Archaeal diversity and community structure in a Swedish barley field: Specificity of the EK510R/(EURY498) 16S rDNA primer

The aim of this study was to analyze a total euryarchaeal community at DNA and RNA levels in a Swedish barley field with relation to soil depth (0-10 and 20-30 cm layers), soil fraction (bulk soil and rhizosphere) and time (August and November sample collection). Amplification of 16S rRNA gene using the archaeal universal A2F and Euryarchaea specific EK510R/(EURY498) primer pair, combined with denaturing gradient gel electrophoresis (DGGE), revealed distinct differences between rDNA and rRNA DGGE profiles. The soil depth, time, or rhizosphere effects did not significantly influence Archaeal community structure. Surprisingly, sequence analysis of DGGE-derived amplicons revealed the presence of Euryarchaea as well as uncultured soil Crenarchaea affiliated with group 1. In agreement, sequence comparison analyses showed that the majority of uncultured Crenarchaea group 1 had almost 100% sequence complementarity to the 3' end of the EK510R/(EURY498) primer. Therefore, we propose that EK510R/(EURY498R) is a universal archaeal primer rather than a Euryarchaea specific SSUrRNA primer. Hence, considerable care should be taken during application of this primer in studies of euryarchaeal biodiversity in soil environments.     

Comparative analysis of codon usage bias in Crenarchaea and Euryarchaea genome reveals differential preference of synonymous codons to encode highly expressed ribosomal and RNA polymerase proteins

The present study was undertaken to investigate the pattern of optimal codon usage in Archaea. Comparative analysis was executed to understand the pattern of codon usage bias between the high expression genes (HEG) and the whole genomes in two Archaeal phyla, Crenarchaea and Euryarchaea. The G + C% of the HEG was found to be less in comparison to the genome G + C% in Crenarchaea, whereas reverse was the case in Euryarchaea. The preponderance of U/A ending codons that code for HEG in Crenarchaea was in sharp contrast to the C/G ended ones in Euryarchaea. The analysis revealed prevalence of U-ending codons even within the WWY (nucleotide ambiguity code) families in Crenarchaea vis-à-vis Euryarchaea, bacteria and Eukarya. No plausible interpretation of the observed disparity could be made either in the context of tRNA gene composition or genome G + C%. The results in this study attested that the preferential biasness for codons in HEG of Crenarchaea might be different from Euryarchaea. The main highlights are (i) varied CUB in the HEG and in the whole genomes in Euryarchaea and Crenarchaea. (ii) Crenarchaea was found to have some unusual optimal codons (OCs) compared to other organisms. (iii) G + C% (and GC₃) of the HEG were different from the genome G + C% in the two phyla. (iv) Genome G + C% and tRNA gene number failed to explain CUB in Crenarchaea. (v) Translational selection is possibly responsible for A + T rich OCs in Crenarchaea.     

Bacterial, archaeal and eukaryotic diversity of smooth and pustular microbial mat communities in the hypersaline lagoon of Shark Bay

The bacterial, archaeal and eukaryotic populations of nonlithifying mats with pustular and smooth morphology from Hamelin Pool, Shark Bay were characterised using small subunit rRNA gene analysis and microbial isolation. A highly diverse bacterial population was detected for each mat, with 16S rDNA clones related to Actinobacteria, Bacteroidetes, Chloroflexi, Cyanobacteria, Gemmatimonas, Planctomycetes, Alphaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Verrucomicrobia and candidate division TM6 present in each mat. Spirochaetes were detected in the smooth mat only, whereas candidate division OP11 was only detected in the pustular mat. Targeting populations with specific primers revealed additional cyanobacterial diversity. The archaeal population of the pustular mat was comprised purely of Halobacteriales, whereas the smooth mat contained 16S rDNA clones from the Halobacteriales, two groups of Euryarchaea with no close characterised matches, and the Thaumarchaea. Nematodes and fungi were present in each mat type, with diatom 18S rDNA clones only obtained from the smooth mat, and tardigrade and microalgae clones only retrieved from the pustular mat. Cultured isolates belonged to the Firmicutes, Gammaproteobacteria, Alphaproteobacteria, Bacteroidetes, Actinobacteria, Cyanobacteria, and Halobacteriales. The mat populations were significantly more diverse than those previously reported for Hamelin Pool stromatolites, suggesting specific microbial populations may be associated with the nonlithifying and lithifying microbial communities of Hamelin Pool.     

Archaeal community diversity in municipal waste landfill sites

Despite the pivotal role of archaea in methane production in landfills, the identity, ecology, and functional diversity of these microorganisms and their link to environmental factors remain largely unknown. We collected 11 landfill leachate samples from six geographically distinct landfills of different ages in China and analyzed the archaeal community by bar-coded 454 pyrosequencing. We retrieved 121,797 sequences from a total of 167,583 sequences (average length of 464 bp). The archaeal community was geographically structured, and nonabundant taxa primarily contributed to the observed dissimilarities. Canonical correlation analysis (CCA) suggested that the total phosphorous (TP), nitrate, and conductivity are important drivers for shaping the archaeal community. The hydrogenotrophic methanogens Methanomicrobiales and Methanobacteriales greatly dominated 9 of 11 samples, ranging from 83.7 to 99.5 %. These methanogens also dominated the remaining two samples, accounting for 70.3 and 58.8 %, respectively. Interestingly, for all of the studied Chinese landfills, 16S rRNA analysis indicated the predominance of hydrogenotrophic methanogens.     

A highly conserved family of inactivated archaeal B family DNA polymerases

   

A widespread and highly conserved family of apparently inactivated derivatives of archaeal B-family DNA polymerases is described. Phylogenetic analysis shows that the inactivated forms comprise a distinct clade among archaeal B-family polymerases and that, within this clade, Euryarchaea and Crenarchaea are clearly separated from each other and from a small group of bacterial homologs. These findings are compatible with an ancient duplication of the DNA polymerase gene followed by inactivation and parallel loss in some of the lineages although contribution of horizontal gene transfer cannot be ruled out. The inactivated derivative of the archaeal DNA polymerase could form a complex with the active paralog and play a structural role in DNA replication.     

Thymidine Kinases in Archaea

Twenty-six fully sequenced archaeal genomes were searched for genes coding for putative deoxyribonucleoside kinases (dNKs). We identified only 5 human-like thymidine kinase 1 genes (TK1s) and none for non-TK1 kinases. Four TK1s were identified in the Euryarchaea and one was found in the Crenarchaea, while none was found in Nanoarchaeum. The identified TK1s have high identity to Gram-positive bacteria TK1s. The TK1s from archaea, Gram-positive bacteria and eukaryotes share the same common ancestor, while the TK1s from Gram-negative bacteria belong to a less-related subgroup. It seems that a functional deoxyribonucleoside salvage pathway is not crucial for the archaeal cell.     

Thymidine kinases in archaea

Twenty-six fully sequenced archaeal genomes were searched for genes coding for putative deoxyribonucleoside kinases (dNKs). We identified only 5 human-like thymidine kinase 1 genes (TK1s) and none for non-TK1 kinases. Four TK1s were identified in the Euryarchaea and one was found in the Crenarchaea, while none was found in Nanoarchaeum. The identified TK1s have high identity to Gram-positive bacteria TK1s. The TK1s from archaea, Gram-positive bacteria and eukaryotes share the same common ancestor, while the TK1s from Gram-negative bacteria belong to a less-related subgroup. It seems that a functional deoxyribonucleoside salvage pathway is not crucial for the archaeal cell.     

Archaeal rRNA diversity and methane production in deep boreal peat

Northern peatlands play a major role in the global carbon cycle as sinks for CO₂ and as sources of CH₄. These diverse ecosystems develop through accumulation of partially decomposed plant material as peat. With increasing depth, peat becomes more and more recalcitrant due to its longer exposure to decomposing processes. Compared with surface peat, deeper peat sediments remain microbiologically poorly described. We detected active archaeal communities even in the deep bottom layers (−220/−280 cm) of two Finnish fen-type peatlands by 16S rRNA-based terminal restriction fragment length polymorphism analysis. In the sediments of the northern study site, all detected archaea were methanogens with Rice Cluster II (RC-II) and Methanosaetaceae as major groups. In southern peatland, Crenarchaeota of a rare unidentified cluster were present together with mainly RC-II methanogens. RNA profiles showed a larger archaeal diversity than DNA-based community profiles, suggesting that small but active populations were better visualized with rRNA. In addition, potential methane production measurements indicated methanogenic activity throughout the vertical peat profiles.     

Phylogenetic Diversity of the Archaeal Community in a Continental High-Temperature,Water-Flooded Petroleum Reservoir

The diversity of an archaeal community was analyzed in the water from a continental high-temperature, long-term water-flooded petroleum reservoir in Huabei Oilfield in China. The archaea were characterized by their 16S rRNA genes. An archaeal 16S rDNA clone library was constructed from the DNA isolated from the formation water, and 237 randomly selected positive clones were clustered in 28 phylotypes by sequencing analyses. Phylogenetic analysis of these sequences indicated that the dominant members of the archaeal phylotypes were affiliated with the order Methanomicrobiales. Totally, the archaeal community was composed of methanogens belonging to four orders: Methanobacteriales, Methanococcales, Methanomicrobiales, and Methanosarcinales. Most of the clones clustered with sequences previously described for methanogens, but there was a difference in the relative distribution of sequences detected here as compared to that of previous studies. Some thermophilic methanogens detected had been previously isolated from a number of high-temperature petroleum reservoirs worldwide; thus, they might exhibit adaptations to the environments and be the common habitants of geothermally heated subsurface environments.     

Local Conditions Structure Unique Archaeal Communities in the Anoxic Sediments of Meromictic Lake Kivu

Meromictic Lake Kivu is renowned for its enormous quantity of methane dissolved in the hypolimnion. The methane is primarily of biological origin, and its concentration has been increasing in the past half-century. Insight into the origin of methane production in Lake Kivu has become relevant with the recent commercial extraction of methane from the hypolimnion. This study provides the first culture-independent approach to identifying the archaeal communities present in Lake Kivu sediments at the sediment-water interface. Terminal restriction fragment length polymorphism analysis suggests considerable heterogeneity in the archaeal community composition at varying sample locations. This diversity reflects changes in the geochemical conditions in the sediment and the overlying water, which are an effect of local groundwater inflows. A more in-depth look at the archaeal community composition by clone library analysis revealed diverse phylogenies of Euryarchaeota and Crenarachaeota. Many of the sequences in the clone libraries belonged to globally distributed archaeal clades such as the rice cluster V and Lake Dagow sediment environmental clusters. Several of the determined clades were previously thought to be rare among freshwater sediment Archaea (e.g., sequences related to the SAGMEG-1 clade). Surprisingly, there was no observed relation of clones to known hydrogentrophic methanogens and less than 2 % of clones were related to acetoclastic methanogens. The local variability, diversity, and novelty of the archaeal community structure in Lake Kivu should be considered when making assumptions on the biogeochemical functioning of its sediments.     

The impact of aridification and vegetation type on changes in the community structure of methane-cycling microorganisms in Japanese wetland soils

Over the years, the wetlands covered by Sphagnum in Bibai, Japan have been turning into areas of aridity, resulting in an invasion of Sasa into the bogs. Yet little is known about the methane-cycling microorganisms in such environments. In this study, the methanotrophic, methanogenic, and archaeal community structures within these two types of wetland vegetation were studied by phylogenetic analysis targeting particulate methane monooxygenase (pmoA), methyl coenzyme M reductase (mcrA), and the archaeal 16S rRNA gene. The pmoA library indicated that Methylomonas and Methylocystis predominated in the Sphagnum-covered and Sasa-invaded areas, respectively. The mcrA and 16S rRNA libraries indicated that Methanoregula were abundant methanogens in the Sphagnum-covered area. In the Sasa-invaded area, by contrast, mcrA genes were not detected, and no 16S rRNA clones were affiliated with previously known methanogens. Because the Sasa-invaded area still produced methane, of the various uncultured populations detected, novel euryarchaeotal lineages are candidate methane producers.     

The Thaumarchaeota: an emerging view of their phylogeny and ecophysiology

Thaumarchaeota range among the most abundant archaea on Earth. Initially classified as 'mesophilic Crenarchaeota', comparative genomics has recently revealed that they form a separate and deep-branching phylum within the Archaea. This novel phylum comprises in 16S rRNA gene trees not only all known archaeal ammonia oxidizers but also several clusters of environmental sequences representing microorganisms with unknown energy metabolism. Ecophysiological studies of ammonia-oxidizing Thaumarchaeota suggest adaptation to low ammonia concentrations and an autotrophic or possibly mixotrophic lifestyle. Extrapolating from the wide substrate range of copper-containing membrane-bound monooxygenases, to which the thaumarchaeal ammonia monooxygenases belong, the use of substrates other than ammonia for generating energy by some members of the Thaumarchaeota seems likely.     

Phylogenetic Diversity of Archaea and Bacteria in a Deep Subsurface Paleosol

Abstract A low-biomass paleosol 188 m below the ground surface at the Department of Energy's Hanford Site in south-central Washington State was recovered and maintained at the in situ temperature (17°C) as an intact core or homogenized sediment for 0, 1, 3, 10, and 21 weeks post-sampling. Bacterial and archaeal 16S rRNA genes were amplified by PCR and cloned. Of 746 bacterial and 190 archaeal clones that were categorized by restriction fragment length polymorphism (RFLP), 242 bacterial and 16 archaeal clones were partially sequenced and compared against the small subunit ribosomal RNA database (RDP) and GenBank. Six bacterial and 16 archaeal clones sequences, with little similarity to those in public databases, were sequenced in their entirety, and subjected to more detained phylogenetic analysis. The most frequently occurring clones types were related to Pseudomonas, Bacillus, Micrococcus, Clavibacter, Nocardioides, Burkholderia, Comamonas, and Erythromicrobium. Clone sequences whose RDP similarity value was ≥0.6 consistently grouped with their nearest RDP neighbor during phylogenetic analysis. Six truly novel eubacterial sequences were identified; they consistently cluster with or near the Chloroflexaceae and sequences recovered from the Sargasso Sea. Sixteen unique archaeal RFLP groups were identified from 190 randomly-sampled clones. The novel archaeal rDNA clones formed a coherent clade along the major Crenarchaea branch containing all previously described mesophilic crenarchae clones, but remained firmly associated with 16S rDNA clones previously obtained from a thermal Fe/S spring in Yellowstone National Park. The wealth of group-specific genetic information identified during this study will now allow us to address specific hypotheses related to in situ stimulation of these deep subsurface microorganisms and changes in microbial community composition resulting from subsurface contamination or remediation processes at the Hanford Site. Revised: 21 October 1997; Accepted: 20 November 1997     

Molecular characterization of microbial communities in deep coal seam groundwater of northern Japan

We investigated microbial methanogenesis and community structure based on 16S rRNA gene sequences from a coal seam aquifer located 843–907 m below ground level in northern Japan; additionally, we studied the δ¹³C and δ²H (δD) of coal‐bed gases and other physicochemical parameters. Although isotopic analysis suggested a thermocatalytic origin for the gases, the microbial activity and community structure strongly implied the existence of methanogenic microbial communities in situ. Methane was generated in the enrichment cultures of the hydrogenotrophic and methylotrophic microorganisms obtained from coal seam groundwater. Methanogen clones dominated the archaeal 16S rRNA gene libraries and were mostly related to the hydrogenotrophic genus Methanoculleus and the methylotrophic genus Methanolobus. Bacterial 16S rRNA gene libraries were dominated by the clones related to the genera Acetobacterium and Syntrophus which have a symbiotic association with methanogens. LIBSHUFF analysis revealed that N₂ gas injected into the coal seam (for enhanced methane production) does not affect the coverage of archaeal and bacterial populations. However, amova analysis does provide evidence for a change in the genetic diversity of archaeal populations that are dominated by methanogens. Therefore, N₂ injection into the coal seam might affect the cycling of matter by methanogens in situ.     

Thermophilic methanogens in rice field soil

The soil temperature in flooded Italian rice fields is generally lower than 30°C. However, two temperature optima at ≈ 41°C and 50°C were found when soil slurries were anoxically incubated at a temperature range of 10–80°C. The second temperature optimum indicates the presence of thermophilic methanogens in the rice field soil. Experiments with ¹⁴C-labelled bicarbonate showed that the thermophilic CH₄ was exclusively produced from H₂/CO₂. Terminal restriction fragment length polymorphism (T-RFLP) of archaeal SSU rRNA gene fragments revealed a dramatic change in the archaeal community structure at temperatures > 37°C, with the euryarchaeotal rice cluster I becoming the dominant group (about 80%). A clone library of archaeal SSU rRNA gene fragments generated at 49°C was also dominated (10 out of 11 clones) by rice cluster I. Our results demonstrate that Italian rice field soil contains thermophilic methanogenic activity that was most probably a result of members of the as yet uncultivated euryarchaeotal rice cluster I.     

Archaeal DNA replication: spotlight on a rapidly moving field

The replication of DNA is a fundamental step in the cell cycle, which must be coordinated with cell division to ensure that the daughter cells inherit the same genomic material as the parental cell. The recently published complete genome sequences of some archaeal species together with preliminary biochemical studies suggest that the Archaea quite likely duplicate their chromosome by using replication machinery that seems to be a simplified version of the eukaryotic machinery, although their metabolic facets and their cellular morphology are prokaryotic-like. This review is focused on recent progress on the structural and functional analysis of proteins and enzymes involved in the initiation and elongation steps of DNA replication in Archaea. Differences between the genome replication apparatus of the Euryarchaea and the Crenarchaea (the two main phylogenetic divisions of the Archaea domain) are highlighted.     

Diversity of prokaryotes associated with soils around coal-fire gas vents in MaNasi county of Xinjiang, China

Bacterial and archaeal diversity in surface soils of three coal-fire vents was investigated by T-RFLP analysis and clone libraries of 16S rRNA genes. Soil analysis showed that underground coal fires significantly influenced soil pH, moisture and NO₃ ^? content but had little effect on other elements, organic matter and available nutrients. Hierarchical cluster analysis showed that bacterial community patterns in the soils were very similar, but abundance varied with geographic distance. A clone library from one soil showed that the bacterial community was mainly composed of Firmicutes, Proteobacteria, Acidobacteria, Bacteroidetes, Planctomycetes, Actinobacteria, and unidentified groups. Of these, Firmicutes was the most abundant, accounting for 71.4 % of the clones, and was mainly represented by the genera Bacillus and Paenibacillus. Archaeal phylotypes were closely related to uncultivated species of the phyla Crenarchaeota (97.9 % of clones) and Thaumarchaeota (2.1 %). About 28 % of archaeal phylotypes were associated with ammonia oxidization, especially phylotypes that were highly related to a novel, ammonia-oxidizing isolate from the phylum Thaumarchaeota. These results suggested that microbial communities in the soils were diverse and might contain a large number of novel cultivable species with the potential to assimilate materials by heterotrophic metabolism at high temperature.     

Prokaryotic diversity in Zostera noltii-colonized marine sediments

The diversity of microorganisms present in a sediment colonized by the phanerogam Zostera noltii has been analyzed. Microbial DNA was extracted and used for constructing two 16S rDNA clone libraries for Bacteria and Archaea. Bacterial diversity was very high in these samples, since 57 different sequences were found among the 60 clones analyzed. Eight major lineages of the Domain Bacteria were represented in the library. The most frequently retrieved bacterial group (36% of the clones) was delta-Proteobacteria related to sulfate-reducing bacteria. The second most abundant group (27%) was gamma-Proteobacteria, including five clones closely related to S-oxidizing endosymbionts. The archaeal clone library included members of Crenarchaeota and Euryarchaeota, with nine different sequences among the 15 analyzed clones, indicating less diversity when compared to the Bacteria organisms. None of these sequences was closely related to cultured Archaea organisms.     

Diversity and ubiquity of thermophilic methanogenic archaea in temperate anoxic soils

Temperate rice field soil from Vercelli (Italy) contains moderately thermophilic methanogens of the yet uncultivated rice cluster I (RC-I), which become prevalent upon incubation at temperatures of 45-50 degrees C. We studied whether such thermophilic methanogens were ubiquitously present in anoxic soils. Incubation of different rice field soils (from Italy, China and the Philippines) and flooded riparian soils (from the Netherlands) at 45 degrees C resulted in vigorous CH(4) production after a lag phase of about 10 days. The archaeal community structure in the soils was analysed by terminal restriction fragment length polymorphism (T-RFLP) targeting the SSU rRNA genes retrieved from the soil, and by cloning and sequencing. Clones of RC-I methanogens mostly exhibited T-RF of 393 bp, but also terminal restriction fragment (T-RF) of 158 and 258 bp length, indicating a larger diversity than previously assumed. No RC-I methanogens were initially found in flooded riparian soils. However, these archaea became abundant upon incubation of the soil at 45 degrees C. Thermophilic RC-I methanogens were also found in the rice field soils from Pavia, Pila and Gapan. However, the archaeal communities in these soils also contained other methanogenic archaea at high temperature. Rice field soil from Buggalon, on the other hand, only contained thermophilic Methanomicrobiales rather than RC-I methanogens, and rice field soil from Jurong mostly Methanomicrobiales and only a few RC-I methanogens. The archaeal community of rice field soil from Zhenjiang almost exclusively consisted of Methanosarcinaceae when incubated at high temperature. Our results show that moderately thermophilic methanogens are common in temperate soils. However, RC-I methanogens are not always dominating or ubiquitous.     

Diversity of bovine rumen methanogens In vitro in the presence of condensed tannins, as determined by sequence analysis of 16S rRNA gene library

Molecular diversity of rumen archaeal populations from bovine rumen fluid incubated with or without condensed tannins was investigated using 16S rRNA gene libraries. The predominant order of rumen archaea in the 16S rRNA gene libraries of the control and condensed tannins treatment was found to belong to a novel group of rumen archaea that is distantly related to the order Thermoplasmatales, with 59.5% (15 phylotypes) and 81.43% (21 phylotypes) of the total clones from the control and treatment clone libraries, respectively. The 16S rRNA gene library of the control was found to have higher proportions of methanogens from the orders Methanomicrobiales (32%) and Methanobacteriales (8.5%) as compared to those found in the condensed tannins treatment clone library in both orders (16.88% and 1.68% respectively). The phylotype distributed in the order Methanosarcinales was only found in the control clone library. The study indicated that condensed tannins could alter the diversity of bovine rumen methanogens.