Microbiology: eukaryotic diversity in Spain's River of Fire

The Rio Tinto, known by the Phoenicians as 'Ur-yero', or 'River of Fire', because of its deep red colour and high acidity, flows through the world's largest pyritic belt in southwestern Spain. Surprisingly, eukaryotic microbes are the principal contributors of biomass in this hostile river, which has a pH of 2 and contains much higher concentrations of heavy metals than are typically found in fresh waters. Here we show that the Rio Tinto shows an unexpected degree of eukaryotic diversity and includes new lineages that we have identified by sequence analysis of genes encoding small-subunit ribosomal RNAs. The diversity of these eukaryotes is much greater than that of prokaryotes, whose metabolism is responsible for the extreme environment.     

Origin of the algae

Eukaryotic algae are traditionally separated into three broad divisions: the rhodophytes, the chromophytes and the chlorophytes. The evolutionary relationships between these groups, their links with other eukaryotes and with other photosynthetic groups, such as euglenophytes and cryptophytes, have been the subject of much debate and speculation. Here we analyse partial sequences of the large (28S) cytoplasmic ribosomal RNA from ten new species of protists belonging to various groups of unicellular algae. By combining them with the homologous sequences from 14 other unicellular and multicellular eukaryotes, we show that rhodophytes, chromophytes and chlorophytes emerge as three distinct groups late among eukaryotes, that is, close to the metazoa-metaphytes radiation. This implies a relatively late occurrence of eukaryotic photosynthetic symbiosis. We also provide details of intra- and inter-phyla relationships.     

Morphological and ecological complexity in early eukaryotic ecosystems.

Molecular phylogeny and biogeochemistry indicate that eukaryotes differentiated early in Earth history. Sequence comparisons of small-subunit ribosomal RNA genes suggest a deep evolutionary divergence of Eukarya and Archaea; C27-C29 steranes (derived from sterols synthesized by eukaryotes) and strong depletion of 13C (a biogeochemical signature of methanogenic Archaea) in 2,700 Myr old kerogens independently place a minimum age on this split. Steranes, large spheroidal microfossils, and rare macrofossils of possible eukaryotic origin occur in Palaeoproterozoic rocks. Until now, however, evidence for morphological and taxonomic diversification within the domain has generally been restricted to very late Mesoproterozoic and Neoproterozoic successions. Here we show that the cytoskeletal and ecological prerequisites for eukaryotic diversification were already established in eukaryotic microorganisms fossilized nearly 1,500 Myr ago in shales of the early Mesoproterozoic Roper Group in northern Australia.     

Discovery of novel intermediate forms redefines the fungal tree of life

Fungi are the principal degraders of biomass in terrestrial ecosystems and establish important interactions with plants and animals. However, our current understanding of fungal evolutionary diversity is incomplete and is based upon species amenable to growth in culture. These culturable fungi are typically yeast or filamentous forms, bound by a rigid cell wall rich in chitin. Evolution of this body plan was thought critical for the success of the Fungi, enabling them to adapt to heterogeneous habitats and live by osmotrophy: extracellular digestion followed by nutrient uptake. Here we investigate the ecology and cell biology of a previously undescribed and highly diverse form of eukaryotic life that branches with the Fungi, using environmental DNA analyses combined with fluorescent detection via DNA probes. This clade is present in numerous ecosystems including soil, freshwater and aquatic sediments. Phylogenetic analyses using multiple ribosomal RNA genes place this clade with Rozella, the putative primary branch of the fungal kingdom. Tyramide signal amplification coupled with group-specific fluorescence in situ hybridization reveals that the target cells are small eukaryotes of 3-5?μm in length, capable of forming a microtubule-based flagellum. Co-staining with cell wall markers demonstrates that representatives from the clade do not produce a chitin-rich cell wall during any of the life cycle stages observed and therefore do not conform to the standard fungal body plan. We name this highly diverse clade the cryptomycota in anticipation of formal classification.     

Oceanic 18S rDNA sequences from picoplankton reveal unsuspected eukaryotic diversity

Picoplankton--cells with a diameter of less than 3 microm--are the dominant contributors to both primary production and biomass in open oceanic regions. However, compared with the prokaryotes, the eukaryotic component of picoplankton is still poorly known. Recent discoveries of new eukaryotic algal taxa based on picoplankton cultures suggest the existence of many undiscovered taxa. Conventional approaches based on phenotypic criteria have limitations in depicting picoplankton composition due to their tiny size and lack of distinctive taxonomic characters. Here we analyse, using an approach that has been very successful for prokaryotes but has so far seldom been applied to eukaryotes, 35 full sequences of the small-subunit (18S) ribosomal RNA gene derived from a picoplanktonic assemblage collected at a depth of 75 m in the equatorial Pacific Ocean, and show that there is a high diversity of picoeukaryotes. Most of the sequences were previously unknown but could still be assigned to important marine phyla including prasinophytes, haptophytes, dinoflagellates, stramenopiles, choanoflagellates and acantharians. We also found a novel lineage, closely related to dinoflagellates and not previously described.     

Genetic diversity in Sargasso Sea bacterioplankton

Bacterioplankton are recognized as important agents of biogeochemical change in marine ecosystems, yet relatively little is known about the species that make up these communities. Uncertainties about the genetic structure and diversity of natural bacterioplankton populations stem from the traditional difficulties associated with microbial cultivation techniques. Discrepancies between direct counts and plate counts are typically several orders of magnitude, raising doubts as to whether cultivated marine bacteria are actually representative of dominant planktonic species. We have phylogenetically analysed clone libraries of eubacterial 16S ribosomal RNA genes amplified from natural populations of Sargasso Sea picoplankton by the polymerase chain reaction. The analysis indicates the presence of a novel microbial group, the SAR11 cluster, which appears to be a significant component of this oligotrophic bacterioplankton community. A second cluster of lineages related to the oxygenic phototrophs--cyanobacteria, prochlorophytes and chloroplasts--was also observed. However, none of the genes matched the small subunit rRNA sequences of cultivated marine cyanobacteria from similar habitats. The diversity of 16S rRNA genes observed within the clusters suggests that these bacterioplankton may be consortia of independent lineages sharing surprisingly distant common ancestors.     

Cultivation of the ubiquitous SAR11 marine bacterioplankton clade

The alpha-proteobacterial lineage that contains SAR11 and related ribosomal RNA gene clones was among the first groups of organisms to be identified when cultivation-independent approaches based on rRNA gene cloning and sequencing were applied to survey microbial diversity in natural ecosystems. This group accounts for 26% of all ribosomal RNA genes that have been identified in sea water and has been found in nearly every pelagic marine bacterioplankton community studied by these methods. The SAR11 clade represents a pervasive problem in microbiology: despite its ubiquity, it has defied cultivation efforts. Genetic evidence suggests that diverse uncultivated microbial taxa dominate most natural ecosystems, which has prompted widespread efforts to elucidate the geochemical activities of these organisms without the benefit of cultures for study. Here we report the isolation of representatives of the SAR11 clade. Eighteen cultures were initially obtained by means of high-throughput procedures for isolating cell cultures through the dilution of natural microbial communities into very low nutrient media. Eleven of these cultures have been successfully passaged and cryopreserved for future study. The volume of these cells, about 0.01 micro m(3), places them among the smallest free-living cells in culture.     

Phylogenetic analysis based on rRNA sequences supports the archaebacterial rather than the eocyte tree

How many primary lineages of life exist and what are their evolutionary relationships? These are fundamental but highly controversial issues. Woese and co-workers propose that archaebacteria, eubacteria and eukaryotes are the three primary lines of descent and their relationships can be represented by Fig. 1a (the 'archaebacterial tree') if one neglects the root of the tree. In contrast, Lake claims that archaebacteria are paraphyletic, and he groups eocytes (extremely thermophilic, sulphur-dependent bacteria) with eukaryotes, and halobacteria with eubacteria (the 'eocyte tree', Fig. 1b). Lake's view has gained considerable support as a result of an analysis of small subunit ribosomal RNA sequence data by a new approach, the evolutionary parsimony method. Here we report that analysis of small subunit data by the neighbour-joining and maximum parasimony methods favours the archaebacterial tree and that computer simulations using either the archaebacterial or the eocyte tree as a model tree show that the probability of recovering the model tree is very high (greater than 90 per cent) for both the neighbour-joining and maximum parsimony methods but is relatively low for the evolutionary parsimony method. Moreover, analysis of large subunit rRNA sequences by all three methods strongly favours the archaebacterial tree.     

Analysis of the genome sequence of the flowering plant Arabidopsis thaliana

The flowering plant Arabidopsis thaliana is an important model system for identifying genes and determining their functions. Here we report the analysis of the genomic sequence of Arabidopsis. The sequenced regions cover 115.4 megabases of the 125-megabase genome and extend into centromeric regions. The evolution of Arabidopsis involved a whole-genome duplication, followed by subsequent gene loss and extensive local gene duplications, giving rise to a dynamic genome enriched by lateral gene transfer from a cyanobacterial-like ancestor of the plastid. The genome contains 25,498 genes encoding proteins from 11,000 families, similar to the functional diversity of Drosophila and Caenorhabditis elegans--the other sequenced multicellular eukaryotes. Arabidopsis has many families of new proteins but also lacks several common protein families, indicating that the sets of common proteins have undergone differential expansion and contraction in the three multicellular eukaryotes. This is the first complete genome sequence of a plant and provides the foundations for more comprehensive comparison of conserved processes in all eukaryotes, identifying a wide range of plant-specific gene functions and establishing rapid systematic ways to identify genes for crop improvement.     

The genome sequence of Schizosaccharomyces pombe

We have sequenced and annotated the genome of fission yeast (Schizosaccharomyces pombe), which contains the smallest number of protein-coding genes yet recorded for a eukaryote: 4,824. The centromeres are between 35 and 110 kilobases (kb) and contain related repeats including a highly conserved 1.8-kb element. Regions upstream of genes are longer than in budding yeast (Saccharomyces cerevisiae), possibly reflecting more-extended control regions. Some 43% of the genes contain introns, of which there are 4,730. Fifty genes have significant similarity with human disease genes; half of these are cancer related. We identify highly conserved genes important for eukaryotic cell organization including those required for the cytoskeleton, compartmentation, cell-cycle control, proteolysis, protein phosphorylation and RNA splicing. These genes may have originated with the appearance of eukaryotic life. Few similarly conserved genes that are important for multicellular organization were identified, suggesting that the transition from prokaryotes to eukaryotes required more new genes than did the transition from unicellular to multicellular organization.     

Nuclear export dynamics of RNA-protein complexes

The central dogma of molecular biology - DNA makes RNA makes proteins - is a flow of information that in eukaryotes encounters a physical barrier: the nuclear envelope, which encapsulates, organizes and protects the genome. Nuclear-pore complexes, embedded in the nuclear envelope, regulate the passage of molecules to and from the nucleus, including the poorly understood process of the export of RNAs from the nucleus. Recent imaging approaches focusing on single molecules have provided unexpected insight into this crucial step in the information flow. This review addresses the latest studies of RNA export and presents some models for how this complex process may work.     

Archaeal dominance in the mesopelagic zone of the Pacific Ocean

The ocean's interior is Earth's largest biome. Recently, cultivation-independent ribosomal RNA gene surveys have indicated a potential importance for archaea in the subsurface ocean. But quantitative data on the abundance of specific microbial groups in the deep sea are lacking. Here we report a year-long study of the abundance of two specific archaeal groups (pelagic euryarchaeota and pelagic crenarchaeota) in one of the ocean's largest habitats. Monthly sampling was conducted throughout the water column (surface to 4,750 m) at the Hawai'i Ocean Time-series station. Below the euphotic zone (> 150 m), pelagic crenarchaeota comprised a large fraction of total marine picoplankton, equivalent in cell numbers to bacteria at depths greater than 1,000 m. The fraction of crenarchaeota increased with depth, reaching 39% of total DNA-containing picoplankton detected. The average sum of archaea plus bacteria detected by rRNA-targeted fluorescent probes ranged from 63 to 90% of total cell numbers at all depths throughout our survey. The high proportion of cells containing significant amounts of rRNA suggests that most pelagic deep-sea microorganisms are metabolically active. Furthermore, our results suggest that the global oceans harbour approximately 1.3 x 10(28) archaeal cells, and 3.1 x 10(28) bacterial cells. Our data suggest that pelagic crenarchaeota represent one of the ocean's single most abundant cell types.     

Alu sequences are processed 7SL RNA genes

7SL RNA is an abundant cytoplasmic RNA which functions in protein secretion as a component of the signal recognition particle. Alu sequences are the most abundant family of human and rodent middle repetitive DNA sequences (reviewed in ref. 2). The primary structure of human 7SL RNA consists of an Alu sequence interrupted by a 155-base pair (bp) sequence that is unique to 7SL RNA. In order to obtain information about the evolution of the Alu domain of 7SL RNA, we have determined the nucleotide sequence of a cDNA copy of Xenopus laevis 7SL RNA and of the 7SL RNA gene of Drosophila melanogaster. We find that the Xenopus sequence is 87% homologous with its human counterpart and the Drosophila 7SL RNA is 64% homologous to both the human and amphibian molecules. Despite the evolutionary distance between the species, significant blocks of homology to both the Alu and 7SL-specific portions of mammalian 7SL RNA can be found in the insect sequence. These results clearly demonstrate that the Alu sequence in 7SL RNA appeared in evolution before the mammalian radiation. We suggest that mammalian Alu sequences were derived from 7SL RNA (or DNA) by a deletion of the central 7SL-specific sequence, and are therefore processed 7SL RNA genes.     

Genome sequence of the ultrasmall unicellular red alga Cyanidioschyzon merolae 10D

Small, compact genomes of ultrasmall unicellular algae provide information on the basic and essential genes that support the lives of photosynthetic eukaryotes, including higher plants. Here we report the 16,520,305-base-pair sequence of the 20 chromosomes of the unicellular red alga Cyanidioschyzon merolae 10D as the first complete algal genome. We identified 5,331 genes in total, of which at least 86.3% were expressed. Unique characteristics of this genomic structure include: a lack of introns in all but 26 genes; only three copies of ribosomal DNA units that maintain the nucleolus; and two dynamin genes that are involved only in the division of mitochondria and plastids. The conserved mosaic origin of Calvin cycle enzymes in this red alga and in green plants supports the hypothesis of the existence of single primary plastid endosymbiosis. The lack of a myosin gene, in addition to the unexpressed actin gene, suggests a simpler system of cytokinesis. These results indicate that the C. merolae genome provides a model system with a simple gene composition for studying the origin, evolution and fundamental mechanisms of eukaryotic cells.     

土壤细菌基因资源的直接分离——16S核糖体RNA基因模式

细菌遗传资源的开发利用必须先分离纯培养物,但绝大多数环境细菌无法人工培养。由于基因工程技术能直接利用基因元件,为绕过人工培养的困难,我们以细菌１６Ｓ核糖体ＲＮＡ基因为模式,建立了直接从土壤中分离基因元件的方法,该方法包括直接从土壤中分离ＤＮＡ,ＰＣＲ扩增基因和ＰＣＲ产物克隆等步骤,为直接收集,利用土壤细菌遗传资源奠定了基础。     

Structure of the Escherichia coli ribosomal termination complex with release factor 2

Termination of protein synthesis occurs when the messenger RNA presents a stop codon in the ribosomal aminoacyl (A) site. Class I release factor proteins (RF1 or RF2) are believed to recognize stop codons via tripeptide motifs, leading to release of the completed polypeptide chain from its covalent attachment to transfer RNA in the ribosomal peptidyl (P) site. Class I RFs possess a conserved GGQ amino-acid motif that is thought to be involved directly in protein-transfer-RNA bond hydrolysis. Crystal structures of bacterial and eukaryotic class I RFs have been determined, but the mechanism of stop codon recognition and peptidyl-tRNA hydrolysis remains unclear. Here we present the structure of the Escherichia coli ribosome in a post-termination complex with RF2, obtained by single-particle cryo-electron microscopy (cryo-EM). Fitting the known 70S and RF2 structures into the electron density map reveals that RF2 adopts a different conformation on the ribosome when compared with the crystal structure of the isolated protein. The amino-terminal helical domain of RF2 contacts the factor-binding site of the ribosome, the 'SPF' loop of the protein is situated close to the mRNA, and the GGQ-containing domain of RF2 interacts with the peptidyl-transferase centre (PTC). By connecting the ribosomal decoding centre with the PTC, RF2 functionally mimics a tRNA molecule in the A site. Translational termination in eukaryotes is likely to be based on a similar mechanism.