The Phylogeny, Metabolic Potentials, and Environmental Adaptation of an Anaerobe, Abyssisolibacter sp. M8S5, Isolated from Cold Seep Sediments of the South China Sea

Bacillota are widely distributed in various environments, owing to their versatile metabolic capabilities and remarkable adaptation strategies. Recent studies reported that Bacillota species were highly enriched in cold seep sediments, but their metabolic capabilities, ecological functions, and adaption mechanisms in the cold seep habitats remained obscure. In this study, we conducted a systematic analysis of the complete genome of a novel Bacillota bacterium strain M8S5, which we isolated from cold seep sediments of the South China Sea at a depth of 1151 m. Phylogenetically, strain M8S5 was affiliated with the genus Abyssisolibacter within the phylum Bacillota. Metabolically, M8S5 is predicted to utilize various carbon and nitrogen sources, including chitin, cellulose, peptide/oligopeptide, amino acids, ethanolamine, and spermidine/putrescine. The pathways of histidine and proline biosynthesis were largely incomplete in strain M8S5, implying that its survival strictly depends on histidine- and proline-related organic matter enriched in the cold seep ecosystems. On the other hand, strain M8S5 contained the genes encoding a variety of extracellular peptidases, e.g., the S8, S11, and C25 families, suggesting its capabilities for extracellular protein degradation. Moreover, we identified a series of anaerobic respiratory genes, such as glycine reductase genes, in strain M8S5, which may allow it to survive in the anaerobic sediments of cold seep environments. Many genes associated with osmoprotectants (e.g., glycine betaine, proline, and trehalose), transporters, molecular chaperones, and reactive oxygen species-scavenging proteins as well as spore formation may contribute to its high-pressure and low-temperature adaptations. These findings regarding the versatile metabolic potentials and multiple adaptation strategies of strain M8S5 will expand our understanding of the Bacillota species in cold seep sediments and their potential roles in the biogeochemical cycling of deep marine ecosystems.


Introduction
Cold seeps, which are widely distributed on the seafloor of the world's oceans, play a crucial role in the global marine geochemical cycling of carbon and sulfur [1][2][3].Cold seep environments are characterized by methane-rich fluid emissions and distinctive sulfur oxidation-reduction reactions and support seabed oasis ecosystems composed of various kinds of high-productive microbial and faunal assemblages [2,[4][5][6][7].Therefore, diverse and abundant organic matter exists in cold seep environments, including gaseous and liquid hydrocarbons, aromatic compounds, polysaccharides, and biomass from chemosynthetic microorganisms and invertebrates [3,[8][9][10][11][12].However, much less is known about which microorganisms are capable of degrading the organic matter and how these degradation processes happen in cold seeps.
The phylum Bacillota is predominantly low G+C content, gram-positive, rod-shaped, obligate anaerobic bacteria capable of forming spores to resist extreme environmental conditions [13][14][15].Bacillota is one of the most abundant bacterial groups and is widely distributed in a variety of environments, including cold seeps [5,16], marine environments [17][18][19][20][21], soils [22][23][24][25], oil fields [15,26], freshwater lakes [27], the human gut [28], plateau anaerobic batch reactors [29], and the rhizosphere of plants [30].Notably, Cui et al. (2019) found that Bacillota was the dominant bacterial group in the cold seep sediments of the South China Sea (SCS), with up to 37% of the relative abundance in the total bacterial communities [5].Metabolically, many members of Bacillota play an essential role in the biodegradation of lignocellulosic and carbohydrate polymers [23], polychlorinated biphenyl [29], and petroleum hydrocarbons [31].Other studies have shown that Clostridia (assigned to the phylum Bacillota) has the capability to utilize various complex carbohydrates, such as starch, cellulose, chitin, and xylan, and the Bacillus sp. of this phylum may play an important role in long-chain alkane biodegradation [32,33].Together, these findings suggest that Bacillota plays a crucial role in biogeochemical cycles.
The genus Abyssisolibacter belongs to the order Clostridiales and the family Clostridiaceae within the phylum Bacillota.At present, the genus Abyssisolibacter contains only one isolated strain, Abyssisolibacter fermentans MCWD3, which was isolated from deep-sea sediment (depth 2150 m) obtained from the Ulleung Basin, East Sea, Korea [34].Strain MCWD3 was a gram-staining-negative and anaerobic bacterium and could utilize fructose, mannose, cellobiose, and methionine only in the presence of yeast extract [34].Therefore, the characteristics of the substrate requirements of strain MCWD3 remain largely uncertain, and little is known about the metabolic potential and ecological functions of the species in the genus Abyssisolibacter.
Hitherto, more than 40 cold seep sites have been detected in the South China Sea (SCS), only two of which are active: the Haima cold seep and the Site F (or Formosa Ridge) cold seep [2,4,12,[35][36][37].The cold seep at Site F was found in 2007 and is located at a depth of 1120 m on the Formosa Ridge of the northeastern SCS, offshore of southwestern Taiwan [36,[38][39][40].This cold seep is enriched by chemosynthetic communities, such as deep-sea mussels and galatheid crabs, which may provide various types of organic matter to support the growth of heterotrophic microbial communities [41].In this study, we report a novel anaerobic strain, Abyssisolibacter sp.M8S5, which was isolated from the cold seep sediments of Site F at a depth of 1151 m.Our analysis of the complete genome sequence of strain M8S5 revealed the phylogeny, metabolic versatility, and multiple adaptive strategies of M8S5 in the high-pressure, low-temperature, and anaerobic cold seep environment.

Sample Description and Strain Isolation
Remotely operated vehicle (ROV) sediment push cores were collected at Site F (22 • 07 N, 119 • 17 E) at a depth of 1151 m from the cold seep of the South China Sea (Figure S1) in May 2018.After recovering the cores onboard, the sediment samples were anaerobically preserved in sterilized seawater at 4 • C.
Once in the lab, a subsample was used to inoculate M89 medium, prepared with a gas phase of N 2 (200 kPa), and incubated anaerobically at 35 • C. The M89 medium contained (L −1 ) 700 mL seawater, 300 mL distilled water, 1 g tryptone, 2 g yeast extract, 2 g casamino acid, 2 g 4-aminobutyric acid, 3 g HEPES buffer, 0.25 g L-cysteine hydrochloride dihydrate (as a reducing agent), and 1 mg sodium resazurin (as a redox indicator).The pH of the medium was adjusted to 7.0 with 5 M NaOH at room temperature.After autoclaving, 1 mL of vitamin solution [42] and 1 mL of selenite-tungstate solution [43] were added.After 10 days of incubation, the enrichments were sub-cultured under the same conditions and purified by eight repeated dilutions-to-extinction series.One isolate, designated as M8S5, was obtained.Stock cultures were stored at −80 • C with 5% (v/v) DMSO.

DNA Extraction, Genome Sequencing, and Assembly
The cells of strain M8S5 were cultured in M89 medium at 35 • C for 5 days.The genomic DNA of strain M8S5 was extracted using the Qiagen DNA extraction kit (Qiagen, Hilden, Germany).The total DNA was used for quality control and was obtained by gel electrophoresis and further quantified by a Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA, USA).
The complete genome sequence of strain M8S5 was sequenced using Oxford Nanopore Sequencing Technologies at Nextomics Biosciences Co., Ltd.(Wuhan, China).Oxford Nanopore Technologies sequencing libraries were prepared using the manufacturer's ligation sequencing kit (SQK-LSK109), and sequencing was carried out on a PromethION device using flow cells (FLO-PRO002) without fragmentation.Finally, Nanopore reads were assembled using Canu (version 1.7) [44] in the normal assembly mode.

Gene Annotation and Metabolic Pathway Reconstruction
Genome annotation and ORF prediction in M8S5 and the other four strains were performed by the NCBI prokaryotic genome annotation pipeline (PGAP) [45].The whole genome map of strain M8S5 was generated using the Circos visualization tool [46].Metabolic pathway analysis of M8S5 and the other four related strains was performed by searching the KEGG GENES database [47] using BlastKOALA [48].The gene functional annotation of M8S5 and MCWD3 was predicted by a BLASTP search in the Clusters of Orthologous Groups of proteins (COGs) database [49].Carbohydrate hydrolases were identified in the genomes of M8S5 and the other four strains based on BLASTP searches against the CAZy database (Carbohydrate-Active Enzymes database) [50].The number and size of the genomic islands in strain M8S5 were determined using the IslandViewer 4 server [51].The transporters of M8S5 and the other four strains were determined by BLASTP against TransportDB 2.0 [52].The peptidases of M8S5 and the other four strains were predicted by BLASTP against MEROPS [53].The signal peptides of M8S5 and the other four strains were predicted by SignalP v.5.0 [54].

Phylogenetic Tree Construction
The taxonomy annotation of strain M8S5 and the related species was performed using the Genome Taxonomy Database Toolkit (GTDB-Tk, version 3) [55] with the Genome Taxonomy Database (GTDB, release R08-RS214).Then, these protein sequences were separately aligned using Clustal Omega [56], and all the aligned sequences were manually degapped and tandemly connected.The phylogenetic tree was constructed using FastTree 2 [57] with the neighbor-joining method, and the robustness of the tree was evaluated by bootstrap analysis with 1000 replicates.Finally, the phylogenetic tree based on 120 bacterial conserved marker genes generated by GTDB-Tk was visualized using iTOL [58].

Comparative Genomic Analyses
The ribosomal RNAs (rRNAs) of strain M8S5 and the other four strains were predicted using Barrnap version 0.9 with the default parameters [59].Then, the 16S rRNA gene sequences of the five strains were extracted and compared with each other using BLASTn [60].Finally, the values of 16S rRNA gene similarity between strain M8S5 and the other four species were obtained.
The protein sequence alignment was conducted by BLASTp with the following cut-off values: identity, 50%; query coverage, 50%; E-value, 1 × 10 −10 .The proteins employed by strain M8S5 but not by strain MCWD3 were considered to be strain-specific.On the other hand, the proteins shared by strain M8S5 and MCWD3 but absent in the other three species (Senegalia massiliensis SIT17, Senegalia massiliensis 583, and Clostridiisalibacter paucivorans DSM 22131) were considered to be genus-specific.
The average nucleotide identity (ANI) is referred to as the mean of the identity values between homologous genomic regions shared by two genomes [61].The ANI is used to compare the closely related genomes of strains [62].The ANI was calculated between M8S5 and the other four species using the JSpeciesWS server with the BLAST algorithm [63].
The average amino acid identity (AAI) can provide better resolution when comparing highly different bacterial species, such as species from different genera [62,64].The AAI was estimated between M8S5 and the other four strains using compareM v0.1.0.

16S rRNA gene Distribution Analysis of Strain M8S5
The full-length 16S rRNA gene sequences of strain M8S5 were used to screen for the presence of closely related sequences in all publicly available Short Read Archive (SRA, www.ncbi.nlm.nih.gov/sra,accessed on 1 May 2023) datasets with Integrated Microbial Next Generation Sequencing (IMNGS, 99% identity threshold and size over 200 nucleotides) [65], the metadata for the matched samples was obtained from NCBI, and the latitude/longitude coordinates were plotted using Ocean Data View [66].

The Genomic Features of Strain M8S5
The complete genome of strain M8S5 contains one single chromosome with a total length of 4,683,267 base pairs (bp), which is slightly smaller than the scaffold genome of Abyssisolibacter fermentans MCWD3 (4,789,356 bp).The G+C content of the M8S5 genome is 30.3%.The genome of strain M8S5 consists of 4376 genes, including 4250 protein-coding genes, 7 rRNA operons (4 with the pattern of 16S-23S-5S, and 3 with the pattern of 16S-tRNA Ala -23S-5S), 77 tRNAs, 4 ncRNAs, and 24 pseudogenes (Table 1).For COG classification, about 1575 (35.9%) and 1729 (40.8%) protein-coding genes of strains M8S5 and MCWD3, respectively, were assigned to 23 different clusters of orthologous groups (COGs) categories (Supplementary Table S1).Such a low percentage of COG annotation in the two strains may indicate that the genome of Abyssisolibacter species contains a high proportion of novel genes and may be valuable for further studies.The top five COG categories in strain M8S5 included translation, ribosomal structure and biogenesis (COG-J, 11.14%), amino acid transport and metabolism (COG-E, 10.91%), transcription (COG-K, 9.02%), general function prediction only (COG-R, 7.48%), and energy production and conversion (COG-C, 6.68%) (Supplementary Table S1).A graphical map of the complete genome of strain M8S5 is shown in Figure 1.
biogenesis (COG-J, 11.14%), amino acid transport and metabolism (COG-E, 10.91%), transcription (COG-K, 9.02%), general function prediction only (COG-R, 7.48%), and energy production and conversion (COG-C, 6.68%) (Supplementary Table S1).A graphical map of the complete genome of strain M8S5 is shown in Figure 1.The proteins employed by strain M8S5 but not by strain MCWD3 were considered to be strain-specific.On the other hand, the proteins shared by strain M8S5 and MCWD3 but absent in the other three species (Senegalia massiliensis SIT17, Senegalia massiliensis 583, and Clostridiisalibacter paucivorans DSM 22131) were considered to be genus-specific.As many as 20 genomic islands (GIs) were predicted in strain M8S5, and, in particular, seven of these GIs were related to nitrogenase and endospore germination permease, suggesting that this genome harbors obvious evidence of horizontal gene transfer (Supplementary Table S2).Moreover, the genes encoding six transposases, nine recombinases, one sitespecific tyrosine recombinase, and three tyrosine-type recombinases/integrases were identified in strain M8S5, and more than half of the enzymes (10, 52.6%) were specific in strain M8S5 (Supplementary Table S3).The proteins employed by strain M8S5 but not by strain MCWD3 were considered to be strain-specific.On the other hand, the proteins shared by strain M8S5 and MCWD3 but absent in the other three species (Senegalia massiliensis SIT17, Senegalia massiliensis 583, and Clostridiisalibacter paucivorans DSM 22131) were considered to be genus-specific.As many as 20 genomic islands (GIs) were predicted in strain M8S5, and, in particular, seven of these GIs were related to nitrogenase and endospore germination permease, suggesting that this genome harbors obvious evidence of horizontal gene transfer (Supplementary Table S2).Moreover, the genes encoding six transposases, nine recombinases, one site-specific tyrosine recombinase, and three tyrosine-type recombinases/integrases were identified in strain M8S5, and more than half of the enzymes (10, 52.6%) were specific in strain M8S5 (Supplementary Table S3).

The Phylogeny of Strain M8S5
The alignment of the 16S rRNA gene sequences revealed that strain M8S5 was closely related to Abyssisolibacter fermentans MCWD3, followed by Senegalia massiliensis SIT17, Senegalia massiliensis 583, and Clostridiisalibacter paucivorans DSM 22131, with identities of 96.87%, 94.75%, 91.92%, and 89.74%, respectively (Supplementary Table S4).Nevertheless, most of the species in the phylum Bacillota have 16S rRNA gene sequence heterogeneity [67], and the seven 16S rRNA gene sequences in strain M8S5 were different from each other (with the identity lower than 100%).Therefore, phylogenetic analysis based on the 16S rRNA gene may result in biases in the phylogeny of strain M8S5.
In this study, we constructed a phylogenetic tree based on 120 conserved protein sequences (known as GTDB taxonomy) (Figure 2).It showed that strain M8S5 is affiliated with the genus Abyssisolibacter and is closely related to Abyssisolibacter fermentans MCWD3 (Figure 2), which was isolated from deep-sea sediments of the Ulleung Basin, East Sea, Korea [34].

The Phylogeny of Strain M8S5
The alignment of the 16S rRNA gene sequences revealed that strain M8S5 was closely related to Abyssisolibacter fermentans MCWD3, followed by Senegalia massiliensis SIT17, Senegalia massiliensis 583, and Clostridiisalibacter paucivorans DSM 22131, with identities of 96.87%, 94.75%, 91.92%, and 89.74%, respectively (Supplementary Table S4).Nevertheless, most of the species in the phylum Bacillota have 16S rRNA gene sequence heterogeneity [67], and the seven 16S rRNA gene sequences in strain M8S5 were different from each other (with the identity lower than 100%).Therefore, phylogenetic analysis based on the 16S rRNA gene may result in biases in the phylogeny of strain M8S5.
In this study, we constructed a phylogenetic tree based on 120 conserved protein sequences (known as GTDB taxonomy) (Figure 2).It showed that strain M8S5 is affiliated with the genus Abyssisolibacter and is closely related to Abyssisolibacter fermentans MCWD3 (Figure 2), which was isolated from deep-sea sediments of the Ulleung Basin, East Sea, Korea [34].In addition, the average amino acid identity (AAI) evaluates genome-wide identity between distance species.Our results showed that the AAI values between M8S5 and the related four species ranged from 57.27 to 69.97%, and the best-matched strain was A. fermentans MCWD3 (Supplementary Table S5).The average nucleotide identity (ANI) estimates genome-wide identity between closely related species.Our results revealed that the ANI value between strain M8S5 and A. fermentans MCWD3 was 77.85% (Supplementary Table S5), suggesting the closest evolution distance between the two strains.The ANI In addition, the average amino acid identity (AAI) evaluates genome-wide identity between distance species.Our results showed that the AAI values between M8S5 and the related four species ranged from 57.27 to 69.97%, and the best-matched strain was A. fermentans MCWD3 (Supplementary Table S5).The average nucleotide identity (ANI) estimates genome-wide identity between closely related species.Our results revealed that the ANI value between strain M8S5 and A. fermentans MCWD3 was 77.85% (Supplementary Table S5), suggesting the closest evolution distance between the two strains.The ANI values between M8S5 and the other three species could not be calculated due to the relatively distant evolution relationships.These pieces of evidence indicated that strain M8S5 is affiliated with the genus Abyssisolibacter, the family Clostridiaceae, and the order Clostridiales within the phylum Bacillota.

The Environmental Distribution of Abyssisolibacter Species
To study the environmental distribution of this genus with very few isolates, we searched and collected a total of 16 16S rDNA amplicons, which showed >99% identity with strain M8S5 (Supplementary Table S6).The possible environmental distribution of strain Abyssisolibacter M8S5 is shown in Figure 3.It shows that Abyssisolibacter species are widely distributed in various marine environments, including seawater (18 of the sample locations), marine sediments (10), coral reefs (9), sponges (7), and an oyster gut (1) (dots with blue color in Supplementary Table S6).Therefore, we propose that the genus Abyssisolibacter may play an important role in biogeochemical cycling in marine ecosystems.
tively distant evolution relationships.These pieces of evidence indicated that strain M8S5 is affiliated with the genus Abyssisolibacter, the family Clostridiaceae, and the order Clostridiales within the phylum Bacillota.

The Environmental Distribution of Abyssisolibacter Species
To study the environmental distribution of this genus with very few isolates, we searched and collected a total of 16 16S rDNA amplicons, which showed >99% identity with strain M8S5 (Supplementary Table S6).The possible environmental distribution of strain Abyssisolibacter M8S5 is shown in Figure 3.It shows that Abyssisolibacter species are widely distributed in various marine environments, including seawater (18 of the sample locations), marine sediments (10), coral reefs (9), sponges (7), and an oyster gut (1) (dots with blue color in Supplementary Table S6).Therefore, we propose that the genus Abyssisolibacter may play an important role in biogeochemical cycling in marine ecosystems.

Metabolic Characteristics of Strain M8S5
Cold seep habitats possess diverse abundant organic matter, such as methane, petroleum, microorganisms, and macrofauna [1].Previous studies showed that Bacillota could utilize various kinds of organic matter [68].To study the metabolic characteristics and possible ecological functions of strain M8S5 in cold seep habitats, the metabolic pathways of strain M8S5 were reconstructed and compared to those of four neighboring strains (Figure 4).

Metabolic Characteristics of Strain M8S5
Cold seep habitats possess diverse abundant organic matter, such as methane, petroleum, microorganisms, and macrofauna [1].Previous studies showed that Bacillota could utilize various kinds of organic matter [68].To study the metabolic characteristics and possible ecological functions of strain M8S5 in cold seep habitats, the metabolic pathways of strain M8S5 were reconstructed and compared to those of four neighboring strains (Figure 4).
(1) Carbohydrate metabolism Metabolic analysis showed that strain M8S5 has the potential to utilize various kinds of carbohydrates, including chitin, cellulose, trehalose, arabinogalactan oligomer/ maltooligosaccharide, maltose, sucrose, mannose, glucose, sugar, cellobiose, and galactitol (Figure 4 and Supplementary Table S7).Among them, the genes encoding the sucrose PTS system (scrA) and mannose (manA) were detected in the two isolated Abyssisolibacter strains (M8S5 and MCWD3) but not in the three neighboring species (S. massiliensis SIT17, S. massiliensis 583, and C. paucivorans DSM 22131).Moreover, strain M8S5 harbored eight genes encoding fructose transporter, including a strain-specific gene (fruAb) (Figure 4 and Supplementary Table S7).These results suggest that sucrose and fructose might be important carbon sources for strain M8S5.In addition, the genes encoding maltose (mapA), trehalose transporter (treBC and malX), and arabinogalactan oligomer/maltooligosaccharide transporters (ganOPQ) were found in strain M8S5 but not in strain MCWD3, implying that the utilization of these sugars may contribute to the survival of strain M8S5 to the in situ cold seep environment.(1) Carbohydrate metabolism Metabolic analysis showed that strain M8S5 has the potential to utilize various kinds of carbohydrates, including chitin, cellulose, trehalose, arabinogalactan oligomer/maltooligosaccharide, maltose, sucrose, mannose, glucose, sugar, cellobiose, and galactitol (Figure 4 and Supplementary Table S7).Among them, the genes encoding the sucrose PTS system (scrA) and mannose (manA) were detected in the two isolated Abyssisolibacter strains (M8S5 and MCWD3) but not in the three neighboring species (S. massiliensis SIT17, S. massiliensis 583, and C. paucivorans DSM 22131).Moreover, strain M8S5 harbored eight genes encoding fructose transporter, including a strain-specific gene (fruAb) (Figure 4 and Supplementary Table S7).These results suggest that sucrose and fructose might be important carbon sources for strain M8S5.In addition, the genes encoding maltose (mapA), trehalose transporter (treBC and malX), and arabinogalactan oligomer/maltooligosaccharide transporters (ganOPQ) were found in strain M8S5 but not in strain MCWD3, implying that the utilization of these sugars may contribute to the survival of strain M8S5 to the in situ cold seep environment.
(2) Amino acid metabolism and extracellular protein degradation Regarding the biosynthesis of amino acids, our genomic survey showed that the strain M8S5 preoccupies the complete pathways for the biosynthesis of 18 kinds of amino acids, whereas the biosynthesis pathways of histidine and proline were largely incomplete (Figure 4 and Supplementary Table S8).Similarly, strain MCWD3 could also disable to biosynthesize histidine.The absence of these amino acid synthetic genes indicates that the strains M8S5 and MCWD3 are dependent on the intake of histidine and proline, which may originate from the mussels, clams, and tubeworms that thrive in cold seep environments [1,12,32,38].
On the other hand, strain M8S5 possesses a larger number of genes encoding amino acid and peptide transporters, which may help the intake of peptide (ddpDF), oligopeptide (oppABCDF), arginine/lysine/histidine (artQPR), alanine, proline/glycine (opuA), glutamate/aspartate, and polar amino acid from the extracellular cold seep environments (Figure 4 and Supplementary Table S8).Moreover, considering that the prosperity of macrofauna (such as mussels, clams, and tubeworms) in the cold seep contains abundant proteins [1,12,38], we propose that the secretion of extracellular peptidases is an important physiological characteristic of M8S5.In this study, approximately 28 different genes of SignalP-fused peptidases belonging to 17 peptidase subfamilies (C25, C39, M1, M4, M12, M23B, M28, M41, M50B, S8A, S8B, S9B, S11, S12, S41A, S41B, and S49C) were predicted in M8S5, among which S8 peptidase was found to be remarkably abundant (Supplementary Table S9).The S8 family, generally consisting of the S8A (subtilisin) and S8B (kexin) subfamilies, is the second largest family of serine-type peptidases, which has a catalytic triad containing aspartate, histidine, and serine.Most members of the S8 peptidase family are secreted and non-specific and may be involved in nutrition intake and further meet the protein requirements of microorganisms [69].A total of seven extracellular S8 family peptidases were detected, six of which were genus-specific, and one was strain-specific (Supplementary Table S9).Interestingly, three genes of the S8A family peptidase form an operon in strains M8S5 and MCWD3 (Supplementary Table S9).Such organization was previously observed in several Kangiella strains, which were proposed as extraordinary extracellular protein degraders [70].These findings suggest that the secretion of extracellular proteases may be an important survival strategy for Abyssisolibacter species.
(3) D-amino acid metabolism It is well known that bacteria produce diverse D-amino acids (DAAs), which are essential components of bacterial cell wall peptidoglycan and are also found in various non-ribosomal peptides produced by microbes [71][72][73].A high proportion of DAAs may indicate that abundant refractory organic matter exists in the cold seep environment [74].
(4) Nitrogen metabolism Cold seep ecosystems are mainly supported by anaerobic methanotrophy, and the abundant input of methane (a non-nitrogenous carbon source) may drive the cold seep microbial community to nitrogen limitation [75,76].In this study, various kinds of transporters and metabolic pathways of nitrogen sources were predicted in strain M8S5, including ethanolamine, spermidine, putrescine, nitrogenase, and ammonia (Figure 4 and Supplementary Table S11).
Ethanolamine (EA), derived from the membrane phospholipid phosphatidyl-ethanolamine of bacterial cells, can be used as a valuable source of carbon and/or nitrogen by a variety of bacteria (including Bacillota, Flavobacterium, and Salmonella) from the environments [77][78][79].
In the present study, 17 genes encoding ethanolamine transporter (encoding by eutH) and utilization (encoding by eutABCJLMNPQS) were predicted in strain M8S5 (Figure 4 and Supplementary Table S11).Among these genes, eutB and eutC are the central ethanolamine utilization genes, which encode the large and small subunits of ethanolamine ammonia-lyase (EC:4.3.1.7),respectively [78].Intriguingly, only strain M8S5 possessed the genes of eutB and eutC, which catabolize ethanolamine into ammonia and acetaldehyde by an ethanolamine ammonia lyase (Figure 4 and Supplementary Table S11).Therefore, these findings suggest that the utilization of ethanolamine may be an important metabolic characteristic for strain M8S5 to survive in cold seep ecosystems.
Polyamine compounds, including spermidine and putrescine, are a group of ubiquitous polycations necessary for cell growth and play essential roles in the biosynthesis of nucleic acids and proteins in microorganisms [80][81][82].Moreover, recent studies reported that diverse kinds of epifauna thrived in the cold seep environment of the South China Sea [38], which may produce a large number of spermidines and putrescines.Our results showed that a total of nine genes encoding spermidine and putrescine transporters (potABCD) were identified in strain M8S5 (Figure 4 and Supplementary Table S11).Moreover, the enzymes of spermidine and putrescine degradation were also predicted, which finally transform polyamine compounds into ammonia (Figure 4).Among the transport genes of spermidine and putrescine, four were found to be absent in strain MCWD3, indicating that M8S5 may gain more genes via horizontal gene transfer (HGT) for the utilization of the polyamine compounds enriched in cold seep environments.
Biological nitrogen fixation (referred to as diazotrophy), which is the reduction of atmospheric dinitrogen to ammonia, is an important way to obtain a nitrogen source for microorganisms [83].Previous studies reported that members of Bacillota were detected as diazotrophs, which contributed to the nitrogen balance of deep-sea sediments [84].In our study, we identified the complete pathways of nitrogen fixation (catalyzed by nitrogenase and encoded by nifEH) in strain M8S5, but these were absent in the four phylogenetically related strains: MCWD3, S. massiliensis SIT17, S. massiliensis 583, and C. paucivorans DSM 22131 (Figure 4 and Supplementary Table S11).These findings may imply that strain M8S5 has gained the capability to transform inorganic nitrogen to organic nitrogen via horizontal gene transfer (HGT) to survive in nitrogen-limited cold seep environments.
(5) Nucleic acid metabolism For nucleic acid metabolism, four genes encoding nucleoside transporters were identified in strain M8S5 (Figure 4 and Supplementary Table S12), and the biosynthetic pathways of purine and pyrimidine were complete.However, the degradation pathways of nucleotides were absent in strain M8S5, indicating that these nucleotides may constitute DNA, RNA, and other cellular components but may not be utilized as carbon and energy sources by strain M8S5.

Genes Involved in High-Pressure and Low-Temperature Adaptation
To uncover how strain M8S5 adapted to the cold seep environments, we analyzed its genome and identified a large number of genes that were potentially involved in high hydrostatic pressure (HHP) and low-temperature adaptation (Supplementary Table S14), including the genes in osmoprotectants, molecular chaperones, antioxidation, and sporulation.

Genes Involved in High-Pressure and Low-Temperature Adaptation
To uncover how strain M8S5 adapted to the cold seep environments, we analyzed its genome and identified a large number of genes that were potentially involved in high hydrostatic pressure (HHP) and low-temperature adaptation (Supplementary Table S14), including the genes in osmoprotectants, molecular chaperones, antioxidation, and sporulation.
Compatible solutes, commonly referred to as osmoprotectants, are low molecular weight, highly soluble organic compounds that mainly protect microorganisms from a variety of environmental stresses, such as drought, high salt, low or high temperature, HHP, and heavy metals [87][88][89][90][91][92][93].In this study, we found that strain M8S5 possesses 21 genes to transport diverse kinds of compatible solutes, including glycine betaine/carnitine/choline, proline, sarcosine, and trehalose (Figure 4 and Supplementary Table S14).Glycine betaine is widespread in a variety of microorganisms and is thought to primarily serve as a potent osmoprotectant that may stabilize proteins and membrane structures, maintain cell turgor and volume, and act as a cryoprotectant under extreme environments [87][88][89]91,94].Strain M8S5 possessed glycine/betaine/proline (encoding by opuABCD) and three BCCT family transporters (Figure 4 and Supplementary Table S14).Likewise, trehalose, which also acts as an important osmoprotectant, may stabilize membranes at low temperatures, protect proteins from thermal denaturation, enhance resistance to hypoxia, and serve as a source of energy [87,88,94,95].A strain-specific gene (treB) encoding trehalose transporter was also detected in strain M8S5 (Figure 4 and Supplementary Table S14).These findings suggest that the intake of osmoprotectants might be a crucial strategy for M8S5 to adapt to local cold seep environments.
Moreover, a total of five genes encoding molecular chaperones, including dnaJK, grpE, htpG, and hslO, were predicted in strain M8S5 (Supplementary Table S14).These molecular chaperones may be induced by high-pressure and low-temperature stresses, help proteins to fold correctly, and maintain protein function [87,92,95].Also, two genes related to cold shock proteins (cspA) were detected in the genome of strain M8S5 (Supplementary Table S14), which may serve as cryoprotectants in low-temperature adaptation Compatible solutes, commonly referred to as osmoprotectants, are low molecular weight, highly soluble organic compounds that mainly protect microorganisms from a variety of environmental stresses, such as drought, high salt, low or high temperature, HHP, and heavy metals [87][88][89][90][91][92][93].In this study, we found that strain M8S5 possesses 21 genes to transport diverse kinds of compatible solutes, including glycine betaine/carnitine/choline, proline, sarcosine, and trehalose (Figure 4 and Supplementary Table S14).Glycine betaine is widespread in a variety of microorganisms and is thought to primarily serve as a potent osmoprotectant that may stabilize proteins and membrane structures, maintain cell turgor and volume, and act as a cryoprotectant under extreme environments [87][88][89]91,94].Strain M8S5 possessed glycine/betaine/proline (encoding by opuABCD) and three BCCT family transporters (Figure 4 and Supplementary Table S14).Likewise, trehalose, which also acts as an important osmoprotectant, may stabilize membranes at low temperatures, protect proteins from thermal denaturation, enhance resistance to hypoxia, and serve as a source of energy [87,88,94,95].A strain-specific gene (treB) encoding trehalose transporter was also detected in strain M8S5 (Figure 4 and Supplementary Table S14).These findings suggest that the intake of osmoprotectants might be a crucial strategy for M8S5 to adapt to local cold seep environments.
Moreover, a total of five genes encoding molecular chaperones, including dnaJK, grpE, htpG, and hslO, were predicted in strain M8S5 (Supplementary Table S14).These molecular chaperones may be induced by high-pressure and low-temperature stresses, help proteins to fold correctly, and maintain protein function [87,92,95].Also, two genes related to cold shock proteins (cspA) were detected in the genome of strain M8S5 (Supplementary Table S14), which may serve as cryoprotectants in low-temperature adaptation [96].In addition, HHP and low temperatures also produce reactive oxygen species (ROS), which are harmful to anaerobic microbes [97].We identified three peroxidases and one superoxide dismutaserelated gene in strain M8S5 (Supplementary Table S14), which may serve as antioxidants and help to scavenge intracellular ROS and, therefore, protect the cells from cold and/or HHP stress.
Finally, spores are metabolically dormant and highly resistant structures formed by Bacillota that enhance their survival in adverse environmental conditions, such as HHP and low temperatures.Spore formation is essential to the survival of some obligate anaerobic Bacillota bacteria [98,99].In this study, a total of 69 spore-related genes were identified in strain M8S5, mainly including spore germination protein (encoding by gerKABC, gerM, yaaH, yndDE, and ypeB), spore maturation protein (cgeBD and spmAB), sporulation protein (encoding by spoIIABDEGMPR, spoIIIAABCDEFGH, spoIVABF, and spoVABCDEGS, respectively), and spore coat protein (cotAEHIJS) (Supplementary Table S15).These findings may indicate that sporulation may help strain M8S5 to adapt to many extremely unfavorable environments.

Figure 1 .
Figure 1.Graphical representation of M8S5 genome.Genes on the forward (shown in the outer circle) and reverse (shown in the inner circle) strands are colored according to their cluster of orthologous gene (COG) categories (except those colored in black for no hits); tRNA and rRNA genes are highlighted with blue and red colors, respectively; gene islands are shown in green; genes encoding D-amino acids and peptidases are shown in green and orange, respectively; genes encoding polysaccharides are shown in blue color; GC content is shown in yellow and blue; and GC skew is shown in orange and purple (window, 5000 bp; step, 2500 bp).

Figure 1 .
Figure 1.Graphical representation of M8S5 genome.Genes on the forward (shown in the outer circle) and reverse (shown in the inner circle) strands are colored according to their cluster of orthologous gene (COG) categories (except those colored in black for no hits); tRNA and rRNA genes are highlighted with blue and red colors, respectively; gene islands are shown in green; genes encoding D-amino acids and peptidases are shown in green and orange, respectively; genes encoding polysaccharides are shown in blue color; GC content is shown in yellow and blue; and GC skew is shown in orange and purple (window, 5000 bp; step, 2500 bp).

Figure 2 .
Figure 2. The phylogenetic tree of strain M8S5 was constructed based on 120 concentration proteins.Strains M8S5 and MCWD3 are highlighted in red and blue color, respectively.The black nodes represent the bootstrap values ≥ 80.

Figure 2 .
Figure 2. The phylogenetic tree of strain M8S5 was constructed based on 120 concentration proteins.Strains M8S5 and MCWD3 are highlighted in red and blue color, respectively.The black nodes represent the bootstrap values ≥ 80.

Figure 3 .
Figure 3. Environmental distribution of the strain M8S5.The sampling site F in this study is highlighted in red color.

Figure 3 .
Figure 3. Environmental distribution of the strain M8S5.The sampling site F in this study is highlighted in red color.

Figure 4 .
Figure 4. Reconstructed metabolic pathways of strain M8S5.X marks in the red color represent the genes absent in M8S5.

Figure 4 .
Figure 4. Reconstructed metabolic pathways of strain M8S5.X marks in the red color represent the genes absent in M8S5.

Figure 5 .
Figure 5. Two gene clusters of type glycine reductase in the genome of strain M8S5.(A) glycine reductase I, (B) glycine reductase II.

Figure 5 .
Figure 5. Two gene clusters of type glycine reductase in the genome of strain M8S5.(A) glycine reductase I, (B) glycine reductase II.