Eukaryotic Small Subunit Ribosomal RNA Research Articles

Next-Generation Sequencing Assessment of Eukaryotic Diversity in Oil Sands Tailings Ponds Sediments and SurfaceWater.

Tailings ponds in the Athabasca oil sands (Canada) contain fluid wastes, generated by the extraction of bitumen from oil sands ores. Although the autochthonous prokaryotic communities have been relatively well characterized, almost nothing is known about microbial eukaryotes living in the anoxic soft sediments of tailings ponds or in the thin oxic layer of water that covers them. We carried out the first next-generation sequencing study of microbial eukaryotic diversity in oil sands tailings ponds. In metagenomes prepared from tailings sediment and surface water, we detected very low numbers of sequences encoding eukaryotic small subunit ribosomal RNA representing seven major taxonomic groups of protists. We also produced and analysed three amplicon-based 18S rRNA libraries prepared from sediment samples. These revealed a more diverse set of taxa, 169 different OTUs encompassing up to eleven higher order groups of eukaryotes, according to detailed classification using homology searching and phylogenetic methods. The 10 most abundant OTUs accounted for >90% of the total of reads, vs. large numbers of rare OTUs (<1% abundance). Despite the anoxic and hydrocarbon-enriched nature of the environment, the tailings ponds harbour complex communities of microbial eukaryotes indicating that these organisms should be taken into account when studying the microbiology of the oil sands.

Quantification of microbial communities in subsurface marine sediments of the Black Sea and off Namibia

Organic-rich subsurface marine sediments were taken by gravity coring up to a depth of 10 m below seafloor at six stations from the anoxic Black Sea and the Benguela upwelling system off Namibia during the research cruises Meteor 72-5 and 76-1, respectively. The quantitative microbial community composition at various sediment depths was analyzed using total cell counting, catalyzed reporter deposition – fluorescence in situ hybridization (CARD–FISH) and quantitative real-time PCR (Q-PCR). Total cell counts decreased with depths from 109 to 1010 cells/mL at the sediment surface to 107–109 cells/mL below one meter depth. Based on CARD–FISH and Q-PCR analyses overall similar proportions of Bacteria and Archaea were found. The down-core distribution of prokaryotic and eukaryotic small subunit ribosomal RNA genes (16S and 18S rRNA) as well as functional genes involved in different biogeochemical processes was quantified using Q-PCR. Crenarchaeota and the bacterial candidate division JS-1 as well as the classes Anaerolineae and Caldilineae of the phylum Chloroflexi were highly abundant. Less abundant but detectable in most of the samples were Eukarya as well as the metal and sulfate-reducing Geobacteraceae (only in the Benguela upwelling influenced sediments). The functional genes cbbL, encoding for the large subunit of RuBisCO, the genes dsrA and aprA, indicative of sulfate-reducers as well as the mcrA gene of methanogens were detected in the Benguela upwelling and Black Sea sediments. Overall, the high organic carbon content of the sediments goes along with high cell counts and high gene copy numbers, as well as an equal abundance of Bacteria and Archaea.

The Deep Biosphere in Terrestrial Sediments in the Chesapeake Bay Area, Virginia, USA

For the first time quantitative data on the abundance of Bacteria, Archaea, and Eukarya in deep terrestrial sediments are provided using multiple methods (total cell counting, quantitative real-time PCR, Q-PCR and catalyzed reporter deposition–fluorescence in situ hybridization, CARD–FISH). The oligotrophic (organic carbon content of ∼0.2%) deep terrestrial sediments in the Chesapeake Bay area at Eyreville, Virginia, USA, were drilled and sampled up to a depth of 140 m in 2006. The possibility of contamination during drilling was checked using fluorescent microspheres. Total cell counts decreased from 109 to 106 cells/g dry weight within the uppermost 20 m, and did not further decrease with depth below. Within the top 7 m, a significant proportion of the total cell counts could be detected with CARD–FISH. The CARD–FISH numbers for Bacteria were about an order of magnitude higher than those for Archaea. The dominance of Bacteria over Archaea was confirmed by Q-PCR. The down core quantitative distribution of prokaryotic and eukaryotic small subunit ribosomal RNA genes as well as functional genes involved in different biogeochemical processes was revealed by Q-PCR for the uppermost 10 m and for 80–140 m depth. Eukarya and the Fe(III)- and Mn(IV)-reducing bacterial group Geobacteriaceae were almost exclusively found in the uppermost meter (arable soil), where reactive iron was detected in higher amounts. The bacterial candidate division JS-1 and the classes Anaerolineae and Caldilineae of the phylum Chloroflexi, highly abundant in marine sediments, were found up to the maximum sampling depth in high copy numbers at this terrestrial site as well. A similar high abundance of the functional gene cbbL encoding for the large subunit of RubisCO suggests that autotrophic microorganisms could be relevant in addition to heterotrophs. The functional gene aprA of sulfate reducing bacteria was found within distinct layers up to ca. 100 m depth in low copy numbers. The gene mcrA of methanogens was not detectable. Cloning and sequencing data of 16S rRNA genes revealed sequences of typical soil Bacteria. The closest relatives of the archaeal sequences were Archaea recovered from terrestrial and marine environments. Phylogenetic analysis of the Crenarchaeota and Euryarchaeota revealed new members of the uncultured South African Gold Mine Group, Deep Sea Hydrothermal Vent Euryarchaeotal Group 6, and Miscellaneous Crenarcheotic Group clusters.

Molecular Evidence that Phylogenetically Diverged Ciliates Are Active in Microbial Mats of Deep‐Sea Cold‐Seep Sediment

Cold seeps are areas of the seafloor where hydrogen sulfide- and methane-rich fluid seepage occurs, often sustaining chemosynthetic ecosystems. It is well known that both archaea and bacteria oxidize sulfides and methane to produce chemical energy and that several endemic animals use this energy to thrive in cold seeps. On the other hand, there is little knowledge regarding diversity and ecology of microbial eukaryotes in this ecosystem. In this study we isolated environmental RNA and DNA from microbial mats of cold-seep sediment in Sagami Bay, Japan, and retrieved eukaryotic small-subunit ribosomal RNA sequences with polymerase chain reaction methods followed by clone library construction. Most RNA-derived clones obtained were from ciliates, although DNA-derived clones were mainly from the fungus Cryptococcus curvatus, suggesting that ciliates are active in the environment. The ciliate sequences were phylogenetically diverse, and represented eight known class lineages as well as undesignated lineages. Because most ciliates are bacterivorous, it is highly likely that the ciliates for which sequences were recovered play a role in the food web of this ecosystem as grazers of microbial mats. In addition, given that the environment studied is under highly reduced (anoxic) conditions, based on the prokaryotic community structure deduced from T-RFLP profiles, the ciliates detected may be obligatory or facultative anaerobes.

Rapid quantification and taxonomic classification of environmental DNA from both prokaryotic and eukaryotic origins using a microarray

A microarray has been designed using 62,358 probes matched to both prokaryotic and eukaryotic small-subunit ribosomal RNA genes. The array categorized environmental DNA to specific phylogenetic clusters in under 9 h. To a background of DNA generated from natural outdoor aerosols, known quantities of rRNA gene copies from distinct organisms were added producing corresponding hybridization intensity scores that correlated well with their concentrations ( r = 0.917). Reproducible differences in microbial community composition were observed by altering the genomic DNA extraction method. Notably, gentle extractions produced peak intensities for Mycoplasmatales and Burkholderiales, whereas a vigorous disruption produced peak intensities for Vibrionales, Clostridiales, and Bacillales.

Transition of Plasmodium vivax ribosome types corresponds to sporozoite differentiation in the mosquito

Two distinct small subunit ribosomal RNA (SSUrRNA) genes were amplified from the genomic DNA of Plasmodium vivax. Comparison of the two coding sequences reveals an overall divergence of 14.5% and most differences are clustered into the regions known to diverge rapidly in all eukaryotic SSUrRNAs. Oligonucleotides complementary to unique sequences of each gene have been used to distinguish the transcripts expressed either at schizogony in human blood (A gene) or at sporogony in the mosquito (C gene). These oligonucleotides were also used to monitor turnover of ribosomes during parasite development in mosquitoes. Transcripts of the A gene were predominant in the infected human blood and engorged mosquitoes but disappeared within 24 h after feeding. Expression of the C gene in mosquitoes was not detected until day 6 after the blood meal. A period of rapid accumulation of the C type rRNA from day 6 today 8 corresponds to differentiation of individual sporozoites within the oocyst. Possible functional implications relating to the timing of this transition are discussed.

A parsimony analysis of eukaryotic small subunit ribosomal RNA (18S) sequences

Using the principles of Felsentein's DNA parsimony analysis program (package PHYLIP 3.2), a program with the capacity to handle data sets of the magnitude of ss rRNA has been developed. With this program we applied an heuristic approach to 83 ss rRNA sequences from the Antwerp data bank, compared along 4230 positions of their alignment. Branch and bound can also be applied to a smaller number of sequences. We suggest a heuristic tree for a large number of species might be more interesting than a proven minimal tree for a smaller set of species. Comparisons are also made with earlier studies using distance matrix methods; caution is urged for deciding on the evolutionary position of widely divergent groups, whatever the tree building technique used.

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.

Demonstration of nucleomorph-encoded eukaryotic small subunit ribosomal RNA in cryptomonads

In cryptomonads, unicellular phototrophic flagellates, the plastid(s) is (are) located in a special narrow compartment which is bordered by two membranes; it harbours neither mitochondria nor Golgi dictyosomes but comprises eukaryotic ribosomes and starch grains together with a small organelle called the nucleomorph. The nucleomorph contains DNA and is surrounded by a double membrane with pores. It is thought to be the vestigial nucleus of a phototrophic eukaryotic endosymbiont. Cryptomonads are therefore supposed to represent an intermediate state in the evolution of complex plastids from endosymbionts. We have succeeded in isolating pure nucleomorph fractions, and can thus provide, using pulsed field gel electrophoresis, polymerase chain reaction and sequence analysis, definitive proof for the eukaryotic nature of the symbiont and its phylogenetic origin.

An overview of the secondary structure of the V4 region of eukaryotic small-subunit ribosomal RNA

The V4 region of the small subunit (18S) ribosomal RNA was examined in 72 different sequences representing a broad sample eukaryotic diversity. This domain is the most variable region of the 18S rRNA molecule and ranges in length from ca. 230 to over 500 bases. Based upon comparative analysis, secondary structural models were constructed for all sequences and the resulting generalized model shows that most organisms possess seven helices for this region. The protists and two insects show from one to as many as four helices in addition to the above seven. In this report, we summarize secondary structure information presented elsewhere for the V4 region, describe the general features for helical and apical regions, and identify signature sequences useful in helix identification. Our model generally agrees with other current concepts; however, we propose modifications or alternative structures for the start of the V4 region, the large protist inserts, and the sector that may possibly contain a pseudoknot.