Microbial Communities of Deep Marine Subsurface Sediments: Molecular and Cultivation Surveys

Recent molecular analyses show that microbial communities of deep marine sediments harbor members of distinct, uncultured bacterial and archaeal lineages, in addition to Gram-positive bacteria and Proteobacteria that are detected by cultivation surveys. Several of these subsurface lineages show cosmopolitan occurrence patterns; they can be found in cold marine sediments and also in hydrothermal habitats, suggesting a continuous deep subsurface and hydrothermal biosphere with shared microbiota. The physiologies and activities of these uncultured subsurface lineages remain to be explored by innovative combinations of genomic and biogeochemical approaches.     

暗黑菌(Atribacteria)的环境分布和功能特征

暗黑菌门包括OP9和JS1两大分支,成员大多为未培养微生物,在自然环境中广泛分布,并且在部分环境如厌氧海洋沉积物、地热环境以及油藏中为优势微生物。基于基因组信息的研究表明,暗黑菌为严格的厌氧微生物,同时具有降解糖类、小分子酸、短链正构烷烃的能力,在地球碳循环过程中可能扮演着重要的角色。然而,由于缺乏相应代表性的纯培养菌株,对于暗黑菌的生理生化功能推测仍有待验证。文章概述了暗黑菌的发现及发展历史,分析了其环境分布和多样性,简述了目前提出的三种代谢方式,提出了未来暗黑菌的研究发展方向。     

Mineralization of Alvinella polychaete tubes at hydrothermal vents

Alvinellid polychaete worms form multilayered organic tubes in the hottest and most rapidly growing areas of deep‐sea hydrothermal vent chimneys. Over short periods of time, these tubes can become entirely mineralized within this environment. Documenting the nature of this process in terms of the stages of mineralization, as well as the mineral textures and end products that result, is essential for our understanding of the fossilization of polychaetes at hydrothermal vents. Here, we report in detail the full mineralization of Alvinella spp. tubes collected from the East Pacific Rise, determined through the use of a wide range of imaging and analytical techniques. We propose a new model for tube mineralization, whereby mineralization begins as templating of tube layer and sublayer surfaces and results in fully mineralized tubes comprised of multiple concentric, colloform, pyrite bands. Silica appeared to preserve organic tube layers in some samples. Fine‐scale features such as protein fibres, extracellular polymeric substances and two types of filamentous microbial colonies were also found to be well preserved within a subset of the tubes. The fully mineralized Alvinella spp. tubes do not closely resemble known ancient hydrothermal vent tube fossils, corroborating molecular evidence suggesting that the alvinellids are a relatively recent polychaete lineage. We also compare pyrite and silica preservation of organic tissues within hydrothermal vents to soft tissue preservation in sediments and hot springs.     

Cosmopolitanism of microbial eukaryotes in the global deep seas

Deep sea environments cover more than 65% of the earth’s surface and fulfil a range of ecosystem functions, yet they are also amongst the least known habitats on earth. Whilst the discovery of key geological processes, combined with technological developments, has focused interest onto geologically active areas such as hydrothermal vents, most abyssal biodiversity remains to be discovered ( Danovaro et al. 2010 ). However, as for terrestrial reservoirs of biodiversity, the world’s largest biome is under threat from anthropogenic activities ranging from environmental change to the exploitation of minerals and rare‐earth elements ( Kato et al. 2011 ). It is therefore important to understand the magnitude, nature and composition of deep sea biological communities to inform us of levels of local adaptation, functionality and resilience with respect to future environmental perturbation. In this issue of Molecular Ecology, Bik et al. utilize 454 Roche metagenetic environmental sequencing to assess microbial metazoan community composition and phylogenetic identity across deep sea depth gradients and between ocean basins. The analyses suggest that although the majority of microbial eukaryotic taxa are regionally restricted, a small percentage might maintain cosmopolitan deep sea distributions, and an even smaller fraction appear to be eurybathic (live across depth gradients).     

Novel Cyanobacterial Diversity Found in Costa Rican Thermal Springs Associated with Rincon de la Vieja and Miravalles Volcanoes: A Polyphasic Approach

Central America is one of the most important biodiversity hot spots in the world, and Costa Rican microbial communities from thermal springs are the best characterized in the isthmus. Miravalles is an inactive quaternary stratovolcano, and the Rincón de la Vieja is a unique active volcano, in whose slopes diverse hydrothermal springs, such as Las Lilas, are located. These springs harbor extensive microbial mats, whose diversity has been studied. Based on their importance as primary producers, in this study we focused on cultured cyanobacterial diversity from two geothermal environments of northern Costa Rica. Several cultural, molecular and taxonomic techniques were employed to maximize the results of a polyphasic approach. Sample collection sites were physicochemically described, and strains were isolated and characterized by light and electron microscopy. Phylogenetic analysis was performed using 16S rRNA gene sequences and amplified ribosomal DNA restriction analysis (ARDRA). Fifty‐six phylotypes were isolated and classified into 21 morphotypes and identified in 14 genera, some of them might be new species within these genera. Furthermore, according to phylogenetic analysis, there are three possible new genera in our collection. Miravalles and Las Lilas thermal springs are reservoirs of novel phylogeographic lineages of phototrophic microorganisms. This study is the first report of strains that belong to the genera Gloeocapsa, Stanieria, Microseira, Klisinema and Oculatella isolated from thermal springs and growing at temperatures above 50°C. We also obtained isolates assigned to Synechococcus, Leptolyngbya spp., and Fischerella, which are considered typical strains in these environments.     

Sediment cooling triggers germination and sulfate reduction by heat-resistant thermophilic spore-forming bacteria

Thermophilic endospores are widespread in cold marine sediments where the temperature is too low to support growth and activity of thermophiles in situ. These endospores are likely expelled from warm subsurface environments and subsequently dispersed by ocean currents. The endospore upper temperature limit for survival is 140°C, which can be tolerated in repeated short exposures, potentially enabling transit through hot crustal fluids. Longer-term thermal tolerance of endospores, and how long they could persist in an environment hotter than their maximum growth temperature, is less understood. To test whether thermophilic endospores can survive prolonged exposure to high temperatures, sediments were incubated at 80–90°C for 6, 12 or 463 days. Sediments were then cooled by 10–40°C, mimicking the cooling in subsurface oil reservoirs subjected to seawater injection. Cooling the sediments induced sulfate reduction, coinciding with an enrichment of endospore-forming Clostridia. Different Desulfofundulus, Desulfohalotomaculum, Desulfallas, Desulfotomaculum and Desulfofarcimen demonstrated different thermal tolerances, with some Desulfofundulus strains surviving for >1 year at 80°C. In an oil reservoir context, heat-resistant endospore-forming sulfate-reducing bacteria have a survival advantage if they are introduced to, or are resident in, an oil reservoir normally too hot for germination and growth, explaining observations of reservoir souring following cold seawater injection.     

EVOLUTION OF ASEXUALITY IN THE COSMOPOLITAN MARINE CLAM LASAEA

The marine clam genus Lasaea is unique among marine bivalves in that it contains both sexual and asexual lineages. We employed molecular tools to infer intrageneric relationships of geographically restricted sexual versus cosmopolitan asexual forms. Polymerase chain reaction primers were used to amplify and sequence homologous 624 nucleotide fragments of COIII from polyploid, asexual, direct-developing individuals representing northeastern Pacific, northeastern Atlantic, Mediterranean, southern Indian Ocean, and Australian populations. DNA sequences also were obtained from the two known diploid congeners, the Australian sexual, indirect developer, Lasaea australis, and an undescribed meiotic Australian direct developer. Estimated tree topologies did not support monophyly for polyploid asexual Lasaea lineages. A robust dichotomy was evident in all phylogenetic trees and each of the two main branches included one of the diploid meitoic Australian congeners. Lasaea australis clustered with two of the direct-developing, polyploid asexual haplotypes, one from Australia, the other from the northeastern Atlantic. Monophyly is supported for the diploid Australian direct-developing lineage together with the remaining polyploid asexual lineages from the northeastern Pacific, northeastern Atlantic, Mediterranean, and southern Indian Ocean. These results indicate that asexual Lasaea lineages are polyphyletic and may have resulted from multiple hybridization events. The high degree of genetic divergence of asexual lineages from co-clustering meiotic congeners (16%–22%) and among geographically restricted monophyletic clones (9%–11%) suggests that asexual Lasaea lineages may be exceptionally long lived.     

Bacterial and archaeal diversities in Yunnan and Tibetan hot springs,China

Thousands of hot springs are located in the north‐eastern part of the Yunnan–Tibet geothermal zone, which is one of the most active geothermal areas in the world. However, a comprehensive and detailed understanding of microbial diversity in these hot springs is still lacking. In this study, bacterial and archaeal diversities were investigated in 16 hot springs (pH 3.2–8.6; temperature 47–96°C) in Yunnan Province and Tibet, China by using a barcoded 16S rRNA gene‐pyrosequencing approach. Aquificae, Proteobacteria, Firmicutes, Deinococcus‐Thermus and Bacteroidetes comprised the large portion of the bacterial communities in acidic hot springs. Non‐acidic hot springs harboured more and variable bacterial phyla than acidic springs. Desulfurococcales and unclassified Crenarchaeota were the dominated groups in archaeal populations from most of the non‐acidic hot springs; whereas, the archaeal community structure in acidic hot springs was simpler and characterized by Sulfolobales and Thermoplasmata. The phylogenetic analyses showed that Aquificae and Crenarchaeota were predominant in the investigated springs and possessed many phylogenetic lineages that have never been detected in other hot springs in the world. Thus findings from this study significantly improve our understanding of microbial diversity in terrestrial hot springs.     

Bacterial Communities Associated with Subsurface Geochemical Processes in Continental Serpentinite Springs

Reactions associated with the geochemical process of serpentinization can generate copious quantities of hydrogen and low-molecular-weight organic carbon compounds, which may provide energy and nutrients to sustain subsurface microbial communities independently of the photosynthetically supported surface biosphere. Previous microbial ecology studies have tested this hypothesis in deep sea hydrothermal vents, such as the Lost City hydrothermal field. This study applied similar methods, including molecular fingerprinting and tag sequencing of the 16S rRNA gene, to ultrabasic continental springs emanating from serpentinizing ultramafic rocks. These molecular surveys were linked with geochemical measurements of the fluids in an interdisciplinary approach designed to distinguish potential subsurface organisms from those derived from surface habitats. The betaproteobacterial genus Hydrogenophaga was identified as a likely inhabitant of transition zones where hydrogen-enriched subsurface fluids mix with oxygenated surface water. The Firmicutes genus Erysipelothrix was most strongly correlated with geochemical factors indicative of subsurface fluids and was identified as the most likely inhabitant of a serpentinization-powered subsurface biosphere. Both of these taxa have been identified in multiple hydrogen-enriched subsurface habitats worldwide, and the results of this study contribute to an emerging biogeographic pattern in which Betaproteobacteria occur in near-surface mixing zones and Firmicutes are present in deeper, anoxic subsurface habitats.     

Starvation survivability of Thermococcus strains isolated from Japanese oil reservoirs

The starvation survivability of seven Thermococcus strains isolated from four Japanese oil reservoirs was compared with that of Thermococcus strains from marine hydrothermal fields. 16S rDNA analyses showed the isolates to be closely related to Thermococcus litoralis. Growth of the isolates was dependent on amino acids, which were present at low concentrations in the oil reservoirs. At 80 degrees C in the formation water, strain CKU-1 from the oil reservoir showed a higher starvation survivability than strain KS-1 from the marine hydrothermal field. Crude oil did not affect the starvation survivability of strain CKU-1, but it reduced that of strain KS-1. These results indicate that strain CKU-1 could survive longer than stain KS-1 under the conditions of an oil reservoir. At 90 degrees C in artificial seawater without organic nutrients, the half-lives of the isolates were between 7.7 and 25.1 days. However, those of the strains from marine hydrothermal fields, except Thermococcus litoralis and Thermococcus chitonophagus, were less than 1.0 day. The higher starvation survivability is probably important for the hyperthermophiles to continue to exist in a hot subterranean oil reservoir where the supply of nutrients seems to be limited.     

Morphological and genetic diversity of the thermophilic cyanobacterium,Mastigocladus laminosus (Stigonematales,Cyanobacteria) from Japan and Myanmar

The morphology and phylogeny of 13 strains of a thermophilic cyanobacterium, Mastigocladus laminosus Cohn, isolated from hot springs in Japan and Myanmar were analyzed to determine taxonomy and biogeography. From the morphological observations of cell size, there were significant differences among strains. Phylogenetic analyses based on 16S rRNA gene sequences revealed two lineages: Lineages I and II. Lineage I consisted of strains collected in Japan and reference strains from a previous study (CCMEE 5329 and 5331, Hakone, Japan); Lineage II included all of the Myanmar strains and one Japanese strain, and was a novel lineage in phylogeographic studies on M. laminosus. Since strains in the Lineage II tended to have larger cells than those in the Lineage I, the morphological and phylogenetic lineages corresponded well with each other.     

Evidence for extensive gene flow and Thermotoga subpopulations in subsurface and marine environments

Oil reservoirs represent a nutrient-rich ecological niche of the deep biosphere. Although most oil reservoirs are occupied by microbial populations, when and how the microbes colonized these environments remains unanswered. To address this question, we compared 11 genomes of Thermotoga maritima-like hyperthermophilic bacteria from two environment types: subsurface oil reservoirs in the North Sea and Japan, and marine sites located in the Kuril Islands, Italy and the Azores. We complemented our genomes with Thermotoga DNA from publicly available subsurface metagenomes from North America and Australia. Our analysis revealed complex non-bifurcating evolutionary history of the isolates'' genomes, suggesting high amounts of gene flow across all sampled locations, a conjecture supported by numerous recombination events. Genomes from the same type of environment tend to be more similar, and have exchanged more genes with each other than with geographically close isolates from different types of environments. Hence, Thermotoga populations of oil reservoirs do not appear isolated, a requirement of the ‘burial and isolation'' hypothesis, under which reservoir bacteria are descendants of the isolated communities buried with sediments that over time became oil reservoirs. Instead, our analysis supports a more complex view, where bacteria from subsurface and marine populations have been continuously migrating into the oil reservoirs and influencing their genetic composition. The Thermotoga spp. in the oil reservoirs in the North Sea and Japan probably entered the reservoirs shortly after they were formed. An Australian oil reservoir, on the other hand, was likely colonized very recently, perhaps during human reservoir development.     

Cryptic diversity and comparative phylogeography of the estuarine copepod Acartia tonsa on the US Atlantic coast

Unexpectedly strong geographic structures in many cosmopolitan species of marine holoplankton challenge the traditional view of their unrestrained dispersal and presumably high gene flow. We investigated cryptic lineage diversity and comparative phylogeography of a common estuarine copepod, Acartia tonsa, on the US Atlantic coast, using mitochondrial (mtCOI) and nuclear (nITS) gene markers. Three broadly sympatric lineages (F, S, X) were defined by genealogically concordant clades across both gene trees, strongly supporting recognition as reproductively isolated species. Limited dispersal seems to have had a major role in population differentiation of A. tonsa in general, with gene flow propensities rank ordered X > S > F. Geographic structure was found only at large scales (1000–2000 km) in X and S. Phylogeographic patterns in all three lineages were mostly concordant with previously recognized zoogeographic provinces but a large mid‐Atlantic gap in the occurrence of lineage X, coupled with its presence in Europe, suggests possible nonindigenous origins. For lineage F, physiological adaptation to low‐salinity environments is likely to have accentuated barriers to gene flow and allopatric differentiation at both regional and continental scales. Three allopatric F sublineages inferred a southern centre of origin and a stepwise northward diversification history at the continental scale. The most recently derived F sublineages, in the mid‐Atlantic Bight, showed strong phylogeographic patterns at nITS albeit weaker at mtCOI. Applying a crustacean mtCOI molecular clock suggests that A. tonsa lineages diverged pre‐Pleistocene but mid‐Atlantic F lineage diversification may be post‐Pleistocene.     

我国产甲烷古菌研究进展与展望

产甲烷古菌广泛分布在湿地、水稻田、动物瘤胃、油藏、海洋和热液等缺氧环境,在全球碳素循环、气候变化和清洁能源生产等领域发挥着重要作用,一直是国内外的研究热点。本文简要回顾了我国产甲烷古菌的研究进展,重点阐述了产甲烷古菌的资源与分类、生理生化、分子生物学、生态学功能和应用等方面的研究进展,并展望了产甲烷古菌的未来研究趋势。     

Bacterial Populations (First Record) at Two Shallow Hydrothermal Vents of the Mexican Pacific West Coast

Thermophilic and metal-oxidizing bacteria were identified in shallow hydrothermal vents on the western Mexican coast. The role of these bacteria in biomineralization processes observed in the vents is explained, and the effect of the vents on biodiversity of prokaryotes is discussed. Research was done at two shallow hydrothermal vent sites: Bahía Concepción (BC) in the Baja California Peninsula and Punta Mita (PM), on the central Pacific coast. Temperature at the sediments proximal to the vents was similar, but the redox potentials (0.5 V in BC and ?0.3 V in PM) and pH (6.2 in BC and as low as 4.5 in PM) differed. The composition of the discharged water ranged from nearly seawater to lower-salinity fluids, and the gas phase was mainly CO₂ at BC and N₂ and CH₄ at PM. The study focuses on the biogeochemical processes related to the different species of bacteria present in the studied sites, which are involved in the anaerobic oxidation of methane (AOM), seawater sulfate reduction, and metal oxidation. The detected bacterial lineages represented typical deep vent species, which disproves a previous hypothesis that proposed that different consortia were populating deep and shallow hydrothermal vents. The results obtained here show that the main parameter affecting the bacterial groups present in shallow vents was the redox potential: gamma-, delta-, and epsilon proteobacteria as well as Bacteriodetes are present under the oxidizing conditions of BC, and Thermotogae, Aquificae, and Planctomycetes are present in PM. Sunlight abundance favored the prevalence of halophilic and Chlorofleaxae bacteria in both areas.     

Rapid Diversification of Marine Picophytoplankton with Dissimilar Light-Harvesting Structures Inferred from Sequences of Prochlorococcus and Synechococcus (Cyanobacteria)

Cultured isolates of the unicellular planktonic cyanobacteria Prochlorococcus and marine Synechococcus belong to a single marine picophytoplankton clade. Within this clade, two deeply branching lineages of Prochlorococcus, two lineages of marine A Synechococcus and one lineage of marine B Synechococcus exhibit closely spaced divergence points with low bootstrap support. This pattern is consistent with a near-simultaneous diversification of marine lineages with divinyl chlorophyll b and phycobilisomes as photosynthetic antennae. Inferences from 16S ribosomal RNA sequences including data for 18 marine picophytoplankton clade members were congruent with results of psbB and petB and D sequence analyses focusing on five strains of Prochlorococcus and one strain of marine A Synechococcus. Third codon position and intergenic region nucleotide frequencies vary widely among members of the marine picophytoplankton group, suggesting that substitution biases differ among the lineages. Nonetheless, standard phylogenetic methods and newer algorithms insensitive to such biases did not recover different branching patterns within the group, and failed to cluster Prochlorococcus with chloroplasts or other chlorophyll b-containing prokaryotes. Prochlorococcus isolated from surface waters of stratified, oligotrophic ocean provinces predominate in a lineage exhibiting low G + C nucleotide frequencies at highly variable positions. Received: 18 January 1997 / Accepted: 18 May 1997     

Investigation of structure and antigenic capacities of Thermococcales cell envelopes and reclassification of "Caldococcus litoralis" Z-1301 as Thermococcus litoralis Z-1301

Fourteen strains of hyperthermophilic organotrophic anaerobic marine Archaea were isolated from shallow water and deep-sea hot vents, and four of them were characterized. These isolates, eight previously published strains, and six type strains of species of the order Thermococcales were selected for the study of cell wall components by means of thin sectioning or freeze-etching electron microscopy. The cell envelopes of most isolates were shown to consist of regularly arrayed surface protein layers, either single or double, with hexagonal lattice (p6) symmetry, as the exclusive constituents outside the cytoplasmic membrane. The S-layers studied differed in center-to-center spacing and molecular mass of the constituent protein subunits. Polyclonal antisera raised against the cells of 10 species were found to be species-specific and allowed 12 new isolates from shallow water hot vents to be identified as representatives of the species Thermococcus litoralis, Thermococcus stetteri, Thermococcus chitonophagus, and Thermococcus pacificus. Of the 7 deep-sea isolates, only 1 was identified as a T. litoralis strain. Thus, hyperthermophilic marine organotrophic isolates obtained from deep-sea hot vents showed greater diversity with regard to their S-layer proteins than shallow water isolates. Received: February 5, 1999 / Accepted: May 11, 1999     

Archaeal phylotypes in a metal-rich and low-activity deep subsurface sediment of the Peru Basin, ODP Leg 201, Site 1231

Site 1231 of the Ocean Drilling Project (ODP) was characterized by low concentrations of organic carbon, as well as low cell numbers and biological activity rates. A 16S rRNA survey was performed in order to analyse the microbial community composition of these central oceanic sediments. Archaeal 16S rRNA genes from subsurface sediments at Site 1231 (1.8, 9.0, and 43 mbsf) were affiliated with uncultured lineages from subsurface or hydrothermal vent habitats. Members of the Marine Group I (MGI) found in the 1.8 mbsf sediment formed distinct clusters, some dominated by phylotypes from Site 1231 and other subsurface environments. The archaeal community survey at Site 1231 indicated that several archaeal lineages were widespread in subsurface environments, marine sediments as well as hydrothermal habitats.