Isolation and phylogenetic diversity of members of previously uncultivated epsilon-Proteobacteria in deep-sea hydrothermal fields

We report the successful cultivation and partial characterization of novel members of epsilon-Proteobacteria, which have long been recognized solely as genetic signatures of small subunit ribosomal RNA genes (rDNA) from a variety of habitats occurring in deep-sea hydrothermal fields. A newly designed microhabitat designated 'in situ colonization system' was used for enrichment. Based on phylogenetic analysis of the rDNA of the isolates, most of these represent the first cultivated members harboring previously uncultivated phylotypes classified into the Uncultivated epsilon-Proteobacteria Groups A, B, F and G, as well as some novel members of Group D. Preliminary characterization of the isolates indicates that all are mesophilic or thermophilic chemolithoautotrophs using H(2) or reduced sulfur compounds (elemental sulfur or thiosulfate) as an electron donor and O(2), nitrate or elemental sulfur as an electron acceptor. The successful cultivation will enable the subsequent characterization of physiological properties and ecological impacts of a diversity of epsilon-Proteobacteria in the deep-sea hydrothermal environments.     

Thermococcus siculi sp. nov., a novel hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent at the Mid-Okinawa Trough

A novel coccoid-shaped, hyperthermophilic, anaerobic archaeon, strain RG-20, was isolated from a deep-sea hydrothermal vent fluid sample taken at 1394-m depth at the Mid-Okinawa Trough (27°32.7′N, 126°58.5′E). Cells of this isolate occur singly or in pairs and are about 0.8 to 2 μm in diameter. Growth was observed at temperatures between 50° and 93°C, with an optimum at 85°C. The pH range for growth is 5.0–9.0, with an optimum around 7.0. Strain RG-20 requires 1%–4% of NaCl for growth, and cell lysis occurs at concentrations below 1%. The newly isolated strain grows preferentially in the presence of elemental sulfur on proteinaceous substrates such as yeast extract, peptone, or tryptone, and no growth was observed on carbohydrates, carboxylic acids, alcohols, or lipids. This microorganism is resistant to streptomycin, chloramphenicol, ampicillin, and kanamycin at concentrations up to 150 μg/ml, but is susceptible to rifampicin. Analysis of the hydrolyzed core lipids by thin-layer chromatography (TLC) revealed the presence of archaeol and caldarchaeol. The mol% G+C content of the DNA is 55.8. Partial sequencing of the 16S rDNA indicates that strain RG-20 belongs to the genus Thermococcus. Considering these data and on the basis of the results from DNA-DNA hybridization studies, we propose that this strain should be classified as a new species named Thermococcus siculi (si′cu.li. L. gen. n. siculi, of the deep-sea [siculum, deep-sea in literature of Ovid], referring to the location of the sample site, a deep-sea hydrothermal vent). The type strain is isolate RG-20 (DSM No. 12349). Received: May 11, 1998 / Accepted: July 24, 1998     

Molecular characteristics of the tubeworm, Ridgeia piscesae, from the deep-sea hydrothermal vent

Ridgeia piscesae, living around the extremely harsh hydrothermal vent in deep sea, is an ideal model for studying the adaptative mechanism to extreme environment. For insights of its molecular characteristics, a cDNA library of R. piscesae was constructed. A total of 879 expressed sequence tags (ESTs) were sequenced and 199 genes were identified for the first time. They were found to be involved in basal metabolism, adaptation and defense, or signal transduction. Among them, we found 23 various chitin-binding proteins, which are the major component of the chitinous tube that prevents the tubeworms from predators and surrounding extreme environment. Additionally, high polymorphism also exists in other genes, such as myohemerythrin, lysozyme. The gene-expression profile might help to further understand the molecular basis of tubeworm physiology. It will also lay a good foundation for functional studies on the adaptation to extreme environments.     

Halomonas profundus sp. nov., a new PHA-producing bacterium isolated from a deep-sea hydrothermal vent shrimp

Aims:  The objective of the present work was to describe a new deep-sea, aerobic, mesophilic and heterotrophic bacterium, referenced as strain AT1214, able to produce polyhydroxyalkanoates (PHAs) under laboratory conditions. This bacterium was isolated from a shrimp collected nearby a hydrothermal vent located on the Mid-Atlantic Ridge. Methods and Results:  This micro-organism, on the basis of the phenotypical features and genotypic investigations, can be clearly assigned to the Halomonas genus and the name of Halomonas profundus is proposed. Optimal growth occurred between 32 and 37°C at a pH between 8 and 9 and at ionic strength between 20 and 30 g l⁻¹ of sea salts. The G + C content of DNA was 58·6%. This bacterium produced PHAs of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from different carbon sources. Conclusions:  The bacterium H. profundus produces PHA of 3HB and 3HV monomers from different carbon sources. Significance and Impact of the Study:  PHAs share physical and material properties that suggest them for application in various areas, and are considered as an alternative to nonbiodegradable plastics produced from fossil oils. In this study, we describe a new bacteria isolated from a deep-sea hydrothermal vent with the capability to produce polyesters of biotechnological interest.     

Shell nacre ultrastructure and depressurisation dissolution in the deep-sea hydrothermal vent mussel Bathymodiolus azoricus

This study describes the micro-morphological features of the shell nacre in the vent mytilid Bathymodiolus azoricus collected along a bathymetric gradient of deep-sea hydrothermal vents of the mid-Atlantic ridge (MAR). Pressure-dependent crystallisation patterns were detected in animals subjected to post-capture hydrostatic simulations. We provide evidence for the following: (1) shell micro morphology in B. azoricus is similar to that of several vent and cold-seep species, but the prismatic shell layers may vary among bathymodiolids; (2) nacre micro-morphology of mussels from three vent sites of the MAR did not differ significantly; minor differences do not appear to be related to hydrostatic pressure, but rather to calcium ion availability; (3) decompression stress may cause drop off in pH of the pallial fluid that damages nascent crystals, and in a more advanced phase, the aragonite tablets as well as the continuous layer of mature nacre; and (4) adverse effects of decompression on calcium salt deposition in shells was diminished by re-pressurisation of specimens. The implications of the putative influence of hydrostatic pressure on biomineralisation processes in molluscs are discussed. An erratum to this article can be found at     

Isolation and characterization of Thermus thermophilus Gy1211 from a deep-sea hydrothermal vent

We examined a single, non-spore-forming, aerobic, thermophilic strain that was isolated from a deep-sea hydrothermal vent in the Guaymas Basin at a depth of 2000 m and initially placed in a phenetic group with Thermus scotoductus (X-1). We identified this deep-sea isolate as a new strain belonging to Thermus thermophilus using several parameters. DNA–DNA hybridization under stringent conditions showed 74% similarity between the deep-sea isolate and T. thermophilus HB-8^T (T = type strain). Phenotypic characteristics, such as the utilization of carbon sources, hydrolysis of different compounds, and antibiotic sensitivity were identical in the two strains. The polar lipids composition showed that strain Gy1211 belonged to the genus Thermus. The fatty acids composition indicated that this strain was related to the marine T. thermophilus strain isolated from the Azores. The new isolate T. thermophilus strain Gy1211 grew optimally at 75°C, pH 8.0, and 2% NaCl. A hydrostatic pressure of 20 MPa, similar to the in situ hydrostatic pressure of the deep-sea vent from which the strain was isolated, had no effect on growth. Strain HB-8^T, however, showed slower growth under these conditions. Received: November 26, 1997 / Accepted: May 20, 1999     

<Emphasis Type="Italic">Pyrolycus manusanus</Emphasis>, a new genus and species of deep-sea eelpout from a hydrothermal vent field in the Manus Basin,Papua New Guinea (Zoarcidae,Lycodinae)

A new genus and species of deep-sea zoarcid fish, Pyrolycus manusanus, is described on the basis of eight specimens collected from a hydrothermal vent field in the Manus Basin, Papua New Guinea, at depths of 1623–1971 m. This genus is distinguished from other scaleless eelpout genera in the subfamily Lycodinae by the following combination of characters: six suborbital bones; six to seven suborbital pores; flesh gelatinous; gill slit large, extending ventrally beyond pectoral fin base; three postorbital pores; single occipital pore; two to three pelvic fin rays; palatopterygoid series weak; vomer and palatine teeth present; pseudobranch, scales, lateral line absent; pyloric caeca present; 16–17 pectoral fin rays; 22–23 + 56–59 = 78–81 vertebrae. Received: August 5, 1999 / Revised: July 5, 2001 / Accepted: August 26, 2001     

西藏羊八井废弃热井沉积物中的真核微生物多样性

【目的】研究西藏羊八井废弃热井沉积物中的真核微生物多样性。【方法】采用ITS-rRNA基因分析法构建了沉积物中真核微生物的ITS-rRNA基因文库, 随机抽取文库中的克隆子进行扩增rDNA限制性酶切片段分析(ARDRA), 并对ITS-rRNA基因进行了系统发育分析。【结果】沉积物中主要的真核微生物有子囊菌、壶菌、担子菌、丝足虫、后生动物和非培养真菌。其中壶菌Chytridiales sp.、Physoderma maydis和Powellomyces sp., 子囊菌的假丝酵母(Candida parapsilosis)、鲁氏接合酵母(Zygosaccharomyces rouxii)和短梗霉(Aureobasidium pullulans)以及后生动物的轮虫(Lecane bulla)均未有报导存在于热泉或热井中。【结论】羊八井热井沉积物中具有丰富的真核微生物多样性。     

Prolonged recovery time after eruptive disturbance of a deep-sea hydrothermal vent community

Deep-sea hydrothermal vents are associated with seafloor tectonic and magmatic activity, and the communities living there are subject to disturbance. Eruptions can be frequent and catastrophic, raising questions about how these communities persist and maintain regional biodiversity. Prior studies of frequently disturbed vents have led to suggestions that faunal recovery can occur within 2–4 years. We use an unprecedented long-term (11-year) series of colonization data following a catastrophic 2006 seafloor eruption on the East Pacific Rise to show that faunal successional changes continue beyond a decade following the disturbance. Species composition at nine months post-eruption was conspicuously different than the pre-eruption ‘baseline'' state, which had been characterized in 1998 (85 months after disturbance by the previous 1991 eruption). By 96 months post-eruption, species composition was approaching the pre-eruption state, but continued to change up through to the end of our measurements at 135 months, indicating that the ‘baseline'' state was not a climax community. The strong variation observed in species composition across environmental gradients and successional stages highlights the importance of long-term, distributed sampling in order to understand the consequences of disturbance for maintenance of a diverse regional species pool. This perspective is critical for characterizing the resilience of vent species to both natural disturbance and human impacts such as deep-sea mining.     

Microbial diversity of a sulphide spire located in the Edmond deep-sea hydrothermal vent field on the Central Indian Ridge

A culture‐independent molecular phylogenetic survey was carried out for a bacterial and archaeal community of a mineralized crust coating a sulphide spire, which was collected from the Edmond vent field (23° S, 69° E, 3300 m depth) on the Central Indian Ridge. Small‐subunit rRNA genes (16S rDNA) were amplified from environmental DNA by PCR utilizing Bacteria‐specific, and Archaea‐specific 16S rDNA primers. PCR products were cloned and 26 bacterial and nine archaeal unique sequence types (phylotypes) were identified from 150 clones analysed by restriction fragment length polymorphism, representing eight and four distinct lineages, respectively. The majority (>90%) of the bacterial phylotypes group with the ?‐Proteobacteria and confirms the global prevalence of ?‐Proteobacteria in deep‐sea hydrothermal environments. Among the ?‐Proteobacteria, >40% of the phylotypes were closely related to the recently isolated deep‐sea vent thermophilic chemolithoautotrophic sulphur‐reducer, Nautilia lithotrophica. A single bacterial sequence was nearly identical (99% similarity) to the thermophilic hydrogen‐oxidizing Hydrogenobacter thermolithotrophum, and is the first report of Hydrogenobacter at deep‐sea hydrothermal vents. A majority (97%) of the archaeal phylotypes grouped with the ‘Deep‐sea Hydrothermal Vent Euryarchaeotal Group’, a phylogenetic lineage of uncultured Archaea that have only been reported from other deep‐sea hydrothermal vents on the Mid‐Atlantic Ridge, East Pacific Rise, Juan de Fuca Ridge, Isu–Ogasawara Arc, Okinawa Trough and the Manus Basin. A single sequence was closely related to the hyperthermophilic sulphur‐reducing Thermococcales frequently found in diverse deep‐sea vent environments. Scanning electron micrographs of the mineralized crust reveal abundant filamentous, rod and coccoidal forms encased in sulphur and sulphide mineral precipitate, suggesting that the thermophilic chemolithoautorophs and sulphide‐producing heterotrophs may influence the architecture and sulphur cycling of the sulphide spire.     

Genetic diversity and connectivity of deep‐sea hydrothermal vent metapopulations

Deep‐sea hydrothermal vents provide ephemeral habitats for animal communities that depend on chemosynthetic primary production. Sporadic volcanic and tectonic events destroy local vent fields and create new ones. Ongoing dispersal and cycles of extirpation and colonization affect the levels and distribution of genetic diversity in vent metapopulations. Several species exhibit evidence for stepping‐stone dispersal along relatively linear, oceanic, ridge axes. Other species exhibit very high rates of gene flow, although natural barriers associated with variation in depth, deep‐ocean currents, and lateral offsets of ridge axes often subdivide populations. Various degrees of impedance to dispersal across such boundaries are products of species‐specific life histories and behaviours. Though unrelated to the size of a species range, levels of genetic diversity appear to correspond with the number of active vent localities that a species occupies within its range. Pioneer species that rapidly colonize nascent vents tend to be less subdivided and more diverse genetically than species that are slow to establish colonies at vents. Understanding the diversity and connectivity of vent metapopulations provides essential information for designing deep‐sea preserves in regions that are under consideration for submarine mining of precious metals.     

Phylogenetic relationships ofThiomicrospira species and their identification in deep-sea hydrothermal vent samples by denaturing gradient gel electrophoresis of 16S rDNA fragments

Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA fragments was used to explore the genetic diversity of hydrothermal vent microbial communities, specifically to determine the importance of sulfur-oxidizing bacteria therein. DGGE analysis of two different hydrothermal vent samples revealed one PCR band for one sample and three PCR bands for the other sample, which probably correspond to the dominant bacterial populations in these communities. Three of the four 16S rDNA fragments were sequenced. By comparison with 16S rRNA sequences of the Ribosomal Database Project, two of the DGGE-separated fragments were assigned to the genusThiomicrospira. To identify these ‘phylotypes’ in more detail, a phylogenetic framework was created by determining the nearly complete 16S rRNA gene sequence (approx. 1500 nucleotides) from three describedThiomicrospira species, viz.,Tms. crunogena, Tms. pelophila, Tms. denitrificans, and from a new isolate,Thiomicrospira sp. strain MA2-6. AllThiomicrospira species exceptTms. denitrificans formed a monophyletic group within the gamma subdivision of the Proteobacteria.Tms. denitrificans was assigned as a member of the epsilon subdivision and was distantly affiliated withThiovulum, another sulfur-oxidizing bacterium. Sequences of two dominant 16S rDNA fragments obtained by DGGE analysis fell into the gamma subdivisionThiomicrospira. The sequence of one fragment was in all comparable positions identical to the 16S rRNA sequence ofTms. crunogena. Identifying a dominant molecular isolate asTms. crunogena indicates that this species is a dominant community member of hydrothermal vent sites. Another ‘phylotype’ represented a newThiomicrospira species, phylogenetically in an intermediate position betweenTms. crunogena andTms. pelophila. The third ‘phylotype’ was identified as aDesulfovibrio, indicating that sulfate-reducing bacteria, as sources of sulfide, may complement sulfur- and sulfide-oxidizing bacteria ecologically in these sulfide-producing hydrothermal vents.     

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。     

Development and characterization of 12 microsatellite markers from the deep-sea hydrothermal vent siboglinid Riftia pachyptila

Ecological processes at deep‐sea hydrothermal vents on fast‐spreading mid‐ocean ridges are punctuated by frequent physical disturbance, often accompanied by a high occurrence of population turnover. To persist through local extinction events, sessile invertebrate species living in these geologically and chemically dynamic habitats depend on larval dispersal. We characterized 12 polymorphic microsatellite loci from one such species, the siboglinid tubeworm Riftia pachyptila. All loci conformed to Hardy–Weinberg expectations without linkage (mean H_E = 0.9405, mean N_A = 20.25). These microsatellites are being employed in the investigation of spatial and temporal population genetic structure in the eastern Pacific Ocean.     

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.     

Thermosipho japonicus sp. nov., an extremely thermophilic bacterium isolated from a deep-sea hydrothermal vent in Japan

A novel barophilic, extremely thermophilic bacterium was isolated from a deep-sea hydrothermal vent chimney at the Iheya Basin, in the Okinawa area, Japan. The cells were found to be rod shaped and surrounded by a sheath-like outer structure; the organism did not possess flagella and was not motile. Growth was observed between 45° and 80°C (optimum, 72°C, 45 min doubling time), pH 5.3 and 9.3 (optimum, pH 7.2–7.6), 6.6 and 79 g/l sea salts (optimum, 40 g/l), and 0.1 and 60 MPa (optimum, 20 MPa). Strain IHB1 was found to be a strictly anaerobic chemoorganotroph capable of utilizing yeast extract and proteinaceous substrates such as peptone and tryptone. Elemental sulfur or thiosulfate acted as electron acceptors improving growth. The isolate was able to utilize casein as a sole carbon and energy source in the presence of thiosulfate. The G + C content of the genomic DNA was 31.4 mol%. Phylogenetic analysis based on 16S rDNA sequences and DNA–DNA hybridization analysis indicated that the isolate is closely related to Thermosipho africanus; however, it represents a species distinct from the previously described members of the genus Thermosipho. On the basis of the physiological and molecular properties, we propose that the new isolate represents a new species, which we name Thermosipho japonicus sp. nov. (type strain: IHB1; JCM10495). Received: May 26, 1999 / Accepted: August 7, 1999