising that *nif* genes are not present in the genome of this bacterium. The strain does utilize ammonia as a source of cellular nitrogen and has an ammonium transporter gene *amt*, glutamine synthetase/glutamate synthase (bacteria-like GS-GOGAT cycle) and glutamate dehydrogenase for the assimilation of ammonia. The genes *nasDE* are localized immediately upstream of the nitrite transporter gene *nirC*. The *nasDE* cluster codes for a nitrite reductase that is involved in both assimilatory and dissimilatory pathways and is involved in the reduction of nitrite to ammonia. Assimilatory nitrate reductase genes *nasBC* were not identified. Together, these latter results suggest that strain SS-5T may be capable of using nitrite but not nitrate as a source of cellular nitrogen although this remains to be tested in the laboratory. Strain SS-5T possesses all the genes for the entire denitrification pathway including those

encoding the nitrate reductases *NarGHJC*, the nitrite reductases *NirSK*, *NasDE* and cytochrome *cd1*, the nitric oxide reductase *NorBC*, and the nitrous oxide reductase *NosZ*. Additionally, the genes encoding the enzymes for the reduction of nitrate to nitrite through a periplasmic respiratory nitrate reductase system, *napABCDFGH*, were identified in two gene clusters ~55 kb apart. However, nitrate transporters have not been identified in SS-5T genome and despite having these nitrate reducing genes, attempts to grow strain SS-5T anaerobically with nitrate as a terminal electron acceptor were unsuccessful (Table 1).

### Sulfur metabolism

As a sulfide- and thiosulfate-oxidizing bacterium, strain SS-5T has several genes related to oxidation of sulfide and thiosulfate scattered throughout its genome. This includes *soxAX*, *soxYZ*, *soxB*, *soxW* and *soxF*, all in single copies. Strain SS-5T seems to be missing *soxCD*. Subsequent oxidation can be achieved by proteins encoded by the reverse-acting (oxidizing) Dsr system that includes proteins closely related to dissimilatory sulfite reductases operating to reduce sulfite. Strain SS-5T has the complete set of 16 Dsr encoding genes including the membrane complex *dsrMKJOP* and the reverse acting dissimilatory sulfite reductase *dsrAB*.

No genes for sulfate reduction, either assimilatory or dissimilatory, were identified, indicating a reliance on exogenous reduced sulfur sources as a source of cellular S directed to anabolic pathways.

Based on the data presented here, strain SS-5T is considered to represent a novel species of a new genus, for which the name *Magnetovirga frankeli* gen. Nov., sp. nov. is proposed. The protologue of the proposed new taxa is presented in the Table 4. *Magnetovirga* represents the third genus of the *Sedimenticolaceae* family along with *Thiolapillus* (Nunoura et al., 2014) and *Sedimenticola* (Narasingarao and Häggblom, 2006; Flood et al., 2015; Slobodkina et al., 2023). *Magnetovirga frankeli* represents the fifth type strain described in the *Sedimenticolaceae* family. The definition of the *Sedimenticolaceae* proposed by Slobodkina et al. in 2023 (Slobodkina et al., 2023) is also valid for *Magnetovirga frankeli*.

Supplementary data to this article can be found online at https://doi.org/10.1016/j.syapm.2025.126621.

### CRediT authorship contribution statement

**Mila Sirinelli-Kojadinovic:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Elsa C.A. Turrini:** Investigation, Formal analysis. **Emma Ropion:** Formal analysis. **Béatrice Alonso:** Formal analysis. **Marine Bergot:** Methodology, Investigation, Formal analysis, Data curation. **Emilie Gachon:** Validation, Methodology, Investigation, Formal analysis. **Philippe Ortet:** Methodology, Investigation, Formal analysis. **Paul E.D. Soto-Rodriguez:** Methodology, Formal analysis. **Caroline L. Monteil:** Validation, Supervision, Methodology, Formal analysis. **Christopher T. Lefèvre:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.
<table><caption>**Table 4**

Description of Magnetovirga gen. Nov. and Magnetovirga frankeli sp. nov.</caption><thead><tr><th>Parameter</th><th>Genus: Magnetovirga gen. Nov.</th><th>Species: Magnetovirga frankeli sp. nov.</th></tr></thead><tbody><tr><td>Genus name</td><td>Magnetovirga</td><td>Magnetovirga frankeli</td></tr><tr><td>Species name</td><td></td><td>frankeli</td></tr><tr><td>Specific epithet</td><td></td><td>sp. nov.</td></tr><tr><td>Status</td><td>gen. Nov.</td><td>fran'kel.i. N.L. gen. n.</td></tr><tr><td>Etymology</td><td>Ma.gne.to.vi'rga.: Gr. masc. n. magnes, magnet; L. fem. n. virga, rod; N.L. fem. n. Magnetovirga, a magnetic rod.</td><td>frankeli, of Frankel, named in honor of US physicist Richard B. Frankel from California Polytechnic State University, San Luis Obispo, who made major contributions to the study of magnetotactic bacteria. Cells exhibit the following characteristics in addition to those given for the genus. They are 2-3 µm long by 1 µm wide. They are obligately microaerophilic and mesophilic, with a growth temperature range of 13 to 36 °C, and optimal growth temperature of 28 °C to 36 °C. Cells produce internal sulfur globules when grown on S<sub>2</sub>O<sub>3</sub><sup>2-</sup>. The GC content of the genome is 61.65%. The major cellular fatty acids are C<sub>16:1</sub> w7c, C<sub>16:0</sub>, C<sub>18:1</sub> w7c and C<sub>18:0</sub>. Ubiquinone Q-8 is the only respiratory quinones detected in strain SS-5<sup>T</sup>. Aminolipids are the dominant polar lipids. SS-5<sup>T</sup></td></tr><tr><td>Description of the new taxon and diagnostic traits</td><td>Cells are rod-shaped, Gram-negative. They assimilate inorganic carbon (as CO<sub>2</sub>) chemolithoautotrophically with S<sub>2</sub>O<sub>3</sub><sup>2-</sup> as the electron donor by using the CBB cycle. Cells are motile by means of a single polar flagellum (monotrichous). They exhibit a unipolar magnetotaxis directed towards the oxic zone, and biomineralize a single chain of magnetosomes that contain elongated magnetite crystals positioned along the long axis of the cell. Strain SS-5<sup>T</sup> belongs to the Sedimenticolaceae family within the order Chromatiales of the Gammaproteobacteria. The type species is Magnetovirga frankeli strain SS-5<sup>T</sup>.</td><td> (=DSM 116211<sup>T</sup> = JCM 39467<sup>T</sup>) Complete GenBank: GCA_009497875.2 3.7 61.7 HQ595729 USA California Sediment of the Salton Sea</td></tr><tr><td>Designation of the Type Strain</td><td></td><td></td></tr><tr><td>Strain Collection Numbers</td><td></td><td></td></tr><tr><td>Genome status</td><td></td><td></td></tr><tr><td>Genome accession</td><td></td><td></td></tr><tr><td>Genome size</td><td></td><td></td></tr><tr><td>GC mol%</td><td></td><td></td></tr><tr><td>16S rRNA gene accession nr.</td><td></td><td></td></tr><tr><td>Country of origin</td><td></td><td></td></tr><tr><td>Region of origin</td><td></td><td></td></tr><tr><td>Source of isolation</td><td></td><td></td></tr><tr><td>Sampling date</td><td></td><td></td></tr><tr><td>Latitude</td><td></td><td></td></tr><tr><td>Longitude</td><td></td><td></td></tr><tr><td>Number of strains in study</td><td></td><td></td></tr><tr><td>Information related to the Nagoya Protocol</td><td></td><td>Non applicable</td></tr></tbody></table>

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgments

This work was supported by the French National Research Agency (CarboMagnet: ANR-21-01CE-0010). We acknowledge the Institut de

Radioprotection et de Sûreté Nucléaire (IRSN) at CEA Cadarache for the access of the transmission electron microscope Tecnai G<sup>2</sup> BioTWIN. We are grateful to the INRA MIGALE bioinformatics platform (http://migale.jouy.inrae.fr) for providing computational resources. E.G. acknowledges financial support from the French National Research Agency (manacovv: ANR-20-92CE-0051). P.E.D.S.R acknowledges financial support from the Excellence Initiative of Aix-Marseille University – A*Midex, a French “Investissements d’Avenir” program.

## Data availability

Data will be made available on request.

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