深海热液区超嗜热古菌Thermococcus sp. TVG2的培养分离与鉴定

【目的】培养分离大西洋脊热液区的超嗜热古菌,为进一步认识该生态系统中的物种及其特点奠定基础。【方法】将大西洋脊热液区海水样品用YTSV培养基富集培养,选取其中富集效果最佳的TVG2培养物用减绝稀释法分离纯化。对所分离菌株进行形态、生理生化特征等分析,并通过分子生物学手段对其进行初步鉴定。【结果】菌株TVG2属于超嗜热厌氧球菌,直径约1.0 μm;生长温度范围50?88 °C,最适生长温度82 °C;生长pH范围为5.0?9.0,最适生长pH 6.5;生长NaCl浓度为1.0%?4.0% (质量体积比),最适生长浓度为2.5%;元素硫可显著提高菌株TVG2的生物量,但非生长必需;丙酮酸钠能显著促进该菌株生长,但葡萄糖对其生长则有抑制作用。根据其形态特征、生理生化特性及16S rRNA基因序列分析,确定菌株TVG2属于热球菌属。【结论】用YTSV培养基从大西洋脊热液区样品中分离获得超嗜热厌氧菌株TVG2,并确定其为Thermococcus属成员,命名为Thermococcus sp. TVG2。     

Expansion of the geographic distribution of a novel lineage of epsilon-Proteobacteria to a hydrothermal vent site on the Southern East Pacific Rise

The diversity associated with a microbial mat sample collected from a deep-sea hydrothermal vent on the Southern East Pacific Rise was determined using a molecular phylogenetic approach based on the comparison of sequences from the small subunit ribosomal RNA gene (16S rDNA). The DNA was extracted from the sample and the 16S rDNA was amplified by PCR. Sixteen different phylotypes were identified by restriction fragment length polymorphism analysis; four phylotypes were later identified as putative chimeras. Analysis of the 16S rDNA sequences placed all the phylotypes within the Proteobacteria. The majority of the sequences (98%) were most closely related to a new clade of epsilon-Proteobacteria that were initially identified from an in situ growth chamber deployed on a deep-sea hydrothermal vent on the Mid-Atlantic Ridge in 1995. The similarity between phylotypes identified from Atlantic and Pacific deep-sea hydrothermal vent sites indicates that this new clade of Proteobacteria may be endemic to and widely distributed among deep-sea hydrothermal vents.     

基于宏基因组学重建来自深海热液喷口的Epsilonproteobacteria基因组及其代谢分析

【背景】非致病性Epsilonproteobacteria广泛存在于全球各种不同的自然环境中,特别是一些极端生境如深海热液喷口,并且经常在微生物群落中作为优势物种被发现。然而,由于现阶段培养技术的限制,仅有为数不多的深海热液Epsilonproteobacteria被分离培养,极大限制了对其生理特征、代谢方式以及生态功能的深入认识。【目的】研究深海热液未培养Epsilonproteobacteria的进化地位、代谢潜能及其在原位生态系统中可能发挥的作用。【方法】基于宏基因组学Binning的方法,从采集自东太平洋海隆深海热液烟囱体样本中构建4个高质量的Epsilonproteobacteria基因组Bin225、Bin51、Bin54和Bin189,并进行了系统发育和代谢途径的分析。【结果】Bin189在系统发育树上相对独立于其他所有已知的Epsilonproteobacteria类群,而其余3个重构基因组都与Nitratiruptor sp. SB155-2具有较近的亲缘关系。在代谢潜能方面,所有的基因组除了都含有sqr硫氧化和rTCA碳固定途径的基因以外,也同时具有脂多糖输出转运子和多种分泌系统。Bin189显示出与其它基因组显著不同的代谢特征,其中还检测到与有机物和氨基酸转运相关的功能基因。而其他的3个基因组均具有完整的反硝化途径的功能基因,其中2个还具有Sox系统、氢化酶和鞭毛移动系统。【结论】Bin189可能是一种新发现的深海热液兼性化能营养型Epsilonproteobacteria,推测其余的3个类群能够利用硫化物和氢气作为能源进行化能自养生长。考虑到它们多样的代谢潜能,这些Epsilonproteobacteria类群很可能在深海热液微生物群落的形成发展和地球化学元素循环中发挥重要作用。     

The influence of ultramafic rocks on microbial communities at the Logatchev hydrothermal field, located 15 degrees N on the Mid-Atlantic Ridge

The ultramafic-hosted Logatchev hydrothermal field (LHF) on the Mid-Atlantic Ridge is characterized by high hydrogen and methane contents in the subseafloor, which support a specialized microbial community of phylogenetically diverse, hydrogen-oxidizing chemolithoautotrophs. We compared the prokaryotic communities of three sites located in the LHF and encountered a predominance of archaeal sequences affiliated with methanogenic Methanococcales at all three. However, the bacterial composition varied in accordance with differences in fluid chemistry between the three sites investigated. An increase in hydrogen seemed to coincide with the diversification of hydrogen-oxidizing bacteria. This might indicate that the host rock indirectly selects this specific group of bacteria. However, next to hydrogen availability further factors are evident (e.g. mixing of hot reduced hydrothermal fluids with cold oxygenated seawater), which have a significant impact on the distribution of microorganisms.     

一株深海热液口嗜热芽孢杆菌SY27F代谢产物活性的研究

【目的】对一株来源于深海热液口嗜热芽孢杆菌的次生代谢产物进行抑菌活性和抗肿瘤活性的初步研究。【方法】采用纸片法和微量肉汤稀释法检测嗜热芽孢杆菌SY27F次生代谢产物的抑菌活性,采用CCK-8法测定其次生代谢产物的抗肿瘤活性。【结果】抑菌实验表明,嗜热芽孢杆菌代谢产物对大肠杆菌、金黄色葡萄球菌均有抑菌作用,其最低抑菌浓度分别为1.56 mg/mL和3.13 mg/mL;细胞实验表明,其代谢产物对肿瘤细胞A549、HepG2、HeLa、MCF-7均有一定的抑制作用,其半致死浓度分别为0.390、0.451、0.704、1.105 mg/mL;与人肝肿瘤细胞(HepG2)相比,其对人正常肝细胞(L02)表现出良好的生物相容性。【结论】嗜热芽孢杆菌SY27F次生代谢产物具有一定的抑菌和抗肿瘤活性,可为寻找新型抑菌抗肿瘤活性物质提供优质资源。     

Effect of variation of environmental conditions on the microbial communities of deep-sea vent chimneys, cultured in a bioreactor

Both cultivation and molecular techniques were used to investigate the microbial diversity and dynamic of a deep-sea vent chimney. The enrichment cultures performed in a gas-lift bioreactor were inoculated with a black smoker chimney sample collected on TAG site on the mid-Atlantic ridge. To mimic as close as possible environmental conditions, the cultures were performed in oligotrophic medium with nitrogen, hydrogen and carbon dioxide (N₂/H₂/CO₂) gas sweeping. Also, the temperature was first settled at a temperature of 85°C and colloidal sulphur was added. Then, the temperature was lowered to 60°C and sulphur was omitted. Archaeal and bacterial diversity was studied in both culture and natural samples. Through 16S rRNA gene sequences analysis of the enrichment cultures microorganisms affiliated to Archeoglobales, Thermococcales were detected in both conditions while, Deferribacterales and Thermales were detected only at 65°C in the absence of sulphur. Single-stranded conformational polymorphism and quantitative PCR permit to study the microbial community dynamic during the two enrichment cultures. The effect of environmental changes (modification of culture conditions), i.e. temperature, medium composition, electron donors and acceptors availability were shown to affect the microbial community in culture, as this would happen in their environment. The effect of environmental changes, i.e. temperature and medium composition was shown to affect the microbial community in culture, as this could happen in their environment. The modification of culture conditions, such as temperature, organic matter concentration, electron donors and acceptors availability allowed to enrich different population of prokaryotes inhabiting hydrothermal chimneys.     

Bacterial and archaeal phylotypes associated with distinct mineralogical layers of a white smoker spire from a deep-sea hydrothermal vent site (9 degrees N, East Pacific Rise)

A diffusely venting chimney spire from the East Pacific Rise (9 degrees N) was analysed by petrographic thin sectioning and 16S rRNA gene cloning and sequencing in parallel, to correlate microbial community composition with mineralogy and inferred in situ conditions within the chimney mineral matrix. Both approaches indicated a zonation of the chimney spire into distinct microhabitats for different bacteria and archaea. The thermal gradient inferred from the mineral composition and porosity of the chimney was consistent with the distribution of bacterial and archaeal phylotypes in the chimney matrix. A novel phylogenetic lineage of euryarchaeota was found that co-occurred with clones related to cultured hyperthermophilic archaea. A few phylotypes related to mesophilic bacteria were found in the hot core of the chimney, indicating that seawater influx during retrieval and cooling of these highly porous structures can entrain microorganisms into chimney layers that are not their native habitat.     

Lebetimonas natsushimae sp. nov., a novel strictly anaerobic,moderately thermophilic chemoautotroph isolated from a deep-sea hydrothermal vent polychaete nest in the Mid-Okinawa Trough

A moderately thermophilic, strictly anaerobic, chemoautotrophic bacterium, designated strain HS1857^T, was isolated from a deep-sea hydrothermal vent at the Noho site in the Mid-Okinawa Trough. Strain HS1857^T grew between 35 and 63 °C (optimum 55 °C), in the presence of 10–55 g l^?1 NaCl (optimum 25 g l^?1), and pH 5.5–7.1 (optimum 6.4). Growth occurred with molecular hydrogen as the electron donor and elemental sulfur, nitrate, or selenate as the electron acceptors. Formate could serve as an alternative electron donor with nitrate as an electron acceptor. During growth with nitrate as the electron acceptor, strain HS1857^T produced ammonium and formed a biofilm. CO₂ was utilized as the sole carbon source. The G + C content of the genomic DNA was 33.2 mol%. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain HS1857^T is a member of the order Nautiliales, showing a sequence similarity of 95.0% with Lebetimonas acidiphila Pd55^T. The fatty acid composition was similar to that of L. acidiphila, which was dominated by C_18:0 (47.0%) and C_18:1 (23.7%). Based on the genomic, chemotaxonomic, phenotypic characteristics, the name Lebetimonas natsushimae sp. nov., is proposed. The type strain is HS1857^T (= NBRC 112478^T = DSM 104102^T).     

Draft genome sequence of Caminibacter mediatlanticus strain TB-2, an epsilonproteobacterium isolated from a deep-sea hydrothermal vent

Caminibacter mediatlanticus strain TB-2(T) [1], is a thermophilic, anaerobic, chemolithoautotrophic bacterium, isolated from the walls of an active deep-sea hydrothermal vent chimney on the Mid-Atlantic Ridge and the type strain of the species. C. mediatlanticus is a Gram-negative member of the Epsilonproteobacteria (order Nautiliales) that grows chemolithoautotrophically with H(2) as the energy source and CO(2) as the carbon source. Nitrate or sulfur is used as the terminal electron acceptor, with resulting production of ammonium and hydrogen sulfide, respectively. In view of the widespread distribution, importance and physiological characteristics of thermophilic Epsilonproteobacteria in deep-sea geothermal environments, it is likely that these organisms provide a relevant contribution to both primary productivity and the biogeochemical cycling of carbon, nitrogen and sulfur at hydrothermal vents. Here we report the main features of the genome of C. mediatlanticus strain TB-2(T).     

Isolation and characterization of Thiobacillus hydrothermalis sp. nov., a mesophilic obligately chemolithotrophic bacterium isolated from a deep-sea hydrothermal vent in Fiji Basin

A novel obligately chemolithotrophic Thiobacillus species isolated from a deep-sea hydrothermal vent is described. This organism grows lithoautotrophically on thiosulphate, tetrathionate, sulphide and sulphur which are oxidized to sulphate. The isolate is slightly halophilic and markedly halotolerant, showing optimum growth at pH 7.5 and at 35°C. The G+C content of the DNA is 67.1 mol%. The 16S rRNA sequence is distinct from any other Thiobacilli sequences. Phylogenetic analysis shows the organism to be a representative of the -group of proteobacteria and a specific relative of Thiobacillus neapolitanus. The ubiquinone is ubiquinone-8. These characters distinguish the isolate from any other Thiobacillus or Thiomicrospira species previously reported and is a new species described as Thiobacillus hydrothermalis. The type strain is isolate R3, DSM7121.     

Dissimilatory Iron [Fe(III)] Reduction by a Novel Fermentative,Piezophilic Bacterium Anoxybacter fermentans DY22613T Isolated from East Pacific Rise Hydrothermal Sulfides

Dissimilatory iron-reducing microorganisms play an important role in the biogeochemical cycle of iron and influence iron mineral formation and transformation. However, studies on microbial iron-reducing processes in deep-sea hydrothermal fields are limited. A novel piezophilic, thermophilic, anaerobic, fermentative iron-reducing bacteria of class Clostridia, named Anoxybacter fermentans DY22613^T, was isolated from East Pacific Rise hydrothermal sulfides. In this report, we examined its cell growth, fermentative metabolites, and biomineralization coupled with dissimilatory iron reduction. Both soluble ferric citrate (FC) and solid amorphous Fe(III) oxyhydroxide (FO) could promote cell growth of this strain, accompanied by increased peptone consumption. More acetate, butyrate, and CO₂ were produced than without adding FO or FC in the media. The highest yield of H₂ was observed in the Fe(III)-absent control. Coupled to fermentation, magnetite particles, and iron-sulfur complexes were respectively formed by the strain during FO and FC reduction. Under experimental conditions mimicking the pressure prevailing at the deep-sea habitat of DY22613^T (20?MPa), Fe(III)-reduction rates were enhanced resulting in relatively larger magnetite nanoparticles with more crystal faces. These results implied that the potential role of A. fermentans DY22613^T in situ in deep-sea hydrothermal sediments is coupling iron reduction and mineral transformation to fermentation of biomolecules. This bacterium likely contributes to the complex biogeochemical iron cycling in deep-sea hydrothermal fields.     

New species of Stygiopontius (Copepoda, Siphonostomatoida) from a deep–sea hydrothermal vent at the East Pacific Rise

Three dirivultid copepods, Stygiopontius puxillifer sp.n . and S. appositus sp.n., unusual in having pegs along the ventral edges of the cephalosome, and S. lurniger sp.n., distinguiahcd from congeners by the modificd thorn-like seta on the second segment of the maxilliped, arc dcscrihcd from a depth of 2616 m at 21°N in the eastern Pacific.     

Fine-scale vertical distribution of bacteria in the East Pacific deep-sea sediments determined via 16S rRNA gene T-RFLP and clone library analyses

Although the deep-sea sediments harbor diverse and novel bacteria with important ecological and environmental functions, a comprehensive view of their community characteristics is still lacking, considering the vast area and volume of the deep-sea sedimentary environments. Sediment bacteria vertical distribution and community structure were studied of the E272 site in the East Pacific Ocean with the molecular methods of 16S rRNA gene T-RFLP (terminal restriction fragment length polymorphism) and clone library analyses. Layered distribution of the bacterial assemblages was detected by both methods, indicating that the shallow sediments (40 cm in depth) harbored a diverse and distinct bacterial composition with fine-scale spatial heterogeneity. Substantial bacterial diversity was detected and nine major bacterial lineages were obtained, including Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Nitrospirae, Planctomycetes, Proteobacteria, and the candidate divisions OP8 and TM6. Three subdivisions of the Proteobacteria presented in our libraries, including the α-, γ- and δ-Proteobacteria. Most of our sequences have low similarity with known bacterial 16S rRNA genes, indicating that these sequences may represent as-yet-uncultivated novel bacteria. Most of our sequences were related to the GenBank nearest neighboring sequences retrieved from marine sediments, especially from deep-sea methane seep, gas hydrate or mud volcano environments. Several sequences were related to the sequences recovered from the deep-sea hydrothermal vent or basalt glasses-bearing sediments, indicating that our deep-sea sampling site might be influenced to certain degree by the nearby hydrothermal field of the East Pacific Rise at 13°N. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Tepidimonas arfidensis Sp. Nov., a Novel Gram-negative and thermophilic bacterium isolated from the bone marrow of a patient with leukemia in Korea

A Gram-negative bacillus, SMC-6271(T), which was isolated from the bone marrow of a patient with leukemia but could not be identified by a conventional microbiologic method, was characterized by a genotypic analysis of 16S rRNA gene. Sequences of the 16S rRNA gene revealed that this bacterium was closely related to Tepidimonas ignava and other slightly thermophilic isolates but diverged distinctly from them. Analyses of cellular fatty acid composition and performance of biochemical tests confirmed that this bacterium is a distinct species from the other Tepidimonas species. Based on the evaluated phenotypic and genotypic characteristics, it is proposed that SMC-6271T (=ABB 0301T =KCTC 12412T =JCM 13232T) should be classified as a new species, namely Tepidimonas arfidensis sp. nov.     

Population structure, history and gene flow in a group of closely related land snails: genetic variation in Partula from the Society Islands of the Pacific

Previous studies of Partula land snails from the Society Islands, French Polynesia, have shown that populations within species are highly differentiated in terms of their morphology, behaviour, ecology and molecular genetic variation. Despite this level of variability, differences between species are sometimes small, possibly reflecting the fact that reproductive isolation is not always complete and there exists the opportunity for genetic exchange between taxa through hybridization. The present study uses sequence data from a mitochondrial gene to further investigate genetic variation in Society Island Partula. Most populations are found in this study to be highly differentiated, but within individual species there seems to be no simple relationship either between genetic distance and geographical proximity, or between variation in mitochondria and that in allozymes or morphological characteristics. Among species there appears to be no simple correlation between degrees of reproductive isolation and genetic relatedness according to mitochondrial DNA. The results suggest that past events as well as ongoing drift and selection may have been important in affecting patterns of variation. Similarities among species at specific localities suggest that there must have been some genetic exchange in the past, although this may not necessarily reflect ongoing rates of hybridization. The discrepancy between results for different markers probably reflects the differential effects of drift and selection on mitochondrial and nuclear genes.     

Two new species of hydrothermal vent crabs of the genus Bythograea from the southern East Pacific Rise and from the Galapagos Rift (Crustacea Decapoda Brachyura Bythograeidae)

Two new species of Bythograea Williams, B. vrijenhoeki n. sp. and B. galapagensis n. sp., are described based on morphology and mitochondrial DNA comparisons. B. vrijenhoeki was collected on the southern East Pacific Rise, south of the Easter Microplate and B. galapagensis from the Galapagos Rift, from where B. intermedia de Saint Laurent was also described. Our analyses indicate that B. vrijenhoeki is the sister species of B. laubieri Guinot and Segonzac, and B. galapagensis is the sister species of B. thermydron Williams. Bythograea is now composed of six described species, all endemic to hydrothermal vents along the East Pacific Rise and from the Galapagos Rift.     

Description of a new family,new genus,and two new species of deep‐sea Forcipulatacea (Asteroidea), including the first known sea star from hydrothermal vent habitats

Based on a phylogenetic analysis of undescribed taxa within the Forcipulatacea, a new family of deep‐sea forcipulatacean starfishes, Paulasteriidae fam. nov., is described from deep‐sea settings. Paulasterias tyleri gen. et sp. nov. was observed at recently documented hydrothermal vents on the East Scotia Ridge, Southern Ocean. A second species, Paulasterias mcclaini gen. et sp. nov. was observed in deep‐sea settings in the North Pacific, more distant from hydrothermal vents. Both species are multi‐armed (with between six and eight arms), with a fleshy body wall, and a poorly developed or absent adoral carina. Here, we include discussions of pedicellariae morphology, feeding biology, and classification. © 2015 The Linnean Society of London