Nitrogen and sulfur cycling driven by Campylobacterota in the sediment-water interface of deep-sea cold seep: a case in the South China Sea.

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 H2 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. IMPORTANCE 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.

Pontivivens ytuae sp. nov., isolated from deep sea sediment of the Mariana Trench.

A Gram-stain-negative, light pink-coloured, rod-shaped, flagellated and facultative anaerobic bacterial strain, designated MT2928T, was isolated from deep-sea sediment collected from the Mariana Trench. Growth of strain MT2928T occurred optimally at 28 °C, pH 8.0-9.0 and in the presence of 1.0-2.0 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain MT2928T belongs to the genus Pontivivens and has the highest sequence similarity to Pontivivens insulae GYSW-23T (96.6 %). Genomic analysis indicated that strain MT2928T contains a circular chromosome of 4 199 362 bp with G+C content of 67.2 mol%. The strain did not produce bacteriochlorophyll a, but produced carotenoid. The predominant respiratory quinone of MT2928T was ubiquinone-10. The polar lipids of MT2928T contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, two unidentified lipids and two unidentified phospholipids. The major fatty acids of strain MT2928T contained summed feature 8 (C18 : 1 ω7c or/and C18 : 1 ω6c), C18 : 0 and summed feature 2 (iso-C16 : 1 I and/or C14 : 0 3-OH). On the basis of phylogenetic, physiological, biochemical and other phenotypic properties, strain MT2928T represents a novel species of the genus Pontivivens, and the name Pontivivens ytuae sp. nov. is proposed with the type species MT2928T (=MCCC 1K05575T=JCM 34320T).

Psychrosphaera ytuae sp. nov., isolated from the deep-sea cold seep sediment of South China Sea.

In this study, we report a Gram-stain-negative, rod-shaped, non-pigmented, motile and aerobic bacterium named strain MTZ26T, which was isolated from deep-sea sediment sampled at a cold seep in the South China Sea. Growth of strain MTZ26T occurred at 4-40 °C (optimum, 25-30 °C), pH 6.0-10.0 (optimum, 7.0-8.0) and with 1.0-11.0 % (w/v) NaCl (optimum, 6.0-8.0 %). Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain MTZ26T belonged to the genus Psychrosphaera and was closely related to Psychrosphaera aestuarii PSC101T (97.5 % sequence similarity) and Psychrosphaera haliotis KDW4T (97.5 %). Genomic analysis indicated that strain MTZ26T contains a circular chromosome of 3 331 814 bp with G+C content of 42.2 mol%. The predominant respiratory quinone of MTZ26T was ubiquinone-8. The polar lipids of MTZ26T contained phosphatidylglycerol, phosphatidylethanolamine, one unidentified aminophospholipid and one unidentified phospholipid. The major fatty acids of strain MTZ26T contained C15:0, C16:0, C17:0, C17 : 1 ω8c, C10 : 0 3-OH, C11 : 0 3-OH, C15 : 1 ω8c and summed feature 8 (C18 : 1 ω7c or/and C18 : 1 ω6c). Results of phylogenetic, physiological, biochemical and morphological analyses suggested that strain MTZ26T represents a novel species of the genus Psychrosphaera, and the name Psychrosphaera ytuae sp. nov. is proposed with the type strain MTZ26T (=MCCC 1K05568T=JCM 34321T).

Description of Novosphingopyxis iocasae sp. nov., isolated from deep sea sediment from the Mariana Trench, and emended description of the genus Novosphingopyxis.

In this study, we reported a Gram-stain-negative, orange-coloured, rod-shaped, motile and faculatively anaerobic bacterium named strain PB63T, which was isolated from the deep-sea sediment from the Mariana Trench. Growth of PB63T occurred at 10-35 °C (optimum, 28 °C), pH 5.0-8.0 (optimum, 5.0-6.0) and with 0-7 % (w/v) NaCl (optimum, 2-3 %). The results of phylogenetic analysis based on 16S rRNA gene sequences indicated that PB63T represented a member of the genus Novosphingopyxis and was closely related to Novosphingopyxis baekryungensis DSM 16222T (97.9 % sequence similarity). PB63T showed tolerance to a variety of heavy metals, including Co2+, Zn2+, Mn2+ and Cu2+. The complete genome of PB63T was obtained, and many genes involved in heavy metal resistance were found. The genomic DNA G+C content of PB63T was 62.8 mol%. The predominant respiratory quinone of PB63T was ubiquinone-10 (Q-10). The polar lipids of PB63T contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, sphingoglycolipid, glycolipid, phosphatidylcholines and three unidentified lipids. The major fatty acids of PB63T included summed feature 8 (C18 : 1ω7c or/and C18 : 1ω6c), C14 : 0 2-OH, 11-methyl C18 : 1ω7c, C16 : 0, summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) and C17 : 1ω6c. The results of phylogenetic, physiological, biochemical and morphological analyses indicated that strain PB63T represents a novel species of the genus Novosphingopyxis, and the name Novosphingopyxis iocasae sp. nov. is proposed with the type species PB63T (=CCTCC AB 2019195T=JCM 34178T).

Bacillus fonticola sp. nov., isolated from deep sea cold seep sediment.

In this study, we report a novel Gram-positive bacterium, designated as strain CS13T, isolated from deep-sea sediment collected in the cold seep area of the South China Sea. Growth of strain CS13T occurred at 16-37 °C (optimum 25-28 °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 CS13T belonged to the genus Bacillus. The closest phylogenetic neighbors of strain CS13T are Bacillus carboniphilus JCM 9731T (96.0%), Bacillus pakistanensis NCCP-168T (95.7%) and Bacillus acidicola 105-2T (95.6%). The genomic DNA G + C content of strain CS13T is 43.7 mol%. The principal respiratory quinone was menaquinone 7 (MK-7). The polar lipids of CS13T contained diphosphatidylglycerol, phosphatidylglycerol, phospholipid, and glycolipid. The major fatty acids of CS13T contained anteiso-C15:0, anteiso-C17:0, C16:0 and C18:0. Strain CS13T harboured meso-diaminopimelic acid as the diagnostic diamino acid. Phylogenetic, physiological, biochemical, and morphological analyses suggested that strain CS13T represents a novel species of genus Bacillus, and the name Bacillus fonticola sp. nov. is proposed for the type species CS13T (= CCTCC AB 2019194T = JCM 33663T).

High-Throughput Sequencing Reveals a Potentially Novel Sulfurovum Species Dominating the Microbial Communities of the Seawater-Sediment Interface of a Deep-Sea Cold Seep in South China Sea.

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.

Marinobacter fonticola sp. nov., isolated from deep sea cold seep sediment.

In this study, we report a novel Gram-negative bacterium, designated as strain CS412T, isolated from deep-sea sediment collected in a cold seep area of the South China Sea. Growth of strain CS412T occurred at 4-40 °C (optimum, 28 °C), pH 5.0-11.0 (optimum, pH 6.0) and with 0-19 % (w/v) NaCl (optimum, 1-2 %). Phylogenetic analysis based on 16S rRNA gene sequence data indicated that strain CS412T belonged to the genus Marinobacter. The closest phylogenetic neighbours of strain CS412T were Marinobacter pelagius HS225T (96.9 %), Marinobacter szutsaonensis NTU-104T (96.8%), Marinobacter santoriniensis NKSG1T (96.4%) and Marinobacter koreensisdd-M3T (96.3 %). The genomic DNA G+C content of strain CS412T was 58.0 mol%. The principal respiratory quinone was ubiquinone-9 (Q-9). The polar lipids of CS412T contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, aminophospholipidand and four glycolipids. The major fatty acids of CS412T contained cyclo-C19 : 0ω8c, C16 : 0, C18 : 1ω7c and C18 : 1ω7c 11-methyl. The results of phylogenetic, physiological, biochemical and morphological analyses suggested that strain CS412T represents a novel species of the genus Marinobacter, and the name Marinobacter fonticola sp. nov. is proposed with the type species CS412T (=CCTCC AB 2019197T=KCTC 72475T).

A First Study of the Virulence Potential of a Bacillus subtilis Isolate From Deep-Sea Hydrothermal Vent.

Bacillus subtilis is the best studied Gram-positive bacterium, primarily as a model of cell differentiation and industrial exploitation. To date, little is known about the virulence of B. subtilis. In this study, we examined the virulence potential of a B. subtilis strain (G7) isolated from the Iheya North hydrothermal field of Okinawa Trough. G7 is aerobic, motile, endospore-forming, and requires NaCl for growth. The genome of G7 is composed of one circular chromosome of 4,216,133 base pairs with an average GC content of 43.72%. G7 contains 4,416 coding genes, 27.5% of which could not be annotated, and the remaining 72.5% were annotated with known or predicted functions in 25 different COG categories. Ten sets of 23S, 5S, and 16S ribosomal RNA operons, 86 tRNA and 14 sRNA genes, 50 tandem repeats, 41 mini-satellites, one microsatellite, and 42 transposons were identified in G7. Comparing to the genome of the B. subtilis wild type strain NCIB 3610T, G7 genome contains many genomic translocations, inversions, and insertions, and twice the amount of genomic Islands (GIs), with 42.5% of GI genes encoding hypothetical proteins. G7 possesses abundant putative virulence genes associated with adhesion, invasion, dissemination, anti-phagocytosis, and intracellular survival. Experimental studies showed that G7 was able to cause mortality in fish and mice following intramuscular/intraperitoneal injection, resist the killing effect of serum complement, and replicate in mouse macrophages and fish peripheral blood leukocytes. Taken together, our study indicates that G7 is a B. subtilis isolate with unique genetic features and can be lethal to vertebrate animals once being introduced into the animals by artificial means. These results provide the first insight into the potential harmfulness of deep-sea B. subtilis.

High temperature-induced proteomic and metabolomic profiles of a thermophilic Bacillus manusensis isolated from the deep-sea hydrothermal field of Manus Basin.

Thermophiles are organisms that grow optimally at 50 °C-80 °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 °C (Favorable, F) and poorly at 65 °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.

Muricauda iocasae sp. nov., isolated from deep sea sediment of the South China Sea.

In this study, we reported a novel yellow-pigmented, Gram-stain-negative bacterium with appendages, designated as strain L2T, isolated from the South China Sea. Growth of strain L2T occurred at 22-40 °C (optimum, 37 °C), pH 6.0-10.0 (pH 7.0) and with 0-8 % (w/v) NaCl (2 %). Phylogenetic analysis based on its 16S rRNA gene sequence indicated that strain L2T belonged to the genus Muricauda. The close phylogenetic neighbours of strain L2T were Muricauda marina H19-56T, Muricauda ruestringensis B1T, Muricauda antarctica Ar-22T, Muricauda taeanensis 105T and Muricauda flavescens SW-62T (96.4 %, 95.9 %, 95.9 %, 95.8 % and 94.5 % identities, respectively). The genomic DNA G+C content of strain L2T was 51.3±4.6 mol%. Theg major isoprenoid quinone was MK-6 (100.0 %). The polar lipids contained phosphatidylethanolamine and two unidentified lipids. The major fatty acids (>10 % of total fatty acids) were iso-C17 : 0 3-OH (30.3 %), iso-C15 : 1 G (20.6 %) and iso-C15 : 0 (17.6 %). Phylogenetic, physiological, biochemical and morphological analysis suggested that this strain represents a novel species of genus Muricauda, and the name Muricauda iocasae sp. nov. is proposed with the type species L2T (=CCTCC AB 2017193 T=KCTC 62196T).

First characterization of two C-type lectins of the tubeworm Alaysia sp. from a deep-sea hydrothermal vent.

C-type lectins (CTLs) play an important role in innate immune defense. In this study, we identified and characterized two CTLs (Lec1 and Lec2) from the tubeworm Alaysia sp. collected from a hydrothermal vent in Pacmanus. Lec1 and Lec2 possess the typical CTL domain but share low sequence identities (10.8%-20.4%) with known CTLs. Recombinant (r) of Lec1 and Lec2 bound to various PAMPs and a wide arrange of bacteria from neritic and deep-sea environments in a Ca2+-independent manner, but only rLec1 caused agglutination of the bound bacteria. The activities of rLec1 and rLec2 were most stable and highest at 4 °C, the ambient temperature of the hydrothermal vent, and decreased at higher temperatures. Both lectins inhibited bacterial growth in a highly selective manner and agglutinated the erythrocytes of fish, rabbit, and chicken in a Ca2+-dependent manner. These results provided the first insights into the functional properties of CTLs in deep-sea Alaysia sp.

First characterization of an anti-lipopolysaccharide factor (ALF) from hydrothermal vent shrimp: Insights into the immune function of deep-sea crustacean ALF.

Anti-lipopolysaccharide factor (ALF) is a type of antimicrobial peptides (AMPs) with a vital role in antimicrobial defense. Although a large amount of ALFs have been identified from neritic and fresh water crustacean species, no functional investigation of ALFs from deep-sea animals have been documented. In the present study, we characterized the immune function of an ALF molecule (named RspALF1) from the shrimp Rimicaris sp. residing in the deep-sea hydrothermal vent in Desmos, Manus Basin. RspALF1 shares 51.5%-62.4% overall sequence identities with known shrimp ALFs and contains the conserved LPS binding domain (LBD). Both recombinant RspALF1 (rRspALF1) and the LBD-derived peptide (ALF1P1) bound to the cell wall components of Gram-negative and Gram-positive bacteria and killed a wide range of bacteria, especially those from deep-sea hydrothermal field, by damaging bacterial cellular structures. The bactericidal activities of rRspALF1 and ALF1P1 were optimal and stably maintained from 4 °C to 37 °C, which is comparable to the ambient temperature range of the habitat of Rimicaris sp. In addition to bacteria, rRspALF1 and ALF1P1 also exhibited anti-fungal activity. rRspALF1 and ALF1P1 exhibited high killing efficiencies, which, in terms of MIC values, were ranged between 0.25 µM and 4 µM for bacteria and 4 µM-8 µM for fungi. When introduced in vivo, both rRspALF1 and ALF1P1 effectively inhibited bacterial infection in shrimp and reduced the dissemination of bacterial and viral pathogens in fish. Together, these results provide the first insight into the biological property of deep-sea ALF and indicate that RspALF1 very likely plays a significant role in immune defense by functioning as a highly effective antimicrobial with a broad target range.

Description of Bacillus kexueae sp. nov. and Bacillus manusensis sp. nov., isolated from hydrothermal sediments.

Two Gram-staining-positive, strictly aerobic bacilli, designated as strains Ma50-5T and Ma50-6T, were isolated from the hydrothermal sediments of Manus Basin in the western Pacific Ocean. Based on 16S rRNA gene sequence, strains Ma50-5T and Ma50-6T were most closely related to Bacillus alveayuensis (97.0 and 97.2 % identity, respectively). The 16S rRNA gene sequence identity between strains Ma50-5T and Ma50-6T was 97.4 %. The identities between strains Ma50-5T and Ma50-6T and other closely related organisms were below 97.0 %. The G+C contents of the genomic DNA of strains Ma50-5T and Ma50-6T were 43.4 and 47.6 mol%, respectively. The major fatty acids (>10 %) of both strains were iso-C15 : 0 and iso-C17 : 0. The predominant isoprenoid quinone detected in both strains was menaquinone-7. Phylogenetic, physiological, biochemical and morphological analyses suggested that strains Ma50-5T and Ma50-6T represent two novel species of the genus Bacillus, for which the names Bacillus kexueae sp. nov. (type strain Ma50-5T=KCTC 33881T=CCTCC AB 2017020T) and Bacillus manusensis sp. nov. (type strain Ma50-6T=KCTC 33882T=CCTCC AB 2017019T), respectively, are proposed.

Cytochemical identification of turbot myeloperoxidase-positive granulocytes by potassium iodide and oxidized pyronine Y staining.

Cytomorphological and cytochemical staining are important methods for the identification of cell types, in particular in fish which often lack biological tools such as specific antibodies. Myeloperoxidase (MPO) is usually used as an intracellular marker of neutrophil accumulation in tissues and a marker of neutrophil activity in plasma. In this study, we reported a potassium iodide and oxidized pyronine Y (KI-PyY) staining method for rapid and highly sensitive detection of MPO-positive cells in turbot blood, peritoneum, and tissues. MPO-positive cells, which mostly represented neutrophils, were stained brown and clearly distinguished from other cells, such as lymphocytes, monocytes, and macrophages, which were stained pink. Following bacterial stimulation, the proportions of neutrophils were 27.49% and 38.05% in peripheral blood leukocytes and peritoneum, respectively, judging by the stained MPO. Kidney granulocytes contained abundant MPO-positive cells which were probably immature neutrophils with low expression of MPO. It is noteworthy that MPO-positive cells were detected in the tissue sections of kidney, spleen, and gut, with distribution profiles specific to each tissue. However, the cell morphology was not distinct in the stained tissue sections. These results indicate that the KI-PyY staining method is highly sensitive, applicable to different types of samples, and will be useful for the study of neutrophils in different compartments of fish.

A comparative study revealed first insights into the diversity and metabolisms of the microbial communities in the sediments of Pacmanus and Desmos hydrothermal fields.

Currently, little is known about the microbial diversity in the sediments of Pacmanus and Desmos hydrothermal fields in Manus Basin. In this study, Illumina-based sequencing of 16S rRNA gene amplicons and metagenomic analysis were conducted to investigate the microbial populations and metabolic profiles in the sediments from four different regions in Pacmanus and Desmos hydrothermal fields. It was found that Gammaproteobacteria and Thaumarchaeota were the most abundant bacterial and archaeal populations, respectively. The autotrophic prokaryotes in the four communities probably fixed CO2 via four major pathways, i.e. Calvin-Benson-Bassham cycle, reductive acetyl-CoA cycle, rTCA cycle, and 3-hydroxypropionate/4-hydroxybutyrate cycle. Ammonia-oxidizing Thaumarchaeota, nitrifiers, denitrifiers, and sulfur oxidizers belonging to the subgroups of Proteobacteria (e.g., alpha, beta, gamma, and epsilon), Nitrospira, and Nitrospina, and sulfate-reducing Desulfobacterales likely played critical roles in nitrogen and sulfur cycling, in which ammonia, sulfur compounds, and hydrogen could be utilized as potential energy sources. These findings revealed new insights into the operational mechanism of the microbial communities associated with Pacmanus and Desmos hydrothermal fields.

Comparative transcriptome analysis of Rimicaris sp. reveals novel molecular features associated with survival in deep-sea hydrothermal vent.

Shrimp of the family Alvinocarididae are the predominant megafauna of deep-sea hydrothermal vents. However, genome information on this family is currently unavailable. In the present study, by employing Illumina sequencing, we performed the first de novo transcriptome analysis of the gills of the shrimp Rimicaris sp. from the hydrothermal vent in Desmos, Manus Basin. The analysis was conducted in a comparative manner with the shrimp taken directly from the vent (GR samples) and the shrimp that had been maintained for ten days under normal laboratory condition (mGR samples). Among the 128,938 unigenes identified, a large number of differentially expressed genes (DEGs) between the GR and mGR samples were detected, including 2365 and 1607 genes significantly upregulated and downregulated, respectively, in GR. The DEGs covered diverse functional categories. Most of the DEGs associated with immunity were downregulated in GR, while most of the DEGs associated with sulfur metabolism and detoxification were upregulated in GR. These results provide the first comprehensive transcriptomic resource for hydrothermal vent Rimicaris and revealed varied categories of genes likely involved in deep-sea survival.

Description of Algoriphagus iocasae sp. nov., isolated from deep-sea sediment.

In this paper, we report on the identification and species characterization of a novel Gram-staining-negative bacterium, strain S61T, isolated from the deep-sea sediment of the Okinawa Trough. Growth of strain S61T occurred at 4-37 °C (optimum, 25 °C), pH 6.0-10.0 (optimum, pH 7.0) and with 0-8 % (w/v) NaCl (optimum, 2 %). Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain S61T belonged to the genus Algoriphagus. The closest phylogenetic neighbours of strain S61T were Algoriphaguslutimaris S1-3T (98.1 %), Algoriphagushalophilus JC2051T (98.1 %), Algoriphaguschungangensis CAU 1002T (98.0 %) andAlgoriphagusaestuarii MDM-1T (97.4 %). The sequence similarities between strain S61T and other close members of the genus were below 97 %. The values of DNA-DNA relatedness between strain S61T and its closest relatives in the genus Algoriphagus were well below 70 %. The genomic DNA G+C content of strain S61T was 40.2 mol%. The major menaquinone was MK-7. The polar lipids contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and two unidentified phospholipids and one unidentified lipid. The major fatty acids were iso-C15 : 0 and summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c). Phylogenetic, physiological, biochemical, and morphological analyses suggested that this strain represents a novel species of the genus Algoriphagus, for which the name Algoriphagus iocasae sp. nov. is proposed with the type strain S61T (=KCTC 52359T=CCTCC AB 2015446T).

First Comparative Analysis of the Community Structures and Carbon Metabolic Pathways of the Bacteria Associated with Alvinocaris longirostris in a Hydrothermal Vent of Okinawa Trough.

Alvinocaris longirostris is a species of shrimp existing in the hydrothermal fields of Okinawa Trough. To date the structure and function of the microbial community associated with A. longirostris are essentially unknown. In this study, by employment of the techniques of high through-put sequencing and clone library construction and analysis, we compared for the first time the community structures and metabolic profiles of microbes associated with the gill and gut of A. longirostris in a hydrothermal field of Okinawa Trough. Fourteen phyla were detected in the gill and gut communities, of which 11 phyla were shared by both tissues. Proteobacteria made up a substantial proportion in both tissues, while Firmicutes was abundant only in gut. Although gill and gut communities were similar in bacterial diversities, the bacterial community structures in these two tissues were significantly different. Further, we discovered for the first time the existence in the gill and gut communities of A. longirostris the genes (cbbM and aclB) encoding the key enzymes of Calvin-Benson-Bassham (CBB) cycle and the reductive tricarboxylic acid (rTCA) cycle, and that both cbbM and aclB were significantly more abundant in gill than in gut. Taken together, these results provide the first evidence that at least two carbon fixation pathways are present in both the gill and the gut communities of A. longirostris, and that the communities in different tissues likely differ in autotrophic productivity.

Description of Domibacillus iocasae sp. nov., isolated from deep-sea sediment, and emended description of the genus Domibacillus.

In this study, we report a novel Gram-stain-positive bacterium, designated strain S6T, isolated from deep-sea sediment in Okinawa Trough. Growth of strain S6T occurred at 10-45 °C (optimum, 35 °C), at pH 6.0-11.0 (optimum, 8.0) and with 0-8 % (w/v) NaCl (optimum, 3 %). Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain S6T belonged to the genus Domibacillus. The closest phylogenetic neighbours of strain S6T were Domibacillus robiginosus WS 4628T ( = DSM 25058T) (98.6 %16S rRNA gene sequence similarity), D. indicus SD111T ( = DSM 28032T) (97.6 %), D. enclensis NIO-1016T ( = DSM 25145T) (97.6 %), and 'D. tundrae' PAMC 80007 ( = DSM 29572) (97.6 %). Levels of similarity between strain S6T and other close relatives were below 96 %. The levels of DNA-DNA relatedness between strain S6T and its closest relatives in the genus Domibacillus were well below 70 %. The genomic DNA G+C content of strain S6T was 44.4 mol%. The major menaquinone was MK-6. The polar lipids of strain S6T contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol mannosides, phosphatidylethanolamine and three unknown phospholipids. Strain S6T harboured meso-diaminopimelic acid as the diagnostic diamino acid and an A1γ type of peptidoglycan. The major cell-wall sugars of strain S6T were xylose and glucose. Phylogenetic, physiological, biochemical and morphological analyses suggest that this strain represents a novel species of the genus Domibacillus, for which the name Domibacillus iocasae sp. nov. is proposed. The type strain is S6T ( = DSM 29979T = CCTCC AB 2015183T). An emended description of the genus Domibacillus is also provided.

Characteristics of the cultivable bacteria from sediments associated with two deep-sea hydrothermal vents in Okinawa Trough.

In this study, different culture-dependent methods were used to examine the cultivable heterotrophic bacteria in the sediments associated with two deep-sea hydrothermal vents (named HV1 and HV2) located at Iheya Ridge and Iheya North in Okinawa Trough. The two vents differed in morphology, with HV1 exhibiting diffuse flows while HV2 being a black smoker with a chimney-like structure. A total of 213 isolates were identified by near full-length 16S rRNA gene sequence analysis. Of these isolates, 128 were from HV1 and 85 were from HV2. The bacterial community structures were, in large parts, similar between HV1 and HV2. Nevertheless, differences between HV1 and HV2 were observed in one phylum, one class, 4 orders, 10 families, and 20 genera. Bioactivity analysis revealed that 25 isolates belonging to 9 different genera exhibited extracellular protease activities, 21 isolates from 11 genera exhibited extracellular lipase activities, and 13 isolates of 8 genera displayed antimicrobial activities. This is the first observation of a large population of bacteria with extracellular bioactivities existing in deep-sea hydrothermal vents. Taken together, the results of this study provide new insights into the characteristics of the cultivable heterotrophic bacteria in deep-sea hydrothermal ecosystems.