未知古菌基因组

© 2005 Nature Publishing Group *Department of Biology, University of Bergen, Jahnebakken 5, N-5020 Bergen, Norway. ‡Department of Applied Chemistry and Microbiology, Viikinkaari 9, FIN-00014 Helsinki University, Finland. Correspondence to C.S. e-mail: christa.schleper@ bio.uib.no doi:10.1038/nrmicro1159 GENOMIC STUDIES OF UNCULTIVATED ARCHAEA Christa Schleper*, German Jurgens‡ and Melanie Jonuscheit* Abstract | Archaea represent a considerable fraction of the prokaryotic world in marine and terrestrial ecosystems, indicating that organisms from this domain might have a large impact on global energy cycles. However, many novel archaeal lineages that have been detected by molecular phylogenetic approaches have remained elusive because no laboratory-cultivated strains are available. Environmental genomic analyses have recently provided clues about the potential metabolic strategies of several of the uncultivated and abundant archaeal species, including non-thermophilic terrestrial and marine crenarchaeota and methanotrophic euryarchaeota. These initial studies of natural archaeal populations also revealed an unexpected degree of genomic variation that indicates considerable heterogeneity among archaeal strains. Here, we review genomic studies of uncultivated archaea within a framework of the phylogenetic diversity and ecological distribution of this domain. Considering the recently accumulated knowledge of genes and genomes of uncultivated archaea, it is time to refine our perception about the ecology and diversity of this third domain of life. Although archaea have been detected in many moderate environments, they are still primarily considered to be extremists, dominating habitats that define the physical limits for biological systems, such as geothermal hot or acidic springs, deep-sea hydrothermal vents, hypersaline ponds or strictly anoxic ecosystems. From an evolutionary viewpoint, it might be jus- tified to give priority to the extremists: archaea from hot environ ments are phylogenetically diverse and some of them branch close to the root of the archaeal tree1–3 — as inferred from 16S ribosomal RNA (rRNA) analyses (FIG. 1) and from phylogeny based on ribo- somal or tran scrip tional proteins4 — indicating that thermophiles might have arisen first and that some could be progenitors of species that underwent adap- tive radiation into moderate habitats. Support for this speculation has been provided by biogeochemical studies and lipid biomarkers5. However, from an ecological and phylogenetic perspective, we should invert this picture: Archaea are primarily a diverse and widespread group on Earth, which are found in our gardens and forests6–11, in the ocean’s plankton12,13 and sediment14,15, in freshwater lakes16–19 and deep down in the subsurface20. By con- trast, those organisms that have been cultivated in the laboratory and studied in detail, such as methanogens, thermo philes and halophiles, represent only a minority of phylotypes and phenotypes. The broad distribution and abundance of archaea in soils and oceans implies that they contribute to global energy cycles. However, these organisms have been predicted solely from PCR-based surveys and no representatives have been cultivated in the laboratory. Therefore, their specific metabolisms remain elusive. Recent advances in environmental genomic studies show that we now have the necessary technical tools to characterize these organisms in the absence of laboratory cultivation and in the context of indigenous microbial communities21–23. This approach involves direct cloning of genomic DNA from the environment and storing this DNA either in small-insert libraries, for large-scale shotgun-sequencing approaches, or in large-insert libraries, for targeted searches for genomic fragments from specific lineages. Hypotheses about the NATURE REVIEWS | MICROBIOLOGY VOLUME 3 | JUNE 2005 | 479 F O C U S O N M E T A G E N O M I C S © 2005 Nature Publishing Group Euryarchaeota 0.05 Group III DHVE3 PENDANT-33 Methanopyrus kandleri DHVE4 Methanococcales Methanobacteriales Archaeoglobi DHVE1SAGMA1 VADIN W H C A SA1 Halobacteria ANME- 1-AT ANME-1A ANME-2C SAGMA- S/TGroup II Thermoplasmata Thermococcales ANME-1-GBa DHVE6 ANME-1B YNPFFA THSC1 MarBenthGpC MarBenthGpB/DHVC1 FFS ARC1 Methanosaetaceae ANME-2A/B Group I.1A Group I.1B Methanomicrobiales Thermoproteales Group I.2Group I.1C SAGMCG-1 Crenarchaeota Methanosarcina Methanolobus/ Methanohalophylus Korarchaeota AAG Desulfurococcales Sulfolobales 0.01 ANME-1A FOS-GZfos27G5 FOS-GZfos12E1 FOS-GZfos10C7 SB-17a1A2 SB-17a1A11 BS-R-A1 BS-SR-G10 BS-SR-C1-Arch pISA14pISA16 GBa1r013 GBa1r010 0.01 ANME-1B FOS-GZfos34G5 FOS-GZfos25D1(1D1) FOS-GZfos1C11 BS-M-A7 BS-S-E7 Eel-36a2G10 BS-M-D6 HydCal52 BS-S-D7 BS-R-B8 BS-SR-D3 BS-K-411 BS-S-E1 HydBeg01 HydCal61 Thermoplasmata Thermoplasma volcanium 0.05 SG-Ferroplasma sp. Type II SG-Ferroplasma acidarmanus Type I SG-Thermoplasmatales archaeon Gp1 ASL/SC group SC38 ARCP1-60 SC42 MS14 Thermoplasma volcanium Thermoplasma acidophilum Thermoplasma acidophilum Picrophilus torridus Picrophilus oshimae Ferromonas metallovorans Nanoarchaeum equitans 0.05 Axinella symbiont group Group I.1A BAC-74A4 COS- DeepAnt-EC39 FOS-4B7 SG-IBEA_CTG_2146200 Cenarchaeum strain A Cenarchaeum strain B APA/CRA ST-12K2A pIVWA3 pIVWA5 pIVWA11 DCM group PM7 DCM871 CRA7-11cm APA4-0cm pIVWA101 SAGMA-1 CRA36-0cm ST12K 83A10 31B02 19H08 15G10 C20 ST-3K4A Antarctic12 group Rice cluster I 0.05 DCM group Group II SG-IBEA_CTG_2081753 SBAR 16 DH148-W1 DCM875 GIN492 TS10C294 PVA OTU 1 ANTARCTIC 5 WHAR N DCM3921 SB95-72 KTK 31A DCM65231 COS-DeepAnt-JyKC7 0.01 ANME-2C FOS-GZfos26E7 FOS-GZfos18C8 C1_R048 AT_R007 Eel-36a2A1 SB-17a1B11 SB-17a1D3 HydBeg125 HydBeg22 HydBeg05 C1_R019 SB-24a1A12 Eel-36a2A5 CS_R012 C1_R004 0.05SAGMA-W/SCA11 Group I.1B FOS-29i4 FOS-54D9 SCA1145/TRC23 GRU22 Pus4-16 MAL7 GRU16 FSAr20 FSAr24 HTA-C5 HTA-E7 SCA1175 ROB110 HTA-H8 ROB1A11 ROB17 ROBD8 SCA1154 TRC132-3 HTA-D6 S247-3 ROB34 SCA1158/pGrFA4 SCA1150 SCA1173 PET1-19 pOWA133 M arB enth G pE ANME-3/ Methano- coccoides Marine sponge symbiont Soil Acid mine drainage Key to coloured text in insets: Marine plankton Deep-sea methane seeps 480 | JUNE 2005 | VOLUME 3 www.nature.com/reviews/micro R E V I E W S © 2005 Nature Publishing Group DEEP SUBSURFACE Usually 50 m or more below the surface, where the microbiota is not immediately affected by microbial functions or biogeochemical processes of the surface (as opposed to shallow subsurface). BENTHIC Living in or on the bottom of a body of water. specific metabolism of several novel archaeal groups can now be formulated, which supplies the basis for studying the ecological impact of these species in the context of geochemical data and complex microbial communities. The prediction of specific metabolisms might eventually also aid enrichment efforts and enable cultivation of some novel archaeal model organisms. Archaeal diversity based on molecular surveys Among the first discoveries of PCR-based molecular ecological surveys was the detection of 16S rRNA of members of the Crenarchaeota in the marine plank- ton12,13. Since then, novel archaeal ‘phylotypes’ (bacterial phylogenetic types) have been detected in most environ- mental surveys that have targeted archaeal sequences. Three years ago, Philip Hugenholtz dissected 18 differ- ent archaeal phyla (10 of which contained no cultivated representatives) as opposed to 35 phyla of bacteria (13 without cultivated representatives), based on compara- tive analyses of 16S rRNA gene sequences24. Meanwhile, almost 8,000 archaeal 16S rRNA gene sequences from environmental studies have been deposited in public databases, extending the known groups and increasing the number of novel lineages (FIG. 1). Many novel archaeal groups seem (so far) to be confined to specific geographi- cal locations or to ecosystems that have similar geochem- istry, but other groups seem to be widely distributed. For example, the two crenarchaeal lineages, which are mostly defined from sequences of marine plankton (group I.1A, FIG. 1) or soils (group I.1B, FIG. 1), are both also found in freshwater samples and DEEP SUBSURFACES8,20. It is surprising that most of the newly discov- ered lineages seem to expand the two major phyla — Euryarchaeota and Crenarchaeota — that were defined as early as 1986 based on only a few cultured archaeal species25 (FIG. 1). However, the discovery of a few more distant and deeply branching lineages through molecular surveys (Korarchaeota and AAG (ancient archaeal group) in FIG. 1, REFS 1,3) or cultiva- tion (Nanoarchaeota)2, indicates that greater archaeal diversity is to be expected and that more lineages might be recovered through improved molecular ecological searches, more sophisticated cultivation techniques26 and perhaps by metagenomic approaches. Evidence for living archaeal populations Several molecular ecological studies indicate that many of the novel archaeal organisms that are predicted by PCR-based studies do in fact represent metabolically active populations. For example, marine planktonic archaea27, a sponge-associated crenarchaeote28, cre- narchaeota on plant roots10 and euryarchaeota in sediments14 as well as in sulphurous marsh water enriched on polyethylene nets26 have all been visual- ized with fluorescence in situ hybridization (FIG. 2), indicating a distinct morphology and high rRNA content, as expected for living cells. Furthermore, the abundance, distribution and dynamics of several groups shows patterns that are characteristic of active microbial populations. For example, marine plank- tonic archaea, which are found in many different oceanic provinces and represent approximately 20% of the total microbial planktonic population29, vary in relative abundance with respect to water depth and season27,30. Similarly, marine BENTHIC crenarchaeota and euryarchaeaota show depth-related variability in deep-sea sediments31. A specific phylotype of cold-water archaea, the crenarchaeote Cenarchaeum symbiosum, was found in association with a marine sponge28 and related phylotypes were recovered from other sponges in different oceanic regions, indicating specific meta- zoan–archaeal associations32–34. Euryarchaeota species from the three different anaerobic methane oxidation (ANME) lineages are found within the microbial mats of cold seeps, sometimes at high abundance (up to 50% for ANME-1), and have various distributions accord- ing to depth and geographical location35. Furthermore, these methanotrophic archaea form specific aggregates with their syntrophic sulphate-reducing bacterial part- ners14. Crenarchaeota representatives have been found in diverse soil samples, including sandy ecosystems, pristine forest soil, agricultural fields, contaminated soil and the rhizosphere6–10,36, and represent a consider- able fraction (up to 5%) of the total prokaryotic com- munity7,8. They show spatial hetero geneity and changes in abundance and community structure dependent on succession, land-management strategies, heavy-metal contamination or rhizosphere type9,37–39. These find- ings indicate dynamic and active archaeal popula- tions that react according to changing environmental parameters. Despite the accumulated knowledge of several novel archaeal groups and their apparent abundance, none of these archaea have been obtained in pure laboratory cultures. The preliminary evidence for their specific physiologies mostly stems from environmental genomic studies. Genome analyses of cultivated archaea The availability of complete genome sequences from cultivated archaeal species — 45 genomes completed or near completion (listed in REF. 40) — has not only stimulated new research areas that reach far beyond the archaeal community but has served as an impor- tant framework for tracking genomes of uncultivated archaea in complex communities. Figure 1 | The domain Archaea — from diversity to function. A 16S rRNA tree of the Archaea is shown, with groups of uncultivated species that have been targeted in genomic studies emphasized as boxes. Genomic fosmids (prefix FOS), cosmids (COS), bacterial artificial chromosomes (BACs) or sequences that have been assembled from shotgun sequencing projects (SG) are shown in expanded boxes. Triangles in light colours represent branches with exclusively uncultivated species, dark triangles show branches with cultivated species. The size of the triangle is proportional to the number of sequences analysed. The red hexagon in the centre indicates rooting to species in the domain Bacteria. The phylogenetic tree is based on comparisons of 16S rRNA sequences from the Euryarcheota, Crenarcheota and Korarchaeota phyla together with Nanoarchaeum equitans (dotted lines indicate uncertain phylogenetic positions), clone pOWA133 and the ancient archaeal group (AAG), which are not classified within these phyla. In total, 1,344 16S rRNA sequences were included (mostly full- length). Sequence data were analysed with the ARB software package82. The backbone tree was calculated by using maximum likelihood in combination with filters excluding highly variable positions with 55 full-length sequences (1,329 positions in length). Partial sequences were inserted into the reconstructed tree by using parsimony criteria without allowing changes to the overall tree topology. The scale bar for the whole tree represents 0.05 changes per nucleotide. The complete dataset is available from the authors on request. ◀ NATURE REVIEWS | MICROBIOLOGY VOLUME 3 | JUNE 2005 | 481 F O C U S O N M E T A G E N O M I C S © 2005 Nature Publishing Group a b c d 10 µm 10 µm 10 µm 10 µm Mostly through comparative genomic studies, it has become evident that the core components of archaeal information processing systems — replication, tran- scription, translation and DNA repair — show strik- ing similarities to those of eukaryotes (reviewed in REFS 4,40,41). This finding has supported the status of the Archaea as a unique and separate domain, as predicted by Carl Woese more than 25 years ago42. The study of information processing in the simpler, more streamlined systems of the Archaea has become increasingly relevant to understanding cellular evolu- tion and the complex interactions that occur in the eukaryal nucleus43–46. Long before the genomic era, the intriguing relationship with eukaryotes was revealed by Zillig and colleagues, who realized that the archaeal DNA-dependent RNA polymerase and the basal promoter sequences have striking similarities to those of eukaryotes47,48. Nevertheless, archaeal genomes are typically prokaryotic in terms of their small size and organization of genes and operon structures. Archaeal genome diversity reflects the physiological versatility of the Archaea and the wide range of growth condi- tions, as well as frequent gene acquisition by horizontal gene transfer41. Based on the unique archaeal informa- tion-processing machineries, conserved archaeal ‘core’ genes41 and archaeal transcription signals, we can dif- ferentiate archaeal and bacterial genomes, and can even differentiate euryarchaeotes and crenarchaeotes. Even in complex environmental DNA libraries, it is often pos- sible to distinguish large archaeal genome fragments from bacterial fragments, based on gene content and phylogenetic marker genes. However, assigning as-yet- uncultivated archaea to distinct lineages is not easy. As often nothing but the 16S rRNA gene sequence of a novel lineage is known, this information has initially been used as a phylogenetic anchor to retrieve spe- cific genome fragments from complex environmental libraries. More comprehensive environmental genomic studies of specific lineages became possible with large- scale sequence analyses from samples with some highly enriched organisms that allow the subsequent assembly of longer genomic contigs or even almost complete genomes (see below). Genomic studies of marine planktonic archaea The new field of cultivation-independent genomic studies of microorganisms was initiated when Edward DeLong and collaborators attempted to characterize genome fragments of marine planktonic archaea49. Inspired by the rapid advances in genomic techniques applied to cultivated microorganisms, they used a bac- terial artificial chromosome (BAC)-derived fosmid vector to prepare a large-insert library from marine plankton of the North-Eastern Pacific. A 38.5-kb genomic fragment of an uncultivated mesophilic crenarchaeote was identified within 3,552 clones, using archaea-specific 16S rDNA probes (FOS-4B7, Group I.1A in FIG. 1). Even snapshot sequencing of this clone confirmed its archaeal origin49. Further genome fragments of marine archaea have been iso- lated from BAC, fosmid or cosmid libraries of sur- face50,51 and deep waters52,53 of the Antarctic and the North Pacific. Conservation of gene order around the 16S rRNA gene confirmed the close relationship of the planktonic crenarchaeota, even in strains from different oceanic regions50,52 (FIG. 3). By contrast, it is noticeable that considerable genomic variation can be dissected, including microheterogeneity in pro- tein-encoding regions and intergenic spacers, when genome fragments with otherwise identical or almost identical 16S RNA genes were compared from the same DNA library50. The planktonic archaeal clones share several genomic features with their hyperthermophilic relatives, including the estimated low GC content (~32–36%) as well as the gene repertoire and the structure of the rRNA operon. However, some genes that are so far unique to planktonic archaea have also been identified, such as a putative RNA-binding protein that shares fea- tures with the bacterial cold-shock family and a novel zinc-finger protein that was previously only found in eukaryotes50. Given the abundance and ubiquity of marine planktonic archaea, it is plausible that large numbers of archaeal genes would be detected in a random sequencing survey of DNA obtained from filtered sur- face waters. This is in fact the case. The large dataset Figure 2 | Visualization of uncultivated archaea in various environments by fluorescence in situ hybridization (FISH). a | ANME-2C euryarchaeota (red) in association with sulphate-reducing bacteria (green) in sediments above methane hydrates. Image courtesy of T. Lösekann and K. Knittel, Max-Planck Institute for Marine Microbiology, Bremen, Germany. b | The freshwater euryarchaeon SM1 (green; belonging to group Pendant-33 in FIG. 1) in association with Thiotrix (red). Image courtesy of C. Moissl and R. Huber, Univeristy of Regensburg, Germany. c | Cenarchaeum symbiosum (green; belonging to group I.1A in FIG. 1) from the sponge Axinella mexicana (nuclei in red). Image courtesy of C. Preston, Monterey Bay Aquarium Research Institute, Moss Landing, USA. d | Crenarchaeota (red; belonging to group I.1B in FIG. 1) of tomato roots, enriched (associated bacteria in green). Image courtesy of H. Simon, Oregon Health & Science University, Beaverton, USA. 482 | JUNE 2005 | VOLUME 3 www.nature.com/reviews/micro R E V I E W S © 2005 Nature Publishing Group 23S 16SAntarctic deep waters Fosmid EC39 33,347 bp; GC 34% (REF. 52) Unique Homologues in four genome fragments Homologues in three genome fragments Homologues in two genome fragments Miscellaneous feature Sponge symbiont, Pacific coast Fosmid Cenarchaeum symbiosum A 32,998 bp; GC 55% (REF. 55) 23S 16S Sargasso Sea Scaffold CH004643 66,556 bp; GC 35% (REF. 54) Antarctic surface waters Fosmid 74A4 43,902 bp; GC