Eukaryotic Small Subunit rRNA Research Articles

Watershed-Scale Fungal Community Characterization along a pH Gradient in a Subsurface Environment Cocontaminated with Uranium and Nitrate

The objective of this study was to characterize fungal communities in a subsurface environment cocontaminated with uranium and nitrate at the watershed scale and to determine the potential contribution of fungi to contaminant transformation (nitrate attenuation). The abundance, distribution, and diversity of fungi in subsurface groundwater samples were determined using quantitative and semiquantitative molecular techniques, including quantitative PCR of eukaryotic small-subunit rRNA genes and pyrosequencing of fungal internal transcribed spacer (ITS) regions. Potential bacterial and fungal denitrification was assessed in sediment-groundwater slurries amended with antimicrobial compounds and in fungal pure cultures isolated from the subsurface. Our results demonstrate that subsurface fungal communities are dominated by members of the phylum Ascomycota, and a pronounced shift in fungal community composition occurs across the groundwater pH gradient at the field site, with lower diversity observed under acidic (pH <4.5) conditions. Fungal isolates recovered from subsurface sediments, including cultures of the genus Coniochaeta, which were detected in abundance in pyrosequence libraries of site groundwater samples, were shown to reduce nitrate to nitrous oxide. Denitrifying fungal isolates recovered from the site were classified and found to be distributed broadly within the phylum Ascomycota and within a single genus of the Basidiomycota. Potential denitrification rate assays with sediment-groundwater slurries showed the potential for subsurface fungi to reduce nitrate to nitrous oxide under in situ acidic pH conditions.

Differential freshwater flagellate community response to bacterial food quality with a focus on Limnohabitans bacteria

Different bacterial strains can have different value as food for heterotrophic nanoflagellates (HNF), thus modulating HNF growth and community composition. We examined the influence of prey food quality using four Limnohabitans strains, one Polynucleobacter strain and one freshwater actinobacterial strain on growth (growth rate, length of lag phase and growth efficiency) and community composition of a natural HNF community from a freshwater reservoir. Pyrosequencing of eukaryotic small subunit rRNA amplicons was used to assess time-course changes in HNF community composition. All four Limnohabitans strains and the Polynucleobacter strain yielded significant HNF community growth while the actinobacterial strain did not although it was detected in HNF food vacuoles. Notably, even within the Limnohabitans strains we found significant prey-related differences in HNF growth parameters, which could not be related only to size of the bacterial prey. Sequence data characterizing the HNF communities showed also that different bacterial prey items induced highly significant differences in community composition of flagellates. Generally, Stramenopiles dominated the communities and phylotypes closely related to Pedospumella (Chrysophyceae) were most abundant bacterivorous flagellates rapidly reacting to addition of the bacterial prey of high food quality.

Community structure of microorganisms associated with reddish-brown iron-rich snow

Reddish-brown colored snow, containing spherical brown particles, has been observed in several mires in Japan. In order to characterize this remarkable phenomenon, the microbial community and chemical species in snow were analyzed. A core sample of snow which had a colored region was investigated and it revealed vertical shifts in physicochemical characteristics and the microbial community structure. The abundance of particles peaked within the colored layer, and correlated with the amount of reducible Fe(III). The interstitial water of the colored layer was enriched with Fe(II), and characterized by reduced concentration of dissolved methane. The bacterial community in the colored region was characterized by higher relative abundance of iron-reducing bacteria and methanotrophs. Aggregates of the brown particles were found as precipitates in snow melt pools, and were subjected to cloning analyses targeting several different genes. The majority of bacterial 16S rRNA gene clones belonged to the class Betaproteobacteria or the phylum Bacteroidetes. No snow algae were detected in the eukaryotic small subunit rRNA gene clone library. As a possible carbon source to sustain the community in the snow, involvements of carbon dioxide and methane were investigated by analyzing the genes involved in their assimilation. In the analyses of genes for ribulose-1,5-biphosphate carboxylase/oxygenase, clones related to sulfur oxidizers were obtained. The analysis of particulate methane monooxygenase genes indicated dominance of Methylobacter species. These results emphasized the uniqueness of this phenomenon, and iron reducers of the genus Geobacter are suggested to be the key organisms that could be investigated in order to understand the mechanism of this phenomenon.

Protist Genetic Diversity in the Acidic Hydrothermal Environments of Lassen Volcanic National Park, USA

We examined eukaryote genetic diversity in the hydrothermal environments of Lassen Volcanic National Park (LVNP), Northern California. We sampled hydrothermal areas of the Bumpass Hell, Sulfur Works, Devil's Kitchen, and Boiling Springs Lake sites, all of which included diverse acidic pools, mud pots, and streams with visible algal mats and biofilms. Temperatures varied from 15 to 85 degrees C and pH from 1.7 to 5.8. DNA extraction methods compared by denaturing gradient gel electrophoresis fingerprinting exhibited similar patterns, and showed limited diversity of eukaryotic small subunit (SSU) rRNA genes compared with prokaryotes. We successfully amplified eukaryotic SSU rRNA genes from most environments up to 68 degrees C. Cloned rDNA sequences reveal acidophilic protists dominate eukaryotes in LVNP hydrothermal environments. Most sites showed phototrophic assemblages dominated by chlorophytes and stramenopiles (diatoms and chrysophytes). Heterotrophic taxa, though less abundant, included diverse alveolates (ciliates), amoebae, and flagellates. Fungi were also found at most sites, and metazoans (hexapods, nematodes, platyhelminths) were sometimes detected in less acidic environments, especially in algal mats. While many cloned rDNA sequences showed 95%-99% identity to known acidophilic isolates or environmental clones from other acidic sites (Rio Tinto), sequence diversity generally declined both with decreasing pH and increasing temperature, and both were controlling physical variables on the abundance and distribution of organisms at our sites. However, a pool at 68 degrees C with pH 1.7 yielded the greatest number of distinct sequences. While some were likely contaminants from nearby cooler sites, we suggest that Lassen's acidic hydrothermal features may harbor novel protists.

Identities and Phylogenetic Comparisons of Posttranscriptional Modifications in 16 S Ribosomal RNA from Haloferax volcanii

Small subunit (16 S) rRNA from the archaeon Haloferax volcanii, for which sites of modification were previously reported, was examined using mass spectrometry. A census of all modified residues was taken by liquid chromatography/electrospray ionization-mass spectrometry analysis of a total nucleoside digest of the rRNA. Following rRNA hydrolysis by RNase T(1), accurate molecular mass values of oligonucleotide products were measured using liquid chromatography/electrospray ionization-mass spectrometry and compared with values predicted from the corresponding gene sequence. Three modified nucleosides, distributed over four conserved sites in the decoding region of the molecule, were characterized: 3-(3-amino-3-carboxypropyl)uridine-966, N(6)-methyladenosine-1501, and N(6),N(6)-dimethyladenosine-1518 and -1519 (all Escherichia coli numbering). Nucleoside 3-(3-amino-3-carboxypropyl)uridine, previously unknown in rRNA, occurs at a highly conserved site of modification in all three evolutionary domains but for which no structural assignment in archaea has been previously reported. Nucleoside N(6)-methyladenosine, not previously placed in archaeal rRNAs, frequently occurs at the analogous location in eukaryotic small subunit rRNA but not in bacteria. H. volcanii small subunit rRNA appears to reflect the phenotypically low modification level in the Crenarchaeota kingdom and is the only cytoplasmic small subunit rRNA shown to lack pseudouridine.

Comparative sequence analysis and patterns of covariation in RNA secondary structures.

A novel method of RNA secondary structure prediction based on a comparison of nucleotide sequences is described. This method correctly predicts nearly all evolutionarily conserved secondary structures of five different RNAs: tRNA, 5S rRNA, bacterial ribonuclease P (RNase P) RNA, eukaryotic small subunit rRNA, and the 3' untranslated region (UTR) of the Drosophila bicoid (bcd) mRNA. Furthermore, covariations occurring in the helices of these conserved RNA structures are analyzed. Two physical parameters are found to be important determinants of the evolution of compensatory mutations: the length of a helix and the distance between base-pairing nucleotides. For the helices of bcd 3' UTR mRNA and RNase P RNA, a positive correlation between the rate of compensatory evolution and helix length is found. The analysis of Drosophila bcd 3' UTR mRNA further revealed that the rate of compensatory evolution decreases with the physical distance between base-pairing residues. This result is in qualitative agreement with Kimura's model of compensatory fitness interactions, which assumes that mutations occurring in RNA helices are individually deleterious but become neutral in appropriate combinations.

RAGA: RNA sequence alignment by genetic algorithm

We describe a new approach for accurately aligning two homologous RNA sequences when the secondary structure of one of them is known. To do so we developed two software packages, called RAGA and PRAGA, which use a genetic algorithm approach to optimize the alignments. RAGA is mainly an extension of SAGA, an earlier package for multiple protein sequence alignment. In PRAGA several genetic algorithms run in parallel and exchange individual solutions. This method allows us to optimize an objective function that describes the quality of a RNA pairwise alignment, taking into account both primary and secondary structure, including pseudoknots. We report results obtained using PRAGA on nine test cases of pairs of eukaryotic small subunit rRNA sequence (nuclear and mitochondrial).

Evolutionary Relationships Among Higher Fungi Inferred from Small Ribosomal Subunit RNA Sequence Analysis

The primary structure of the small ribosomal subunit RNA. (SSU rRNA) was determined for 13 species belonging to 10 ascomycete families and for the basidiomycetous anamorphic yeast Rhodotorula glutinis. The sequences were fitted into an alignment of all hitherto published complete or nearly complete eukaryotic small subunit rRNA sequences. The evolutionary relationships within the fungi were examined by construction of a tree from 87 SSU rRNA sequences, corresponding to 71 different species, by means of a distance matrix method and bootstrap analysis. It confirms the early divergence of the zygomycetes and the classical division of the higher fungi into basidiomycetes and ascomycetes. The basidiomycetes are divided into true basidiomycetes and ustomycetes. Within the ascomycetes, the major subdivisions hemiascomycetes and euascomycetes can be recognized. However, Schizosaccharomyces pombe does not belong to the cluster of the hemiascomycetes, to which it is assigned in classical taxonomic schemes, but forms a distinct lineage. Among the euascomycetes, the plectomycetes and the pyrenomycetes can be distinguished. Within the hemiascomycetes, the polyphyly of genera like Pichia or Candida and of families like the Dipodascaceae and the Saccharomycetaceae can be observed.

Characterization of the rDNA unit and sequence analysis of the small subunit rRNA and 5.8S rRNA genes from Tritrichomonas foetus

The ribosomal RNA gene unit of the protozoan parasite Tritrichomonas foetus has been cloned and analyzed. Southern blot analysis of the genomic DNA showed that the ribosomal RNA gene unit is organized as a tandem head to tail repeat with a unit length of 6 kb. By Northern analysis a primary transcript of 5.8 kb was detected. Copy number analysis showed the presence of 12 copies of the ribosomal RNA gene unit. The lengths of the small subunit ribosomal RNA and 5.8S ribosomal RNA are 1571 bp and 159 bp, respectively, as determined by sequence analysis. The T. foetus small subunit ribosomal RNA sequence is one of the shortest eukaryotic small subunit rRNA sequences, similar in length to those from 2 other amitochondrial protists. Although shorter than the majority of the eukaryotic small subunit ribosomal RNAs, this sequence maintains the primary and secondary structure common to all eukaryotic small subunit ribosomal RNA structures, while truncating sequences found within the eukaryotic variable regions. The length of the large subunit ribosomal RNA was measured at 2.5 kb.

Functional topography of human ribosomes as studied by affinity labeling with reactive mRNA analogs

Derivatives of 5′- 32P labeled (pU) 3 an (pU) 6 bearing 4-(N-2-chloroethyl-N-methylamino)benzylmethylamine residue attached to 5′-phosphate via phoshamide bond and (Up) 5U[ 32P]pC and (Up) 11U[ 32P]pC bearing 4-(N-2-chlroethyl-N-methylamino)benzyl residue attached to 3′-end via benzylidene bond were applied for the affinity labeling of 80S ribosomes from human placenta in the presence of a cognate tRNA. The derivatives of 32P-labeled pAUG and pAUGU 3 analogous to the 5′-phosphamides of (pU) n were used for affinity labeling of 40S subunits in the presence of ternary complex eIF-2-GTP·Met-tRNA f. The sites of the reagents' attachment to 18S ribosomal RNA were identified by blot-hybridization of the modified 18S rRNA with restriction fragments of the corresponding rDNA. They were found to be located within positions 976–1057 for (pU) 6 and pAUGU 3 derivatives and within 976–1164 for (pU) 3 and pAUG ones. The sites of 18S rRNA modification with the derivatives of (Up) 5UpC and (Up) 11UpC were found within positions 1610–1869 at 3′-end of the molecule. All the sites identified here are located presumably within highly conserved parts of the eukaryotic small subunit rRNA secondary structure.

Evolutionary diversity of eukaryotic small-subunit rRNA genes.

The small-subunit rRNA gene sequences of the flagellated protists Euglena gracilis and Trypanosoma brucei were determined and compared to those of other eukaryotes. A phylogenetic tree was constructed in which the earliest branching among the eukaryotes is represented by E. gracilis. The E. gracilis divergence far antedates a period of massive evolutionary radiation that gave rise to the plants, animals, fungi, and certain groups of protists such as ciliates and the acanthamoebae. The genetic diversity in this collection of eukaryotes is seen to exceed that displayed within either the eubacterial or the archaebacterial lines of descent.