Chemoautotrophs within Campylobacterota, especially Sulfurovum and Sulfurimonas, are abundant in the seawater-sediment interface of the Formosa cold seep in the South China Sea. However, the in situ activity and function of Campylobacterota are unknown. In this study, the geochemical role of Campylobacterota in the Formosa cold seep was investigated with multiple means. Two members of Sulfurovum and Sulfurimonas were isolated for the first time from deep-sea cold seep. These isolates are new chemoautotrophic species that can use molecular hydrogen as an energy source and CO2 as a sole carbon source. Comparative genomics identified an important hydrogen-oxidizing cluster in Sulfurovum and Sulfurimonas. Metatranscriptomic analysis detected high expression of hydrogen-oxidizing gene in the RS, suggesting that H-2 was likely an energy source in the cold seep. Genomic analysis indicated that the Sulfurovum and Sulfurimonas isolates possess a truncated sulfur-oxidizing system, and metatranscriptomic analysis revealed that Sulfurovum and Sulfurimonas with this genotype were active in the surface of RS and likely contributed to thiosulfate production. Furthermore, geochemical and in situ analyses revealed sharply decreased nitrate concentration in the sediment-water interface due to microbial consumption. Consistently, the denitrification genes of Sulfurimonas and Sulfurovum were highly expressed, suggesting an important contribution of these bacteria to nitrogen cycling. Overall, this study demonstrated that Campylobacterota played a significant role in the cycling of nitrogen and sulfur in a deep-sea cold seep. IMPORTANCEChemoautotrophs within Campylobacterota, in particular Sulfurovum and Sulfurimonas, are ubiquitous in deep-sea cold seeps and hydrothermal vents. However, to date, no Sulfurovum or Sulfurimonas has been isolated from cold seeps, and the ecological roles of these bacteria in cold seeps remain to be investigated. In this study, we obtained two isolates of Sulfurovum and Sulfurimonas from Formosa cold seep, South China Sea. Comparative genomics, metatranscriptomics, geochemical analysis, and in situ experimental study indicated collectively that Campylobacterota played a significant part in nitrogen and sulfur cycling in cold seep and was the cause of thiosulfate accumulation and sharp reduction of nitrate level in the sediment-water interface. The findings of this study promoted our understanding of the in situ function and ecological role of deep-sea Campylobacterota.Chemoautotrophs within Campylobacterota, in particular Sulfurovum and Sulfurimonas, are ubiquitous in deep-sea cold seeps and hydrothermal vents. However, to date, no Sulfurovum or Sulfurimonas has been isolated from cold seeps, and the ecological roles of these bacteria in cold seeps remain to be investigated. In this study, we obtained two isolates of Sulfurovum and Sulfurimonas from Formosa cold seep, South China Sea. Comparative genomics, metatranscriptomics, geochemical analysis, and in situ experimental study indicated collectively that Campylobacterota played a significant part in nitrogen and sulfur cycling in cold seep and was the cause of thiosulfate accumulation and sharp reduction of nitrate level in the sediment-water interface. The findings of this study promoted our understanding of the in situ function and ecological role of deep-sea Campylobacterota.

Cold seeps are globally widespread seafloor ecosystems that feature abundant methane production and flourishing chemotrophic benthic communities. Chemical evidence indicates that cold seep methane is largely biogenic; however, the primary methane-producing organisms and associated pathways involved in methano-genesis remain elusive. This work detected methane production when glycine betaine (GBT) or trimethylamine (TMA) was added to the sediment microcosms of the Formosa cold seep, South China Sea. The methane production was suppressed by antibiotic inhi-bition of bacteria, while GBT was accumulated. This suggests that the widely used osmoprotectant GBT could be converted to cold seep biogenic methane via the syner-gistic activity of bacteria and methanogenic archaea because archaea are not sensitive to antibiotics and no bacteria are known to produce ample methane (mM). 16S rRNA gene diversity analyses revealed that the predominant bacterial and archaeal gen-era in the GBT-amended methanogenic microcosms included Oceanirhabdus and Methanococcoides. Moreover, metagenomic analyses detected the presence of grdH and mtgB genes that are involved in GBT reduction and demethylation, respectively. Two novel species were obtained, including bacterium Oceanirhabdus seepicola, which reduces GBT to TMA, and a methanogenic archaeon, Methanococcoides seepicolus, which produces methane from TMA and GBT. The two strains reconstituted coculture efficiently converted GBT to methane at 18 degrees C; however, at 4 degrees C addition of dimethylgly-cine (DMG), the GBT demethylation product, was necessary. Therefore, this work dem-onstrated that GBT is the precursor not only of the biogenic methane but also of the cryoprotectant DMG to the microorganisms at the Formosa cold seep.IMPORTANCE Numerous cold seeps have been found in global continental margins where methane is enriched in pore waters that are forced upward from sediments. Therefore, high concerns have been focused on the methane-producing organisms and the metabolic pathways in these environments because methane is a potent greenhouse gas. In this study, GBT was identified as the main precursor for methane in the Formosa cold seep of the South China Sea. Further, synergism of bacteria and methanogenic archaea was identified in GBT conversion to methane via the GBT reduction pathway, while methanogen-mediated GBT demethylation to methane was also observed. In addition, GBT-demethylated product dimethyl glycine acted as a cryoprotectant that promoted the cold seep microorganisms at cold temperatures. GBT is an osmoprotectant that is widely used by marine organisms, and therefore, the GBT-derived methanogenic pathway reported here could be widely distributed among global cold seep environments.

In this study, we report a novel Gram-positive bacterium, designated as strain CS13(T), isolated from deep-sea sediment collected in the cold seep area of the South China Sea. Growth of strain CS13(T) occurred at 16-37 degrees C (optimum 25-28 degrees C), pH 7.0-9.0 (optimum, 7.0), and 0-8% (w/v) NaCl (optimum, 2-3%). Phylogenetic analysis based on 16S rRNA gene sequence indicated that strain CS13(T) belonged to the genus Bacillus. The closest phylogenetic neighbors of strain CS13(T) are Bacillus carboniphilus JCM 9731(T) (96.0%), Bacillus pakistanensis NCCP-168(T) (95.7%) and Bacillus acidicola 105-2(T) (95.6%). The genomic DNA G + C content of strain CS13(T) is 43.7 mol%. The principal respiratory quinone was menaquinone 7 (MK-7). The polar lipids of CS13(T) contained diphosphatidylglycerol, phosphatidylglycerol, phospholipid, and glycolipid. The major fatty acids of CS13(T) contained anteiso-C-15:0, anteiso-C-17:0, C-16:0 and C-18:0. Strain CS13(T) harboured meso-diaminopimelic acid as the diagnostic diamino acid. Phylogenetic, physiological, biochemical, and morphological analyses suggested that strain CS13(T) represents a novel species of genus Bacillus, and the name Bacillus fonticola sp. nov. is proposed for the type species CS13(T) (= CCTCC AB 2019194(T) = JCM 33663(T)).

Archaea are highly diverse and represent a primary life domain, but the majority of them remain uncultured. Currently, 16S rRNA phylogeny is widely used in archaeal taxonomy and diversity surveys. However, highly conserved sequence of 16S rRNA possibly results in generation of chimera in the amplicons and metagenome-assembled genomes (MAGs) and therefore limits its application. The newly developed phylogenomic approach has overcome these flaws, but it demands high-quality MAGs and intensive computation. In this study, we investigated the use of the archaeal transcription termination factor aCPSF1 in archaeal classification and diversity surveys. The phylogenetic analysis of 1,964 aCPSF1 orthologs retrieved from the available archaeal (meta)genomes resulted in convergent clustering patterns with those of archaeal phylogenomics and 16S rRNA phylogeny. The aCPSF1 phylogeny also displayed comparable clustering with the methanoarchaeal McrABG phylogeny and the haloarchaeal phylogenomics. Normalization of 779 aCPSF1 sequences including 261 from cultured archaeal species yielded a taxonomic ranking system with higher resolutions than that obtained with 16S rRNA for genus and species. Using the aCPSF1 taxonomy, 144 unclassified archaea in NCBI database were identified to various taxonomic ranks. Moreover, aCPSF1-and 16S rRNA-based surveys of the archaeal diversity in a sample from a South China Sea cold seep produced similar results. Our results demonstrate that aCPSF1 is an alternative archaeal phylogenetic marker, which exhibits higher resolution than 16S rRNA, and is more readily usable than phylogenomics in the taxonomic study of archaea.IMPORTANCE Archaea represent a unique type of prokaryote, which inhabit in various environments including extreme environments, and so define the boundary of biosphere, and play pivotal ecological roles, particularly in extreme environments. Since their discovery over 40 years ago, environmental archaea have been widely investigated using the 16S rRNA sequence comparison, and the recently developed phylogenomic approach because the majority of archaea are recalcitrant to laboratory cultivation. However, the highly conserved sequence of 16S rRNA and intensive bioinformatic computation of phylogenomics limit their applications in archaeal species delineation and diversity investigations. aCPSF1 is a ubiquitously distributed and vertically inherited transcription termination factor in archaea. In this study, we developed an aCPSF1-based archaeal taxonomic system which exhibits congruent phylogenic clustering patterns with archaeal phylogenomics and higher resolution than 16S rRNA in distinguishing archaea at lower taxonomic ranks. Therefore, aCPSF1 is a new phylogenetic marker in the taxonomic and diversity studies of archaea.

In the Formosa cold seep of the South China Sea (SCS), large amounts of methane and sulfide hydrogen are released from the subseafloor. In this study, we systematically investigated the microbial communities in the seawater-sediment interface of Formosa cold seep using high-throughput sequencing techniques including amplicon sequencing based on next-generation sequencing and Pacbio amplicon sequencing platforms, and metagenomics. We found that Sulfurovum dominated the microbial communities in the sediment-seawater interface, including the seawater close to the seepage, the surface sediments, and the gills of the dominant animal inhabitant (Shinkaia crosnieri). A nearly complete 16S rRNA gene sequence of the dominant operational taxonomic units (OTUs) was obtained from the Pacbio sequencing platforms and classified as OTU-L1, which belonged to Sulfurovum. This OTU was potentially novel as it shared relatively low similarity percentages (<97%) of the gene sequence with its close phylogenetic species. Further, a draft genome of Sulfurovum was assembled using the binning technique based on metagenomic data. Genome analysis suggested that Sulfurovum sp. in this region may fix carbon by the reductive tricarboxylic acid (rTCA) pathway, obtain energy by oxidizing reduced sulfur through sulfur oxidizing (Sox) pathway, and utilize nitrate as electron acceptors. These results demonstrated that Sulfurovum probably plays an important role in the carbon, sulfur, and nitrogen cycles of the Formosa cold seep of the SCS. This study improves our understanding of the diversity, distribution, and function of sulfur-oxidizing bacteria in deep-sea cold seep.

The ribosomal RNA (rRNA) genes are generally organized as an operon and cotranscribed into a polycistronic precursor; therefore, processing and maturation of pre-rRNAs are essential for ribosome biogenesis. However, rRNA maturation pathways of archaea, particularly of methanoarchaea, are scarcely known. Here, we thoroughly elucidated the maturation pathway of the rRNA operon (16S-tRNA(Ala)-23S-tRNA(Cys)-5S) inMethanolobus psychrophilus, one representative of methanoarchaea. Enzymatic assay demonstrated that EndA, a tRNA splicing endoribonuclease, cleaved bulge-helix-bulge (BHB) motifs buried in the processing stems of pre-16S and pre-23S rRNAs. Northern blot and quantitative PCR detected splicing-coupled circularization of pre-16S and pre-23S rRNAs, which accounted for 2% and 12% of the corresponding rRNAs, respectively. Importantly, endoribonuclease Nob1 was determined to linearize circular pre-16S rRNA at the mature 3 ' end so to expose the anti-Shine-Dalgarno sequence, while circular pre-23S rRNA was linearized at the mature 5 ' end by an unknown endoribonuclease. The resultant 5 ' and 3 ' extension in linearized pre-16S and pre-23S rRNAs were finally matured through 5 '-3 ' and 3 '-5 ' exoribonucleolytic trimming, respectively. Additionally, a novel processing pathway of endoribonucleolysis coupled with exoribonucleolysis was identified for the pre-5S rRNA maturation in this methanogen, which could be also conserved in most methanogenic euryarchaea. Based on evaluating the phylogenetic conservation of the key elements that are involved in circularization and linearization of pre-rRNA maturation, we predict that the rRNA maturation mode revealed here could be prevalent among archaea.

冷泉是由深海沉积物中的甲烷及其他有机质流体向海底渗漏或喷发而形成的独特环境，生活着生物多样性丰富、尤其是化能自养微生物，为底栖海洋动物提供碳源和能源，是支撑无光、低氧海底生物圈的基础生物类群。本课题开展我国主要深海冷泉区的微生物多样性、及其特殊代谢功能研究，解析冷泉微生物的特殊环境生存机制及其对深海冷泉地球化学循环中的贡献；发掘其功能基因资源及代谢能力，提供生物技术创新的资源和策略。本年度采集到Formasa和Haima两个冷泉区水样、沉积物及底栖动物样品32份，理化因子分析得知Formasa冷泉区甲烷含量高、硫酸盐和DIC低。对4个冷泉区微生物多样性分析表明,0-20cm的细菌主要是δ-Proteobacteria 和 Chloroflei,古菌主要是ANME-2a/b,甲烷八叠球菌科;40-400cm的细菌主要是Atribacteria和Proteobacteria;古菌以产甲烷菌为主。特别发现Atribacteria和产甲烷古菌分布在同层，暗示二者代谢依存。基于宏基因组分析拼接出50多个冷泉未培养微生物的基因组；分离到冷泉区好氧和厌氧微生物500株左右，保藏198株。这些分离物中有5个潜在的新种，完成了它们的基因组测序及生理生化特征分析。这些分离的物种中具有淀粉酶、明胶酶、脂肪酶等酶活菌株40株。揭示了深海偏顶蛤与其共生微生物关系的建立和维持机制。 Cold seeps constitute unique environmental niches and inhabit diverse organisms in particularly of the chemoautotrophic microorganisms,which provide carbon and energy sources to the benthic marine animals,so are the foundational biological groups in the dark biosphere.This project investigates the microbial species diversity in Chinese cold seeps and the specific metabolic functions,and aims to elucidate the adaptive mechanisms of the cold seep species,and their contributions to the biogeochemical recycling of Deep Ocean;and also to exploit their functions to serve innovative biotechnology developments.In this fiscal year,we have sampled in total of 32 sampling specimens from the Formasa and Haima cold seeps,including water samples,sediments and marine benthic animals.The physicochemical parameter analysis indicated higher contents of methane and sulfate but lower dissolving chemicals(DIC).Microbial diversity analysis for four cold seep samples showed that different species inhabit in the sea floor and the sediments,in specifically,δ-Proteobacteria and Chloroflei,and ANME-2a/b and Methanosarcina are the dominant of Bacteria and Archaea,respectively distributed in 0-20cm depth of the cold seep sediment;while the uncultured Atribacteria and Proteobacteria,and methanoarchaea are the prevalent Bacteria and Archaea in 40-400cm depth of the sediment,respectively.Particularly,co-occurrence of Atribacteria and methanoarchaea was observed,implying that they were metabolically interdependent.Through metagenome sequencing,we have obtained 50 metagenome-assembled genomes,which represent the cold seep specific microbial species.The genomic data expose the gene contents and metabolic potentials of these uncultured microorganisms.By means of a variety of culturing approaches,we have isolated about 500 cold seep microbial strains and identified most of them,and deposited 198 strains in China General Microorganism Culture Collection Center(CGMCC).Among them,five strains likely represent new species,so their genomes are sequenced and characterized for the physiological and biochemical features;40 strains produced amylase,gelatinase and lipase.In addition,we,via differential transcriptomic analysis,revealed the symbiotic development and maintaining mechanisms between the deep sea modiolus(B.platifrons)and the symbionts.

Thermophiles are organisms that grow optimally at 50 degrees C-80 degrees C and studies on the survival mechanisms of thermophiles have drawn great attention. Bacillus manusensis S50-6 is the type strain of a new thermophilic species isolated from hydrothermal vent in Manus Basin. In this study, we examined the growth and global responses of S50-6 to high temperature on molecular level using multi-omics method (genomics, proteomics, and metabolomics). S50-6 grew optimally at 50 degrees C (Favorable, F) and poorly at 65 degrees C (Non-Favorable, NF); it formed spores at F but not at NF condition. At NF condition, S50-6 formed long filaments containing undivided cells. A total of 1621 proteins were identified at F and NF conditions, and 613 proteins were differentially expressed between F and NF. At NF condition, proteins of glycolysis, rRNA mature and modification, and DNA/protein repair were up-regulated, whereas proteins of sporulation and amino acid/nucleotide metabolism were down-regulated. Consistently, many metabolites associated with amino acid and nucleotide metabolic processes were down-regulated at NF condition. Our results revealed molecular strategies of deep-sea B. manusensis to survive at unfavorable high temperature and provided new insights into the thermotolerant mechanisms of thermophiles. Significance: In this study, we systematically characterized the genomic, proteomic and metabolomic profiles of a thermophilic deep-sea Bacillus manusensis under different temperatures. Based on these analysis, we propose a model delineating the global responses of B. manusensis to unfavorable high temperature. Under unfavorable high temperature, glycolysis is a more important energy supply pathway; protein synthesis is subjected to more stringent regulation by increased tRNA modification; protein and DNA repair associated proteins are enhanced in production to promote heat survival. In contrast, energy-costing pathways, such as sporulation, are repressed, and basic metabolic pathways, such as amino acid and nucleotide metabolisms, are slowed down. Our results provide new insights into the thermotolerant mechanisms of thermophilic Bacillus.

Author summary RNAs frequently misfold into stable but biologically inactive structures especially under stresses, while RNA chaperones interact with various RNAs to prevent the structures that may cause premature transcriptional termination or pausing, and affect mRNA decay and translation. This work for the first time reports that an archaeal RNA chaperone TRAM0076 globally affects the transcription of methanogenic Archaea through posttranscriptional actions. TRAM0076 also binds many cellular mRNAs, possibly at the 5 '-untranslated regions. This work uncovers an important regulatory element of ancient life in the RNA world.TRAM is a conserved domain among RNA modification proteins that are widely distributed in various organisms. In Archaea, TRAM occurs frequently as a standalone protein with in vitro RNA chaperone activity; however, its biological significance and functional mechanism remain unknown. This work demonstrated that TRAM0076 is an abundant standalone TRAM protein in the genetically tractable methanoarcheaon Methanococcus maripaludis. Deletion of MMP0076, the gene encoding TRAM0076, markedly reduced the growth and altered transcription of 55% of the genome. Substitution mutations of Phe39, Phe42, Phe63, Phe65 and Arg35 in the recombinant TRAM0076 decreased the in vitro duplex RNA unfolding activity. These mutations also prevented complementation of the growth defect of the MMP0076 deletion mutant, indicating that the duplex RNA unfolding activity was essential for its physiological function. A genome-wide mapping of transcription start sites identified many 5 ' untranslated regions (5 ' UTRs) of 20-60 nt which could be potential targets of a RNA chaperone. TRAM0076 unfolded three representative 5 ' UTR structures in vitro and facilitated the in vivo expression of a mCherry reporter system fused to the 5 ' UTRs, thus behaving like a transcription anti-terminator. Flag-tagged-TRAM0076 co-immunoprecipitated a large number of cellular RNAs, suggesting that TRAM0076 plays multiple roles in addition to unfolding incorrect RNA structures. This work demonstrates that the conserved archaeal RNA chaperone TRAM globally affects gene expression and may represent a transcriptional element in ancient life of the RNA world.