The phylogenetic diversity was determined for a microbial community obtained from an in situ growth chamber placed on a deep-sea hydrothermal vent on the Mid-Atlantic Ridge (23°22 N, 44°57 W). The chamber was deployed for 5 days, and the temperature within the chamber gradually decreased from 70 to 20°C. Upon retrieval of the chamber, the DNA was extracted and the small-subunit rRNA genes (16S rDNA) were amplified by PCR using primers specific for the Archaea or Bacteria domain and cloned. Unique rDNA sequences were identified by restriction fragment length polymorphisms, and 38 different archaeal and bacterial phylotypes were identified from the 85 clones screened. The majority of the archaeal sequences were affiliated with the Thermococcales (71%) and Archaeoglobales (22%) orders. A sequence belonging to the Thermoplasmales confirms that thermoacidophiles may have escaped enrichment culturing attempts of deep-sea hydrothermal vent samples. Additional sequences that represented deeply rooted lineages in the low-temperature eurarchaeal (marine group II) and crenarchaeal clades were obtained. The majority of the bacterial sequences obtained were restricted to the Aquificales (18%), the subclass of the Proteobacteria (-Proteobacteria) (40%), and the genus Desulfurobacterium (25%). Most of the clones (28%) were confined to a monophyletic clade within the -Proteobacteria with no known close relatives. The prevalence of clones related to thermophilic microbes that use hydrogen as an electron donor and sulfur compounds (S 0 , SO 4 , thiosulfate) indicates the importance of hydrogen oxidation and sulfur metabolism at deep-sea hydrothermal vents. The presence of sequences that are related to sequences from hyperthermophiles, moderate thermophiles, and mesophiles suggests that the diversity obtained from this analysis may reflect the microbial succession that occurred in response to the shift in temperature and possible associated changes in the chemistry of the hydrothermal fluid.Despite the diverse geochemical and temperature gradients that are prevalent at deep-sea hydrothermal vents, relatively little is known about the diversity and ecology of the free-living microbial communities that occupy these fluctuating high-temperature niches. The majority of microbial studies at deep-sea vents have relied on enrichment culturing techniques for growing hyperthermophiles (17, 19) and mesophiles (14, 37). Some biogeochemical studies have elucidated the role of microbial populations such as the sulfur-oxidizing bacteria (45, 53), methane-oxidizing mesophiles (10), and endosymbionts (8, 9) in this unusual ecosystem.Recently, molecular phylogenetic approaches studying the small-subunit rRNA gene (16S rDNA) have been used to examine the diversity of different hydrothermal communities. As has been reported for other environments (for example, see references 2 and 28), this approach revealed a plethora of novel diversity, previously unknown to deep-sea hydrothermal vents (24,35,36,50). Novel bacterial and archaeal sequences were rep...

Data from ice 3590 meters below Vostok Station indicate that the ice was accreted from liquid water associated with Lake Vostok. Microbes were observed at concentrations ranging from 2.8 x 10(3) to 3.6 x 10(4) cells per milliliter; no biological incorporation of selected organic substrates or bicarbonate was detected. Bacterial 16S ribosomal DNA genes revealed low diversity in the gene population. The phylotypes were closely related to extant members of the alpha- and beta-Proteobacteria and the Actinomycetes. Extrapolation of the data from accretion ice to Lake Vostok implies that Lake Vostok may support a microbial population, despite more than 10(6) years of isolation from the atmosphere.

The standard dilution technique can provide unbiased estimates of phytoplankton growth and microzooplankton grazing rates only when certain restrictive assumptions are met. The most important of these assumptions -that grazing impact varies in direct proportion to the dilution of grazer population density -can be easily violated when clearance rate of individual grazers and/or growth response of the grazer population vary significantly with food concentration over the course of the incubation. We have developed a modified protocol which now allows the dilution technique to be applied unambiguously, even when its original assumptions may be violated. The new protocol uses flow-cytometry measured disappearance of fluorescently labeled tracer cells (FLB or FLA) as an internal measure of 'relative grazing activity' in each dilution treatment. Coefficients of phytoplankton growth and mortality rates are determined from Model 11 regression analyses of 'net growth' versus 'relative grazing', rather than the usual Model I regressions of 'net growth' versus 'dilution factor'. Tests of this hybrid experimental design in the central equatorial Pacific during an EQPAC cruise in August 1992 gave results essentially identical to the standard dilution interpretation.

To evaluate the effects of local fluid geochemistry on microbial communities associated with active hydrothermal vent deposits, we examined the archaeal and bacterial communities of 12 samples collected from two very different vent fields: the basalt-hosted Lucky Strike (37°17'N, 32°16.3'W, depth 1600-1750 m) and the ultramafic-hosted Rainbow (36°13'N, 33°54.1'W, depth 2270-2330 m) vent fields along the Mid-Atlantic Ridge (MAR). Using multiplexed barcoded pyrosequencing of the variable region 4 (V4) of the 16S rRNA genes, we show statistically significant differences between the archaeal and bacterial communities associated with the different vent fields. Quantitative polymerase chain reaction (qPCR) assays of the functional gene diagnostic for methanogenesis (mcrA), as well as geochemical modelling to predict pore fluid chemistries within the deposits, support the pyrosequencing observations. Collectively, these results show that the less reduced, hydrogen-poor fluids at Lucky Strike limit colonization by strict anaerobes such as methanogens, and allow for hyperthermophilic microaerophiles, like Aeropyrum. In contrast, the hydrogen-rich reducing vent fluids at the ultramafic-influenced Rainbow vent field support the prevalence of methanogens and other hydrogen-oxidizing thermophiles at this site. These results demonstrate that biogeographical patterns of hydrothermal vent microorganisms are shaped in part by large scale geological and geochemical processes.

Deep-sea hydrothermal vents play an important role in global biogeochemicalcycles, providing biological oases at the seafloor that are supported by the thermal and chemical flux from the Earth's interior. As hot, acidic and reduced hydrothermal fluids mix with cold, alkaline and oxygenated seawater, minerals precipitate to form porous sulphide-sulphate deposits. These structures provide microhabitats for a diversity of prokaryotes that exploit the geochemical and physical gradients in this dynamic ecosystem 1 Table 2, Supplementary Fig. S1). The DHVE2 were identified in 36 samples and represented up to 10 and 15% of the archaeal population at EPR and ELSC, respectively (as determined by quantitative PCR; Table 1). Generally, the occurrence of DHVE2 appeared to be associated with the fine-grained white elemental sulphur, S 8 , (identified in three different samples by X-ray diffraction, Supplementary Methods) or iron oxyhydroxide (X-ray amorphous -identified based on orange color and texture, Supplementary Table 3) deposits that often coat the outside of mature actively venting sulphide deposits ( Supplementary Fig. S2). Additionally, due to the high guanine and cytosine content of the 16S rRNA sequence and its occasional co-occurrence with thermophiles such as Thermococcus, it has been suggested that the DHVE2 are thermophiles, perhaps heterotrophs whose growth may be stimulated by sulphur 1,16 . A DHVE2 genome fragment from a metagenome library contained a thermostable DNA polymerase, which was further suggestive of the thermophilic nature of this lineage 17 . However, all attempts to culture this group using standard media and conditions to select for sulphate reducers, methanogens or fermenters at 60, 80 and 90°C and around pH 6.5 have been unsuccessful 5 .Based on the predictions that conditions within many sulphide deposits should be acidic, and due to the presence of novel sequences most closely related to the acidophiles, Thermoplasma, Picrophilus and Ferroplasma (Fig. 1 The cells are pleiomorphic to spherical, about 0.6-1 µm in diameter and are motile with a single flagellum (Fig. 2a, b). Some flagella have their proximal end encased in an unusual complex periodic sheath (Fig. 2c). Like many other Archaea (e.g.Methanococcus and Sulfolobus), they are enveloped by a plasma membrane and a single S-layer (Fig. 2a, e). The S-layer, similar to that of Picrophilus oshimae 23 , is thick (~40nm), with a presumed tetragonal (p4) lattice and resembles a delicate bridal veil bordering the cells (Fig. 2a). Although S-layers are quasi-crystalline this S-layer bends into small highly curved structures (Fig. 2d, vesicles). These vesicles bud from the cell partitioning small quantities of cytoplasm that could then anneal to adjacent cells. This is a process that is common amongst their Gram-negative bacterial counterparts 24 . The ability of DHVE2's S-layer to bend into such high curvature structures as these vesicles,suggests that the bonding forces between S-layer subunits are weak or transient. This is unusual 25 ,...