A Novel Isolate of Spherical Multicellular Magnetotactic Prokaryotes Has Two Magnetosome Gene Clusters and Synthesizes Both Magnetite and Greigite Crystals

Multicellular magnetotactic prokaryotes (MMPs) are a unique group of magnetotactic bacteria that are composed of 10–100 individual cells and show coordinated swimming along magnetic field lines. MMPs produce nanometer-sized magnetite (Fe3O4) and/or greigite (Fe3S4) crystals—termed magnetosomes. Two types of magnetosome gene cluster (MGC) that regulate biomineralization of magnetite and greigite have been found. Here, we describe a dominant spherical MMP (sMMP) species collected from the intertidal sediments of Jinsha Bay, in the South China Sea. The sMMPs were 4.78 ± 0.67 μm in diameter, comprised 14–40 cells helical symmetrically, and contained bullet-shaped magnetite and irregularly shaped greigite magnetosomes. Two sets of MGCs, one putatively related to magnetite biomineralization and the other to greigite biomineralization, were identified in the genome of the sMMP, and two sets of paralogous proteins (Mam and Mad) that may function separately and independently in magnetosome biomineralization were found. Phylogenetic analysis indicated that the sMMPs were affiliated with Deltaproteobacteria. This is the first direct report of two types of magnetosomes and two sets of MGCs being detected in the same sMMP. The study provides new insights into the mechanism of biomineralization of magnetosomes in MMPs, and the evolutionary origin of MGCs.

In addition, it has been reported that the formation of magnetosomes is controlled by a magnetosome gene cluster (MGC) [9,[36][37][38]. Two sets of MGCs thought to be involved in magnetite and/or greigite formation have been identified in several Deltaproteobacteria MTB, including two uncultured sMMPs (the greigite-producing Candidatus (Ca.) Magnetoglobus multicellularis and the magnetite-and greigite-producing Ca. Magnetomorum HK-1) [31,35,39], one uncultured eMMP (the magnetite-producing Ca. Magnetananas rongchenensis RPA) [40], and two cultured unicellular MTB (the magnetite-producing Desulfovibrio magneticus RS-1 and the magnetite-and greigite-producing Desulfamplus magnetomortis BW-1) [41,42]. The corresponding magnetosome composition and morphology were also detected in the spherical Ca. Magnetoglobus multicellularis (irregularly shaped greigite magnetosomes were biomineralized), unicellular Desulfovibrio magneticus RS-1 (bullet-shaped magnetite magnetosomes were biomineralized), and Desulfamplus magnetomortis BW-1 (irregularly shaped greigite and bullet-shaped magnetite magnetosomes were both biomineralized), respectively [16,42,43], except for the ellipsoidal Ca. Magnetananas rongchenensis RPA and the spherical Ca. Magnetomorum HK-1. While the RPA could biomineralize both bullet-shaped magnetite and rectangular greigite crystals, the magnetosomes in the HK-1 were not described, although it showed 99.3% identity to the spherical Ca. Magnetomorum rongchengroseum, which was collected in another region and is reported to produce both magnetite and greigite particles [21]. In brief, there is no direct evidence whether two sets of MGCs regulate the synthesis of the two types of magnetosomes (magnetite and greigite) in the same MMP. In this study, undertaken in the intertidal zone of Jinsha Bay (South China Sea), we found a novel sMMP that contained two paralogous magnetosome gene clusters and could concurrently biomineralize both bullet-shaped magnetite and irregularly shaped greigite magnetosomes. We sequenced the genome of this sMMP, and showed that it was more integrated than previously reported MMPs. It simultaneously contained sets of both mam ('magnetosome membrane') and mad ('magnetosome associated Deltaproteobacteria') gene clusters, which implied the involvement of independent processes for synthesizing the two distinct types of magnetosome.

Sampling and Enrichment of MMPs
Sediment samples were collected from sites in the low-tide region of the intertidal zone in Jinsha Bay (Zhanjiang City, China; 21 • 16.267 N, 110 • 24.067 E) on 30 August 2020 and 7 September 2021. The salinity at these sites was measured (WTW Cond 3210 SET 1; Xylem, Germany), and ranged from 19.5 to 24.2‰ during the sampling periods. Samples of the subsurface sediment and in situ seawater (approximately 1:1) were transferred to the laboratory in sterile plastic bottles, and incubated in dim light at an ambient temperature for subsequent analyses. To enrich MMPs from the sediment, each plastic bottle was shaken to mix the sediment and water, and two magnets were attached externally, one each to opposite sides of the bottle adjacent to the seawater-sediment interface [44]. MMPs attracted to the magnets were removed, and purified magnetically for a subsequent study using the modified racetrack method [45].

Optical and Electron Microscopy
The morphology and motility of the purified MMPs were observed using the hanging drop method using differential interference contrast (DIC) microscopy (Olympus BX51 equipped with a DP80 camera system; Olympus, Tokyo, Japan) [46]. For scanning electron microscopy (SEM) observations, each sample was fixed in 2.5% glutaraldehyde for >3 h at 4 • C, filtered onto a polycarbonate filter (high-density pores, 1 µm diameter; Whatman), dehydrated through a gradient of ethanol concentrations, dried, and gold-coated. The goldcoated samples were observed using a KYKY-2800B SEM (KYKY Technology Development Ltd., Beijing, China) operating at 25 kV. For transmission electron microscopy (TEM) observations of the MMP and magnetosomes morphologies, 10 µL samples of MMPs, which were purified using the racetrack method and concentrated by slight centrifugation, were deposited on formvar carbon-coated copper grids, washed three times with distilled water, and examined using a Hitachi HT7700 TEM (Hitachi Ltd., Tokyo, Japan) operating at 100 kV, and a JEM-2100 TEM (JEOL Ltd., Tokyo, Japan) operating at 200 kV. The composition of magnetosomes was analyzed using high-resolution transmission electron microscopy (HRTEM; JEM-2100 TEM) equipped for energy-dispersive X-ray spectroscopy (EDXS).

Genomic DNA Extraction, Whole Genome Amplification, and Phylogenetic Analysis of 16S rRNA Genes
The sMMPs were sorted using a TransferMan ONM-2D micromanipulator and a CellTram Oil manual hydraulic pressure-control system (IM-9B) installed on a microscope (Olympus IX51; equipped with a 40 × LD objective, Tokyo, Japan) [6,21,26,47]. The microsorted MMPs stored in PBS were repeatedly freeze-thawed, after which whole genome amplification (WGA) of MMPs was performed using multiple displacement amplification (MDA) for 8 h using the REPLI-g Single Cell kit (cat. #150343; Qiagen, Hilden, Germany), according to the manufacturer's instructions [26]. The WGA products were stored at −80 • C for genome sequencing and 16S rRNA gene analysis.
The WGA products were diluted and used for amplification of the 16S rRNA gene. Universal bacterial primers 27F (5 -AGAGTTTGATCCTGGCTCAG-3 ) and 1492R (5 -GGTTACCTTGTTACGACTT-3 ) were used for the polymerase chain reaction (PCR) in a Mastercycler (Eppendorf, Hamburg, Germany). The purified and retrieved PCR products were cloned into the pMD18-T vector (Takara, Shiga, Japan), and transferred into competent Escherichia coli (strain DH5α) (Takara, Japan). Clones were selected randomly and sequenced using the vector primers M-13 and RV-M (Ruibio BioTech Co. Ltd., Qingdao, China). The 16S rDNA sequences obtained for the sMMPs were aligned against the nr/nt database using the BLAST search program (http://www.ncbi.nlm.nih.gov/BLAST/ accessed on 18 November 2021). The 16S rDNA sequences of reference MMPs and unicellular MTB were downloaded from the GenBank database. All sequences were aligned using Clustal W software version 2.1 [48], and a phylogenetic tree was constructed using the maximum likelihood (ML) method using the IQ-TREE software version 2.0.3 under the best-fit GTR+F+R4 model [49]. Bootstrap values were calculated using 1000 replicates. The tree was visualized and adjusted using iTOL webtool version 6.4.3 (https://itol.embl.de/ accessed on 26 November 2021), and was rooted with unicellular Deltaproteobacteria MTB.

Fluorescence In Situ Hybridization (FISH)
A specific oligonucleotide probe JSMW7 (5 -GCCACCTTTCATCTAATCTATC-3 ) was designed for the 16S rDNA sequence corresponding to position 185-206 of the target sMMP, and its specificity was evaluated using the online probe-match tool (http://rdp. cme.msu.edu/probematch/search.jsp, accessed on 19 November 2021) [50]. The specific probe was labeled with hydrophilic sulfoindocyanine Cy3 as the fluorescent dye at the 5 end. The universal probe EUB338 (5 -GCTGCCTCCCGTAGGAGT-3 ) was used as the positive control in hybridization, and was labelled with fluorescein phosphoramidite FAM at the 5 end. Appropriate amounts of E. coli cells were added to the sample and mixed with the target sMMP cells as negative controls. The specimen was treated and prepared as described previously [51,52], and FISH was carried out according to protocols reported early [51,53,54]. The hybridization results were observed using an Olympus BX51 epifluorescence microscope equipped with a DP80 camera system (Olympus, Tokyo, Japan).

Sequencing, Assembly, and Genome Annotation, and Comparative Analysis of Magnetosome Genes and Proteins
Paired-end 100 bp (PE100) libraries were constructed from the produced DNA of the WGA method using the MGI Easy FS DNA Library Prep Set kit (MGI, Shenzhen, China), according to the manufacturer's instructions. The genome was sequenced using the DNBSEQ-T1 platform (BGI-Qingdao, Qingdao, China), using the paired-end 100 bp library. After quality trimming and filtering using SOAPnuke version 2.1.6 [55], the reads were assembled using MEGAHIT version 1.2.8, using k-mer sizes from 27 to 255 by step 20 [56]. Then, the metaWRAP version 1.2.1 pipeline was used for metagenome binning, refinement, and reassembly with default parameters to select the pure genome [57]. The quality of MMP genomes was assessed using QUAST version 5.0.2 [58], and genomic completeness and contamination were estimated using CheckM version 1.1.3 [59]. The genome was annotated using Prokka version 1.14.5 [60]. Several available genomes of Deltaproteobacteria MTB were obtained from the GenBank database, including Desulfovibrio magneticus RS-1 [41], Desulfamplus magnetomortis BW-1 [42], Ca. Magnetoglobus multicellularis [35,39], Ca. Magnetomorum HK-1 [31], and Ca. Magnetananas rongchenensis RPA [40]. A comparative analysis of MGCs was performed using the MagCluster version 0.2.0 [61] and the clinker [62] with a manual inspection following. The putative magnetosome proteins were confirmed using NCBI PSI-BLAST [63], and the annotations were corrected manually. Comparative analyses of putative magnetosome proteins were performed using BLASTP (http://www.ncbi.nlm.nih.gov/BLAST/, accessed on 13 December 2021).
The phylogenetic trees based on the Mam and Mad protein sequences were both constructed using the maximum likelihood (ML) method, using IQ-TREE software version 2.0.3 under the same best-fit LG+F+I+G4 model [49]. Bootstrap values were calculated using 1000 replicates. The tree was visualized and adjusted using iTOL webtool version 6.4.3 (https://itol.embl.de/, accessed on 15 January 2022).

Occurrence, Structure, and Motility of the sMMPs
Unicellular MTB and highly abundant sMMPs were observed in the intertidal sediment from one site in Jinsha Bay (Figure 1a). Yellow and gray layers of sand were present in the sediment, with the yellow layer approximately 1 cm above the gray layer. The sMMPs were present at a maximum abundance of approximately 304 inds./cm 3 in the gray layer. sMMPs were autofluorescent when illuminated with green, blue, violet, or UV light. The cellular interfaces were evident using autofluorescence excitation at 400−410 nm and 330−385 nm wavelengths (Figure 1f,g), but the cellular contours of MMPs were not distinct when the cells were exposed to illumination at 510−550 nm and 450−480 nm (Figure 1d,e).
The average diameter of the sMMPs was 4.78 ± 0.67 μm (n = 237), and the average size of each individual unit was 1.03 ± 0.15 μm (n = 64) in the largest dimension.  An analysis of DIC images ( Figure 1b) and SEM micrographs ( Figure 1c) showed that each sMMP contained approximately 14-40 constituent cells arranged with helical symmetry, which also appeared as a radical symmetry on the section image (n = 24). The sMMPs were autofluorescent when illuminated with green, blue, violet, or UV light. The cellular interfaces were evident using autofluorescence excitation at 400-410 nm and 330-385 nm wavelengths (Figure 1f,g), but the cellular contours of MMPs were not distinct when the cells were exposed to illumination at 510-550 nm and 450-480 nm (Figure 1d,e).
The average diameter of the sMMPs was 4.78 ± 0.67 µm (n = 237), and the average size of each individual unit was 1.03 ± 0.15 µm (n = 64) in the largest dimension.
Use of the hanging drop method showed that >80% of magnetically enriched sMMPs exhibited north-seeking polarity (Figure 1a), and swam along the magnetic field lines with an average speed of approximately 78.0 ± 41.4 µm/s (v, n = 38; maximum velocity, 177.4 µm/s) along a straight or helical trajectory (Figure 2a). Typical "ping-pong" motility was also observed at droplet edges, the north-seeking sMMPs which accumulated at the edge showed an excursion swim against the magnetic field lines away from the droplet edge (Figure 2b), then performed a return swim along the magnetic field lines back to the droplet edge ( Figure 2c). Additionally, the average speeds of excursion and return were 223.9 ± 54.5 µm/s (v 1 , n = 24; maximum velocity, 330.5 µm/s) and 102.2 ± 19.0 µm/s (v 2 , n = 24; maximum velocity, 138.4 µm/s), respectively (Figure 2b,c).

Characterization of Magnetosome Biomineralization in the sMMPs
TEM observations showed the simultaneous presence of both bullet-shaped and irregularly shaped magnetosomes arranged in chains or clusters within constituent cells of the sMMPs from the South China Sea (Figure 3a

Characterization of Magnetosome Biomineralization in the sMMPs
TEM observations showed the simultaneous presence of both bullet-shap regularly shaped magnetosomes arranged in chains or clusters within constitue the sMMPs from the South China Sea (Figure 3a,b). The average number of mag per individual cell was 30 ± 11 (n = 31), with the proportions of bullet-shaped ( red circle) and irregularly shaped crystals (Figure 3b, yellow circle) averaging 56.7%, respectively. HRTEM and EDXS analyses indicated that the bullet-shap tosomes were composed of magnetite (Fe3O4) (Figure 3c-e). The magnetite part 87.0 ± 20.3 × 35.2 ± 3.5 nm in size and had a width/length ratio of 0.42 ± 0.08 (Figure 3f-h). These analyses (HRTEM and EDXS) also showed that the irregula crystals were composed of greigite (Fe3S4) (Figure 3i-k). The greigite particles w 8.7 × 55.2 ± 7.3 nm in size and had a width/length ratio of 0.77 ± 0.11 (n = 215) ( n).

Phylogenetic Analysis of the sMMPs
We isolated two sMMPs using the micromanipulation sorting method, and their genomic DNA was extracted and amplified by WGA using MDA. The 16S rRNA genes were amplified, cloned, and sequenced from the WGA product. Sequences (53) related to the MMPs were obtained from 55 randomly chosen clones. All the MMP sequences shared an identity of at least 99.1%, indicating that the sMMPs represented a single species. FISH was used to corroborate the authenticity of the 16S rRNA gene sequences. Fluorescence microscopy observations showed that all bacterial cells were hybridized with the general probe EUB338 (Figure S1a, green), while only the spherical MMP cells were hybridized with the specific probe JSMW7, designed from the 16S rRNA sequence of the sMMP ( Figure  S1b, red). These results demonstrated the specificity of the FISH analysis.
The 16S rRNA gene sequence of the sMMP was most closely related (93.3% shared sequence identity) to that of the uncultured delta proteobacterium clone SY_48 (MW356768), which was collected from a mangrove area in Sanya [23]. It also showed 6.8-7.0% sequence divergence from the uncultured delta proteobacteria clones mmp2_9 (DQ630712), mmp45 (DQ630684), and mmp12 (DQ630669), from the Little Sippewissett salt marshes in Falmouth [17]. The phylogenetic analysis of the 16S rRNA gene sequence revealed that these five sMMP clones formed another group of spherical-type MMPs, and belong to the Deltaproteobacteria (Figure 4). We tentatively designate this novel isolated sMMP from Jinsha Bay at Zhanjiang City as MMP XL-1 (Figure 4).   (MW356768), which was collected from a mangrove area in Sanya [23]. It also showed 6.8−7.0% sequence divergence from the uncultured delta proteobacteria clones mmp2_9 (DQ630712), mmp45 (DQ630684), and mmp12 (DQ630669), from the Little Sippewissett salt marshes in Falmouth [17]. The phylogenetic analysis of the 16S rRNA gene sequence revealed that these five sMMP clones formed another group of spherical-type MMPs, and belong to the Deltaproteobacteria (Figure 4). We tentatively designate this novel isolated sMMP from Jinsha Bay at Zhanjiang City as MMP XL-1 (Figure 4). The morphologies and characteristics of the sMMPs (labeled by the purple circles) and the eMMPs (labeled by the orange ellipses) have been described.

General Genomic Features of the Proposed MMP XL-1 and Comparative Genomic Analysis of Magnetosome Gene Clusters
Approximately 8.88 µg of genomic DNA was obtained from the two micro-sorted sMMPs following WGA. Following sequencing, assembly, and binning, a draft genome of approximately 8.49 Mb in size and having a GC content of 34.6% was obtained. The genome contained 279 contigs and had a 60,219 bp N50-value. A total of 5329 coding sequences (CDS) were predicted and annotated, including 46 tRNAs, one tmRNA, and one complete rRNA gene operon. The estimated completeness and contamination of the genome were 97.6% and 1.6%, respectively.
The 11 homologous Mam proteins in clusters 1 and 2 had identities ranging from 46.3% to 83.8% (Table 1, in red), and the six homologous Mad proteins detected in clusters 3 and 4 showed identities between 52.1% and 72.2% (Table 1, in black). There may be two sets of homologous magnetosome gene clusters in the genome of sMMP from Jinsha Bay, one responsible for magnetite magnetosome biomineralization (clusters 1 and 3), and another associated with greigite magnetosome biomineralization (clusters 2 and 4) (Table 1 and Figure 5). A strong correlation in the phylogenies involved in magnetosome biomineralization was found, based on the Mam and Mad protein amino acid sequences ( Figure 6). In the phylogenetic tree of Mam proteins, the magnetite-related Mam proteins (including these from XL-1, HK-1, RPA, RS-1, and BW-1) were gathered in a single branch, while greigite-related Mam proteins from HK-1, Ca. Magnetoglobus multicellularis, XL-1, and BW-1 were gathered in a different branch (Figure 6a). Similar clades were present in the phylogenetic tree of Mad proteins. The Mad proteins from XL-1, HK-1, and RPA involved in magnetite biomineralization were clustered in one branch, while the Mad proteins involved in greigite biomineralization were clustered in another branch, including these detected in HK-1, Ca. Magnetoglobus multicellularis, and XL-1 (Figure 6b).

Discussion
In this study, we isolated a dominant type of sMMP from the intertidal sedimen Jinsha Bay, in the South China Sea. Phylogenetically, the sMMPs could be classified a novel species of Deltaproteobacteria using 16S rRNA analysis strongly, which had than 98.65% 16S rRNA gene sequence identity with its closest species [65]. Each aggr contained approximately 14-40 constituent cells and the individual cells of these sM were arranged loosely in helical symmetry, which showed a little difference from the arrangement of other previously reported sMMPs [16,18,20,21]. Their diameter wa proximately 4.78 μm, smaller than most reported sMMPs, except for the sMMPs (4. average diameter) collected from the mangrove habitat in the Sanya River (Tab [16,18,20,21,23,66]. The velocity of the magnetotaxis motility and the ping-pong moti the MMP XL-1 was more similar to that of the ellipsoidal Ca. Magnetananas rongchen than to other sMMPs ( Table 2) [26]. Both bullet-shaped magnetite and irregularly sh greigite crystals were simultaneously biomineralized by this sMMP (Figure 3), whic phenomenon previously observed in sMMPs from Itaipu Lagoon (Brazil), Lake Y (China), and the Sanya mangroves (China) ( Table 2) [21,23,67]. It has been reported MTB show a clear vertical distribution [33] in the oxic−anoxic interface zone and/or a regions [2]. Magnetite-producing MTB usually inhabit the top of the oxic−anoxic inte whereas greigite-producing MTB prefer the reducing environment at the base and sli below the interface [5,68]; this implies that those sMMPs that can biomineralize both netite and greigite may have broader vertical niches.

Discussion
In this study, we isolated a dominant type of sMMP from the intertidal sediments of Jinsha Bay, in the South China Sea. Phylogenetically, the sMMPs could be classified into a novel species of Deltaproteobacteria using 16S rRNA analysis strongly, which had less than 98.65% 16S rRNA gene sequence identity with its closest species [65]. Each aggregate contained approximately 14-40 constituent cells and the individual cells of these sMMPs were arranged loosely in helical symmetry, which showed a little difference from the tight arrangement of other previously reported sMMPs [16,18,20,21]. Their diameter was approximately 4.78 µm, smaller than most reported sMMPs, except for the sMMPs (4.6 µm average diameter) collected from the mangrove habitat in the Sanya River (Table 2) [16,18,20,21,23,66]. The velocity of the magnetotaxis motility and the ping-pong motion of the MMP XL-1 was more similar to that of the ellipsoidal Ca. Magnetananas rongchenensis than to other sMMPs ( Table 2) [26]. Both bullet-shaped magnetite and irregularly shaped greigite crystals were simultaneously biomineralized by this sMMP (Figure 3), which is a phenomenon previously observed in sMMPs from Itaipu Lagoon (Brazil), Lake Yuehu (China), and the Sanya mangroves (China) ( Table 2) [21,23,67]. It has been reported that MTB show a clear vertical distribution [33] in the oxic-anoxic interface zone and/or anoxic regions [2]. Magnetite-producing MTB usually inhabit the top of the oxic-anoxic interface, whereas greigite-producing MTB prefer the reducing environment at the base and slightly below the interface [5,68]; this implies that those sMMPs that can biomineralize both magnetite and greigite may have broader vertical niches.   [41,43] "L" and "W" indicate the length and width of magnetosome crystals, respectively. "-" means no data acquired. The characteristics described above show that MMP XL-1 represented a unique type of sMMP; consequently, it was further investigated using genomic studies. To date, only two draft genomes of sMMPs were obtained, including the Ca. Magnetoglobus multicellularis and Ca. Magnetomorum HK-1 [31,35]. The genome of Ca. Magnetoglobus multicellularis was the first MMP genome to be analyzed; this showed that the genes involved in magnetite biomineralization were homologous with the genes in greigite-producing MTB, suggesting that the magnetotactic trait is monophyletic [35,39]. Two sets of magnetosome genes, one each responsible for magnetite and greigite biosynthesis, were identified in HK-1, providing the first evidence that two divergent magnetosome gene clusters can co-occur in a single MMP genome [31].
Subsequently, comparative genomic analysis was carried out to clarify which genes within XL-1 governed the biomineralization of each magnetite and greigite. Two sets of relatively complete magnetosome gene clusters, located on two contigs, were recognized in the genome (Table S1). One set showed synteny with homologous regions for the magnetite MGC of Ca. Magnetomorum HK-1 and the magnetite-producing Ca. Magnetananas rongchenensis RPA, while another set had a higher similarity with the MGC cluster from the greigite-producing Ca. Magnetoglobus multicellularis and the greigite MGC of Ca. Magnetomorum HK-1 ( Figure 5) [31,39,40]. In addition, two sets of mam genes involved in magnetite and greigite formation were detected in the unicellular Desulfamplus magnetomortis BW-1, and these were homologous with those of MMP XL-1 [42,64]. Synteny of the MGCs between this sMMP (MMP XL-1) and other Deltaproteobacteria bacteria is conserved, providing strong evidence that magnetite and greigite magnetosomes can be biomineralized by XL-1, controlled by two sets of MGCs ( Figure 5). This is the first direct report that an MMP can contain two types of magnetosomes and corresponding MGCs ( Table 2). Only greigite crystals and greigite-related genes were identified in Ca. Magnetoglobus multicellularis [16,35,39]. Although two types of MGCs were detected in the spherical Ca. Magnetomorum HK-1, the description of the magnetosome particles was not included [31]. Among eMMPs, only for one species (Ca. Magnetananas rongchenensis RPA) has MGC information been reported to date, and its involvement was limited to magnetite formation [26,40].
Intriguingly, we found that two sets of genes coding for proteins related to magnetite and greigite formation within XL-1 were paralogous. They shared variable degrees of amino acid similarity 46.3-83.8% (Table 1). The similarity between its own magnetite and greigite proteins was smaller than that between its magnetite proteins and corresponding magnetite proteins of other Deltaproteobacteria MTB. Additionally, a similar phenomenon was shown when using the greigite proteins for comparison (Table S2), which was consistent with the similarity difference of MGCs of the Ca. Magnetomorum HK-1. The greigite genes of HK-1 were more congruent with the greigite genes of other MTB than with its own magnetite genes [31]. This finding may be consistent with the hypothesis that the occurrence of two sets of magnetosome genes in Deltaproteobacteria MTB may have originated from ancient gene duplication and/or mutation in a common ancestor, then distributed to various MTB by HGT [47], and subsequent vertical inheritance by descent [31,70,71].
It is noteworthy that the arrangement and identities of magnetosome genes on clusters 1 and 3 of XL-1 were similar to the corresponding magnetite genes of RPA. The set of magnetosome genes on the clusters 2 and 4 of XL-1 had a similar gene order and high similarity to the greigite gene clusters of Ca. Magnetoglobus multicellularis, which implied that the clusters 1 and 3 were involved in the magnetite biomineralization, while clusters 2 and 4 were responsible for the greigite biomineralization within XL-1, respectively ( Figure 5). Additionally, the degree of the paralogous Mam and Mad proteins similarities mentioned above was almost the same (46.3-83.8% of the Mam proteins and 52.1-72.2% of the Mad proteins) (Table 1). Furthermore, the clades in the phylogenetic trees based on the Mam and Mad protein sequences were closely consistent as well (Figure 6), suggesting these two sets of mad genes (mad23, mad24, mad25, mad26, and mad27) were likely involved in the magnetite and greigite separately, which were the same as the mam genes ( Figure 5, clusters 3 and 4) [31,42,64]. The short gene cluster that included mad23, mad24, mad25, and mad26 has also been identified in all Nitrospirae MTB that synthesize bullet-shaped magnetosomes [72,73], which implies that they are also the core genes for magnetosome biomineralization. In addition, two branches were readily distinguishable on each of the Mam and Mad phylogenetic trees; one of these was involved in magnetite biomineralization and the other with greigite biomineralization (Figure 6). This provides further evidence for the presence of two sets of MGCs within MMP XL-1.

Conclusions
In this study, we firstly isolated sMMPs from the intertidal sediments of Jinsha Bay, in the South China Sea. Using TEM and EDXS analyses, we showed that both bulletshaped magnetites and irregularly shaped greigite crystals were biomineralized within one spherical multicellular aggregation. SEM observations indicated that individual units within the aggregation were arranged with radial symmetry. Phylogenetic analysis showed that the sMMPs formed a new clade with four other MMP clones (MW356768, DQ630712, DQ630684, and DQ630669), and it was affiliated to the Deltaproteobacteria. Two sets of MGCs involved in magnetite and greigite biomineralization were identified in the genome, and these were found to be more integrated than what has previously been reported in MMPs. The two mamAB-like operons coupled with the downstream mad gene cluster have the potential to control magnetosome biomineralization (magnetite or greigite). It has displayed direct evidence that both magnetosome morphologies and the MGCs related to the two types of magnetosome co-occur in the same species, which provides new information relevant to the study of biomineralization mechanisms associated with the magnetosomes of MMPs, and the evolutionary origin of MGCs.