Archaeal Sequences Research Articles

BBProF: An Asynchronous Application Server for Rapid Identification of Proteins Associated with Bacterial Bioleaching

Recovery of metal value, especially from low-grade ores and overburden minerals using acidophilic bacteria through the process of bioleaching is an environmentally benign and commercially scalable biotechnology. In recent years, while the “OMICS” landscape has been witnessing extensive application of computational tools to understand and interpret global biological sequence data, a dedicated bioinformatic server for analysis of bacterial information in the context of its bioleaching ability is not available. We have developed an on-line Bacterial Bioleaching Protein Finder (BBProF) System, which rapidly identifies novel proteins involved in a bacterial bioleaching process and also performs phylogenetic analysis of 16S rRNA genes. BBProF uses the features of Asynchronous Java Script and XML (AJAX) to provide an efficient and fast user experience with minimal requirement of network bandwidth. In the input module the server accepts any bacterial or archaeal complete genome sequence in RAW format and provides a list of proteins involved in the microbial leaching process. BBProF web server is integrated with the European Bioinformatics Institute (EBI) web services such as BLAST for homology search and InterProScan for functional characterization of output protein sequences. Studying evolutionary relationship of bacterial strains of interest using Muscle and ClustalW2 phylogeny web services from EBI is another key feature of our server, where 16S rRNA gene sequences are considered as input through a JQUERY interface along with the sequences present in the BBProF database library. Complete genome sequences of 24 bioleaching microorganism characterized by genomic and physiological study in the laboratory and their respective 16S rRNA gene sequences were stored in the database of the BBProF library. To our knowledge BBProF is the first integrated bioinformatic web server that demonstrates its utility in identifying potential bioleaching bacteria. We hope that the server facilitate ongoing comparative genomic studies on of bioleaching microorganisms and also assist in identification and design of novel microbial consortia that are optimally efficient bioleaching agents.

Multiple approaches to characterize the microbial community in a thermophilic anaerobic digester running on swine manure: A case study

In this study, the first survey of microbial community in thermophilic anaerobic digester using swine manure as sole feedstock was performed by multiple approaches including denaturing gradient gel electrophoresis (DGGE), clone library and pyrosequencing techniques. The integrated analysis of 21 DGGE bands, 126 clones and 8506 pyrosequencing read sequences revealed that Clostridia from the phylum Firmicutes account for the most dominant Bacteria. In addition, our analysis also identified additional taxa that were missed by the previous researches, including members of the bacterial phyla Synergistetes, Planctomycetes, Armatimonadetes, Chloroflexi and Nitrospira which might also play a role in thermophilic anaerobic digester. Most archaeal 16S rRNA sequences could be assigned to the order Methanobacteriales instead of Methanomicrobiales comparing to previous studies. In addition, this study reported that the member of Methanothermobacter genus was firstly found in thermophilic anaerobic digester.

Integrating genomics into the taxonomy and systematics of the Bacteria and Archaea

The polyphasic approach used today in the taxonomy and systematics of the Bacteria and Archaea includes the use of phenotypic, chemotaxonomic and genotypic data. The use of 16S rRNA gene sequence data has revolutionized our understanding of the microbial world and led to a rapid increase in the number of descriptions of novel taxa, especially at the species level. It has allowed in many cases for the demarcation of taxa into distinct species, but its limitations in a number of groups have resulted in the continued use of DNA-DNA hybridization. As technology has improved, next-generation sequencing (NGS) has provided a rapid and cost-effective approach to obtaining whole-genome sequences of microbial strains. Although some 12,000 bacterial or archaeal genome sequences are available for comparison, only 1725 of these are of actual type strains, limiting the use of genomic data in comparative taxonomic studies when there are nearly 11,000 type strains. Efforts to obtain complete genome sequences of all type strains are critical to the future of microbial systematics. The incorporation of genomics into the taxonomy and systematics of the Bacteria and Archaea coupled with computational advances will boost the credibility of taxonomy in the genomic era. This special issue of International Journal of Systematic and Evolutionary Microbiology contains both original research and review articles covering the use of genomic sequence data in microbial taxonomy and systematics. It includes contributions on specific taxa as well as outlines of approaches for incorporating genomics into new strain isolation to new taxon description workflows.

Meta'omic Analytic Techniques for Studying the Intestinal Microbiome

Nucleotide sequencing has become increasingly common and affordable, and is now a vital tool for studies of the human microbiome. Comprehensive microbial community surveys such as MetaHit and the Human Microbiome Project have described the composition and molecular functional profile of the healthy (normal) intestinal microbiome. This knowledge will increase our ability to analyze host and microbial DNA (genome) and RNA (transcriptome) sequences. Bioinformatic and statistical tools then can be used to identify dysbioses that might cause disease, and potential treatments. Analyses that identify perturbations in specific molecules can leverage thousands of culture-based isolate genomes to contextualize culture-independent sequences, or may integrate sequence data with whole-community functional assays such as metaproteomic or metabolomic analyses. We review the state of available systems-level models for studies of the intestinal microbiome, along with analytic techniques and tools that can be used to determine its functional capabilities in healthy and unhealthy individuals.

Metagenomic analysis of microbial consortium from natural crude oil that seeps into the marine ecosystem offshore Southern California.

Crude oils can be major contaminants of the marine ecosystem and microorganisms play a significant role in the degradation of its main constituents. To increase our understanding of the microbial hydrocarbon degradation process in the marine ecosystem, we collected crude oil from an active seep area located in the Santa Barbara Channel (SBC) and generated a total of about 52 Gb of raw metagenomic sequence data. The assembled data comprised ~500 Mb, representing ~1.1 million genes derived primarily from chemolithoautotrophic bacteria. Members of Oceanospirillales, a bacterial order belonging to the Deltaproteobacteria, recruited less than 2% of the assembled genes within the SBC metagenome. In contrast, the microbial community associated with the oil plume that developed in the aftermath of the Deepwater Horizon (DWH) blowout in 2010, was dominated by Oceanospirillales, which comprised more than 60% of the metagenomic data generated from the DWH oil plume. This suggests that Oceanospirillales might play a less significant role in the microbially mediated hydrocarbon conversion within the SBC seep oil compared to the DWH plume oil. We hypothesize that this difference results from the SBC oil seep being mostly anaerobic, while the DWH oil plume is aerobic. Within the Archaea, the phylum Euryarchaeota, recruited more than 95% of the assembled archaeal sequences from the SBC oil seep metagenome, with more than 50% of the sequences assigned to members of the orders Methanomicrobiales and Methanosarcinales. These orders contain organisms capable of anaerobic methanogenesis and methane oxidation (AOM) and we hypothesize that these orders – and their metabolic capabilities – may be fundamental to the ecology of the SBC oil seep.

A meta-analysis of the bacterial and archaeal diversity observed in wetland soils.

This study examined the bacterial and archaeal diversity from a worldwide range of wetlands soils and sediments using a meta-analysis approach. All available 16S rRNA gene sequences recovered from wetlands in public databases were retrieved. In November 2012, a total of 12677 bacterial and 1747 archaeal sequences were collected in GenBank. All the bacterial sequences were assigned into 6383 operational taxonomic units (OTUs 0.03), representing 31 known bacterial phyla, predominant with Proteobacteria (2791 OTUs), Bacteroidetes (868 OTUs), Acidobacteria (731 OTUs), Firmicutes (540 OTUs), and Actinobacteria (418 OTUs). The genus Flavobacterium (11.6% of bacterial sequences) was the dominate bacteria in wetlands, followed by Gp1, Nitrosospira, and Nitrosomonas. Archaeal sequences were assigned to 521 OTUs from phyla Euryarchaeota and Crenarchaeota. The dominating archaeal genera were Fervidicoccus and Methanosaeta. Rarefaction analysis indicated that approximately 40% of bacterial and 83% of archaeal diversity in wetland soils and sediments have been presented. Our results should be significant for well-understanding the microbial diversity involved in worldwide wetlands.

The Archaea Community Associated with Lava-Formed Gotjawal Forest Soil in Jeju, Korea

The abundance and diversity of archaeal assemblages were analyzed in soils collected from Gyorae Gotjawal forest, Jeju, Korea. Gotjawal soil refers to soil derived from a lava-formed forest, characterized by high organic matter content, fertility, and poor rocky soil. Using domain-specific primers, archaeal 16S rRNA gene sequences were PCR amplified for clone library construction, and a total of 185 archaeal clones were examined. The archaeal clones were affiliated with the phyla Thaumarchaeota (96.2%) and Euryarchaeota (3.8%). The most abundant thaumarchaeal group (90.3% of the clones) was the group I.1b clade, which includes soil ammonia-oxidizing archaea. The unique characteristics of Gotjawal soil, including basalt morphology, vegetation, and groundwater aquifer penetration, may be reflected in the archaeal community composition. Further study is necessary to understand the unique factors of Gotjawal soils that influence archaeal abundance, composition, and diversity.

Phylogenetic Diversity of Archaea and the Archaeal Ammonia Monooxygenase Gene in Uranium Mining-Impacted Locations in Bulgaria

Uranium mining and milling activities adversely affect the microbial populations of impacted sites. The negative effects of uranium on soil bacteria and fungi are well studied, but little is known about the effects of radionuclides and heavy metals on archaea. The composition and diversity of archaeal communities inhabiting the waste pile of the Sliven uranium mine and the soil of the Buhovo uranium mine were investigated using 16S rRNA gene retrieval. A total of 355 archaeal clones were selected, and their 16S rDNA inserts were analysed by restriction fragment length polymorphism (RFLP) discriminating 14 different RFLP types. All evaluated archaeal 16S rRNA gene sequences belong to the 1.1b/Nitrososphaera cluster of Crenarchaeota. The composition of the archaeal community is distinct for each site of interest and dependent on environmental characteristics, including pollution levels. Since the members of 1.1b/Nitrososphaera cluster have been implicated in the nitrogen cycle, the archaeal communities from these sites were probed for the presence of the ammonia monooxygenase gene (amoA). Our data indicate that amoA gene sequences are distributed in a similar manner as in Crenarchaeota, suggesting that archaeal nitrification processes in uranium mining-impacted locations are under the control of the same key factors controlling archaeal diversity.

Archaea Appear to Dominate the Microbiome of Inflatella pellicula Deep Sea Sponges

Microbes associated with marine sponges play significant roles in host physiology. Remarkable levels of microbial diversity have been observed in sponges worldwide through both culture-dependent and culture-independent studies. Most studies have focused on the structure of the bacterial communities in sponges and have involved sponges sampled from shallow waters. Here, we used pyrosequencing of 16S rRNA genes to compare the bacterial and archaeal communities associated with two individuals of the marine sponge Inflatella pellicula from the deep-sea, sampled from a depth of 2,900 m, a depth which far exceeds any previous sequence-based report of sponge-associated microbial communities. Sponge-microbial communities were also compared to the microbial community in the surrounding seawater. Sponge-associated microbial communities were dominated by archaeal sequencing reads with a single archaeal OTU, comprising ∼60% and ∼72% of sequences, being observed from Inflatella pellicula. Archaeal sequencing reads were less abundant in seawater (∼11% of sequences). Sponge-associated microbial communities were less diverse and less even than any other sponge-microbial community investigated to date with just 210 and 273 OTUs (97% sequence identity) identified in sponges, with 4 and 6 dominant OTUs comprising ∼88% and ∼89% of sequences, respectively. Members of the candidate phyla, SAR406, NC10 and ZB3 are reported here from sponges for the first time, increasing the number of bacterial phyla or candidate divisions associated with sponges to 43. A minor cohort from both sponge samples (∼0.2% and ∼0.3% of sequences) were not classified to phylum level. A single OTU, common to both sponge individuals, dominates these unclassified reads and shares sequence homology with a sponge associated clone which itself has no known close relative and may represent a novel taxon.

Phylogenetic characterization and quantification of ammonia-oxidizing archaea and bacteria from Lake Kivu in a long-term microcosm incubation.

A microcosm cultivation-based method was set up to investigate the growth of ammonia-oxidizing archaea (AOA), isolated from a water sample acquired at a depth of 50 m from the northern basin of Lake Kivu. For this purpose, both CARD-FISH and qPCR targeting of archaeal 16S rRNA and amoA genes were used. Archaeal cell growth at the end of the 246-day microcosm experiment accounted for 35% of the SybrGold-stained cells, which corresponded to 6.61 x 10(6) cells/ml and 1.76 +/- 0.09 x 10(6) archaeal 16S rRNA gene copies/ml. Clone libraries and DGGE fingerprinting confirmed the dominance of AOA phylotypes in the archaeal community microcosm. The majority of the identified archaeal 16S rRNA gene sequences in the clone libraries were affiliated with Thaumarchaeota Marine Group 1 .1a. Subsequent cultivation of the AOA community on deep-well microtiter plates in medium containing different carbon sources to stimulate archaeal growth failed to show significant differences in archaeal abundance (ANOVA t14 = -1.058, P = 0.308 and ANOVA t14= 1.584, P = 0.135 for yeast extract and simple organic acids, respectively). The lack of growth stimulation by organic compounds is in concordance with the oligotrophic status of Lake Kivu. Finally, the addition of antibiotics to the growth medium resulted in archaeal cell counts that were significantly lower than those obtained from cultures in antibiotic-free medium (ANOVA t14 = 12.12, P < 0.001).

Specific Archaeal Communities are Selected on the Root Surfaces of Ruppia spp. and Phragmites australis

Archaea, although being active members of microbial communities, have been poorly characterized in the rhizoplane of wetland plants. In this study, we compared the archaeal communities found on the root surface of Ruppia spp. and Phragmites australis. Four lagoons, two euhaline and two oligohaline, were sampled during two periods of the year. Archaeal communities were investigated by means of 16S rRNA gene cloning and sequencing. Archaeal sequences clustered in 171 distinct Operational Taxonomic Units affiliated to the Euryarchaeota (43 %), Crenarchaeota (36 %) and Thaumarchaeota (21 %). Communities found in the unvegetated sediment samples showed a high variability mainly due to changes in the pH and conductivity. Ruppia spp. roots exhibited a clear selection effect over Halobacteriales (>80 % of detected sequences) despite the geographical location and sampling time. In contrast, the archaeal community on the root surface of Phragmites australis was largely dependent on the sampling location and time. Methanogenic lineages were not found to be relevant in almost any of the analysed samples. Our findings suggest that archaeal communities in the root surface of the studied macrophytes are mainly composed (40 to 86 %) of putative heterotrophic archaea, such as Halobacteriales, Thermoplasmatales and the Miscellaneous Crenarchaeotic Group.

An analysis of geothermal and carbonic springs in the western United States sustained by deep fluid inputs

Hydrothermal springs harbor unique microbial communities that have provided insight into the early evolution of life, expanded known microbial diversity, and documented a deep Earth biosphere. Mesothermal (cool but above ambient temperature) continental springs, however, have largely been ignored although they may also harbor unique populations of micro-organisms influenced by deep subsurface fluid mixing with near surface fluids. We investigated the microbial communities of 28 mesothermal springs in diverse geologic provinces of the western United States that demonstrate differential mixing of deeply and shallowly circulated water. Culture-independent analysis of the communities yielded 1966 bacterial and 283 archaeal 16S rRNA gene sequences. The springs harbored diverse taxa and shared few operational taxonomic units (OTUs) across sites. The Proteobacteria phylum accounted for most of the dataset (81.2% of all 16S rRNA genes), with 31 other phyla/candidate divisions comprising the remainder. A small percentage (~6%) of bacterial 16S rRNA genes could not be classified at the phylum level, but were mostly distributed in those springs with greatest inputs of deeply sourced fluids. Archaeal diversity was limited to only four springs and was primarily composed of well-characterized Thaumarchaeota. Geochemistry across the dataset was varied, but statistical analyses suggested that greater input of deeply sourced fluids was correlated with community structure. Those with lesser input contained genera typical of surficial waters, while some of the springs with greater input may contain putatively chemolithotrophic communities. The results reported here expand our understanding of microbial diversity of continental geothermal systems and suggest that these communities are influenced by the geochemical and hydrologic characteristics arising from deeply sourced (mantle-derived) fluid mixing. The springs and communities we report here provide evidence for opportunities to understand new dimensions of continental geobiological processes where warm, highly reduced fluids are mixing with more oxidized surficial waters.

Absolute dominance of hydrogenotrophic methanogens in full-scale anaerobic sewage sludge digesters

Anaerobic digestion (AD) is gaining increasing attention due to the ability to covert organic pollutants into energy-rich biogas and, accordingly, growing interest is paid to the microbial ecology of AD systems. Despite extensive efforts, AD microbial ecology is still limitedly understood, especially due to the lack of quantitative information on the structures and dynamics of AD microbial communities. Such knowledge gap is particularly pronounced in sewage sludge AD processes although treating sewage sludge is among the major practical applications of AD. Therefore, we examined the microbial communities in three full-scale sewage sludge digesters using qualitative and quantitative molecular techniques in combination: denaturing gradient gel electrophoresis (DGGE) and real-time polymerase chain reaction (PCR). Eight out of eleven bacterial sequences retrieved from the DGGE analysis were not affiliated to any known species while all eleven archaeal sequences were assigned to known methanogen species. Quantitative real-time PCR analysis revealed that, based on the 16S rRNA gene abundance, the hydrogenotrophic order Methanomicrobiales is the most dominant methanogen group (> 94% of the total methanogen population) in all digesters. This corresponds well to the prevailing occurrence of the DGGE bands related to Methanolinea and Methanospirillum, both belonging to the order Methanomicrobiales, in all sludge samples. It is therefore suggested that hydrogenotrophic methanogens, especially Methanomicrobiales strains, are likely the major players responsible for biogas production in the digesters studied. Our observation is contrary to the conventional understanding that aceticlastic methanogens generally dominate methanogen communities in stable AD environments, suggesting the need for further studies on the dominance relationship in various AD systems.

Molecular phylogenetic analysis of dominant microbial populations in aged refuse

The phylogenetic analysis of dominant microbial populations in 8-year-old refuse samples was done in terms of the whole Bacterial and Archaeal domains. The results indicated that the Bacterial 16S rRNA genes sequences from the aged refuse were largely affiliated with the genus Bacillus, and that more than 60% of the Archaeal sequences were closely related to the methanogenic archaeon. Some inferentially identified extremophilic organisms, particularly alkaliphiles and/or halophiles, were noted to be present in the aged refuse. Moreover, molecular evidence for the occurrence of ammonia-oxidizing Archaea in aged refuse was reported, which opens up avenues for elucidating its role in ammonia transformation in landfill systems. It seems reasonable to assume that the highly complex environment within the landfill systems may select for microbial populations with versatile metabolism and strong adaptation. These findings underline the need for further biochemical and ecological study of these organisms in aged refuse.

Pyrosequencing‐based profiling of archaeal and bacterial 16S rRNA genes identifies a novel archaeon associated with black band disease in corals

Black band disease (BBD) is a microbial consortium that creates anoxic, sulfide-rich microenvironments and kills underlying coral tissues as it rapidly migrates across colonies. Although bacterial communities associated with BBD have been studied extensively, the presence and roles of archaea are unexplored. Using amplicon-pyrosequencing of 16S ribosomal RNA genes, we investigated the community structure of both archaea and bacteria within microbial lesions of BBD and the less-virulent precursor stage, 'cyanobacterial patches' (CP), affecting the coral Montipora hispida. We detected characteristic shifts in microbial communities during the development of BBD from CP, reflecting microenvironmental changes within lesions. Archaeal profiles in CP suggested a diverse assemblage affiliated with the Thaumarchaeota and Euryarchaeota, similar to communities described for oxic marine environments. In contrast, a novel ribotype, distantly affiliated to the Euryarchaeota, dominated up to 94% of archaeal sequences retrieved from BBD. The physiological characteristics of this dominant archaeal ribotype are unknown because of the novelty of its 16S ribosomal RNA gene sequences; however, their prominent associations with BBD lesions suggest the ability to thrive in the organic- and sulfide-rich anoxic microenvironment characteristic of BBD lesions. Discovery of this novel archaeal ribotype provides new insights into the microbial ecology and aetiology of BBD.

Characterization of eubacterial and archaeal community diversity in the pit mud of Chinese Luzhou-flavor liquor by nested PCR–DGGE

The aim of this study was to investigate and compare the microbial community structures of eubacteria and archaea in the pit mud of Chinese Luzhou-flavor liquor from the wall (C(w)) and bottom (C(b)) of cellar through nested PCR-denaturing gradient gel electrophoresis (DGGE). The Shannon-Wiener index (H) calculated from the DGGE profiles showed that the community diversities of eubacteria and archaea in samples from C(b) were almost higher than that from C(w). In addition, cluster analysis of the DGGE profiles revealed that some differences were found in the microbial community structure in samples from different locations. The closely relative microorganisms of all eubacterial 16S rRNA gene sequences fell into four phyla (Firmicutes, Proteobacteria, Bacteroidetes and Actinobacteria), including 12 genera and 2 uncultured eubacteria. Moreover, 37.1% eubacteria were affiliated with Clostridium. Particularly, genus Acinetobacter was absent in all samples from C(b) but present in all samples from C(w). The closely relative microorganisms of all archaeal 16S rRNA gene sequences fell into four genera, which included Methanobrevibacter, Methanoculleus, Methanobacterium and Methanosaeta, while the dominant archaea in samples from C(w) and C(b) were similar. Results presented in this study provide further understanding of the spatial differences in microbial community structure in the pit mud, and is of great importance for the production and quality improvement of Luzhou-flavor liquor.

Bacterial and archaeal communities in the acid pit lake sediments of a chalcopyrite mine

Bacterial and archaeal community structures and diversity of three different sedimentary environments (BH1A, BH2A and BH3A) in the acid pit lake of a chalcopyrite mine at Touro (Spain) were determined by 16S rRNA gene PCR-DGGE and sequencing of clone libraries. DGGE of bacterial and archaeal amplicons showed that the sediments harbor different communities. Bacterial 16S rRNA gene sequences were assigned to Acidobacteria, Actinobacteria, Cyanobacteria, Planctomycetes, Proteobacteria, Chloroflexi and uncultured bacteria, after clustering into 42 operational taxonomic units (OTUs). OTU 2 represented approximately 37, 42 and 37% of all sequences from sediments BH1A, BH2A and BH3A, respectively, and was phylogenetically related to uncultured Chloroflexi. Remaining OTUs were phylogenetically related to heterotrophic bacteria, including representatives of Ferrithrix and Acidobacterium genera. Archaeal 16S rRNA gene sequences were clustered into 54 OTUs. Most of the sequences from the BH1A sediment were assigned to Euryarchaeota, whereas those from BH2A sediment were assigned to Crenarchaeota. The majority of the sequences from BH3A sediment were assigned to unclassified Archaea, and showed similarities to uncultured and unclassified environmental clones. No sequences related to Acidithiobacillus and Leptospirillum, commonly associated with acid mine drainage, were detected in this study.

Structural Mutations That Probe the Interactions between the Catalytic and Dianion Activation Sites of Triosephosphate Isomerase

Triosephosphate isomerase (TIM) catalyzes the isomerization of dihydroxyacetone phosphate to form d-glyceraldehyde 3-phosphate. The effects of two structural mutations in TIM on the kinetic parameters for catalysis of the reaction of the truncated substrate glycolaldehyde (GA) and the activation of this reaction by phosphite dianion are reported. The P168A mutation results in similar 50- and 80-fold decreases in (kcat/Km)E and (kcat/Km)E·HPi, respectively, for deprotonation of GA catalyzed by free TIM and by the TIM·HPO(3)(2-) complex. The mutation has little effect on the observed and intrinsic phosphite dianion binding energy or the magnitude of phosphite dianion activation of TIM for catalysis of deprotonation of GA. A loop 7 replacement mutant (L7RM) of TIM from chicken muscle was prepared by substitution of the archaeal sequence 208-TGAG with 208-YGGS. L7RM exhibits a 25-fold decrease in (kcat/Km)E and a larger 170-fold decrease in (kcat/Km)E·HPi for reactions of GA. The mutation has little effect on the observed and intrinsic phosphodianion binding energy and only a modest effect on phosphite dianion activation of TIM. The observation that both the P168A and loop 7 replacement mutations affect mainly the kinetic parameters for TIM-catalyzed deprotonation but result in much smaller changes in the parameters for enzyme activation by phosphite dianion provides support for the conclusion that catalysis of proton transfer and dianion activation of TIM take place at separate, weakly interacting, sites in the protein catalyst.

Abundance and diversity of archaeal accA gene in hot springs in Yunnan Province, China

It has been suggested that archaea carrying the accA gene, encoding the alpha subunit of the acetyl CoA carboxylase, autotrophically fix CO2 using the 3-hydroxypropionate/4-hydroxybutyrate pathway in low-temperature environments (e.g., soils, oceans). However, little new information has come to light regarding the occurrence of archaeal accA genes in high-temperature ecosystems. In this study, we investigated the abundance and diversity of archaeal accA gene in hot springs in Yunnan Province, China, using DNA- and RNA-based phylogenetic analyses and quantitative polymerase chain reaction. The results showed that archaeal accA genes were present and expressed in the investigated Yunnan hot springs with a wide range of temperatures (66-96 °C) and pH (4.3-9.0). The majority of the amplified archaeal accA gene sequences were affiliated with the ThAOA/HWCG III [thermophilic ammonia-oxidizing archaea (AOA)/hot water crenarchaeotic group III]. The archaeal accA gene abundance was very close to that of AOA amoA gene, encoding the alpha subunit of ammonia monooxygenase. These data suggest that AOA in terrestrial hot springs might acquire energy from ammonia oxidation coupled with CO2 fixation using the 3-hydroxypropionate/4-hydroxybutyrate pathway.

Using stable isotope probing to obtain a targeted metatranscriptome of aerobic methanotrophs in lake sediment

In this study, we demonstrate the possibility of obtaining a targeted metatranscriptome from a functional group of microorganisms using a stable isotope probing (SIP) approach. Methanotrophs in lake sediment were labelled using (13)CH4, and both labelled and unlabelled-RNA were isolated and sequenced by 454 pyrosequencing. The unlabelled metatranscriptome had a large diversity of bacterial, archaeal, eukaryotic and viral sequences as expected from a diverse sediment community. In contrast, the labelled-RNA metatranscriptome was dominated by methanotroph sequences, particularly from Methylococcaceae. Transcripts of the methane monooxygenase genes pmoCAB were the most abundant in this metatranscriptome, and the pathway of methane oxidation to CO2 could be traced, as well as many steps in the ribulose monophosphate pathway for carbon assimilation. A high abundance of mRNA transcripts for proteins related to motility was detected, suggesting an importance for methanotrophs in lake sediments. This combination of SIP and metatranscriptomics should be broadly applicable, and will enhance the detection and identification of mRNA from target organisms.