Terasakiella Sp Sh-

Haijian Du

Wenyan Zhang

Lin Wei

Xiao Tian

Long-Fei Wu

Sh-

Genomic analysis of a pure culture of magnetotactic bacterium

Keywords: magnetotactic bacteria, magnetotaxis, pure culture, comparative genomic analysis

Magnetotactic bacteria (MTB) display magnetotaxis ability because of biomineralization of intracellular nanometer-sized, membrane-bound organelles termed magnetosomes. Despite having been discovered more than half a century, only a few representatives of MTB have been isolated and cultured in the laboratory. In this study, we report the genomic characterization of a novel marine magnetotactic spirillum strain SH-1 belonging to the genus Terasakiella that was recently isolated. A gene encoding haloalkane dehalogenase, which is involved in the degradation of chlorocyclohexane, chlorobenzene, chloroalkane and chloroalkene, was identified. SH-1 genome contained cysCHI and soxBAZYX genes, thus potentially capable of assimilatory sulfate reduction to H2S and using thiosulfate as electron donors and oxidizing it to sulfate. Genome of SH-1 also contained genes encoding periplasmic dissimilatory nitrate reductases (napAB), assimilatory nitrate reductase (nasA) and assimilatory nitrite reductases (nasB), suggesting that it is capable of gaining energy by converting nitrate to ammonia. The pure culture of Terasakiella sp. SH-1 together with its genomic results offers new opportunities to examine biology, physiology and biomineralization mechanisms of MTB.

INTRODUCTION

Magnetotactic bacteria (MTB) are prokaryotes that orient and migrate along the geomagnetic field lines, a behavior referred to as magnetotaxis or microbial magnetoreception. MTB share the capacity to synthesize magnetosomes, which are magnetic crystals of magnetite (Fe3O4) and/or greigite (Fe3S4) and enveloped by a phospholipid bilayer membrane [START_REF] Blakemore | Magnetotactic Bacteria[END_REF][START_REF] Bazylinski | Controlled Biomineralization of Magnetite (Fe3O4) and Greigite (Fe3S4) in a Magnetotactic Bacterium[END_REF][START_REF] Bazylinski | Magnetosome formation in prokaryotes[END_REF]. Many morphotypes of MTB, including coccoid, ovoid, rod, vibrio, spirillum, and multicellular forms have been observed worldwide across from freshwater, brackish and marine waters to waterlogged soils [START_REF] Maratea | Aquaspirillum magnetotacticum sp. nov., a Magnetic Spirillum[END_REF][START_REF] Arakaki | Desulfovibrio magneticus sp. nov., a novel sulfate-reducing bacterium that produces intracellular single-domain-sized magnetite particles[END_REF][START_REF] Hanzlik | Pulsed-field-remanence measurements on individual magnetotactic bacteria[END_REF][START_REF] Lefèvre | Isolation and characterization of a magnetotactic bacterial culture from the Mediterranean Sea[END_REF][START_REF] Wenter | Ultrastructure, tactic behaviour and potential for sulfate reduction of a novel multicellular magnetotactic prokaryote from North Sea sediments[END_REF][START_REF] Zhou | Adaptation of spherical multicellular magnetotactic prokaryotes to the geochemically variable habitat of an intertidal zone[END_REF][START_REF] Lin | A putative greigite-type magnetosome gene cluster from the candidate phylum Latescibacteria[END_REF]Lin et al., 2017).

All known MTB belong to the domain Bacteria, predominantly within the phylum Proteobacteria [START_REF] Simmons | Spatiotemporal distribution of marine magnetotactic bacteria in a seasonally stratified coastal salt pond[END_REF][START_REF] Jogler | Genomics, genetics, and cell biology of magnetosome formation[END_REF][START_REF] Lefèvre | Ecology, diversity, and evolution of magnetotactic bacteria[END_REF][START_REF] Ji | The chimeric nature of the genomes of marine magnetotactic coccoid-ovoid bacteria defines a novel group of Proteobacteria[END_REF][START_REF] Lin | Genomic expansion of magnetotactic bacteria reveals an early common origin of magnetotaxis with lineage-specific evolution[END_REF]. A diverse group of MTB affiliated with the phylum Nitrospirae, the candidate phylum Omnitrophica, the candidate phylum Latescibacteria and the phylum Planctomycetes have been identified [START_REF] Vali | Magnetotactic bacteria and their magnetofossils in sediments[END_REF][START_REF] Lefèvre | Moderately thermophilic magnetotactic bacteria from hot springs in Nevada[END_REF][START_REF] Kolinko | Single-cell analysis reveals a novel uncultivated magnetotactic bacterium within the candidate division OP3[END_REF][START_REF] Lin | Genomic expansion of magnetotactic bacteria reveals an early common origin of magnetotaxis with lineage-specific evolution[END_REF]. More recently, our knowledge of the phylogenetic diversity of MTB has been dramatically expanded and so far these organisms have been found in up to 16 bacterial phylum-level lineages [START_REF] Lin | Expanding magnetic organelle biogenesis in the domain Bacteria[END_REF][START_REF] Uzun | Unravelling the diversity of magnetotactic bacteria through analysis of open genomic databases[END_REF]. Most of the cultured MTB belong to the phylum Proteobacteria, and a large number of these are within the Alphaproteobacteria, such as freshwater Magnetospirillum species MS-1, AMB-1, MSR-1 and XM-1 [START_REF] Blakemore R P | Isolation and pure culture of a freshwater magnetic spirillum in chemically defined medium[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][START_REF] Wang | Complete genome sequence of Magnetospirillum sp. strain XM-1, isolated from the Xi'an City Moat, China[END_REF], marine vibrio strains MV-1 and MV-2 [START_REF] Delong | Multiple Evolutionary Origins of Magnetotaxis in Bacteria[END_REF][START_REF] Bazylinski | Magnetovibrio blakemorei gen. nov., sp. nov., a magnetotactic bacterium (Alphaproteobacteria: Rhodospirillaceae) isolated from a salt marsh[END_REF], marine spirillum strains MMS-1 and QH-2 [START_REF] Zhu | Isolation and characterization of a marine magnetotactic spirillum axenic culture QH-2 from an intertidal zone of the China Sea[END_REF][START_REF] Williams | Magnetospira thiophila gen. nov., sp. nov., a marine magnetotactic bacterium that represents a novel lineage within the Rhodospirillaceae (Alphaproteobacteria)[END_REF], and recently isolated marine spirillum Candidatus Terasakiella magnetica strain PR-1 belonging to the genus Terasakiella [START_REF] Monteil | Genomic study of a novel magnetotactic Alphaproteobacteria uncovers the multiple ancestry of magnetotaxis[END_REF].

Much genomic data on MTB has now been obtained and analyzed. The whole genomes of some cultured MTB (e.g., AMB-1, MSR-1, QH-2, MC-1, MO-1, XM-1, BW-2 and SS-5) have been obtained [START_REF] Matsunaga | Complete genome sequence of the facultative anaerobic magnetotactic bacterium Magnetospirillum sp. strain AMB-1[END_REF][START_REF] Richter | Comparative genome analysis of four magnetotactic bacteria reveals a complex set of group-specific genes implicated in magnetosome biomineralization and function[END_REF][START_REF] Schubbe | Complete genome sequence of the chemolithoautotrophic marine magnetotactic coccus strain MC-1[END_REF][START_REF] Ji | Comparative genomic analysis provides insights into the evolution and niche adaptation of marine Magnetospira sp. QH-2 strain[END_REF][START_REF] Wang | Complete genome sequence of Magnetospirillum sp. strain XM-1, isolated from the Xi'an City Moat, China[END_REF][START_REF] Ji | The chimeric nature of the genomes of marine magnetotactic coccoid-ovoid bacteria defines a novel group of Proteobacteria[END_REF][START_REF] Uebe | Reevaluation of the complete genome sequence of Magnetospirillum gryphiswaldense MSR-1 with single-molecule real-time sequencing data[END_REF][START_REF] Geurink | Complete genomesequence of strain BW-2, a magnetotactic gammaproteobacterium in the family Ectothiorhodospiraceae, isolated from abrackish spring in Death Valley, California[END_REF][START_REF] Trubitsyn | Complete genome sequence of strain SS-5, a Magnetotactic Gammaproteobacterium isolated from the Salton Sea, a Shallow, Saline, Endorheic Rift Lake located on the San Andreas Fault in California[END_REF].

While many other MTB remain to be draft sequenced. After decades of research on MTB genomes, many interesting features have been revealed. In particular amongst these is the presence of clustered genes that control magnetosome biomineralization and arrangement in MTB cells, which are termed magnetosome gene islands (MAIs) [START_REF] Grunberg | A large gene cluster encoding several magnetosome is conserved in different species of magnetotactic bacteria[END_REF][START_REF] Murat | Comprehensive genetic dissection of the magnetosome gene island reveals the step-wise assembly of a prokaryotic organelle[END_REF][START_REF] Lohße | Functional analysis of the magnetosome island in Magnetospirillum gryphiswaldense: the mamAB operon is sufficient for magnetite biomineralization[END_REF] or magnetosome gene clusters (MGCs) (Lin et al., 2017).

We recently isolated a new marine magnetotactic spirillum (designated Terasakiella sp. SH-1) into axenic culture from an intertidal zone in Sanya, China [START_REF] Du | Magnetosome Gene Duplication as an Important Driver in the Evolution of Magnetotaxis in the Alphaproteobacteria[END_REF]. SH-1 belongs to the genus Terasakiella in the Alphaproteobacteria and is closely related to Ca. Terasakiella magnetica strain PR-1 [START_REF] Monteil | Genomic study of a novel magnetotactic Alphaproteobacteria uncovers the multiple ancestry of magnetotaxis[END_REF]. In the previous study, we have reported a duplication event of magnetosome genes within mamAB operon in the genome of SH-1, which suggests that gene duplication event plays a potentially important role in the evolution of magnetotaxis in the Alphaproteobacteria [START_REF] Du | Magnetosome Gene Duplication as an Important Driver in the Evolution of Magnetotaxis in the Alphaproteobacteria[END_REF]. Here, we perform a comparative analysis of the complete genome of SH-1 with representative MTB genomes, which provides novel insights in to metabolic potential and ecosystem function of this newly isolated MTB strains.

MATERIALS AND METHODS

Genome analysis

The whole-genome sequencing of the SH-1 was performed as previously described [START_REF] Du | Magnetosome Gene Duplication as an Important Driver in the Evolution of Magnetotaxis in the Alphaproteobacteria[END_REF]. All genome data used in this study was downloaded from the NCBI site. The gene prediction was performed on the MicroScope platform [START_REF] Vallenet | MicroScope--an integrated microbial resource for the curation and comparative analysis of genomic and metabolic data[END_REF]. The tandem repeats annotation was obtained using the Tandem Repeat Finder (http://tandem.bu.edu/trf/trf.html) [START_REF] Benson | Tandem Repeats Finder: a program to analyze DNA sequences[END_REF], and the minisatellite DNA and microsatellite DNA were selected based on the number and length of repeat units. Prophage regions were predicted using the PHAge Search Tool Enhanced Release (PHASTER) web server (http://phaster.ca/) [START_REF] David | PHASTER: a better, faster version of the PHAST phage search tool[END_REF] and CRISPR identification using CRISPRFinder [START_REF] Ibtissem | CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats[END_REF]. The best hit was performed using the BLAST (Biology, 2012) alignment tool for function annotation. Seven databases including KEGG (Kyoto Encyclopedia of Genes and Genomes) [START_REF] Minoru | KEGG as a reference resource for gene and protein annotation[END_REF], COG (Clusters of Orthologous Groups) [START_REF] Galperin | Expanded microbial genome coverage and improved protein family annotation in the COG database[END_REF]Kira et al., 2015), NR (Non-Redundant Protein Database databases), Swiss-Prot [START_REF] Consortium | UniProt: a hub for protein information[END_REF], GO (Gene Ontology) [START_REF] Ashburner | Gene Ontology: tool for the unification of biology[END_REF], TrEMBL [START_REF] Rolf | UniProt: the Universal Protein knowledgebase[END_REF], and EggNOG [START_REF] Jaime | eggNOG 4.5: a hierarchical orthology framework with improved functional annotations for eukaryotic, prokaryotic and viral sequences[END_REF] were used for general function annotation.

Nitrogen metabolism analysis

Protein sequence similarities in relation to nitrogen metabolism were determined using BLAST on the MicroScope [START_REF] Vallenet | MicroScope--an integrated microbial resource for the curation and comparative analysis of genomic and metabolic data[END_REF] platforms. Proteins in different organisms were defined as orthologs when their alignments met the criteria: E-value < 1e-5, identity > 30%, and query coverage > 50%. The gene markers used to decide whether particular transformation reactions were present were based on a published review (Kuypers et al., 2018) (see Table S1); the markers were for genes involved in 15 reactions involving eight key inorganic nitrogen species having different oxidation states (Fig. 2a, Table S1).

Pan/core genome analysis

A pan/core genome analysis using the MicroScope [START_REF] Vallenet | MicroScope--an integrated microbial resource for the curation and comparative analysis of genomic and metabolic data[END_REF] gene families (MICFAM), computed using the SiLiX software [START_REF] Miele | Ultra-fast sequence clustering from similarity networks with SiLiX[END_REF] (threshold: 50% of amino-acid identity and 80% of align. coverage).

RESULTS AND DISCUSSION

Genome overview of SH-1

The genome of SH-1 comprised 3 832 570 bp in a circular chromosome having the average G+C content of 47.5%. The chromosome contained 3 664 predicted coding sequences (CDS), including those encoding 50 tRNAs and three sets of rRNAs (5S, 16S, and 23S), which corresponded to 90.12% of the genome being coding sequences. Compared to freshwater M. magneticum AMB-1 and M. gryphiswaldense MSR-1, the genome size, G+C content and CDS number of marine magnetospirilla (SH-1, PR-1, MV-1 and QH-2) are smaller (Table 1). The G+C content of SH-1 is smaller than those of MV-1 and QH-2 but higher than that of PR-1 (Table 1). In particular, SH-1 has a higher number of tRNAs and rRNA operons (Table 1). There was no evidence for the presence of extrachromosomal elements such as plasmids for SH-1. A total of 1 847 CDS (50.8%) could be assigned to putative functions, 1 227 CDS (33.8%) represented conserved hypothetical proteins of unknown function, and the remaining 559 CDS (18.4%) show no sequence similarity to any previously reported sequence. We identified 76 tandem repeats in the genome of SH-1, including 51 minisatellite DNAs and four microsatellite DNAs. No CRISPR sequence was found. In addition, three prophages were identified to be distributed throughout the genome.

Carbon metabolism

Genes involved in glycolysis (core module involving three-carbon compounds), the tricarboxylic acid (TCA) cycle, the non-oxidative phase of reductive pentose phosphate pathway, phosphoribosyl diphosphate (PRPP) biosynthesis, the ethylmalonyl pathway, and the synthesis of all 20 essential amino acids were identified in SH-1 genome (Fig. 1). Genes involved in the Embden-Meyerhof pathway in glycolysis including hexokinase (EC: 2.7.1.1), glucokinase (EC: 2.7.1.2) and glyceraldehyde-3-phosphate dehydrogenase (EC: 1.2.7.6 and EC: 1.2.1.9) were missing. Most of the genes involved in reductive tricarboxylic acid (rTCA) cycle were detected, except for ATP-citrate lyase (EC: 2.3.3.8), suggesting that SH-1 is not able to use the rTCA cycle for autotrophy. Protein encoded by gene SH1_v1_2542 was found to be 67.1% identical to a fragment (170 aa/310 aa) of the gene dhlA encoding haloalkane dehalogenase (EC: 3.8.1.5), which was previously identified and characterized in Xanthobacter autotrophicus involved in the degradation of chlorocyclohexane, chlorobenzene, chloroalkane and chloroalkene (Fig. 1). Organisms having haloalkane dehalogenase are able to degrade hexachlorocyclohexane to 2,3,4,5,6-pentachlorocyclohexanol and 2,3,5,6-tetrachloro-1,4-cyclohexanol, and to degrade 1.2-dichloroethane to glyoxylate [START_REF] Janssen | Cloning of 1,2-dichloroethane degradation genes of Xanthobacter autotrophicus GJ10 and expression and sequencing of the dhlA gene[END_REF]. Similar genes have not been detected in the genomes of other MTB or other organisms belonging to genus Terasakiella. Its presence in SH-1 could be a result of horizontal gene transfer (HGT). It should be noted that active-site residues His289 and Asp260 of DHLA are not present in SH1_v1_2542, thus its function awaits further detailed characterization (Fig. S1) [START_REF] Verschueren | Refined X-ray structures of haloalkane dehalogenase at pH 6.2 and pH 8.2 and implications for the reaction mechanism[END_REF]. Furthermore, additional studies are needed to assess whether SH-1 has the ability to degrade chlorocyclohexane, chlorobenzene, chloroalkane, and chloroalkene, and to investigate the broader complexity of carbon metabolism by SH-1.

Sulfur metabolism

Unlike AMB-1, MSR-1 and QH-2, no evidence was found in the genome of SH-1 for the presence of any of the genes for dissimilatory sulfate reduction, including dsr genes or the Apr system. However, cysN and cysD were present in the genome of SH-1, which encode enzymes that activate sulfate and catalyze the synthesis of adenosine-5-phosphosulfate (APS) in the assimilatory sulfate reduction pathway (Fig. 1). Genes of cysC (APS kinase; produces 3-phosphoadenosine-5-phosphosulfate: PAPS), cysH (PAPS reductase; catalyzes the conversion of PAPS to sulfite) and cysI (sulfite reductase) were also identified. Therefore, SH-1 may be capable of assimilatory sulfate reduction to H2S (Pinto and Microbiology, 2004). SH-1 also contained soxBAZYX gene cluster involved in thiosulfate oxidation (Fig. 1). The soxCD genes encoding sulfur dehydrogenase were also found (Fig. 1). These findings suggest that SH-1 could use thiosulfate as an electron donor and oxidize it to sulfate [START_REF] Hensen | Thiosulphate oxidation in the phototrophic sulphur bacterium Allochromatium vinosum[END_REF].

Nitrogen metabolism

Both SH-1 and PR-1 contained genes encoding periplasmic dissimilatory nitrate reductases (napAB), assimilatory nitrate reductase (nasAB), hydroxylamine reductase (hcp) and assimilatory nitrite reductases (nasB) (Fig. 1), suggesting that they can gain energy by converting nitrate to ammonia (Kuypers et al., 2018). nirB is identified in genomes of SH-1 and PR-1, but nirD is not present. Whether NirB alone could carry out the function needs more studies. napAB are present in the genomes of Terasakiella pusilla but nasAB or hcp are not identified (Fig. 2c &d). However, T.

pusilla contains genes encoding cytochrome c-dependent nitric oxide (NO) reductases (cNOR and cnorB), and nitrous oxide reductases (nosZ) (Fig. 2c), which are not present in SH-1 and PR-1.

This may reflect differences in the metabolism of MTB compared with other bacteria belonging to the same genus. Although genes fixS, fixG and fixI were identified, the nif gene cluster responsible for nitrogen fixation was not present in the genomes of genus Terasakiella. Whether the genus Terasakiella can fix nitrogen needs further investigation.

We further compared the microbial transformations of nitrogen among 14 reprentative species of MTB (Terasakiella sp. SH-1, Candidatus Terasakiella magnetica strain PR-1, Magnetospira sp. strain QH-2, Magnetospirillum gryphiswaldense strain MSR-1, Among these enzymes, genes encoding for nitrate reductases, nitrite reductase and nitric oxide reductases were most commonly found. These genes occur widely in genomes of Alphaproteobacteria, Gammaproteobacteria and Etaproteobacteria. Our results show that MTB commonly act as denitrifiers and nitrogen-fixers in nitrogen cycle processes. Previous studies have shown the potential link between denitrification and magnetosome formation in freshwater Magnetospirillum spp. [START_REF] Bazylinski | Denitrification and Assimilatory Nitrate Reduction in Aquaspirillum magnetotacticum[END_REF][START_REF] Matsunaga | Magnetite formation by a magnetic bacterium capable of growing aerobically[END_REF][START_REF] Matsunaga | Respiratory inhibitors of a magnetic bacterium Magnetospirillum sp. AMB-1 capable of growing aerobically[END_REF][START_REF] Yang | Effects of growth composition, iron sources and atmospheric oxygen concentrations on production of luciferase-bacterial magnetic particle complex by a recombinant Magnetospirillum magneticum AMB-1[END_REF][START_REF] Li | The periplasmic nitrate reductase nap is required for anaerobic growth and involved in redox control of magnetite biomineralization in Magnetospirillum gryphiswaldense[END_REF]. Therefore, denitrification should play an important role in redox control for magnetosome formation. However, their role in magnetosome formation has not yet been fully clarified and needs further experimental investigation.

Magnetospirillum

Prophage

Two predicted prophage regions were found in the genome of SH-1, having lengths of 11.0-22.6 kb and G+C contents of 46.1-48.4 % (Table S2 and Fig. S2). Among these, HK-1, Candidatus Magnetoglobus multicellularis str. Araruama, Candidatus Magnetobacterium bavaricum and Candidatus Desulfamplus magnetomortis BW-1) using PHASTER (Table S3).

Most MTB genomes (11 out of 12 genomes) were found to contain predicted prophage regions, with HK-1 being the only exception. Temperate phage genes have been identified in 40-50% of microbial genomes [START_REF] Canchaya | Prophage Genomics[END_REF][START_REF] Casjens | Casjens S. Prophages and bacterial genomic: what have we learned so far?[END_REF][START_REF] Fouts | Phage_Finder: Automated identification and classification of prophage regions in complete bacterial genome sequences[END_REF][START_REF] Paul | Prophages in marine bacteria: dangerous molecular time bombs or the key to survival in the seas?[END_REF][START_REF] Touchon | Genetic and life-history traits associated with the distribution of prophages in bacteria[END_REF], and almost 50% of bacterial genomes contain at least one prophage [START_REF] Touchon | Genetic and life-history traits associated with the distribution of prophages in bacteria[END_REF].

Our results show that prophages may exist in many genomes of MTB. Through lysogenic conversion or transduction, bacteriophages and archaeal viruses contribute to the horizontal transfer of genetic material among microbial genomes [START_REF] Touchon | Genetic and life-history traits associated with the distribution of prophages in bacteria[END_REF][START_REF] Touchon | Embracing the enemy: the diversification of microbial gene repertoires by phage-mediated horizontal gene transfer[END_REF].

Previous studies have shown that HGT plays an important role in the evolution of magnetotaxis in bacteria [START_REF] Rioux | A second actin-like MamK protein in Magnetospirillum magneticum AMB-1 encoded outside the genomic magnetosome island[END_REF][START_REF] Monteil | Genomic study of a novel magnetotactic Alphaproteobacteria uncovers the multiple ancestry of magnetotaxis[END_REF]. Alternatively, being infected by phage may contribute to this process.

Comparative gene content analysis of three Terasakiella strains using reciprocal best matches

We further perform pangenome analysis of SH-1, PR-1 and T. pusilla. As shown in Figure 3, 1 948 gene families were shared among the three species, representing approximately 50% of the proteins in each strain. A total of 428 families were shared by SH-1 and PR-1, but not T. pusilla.

The larger overlap between SH-1 and PR-1 suggests that they are more closely related to each other than to T. pusilla. These are mainly involved in signal transduction mechanisms (29.8%), inorganic ion transport and metabolism (23.5%), intracellular trafficking, secretion and vesicular transport (18.2%) and cell motility (17.1%) (according to Clusters of Orthologous Groups (COG) automatic classification of Microscope). These genes may also be associated with magnetosome biosynthesis.

Fig. 1

 1 Fig. 1 Reconstruction of metabolic pathways in SH-1.

  magneticum strain AMB-1, Magnetococcus marinus MC-1, Magneto-ovoid bacterium MO-1, Ectothiorhodospiraceae bacterium BW-2, Gammaproteobacteria magnetotactic strain SS-5, Desulfovibrio magneticus RS-1, Candidatus Magnetomorum sp. HK-1, Candidatus Magnetoglobus multicellularis str. Araruama, Candidatus Magnetobacterium bavaricum and Candidatus Magnetoovum chiemensis) (Fig. 2a & b). Seven reactions have been found in one or more genomes of the tested microorganisms (Fig. 2b), including nitrate reductase (nasA, narGH and napA), heme-containing nitrite reductases (nirS), nitric oxide reductase (hcp, cnorB and norVW), nitrous oxide reductase (nosZ), assimilatory (nasB and nirBD) and dissimilatory nitrite reductase (nrfAH and OTR), nitrogenases (nifHDK), cyanase (cynS) and urease (ureABC).

Fig. 2

 2 Fig. 2 Microbial transformations of nitrogen compounds (modified from (Kuypers et al., 2018)).

  SH1_v1_1640-1665 comprised 23 CDS containing three predicted phage tail collar domain proteins, two plasmid maintenance system killer proteins (HigB), and seven transposases, one sulfotransferase and seven hypothetical proteins (Fig.S2 a & b1), while SH1_v1_2382 to SH1_v1_2398 comprised 16 CDS, including three putative tail-related proteins, one putative endolysin, one DNA maturase beta subunit, one putative major capsid protein, and one putative GcrA-like cell cycle regulator (Fig.S2 a & b2). No genes encoding lysine and holin, representing the lysis module, were found. We searched genomes of 12 bacteria (Terasakiella sp. SH-1, Candidatus Terasakiella magnetica strain PR-1, Magnetospira sp. strain QH-2, Magnetospirillum gryphiswaldense strain MSR-1, Magnetospirillum magneticum strain AMB-1, Magnetococcus marinus MC-1, Magneto-ovoid bacterium MO-1, Desulfovibrio magneticus RS-1, Candidatus Magnetomorum sp.

Table 1 General features of Alphaproteobacteria MTB genomes of SH-1, PR-1, MV-1, QH-2, AMB-1 and MSR-1.

 1

		SH-1	PR-1	MV-1	QH-2 (Plasmid)	AMB-1	MSR-1
	Size (bp)	3 832 570 3 687 561	3 638 804	4 021 307 (31 063)	4 967 148	4 365 796
	Number of CDS	3 664	3 501	3 529	3 748 (37)	5 105	4 261
	G + C content (%)	47.5	46.0	54.3	59.5 (54.8)	65.1	63.3
	No. of contigs	1	48	91	1 (1)	1	1
	Coding density (%)	90.7	-	-	90.1 (73.3)	91.1	92.4
	Average CDS
		953.8	-	-	972.5 (623.0)	903.3	954.81
	Length (bp)
	tRNAs	52	45	45	47 (-)	49	50
	5/16/23S rRNA operon	3/3/3	1/2/1	1/1/1	2/2/2 (-)	2/2/2	2/2/2

We thank Jianhong Xu and Weijia Zhang for assistance in sampling. This work was supported by the National Natural Science Foundation of China -Shandong Joint Fund (U1706208), National Natural Science Foundation of China (Nos. 41776131 and 41776130).

Interestingly, genes related to iron metabolism have been found in the genome of T. pusilla but not in SH-1 and PR-1 genomes. These genes included fecR (Anti-FecI sigma factor), fbpC (Fe 3+ ion import ATP-binding protein) and bfr (bacterioferritin, iron storage and detoxification protein). Furthermore, we found 41 copies of histidine kinase in the genome of T. pusilla, fewer than that in SH-1 (74) and PR-1 (70). However, more copies of methyl-accepting chemotaxis proteins (MCPs) were found in T. pusilla (69) than in SH-1 (59) and PR-1 (57), consistent with previous report that high numbers of MCPs are not a common feature of MTB [START_REF] Ji | Comparative genomic analysis provides insights into the evolution and niche adaptation of marine Magnetospira sp. QH-2 strain[END_REF].

CONCLUSION

Comparative genomic analysis of SH-1 performed in the present study revealed that genes involved in glycolysis, the TCA cycle, the non-oxidative phase of the reductive pentose phosphate pathway, PRPP biosynthesis, the ethylmalonyl pathway and the synthesis of all 20 essential amino acids were identified. A fragment of gene encoding haloalkane dehalogenase was also identified.

The soxBAZYX gene cluster, soxCD genes, and cysCHI genes were present, indicating that SH-1 is capable of assimilatory sulfate reduction to H2S, and using thiosulfate as electron donor and oxidizing it to sulfate. SH-1 also contained napAB, nasA and nasB, suggesting that it can gain energy by converting nitrate to ammonia. Two predicted prophage regions were found in SH-1, and our results suggest that prophage may be common in MTB. Pangenome analysis showed that differences between T. pusilla and both SH-1 and PR-1 may be related to signal transduction mechanisms, inorganic ion transport and metabolism, intracellular trafficking, secretion, vesicular transport and cell motility.

DATA AVAILABILITY STATEMENT

All data generated and analyzed during the current study are available from the corresponding author upon request.

SUPPLEMENTARY MATERIAL

Table S1 The 15 reactions involving 8 key inorganic nitrogen species having different oxidation states in MTB.

"+": the genes were detected in the genome of MTB. Proteins in different organisms were defined as orthologs when their alignments had an E-value < 1e-5, identity > 30%, and query coverage > 50%.

(see Table S1)