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Historical DNA analysis of archival samples has added new dimensions to population genetic studies, enabling spatiotemporal approaches for reconstructing population history and informing conservation management. Here we tested the efficacy of Genotyping-in-Thousands by sequencing (GT-seq) for collecting targeted single nucleotide polymorphism genotypic data from archival scale samples, and applied this approach to a study of kokanee salmon (Oncorhynchus nerka) in Kluane National Park and Reserve (KNPR; Yukon, Canada) that underwent a severe 12-year population decline followed by a rapid rebound. We genotyped archival scales sampled pre-crash and contemporary fin clips collected post-crash, revealing high coverage (> 90% average genotyping across all individuals) and low genotyping error (< 0.01% within-libraries, 0.60% among-libraries) despite the relatively poor quality of recovered DNA. We observed slight decreases in expected heterozygosity, allelic diversity, and effective population size post-crash, but none were significant, suggesting genetic diversity was retained despite the severe demographic contraction. Genotypic data also revealed the genetic distinctiveness of a now extirpated population just outside of KNPR, revealing biodiversity loss at the northern edge of the species distribution. More broadly, we demonstrated GT-seq as a valuable tool for collecting genome-wide data from archival samples to address basic questions in ecology and evolution, and inform applied research in wildlife conservation and fisheries management., (© 2021. The Author(s).)

With increasing temperature, nucleobases in DNA become increasingly damaged by hydrolysis of exocyclic amines. The most prominent damage includes the conversion of cytosine to uracil and adenine to hypoxanthine. These damages are mutagenic and put the integrity of the genome at risk if not repaired appropriately. Several archaea live at elevated temperatures and thus, are exposed to a higher risk of deamination. Earlier studies have shown that DNA polymerases of archaea have the property of sensing deaminated nucleobases in the DNA template and thereby stalling the DNA synthesis during DNA replication providing another layer of DNA damage recognition and repair. However, the structural basis of uracil and hypoxanthine sensing by archaeal B-family DNA polymerases is sparse. Here we report on three new crystal structures of the archaeal B-family DNA polymerase from Thermococcus kodakarensis (KOD) DNA polymerase in complex with primer and template strands that have extended single stranded DNA template 5'-overhangs. These overhangs contain either the canonical nucleobases as well as uracil or hypoxanthine, respectively, and provide unprecedented structural insights into their recognition by archaeal B-family DNA polymerases., (© 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

An in vivo study was conducted to compare the enteric methane emissions and diversity of ruminal methanogens in cattle and buffaloes kept in the same environment and fed on the same diet. Six cattle and six buffaloes were fed on a similar diet comprising Napier (Pennisetum purpureum) green grass and concentrate in 70:30. After 90 days of feeding, the daily enteric methane emissions were quantified by using the SF6 technique and ruminal fluid samples from animals were collected for the diversity analysis. The daily enteric methane emissions were significantly greater in cattle as compared to buffaloes; however, methane yields were not different between the two species. Methanogens were ranked at different taxonomic levels against the Rumen and Intestinal Methanogen-Database. The archaeal communities in both host species were dominated by the phylum Euryarchaeota; however, Crenarchaeota represented <1% of the total archaea. Methanogens affiliated with Methanobacteriales were most prominent and their proportion did not differ between the two hosts. Methanomicrobiales and Methanomassillicoccales constituted the second largest group of methanogens in cattle and buffaloes, respectively. Methanocellales (Methanocella arvoryza) were exclusively detected in the buffaloes. At the species level, Methanobrevibacter gottschalkii had the highest abundance (55-57%) in both the host species. The relative abundance of Methanobrevibacter wolinii between the two hosts differed significantly. Methanosarcinales, the acetoclastic methanogens were significantly greater in cattle than the buffaloes. It is concluded that the ruminal methane yield in cattle and buffaloes fed on the same diet did not differ. With the diet used in this study, there was a limited influence (<3.5%) of the host on the structure of the ruminal archaea community at the species level. Therefore, the methane mitigation strategies developed in either of the hosts should be effective in the other. Further studies are warranted to reveal the conjunctive effect of diet and geographical locations with the host on ruminal archaea community composition., Competing Interests: No authors have competing interests.

Land use types with different disturbance gradients show many variations in soil properties, but the effects of different land use types on soil nitrifying communities and their ecological implications remain poorly understood. Using 13 CO 2 -DNA-based stable isotope probing (DNA-SIP), we examined the relative importance and active community composition of ammonia-oxidizing archaea (AOA) and bacteria (AOB) and nitrite-oxidizing bacteria (NOB) in soils under three land use types, forest, cropland, and greenhouse vegetable soil, representing three interference gradients. Soil net nitrification rate was in the order forest soil > cropland soil > greenhouse vegetable soil. DNA-SIP showed that active AOA outcompeted AOB in the forest soil, whereas AOB outperformed AOA in the cropland and greenhouse vegetable soils. Cropland soil was richer in NOB than in AOA and AOB. Phylogenetic analysis revealed that ammonia oxidation in the forest soil was predominantly catalyzed by the AOA Nitrosocosmicus franklandus cluster within the group 1.1b lineage. The 13 C-labeled AOB were overwhelmingly dominated by Nitrosospira cluster 3 in the cropland soil. The active AOB Nitrosococcus watsonii lineage was observed in the greenhouse vegetable soil, and it played an important role in nitrification. Active NOB communities were closely affiliated with Nitrospira in the forest and cropland soils, and with Nitrolancea and Nitrococcus in the greenhouse vegetable soil. Canonical correlation analysis showed that soil pH and organic matter content significantly affected the active nitrifier community composition. These results suggest that land use types with different disturbance gradients alter the distribution of active nitrifier communities by affecting soil physicochemical properties. IMPORTANCE Nitrification plays an important role in the soil N cycle, and land use management has a profound effect on soil nitrifiers. It is unclear how different gradients of land use affect active ammonia-oxidizing archaea and bacteria and nitrite-oxidizing bacteria. Our research is significant because we determined the response of nitrifiers to human disturbance, which will greatly improve our understanding of the niche of nitrifiers and the differences in their physiology.

To what extent multi-omic techniques could reflect in situ microbial process rates remains unclear, especially for highly diverse habitats like soils. Here, we performed microcosm incubations using sandy soil from an agricultural site in Midwest USA. Microcosms amended with isotopically labeled ammonium and urea to simulate a fertilization event showed nitrification (up to 4.1 ± 0.87 µg N-NO 3 - g -1 dry soil d -1 ) and accumulation of N 2 O after 192 hours of incubation. Nitrification activity (NH 4 +  → NH 2 OH → NO → NO 2 -  → NO 3 - ) was accompanied by a 6-fold increase in relative expression of the 16S rRNA gene (RNA/DNA) between 10 and 192 hours of incubation for ammonia-oxidizing bacteria Nitrosomonas and Nitrosospira, unlike archaea and comammox bacteria, which showed stable gene expression. A strong relationship between nitrification activity and betaproteobacterial ammonia monooxygenase and nitrite oxidoreductase transcript abundances revealed that mRNA quantitatively reflected measured activity and was generally more sensitive than DNA under these conditions. Although peptides related to housekeeping proteins from nitrite-oxidizing microorganisms were detected, their abundance was not significantly correlated with activity, revealing that meta-proteomics provided only a qualitative assessment of activity. Altogether, these findings underscore the strengths and limitations of multi-omic approaches for assessing diverse microbial communities in soils and provide new insights into nitrification.

The differences in archaeal diversity and community composition in the sediments and waters of the Qinhuangdao coastal aquaculture zone were investigated. Furthermore, the associations between dominant archaeal taxa with geographic and environmental variables were evaluated. High-throughput sequencing of archaeal 16S rRNA genes yielded a total of 176,211 quality-filtered reads and 1,178 operational taxonomic units (OTUs) overall. The most abundant phylum and class among all communities were Thaumarchaeota and Nitrososphaeria, respectively. Beta diversity analysis indicated that community composition was divided into two groups according to the habitat type (i.e., sediments or waters). Only 9.8% OTUs were shared by communities from the two habitats, while 73.9% and 16.3% of the OTUs were unique to sediment or water communities, respectively. Furthermore, the relative abundances of the dominant OTUs differed with habitat type. Investigations of relationships between dominant OTUs and environmental variables indicated that some dominant OTUs were more sensitive to variation in environmental factors, which could be due to individual taxonomic differences in lifestyles and biological processes. Overall, the investigation of archaeal community variation within the Qinhuangdao coastal aquaculture zone provides an important baseline understanding of the microbial ecology in this important ecosystem., Competing Interests: The authors have declared that no competing interests exist.

Sponges harbor characteristic microbiomes derived from symbiotic relationships shaping their lifestyle and survival. Haliclona fulva is encrusting marine sponge species dwelling in coralligenous accretions or semidark caves of the Mediterranean Sea and the near Atlantic Ocean. In this work, we characterized the abundance and core microbial community composition found in specimens of H. fulva by means of electron microscopy and 16S amplicon Illumina sequencing. We provide evidence of its low microbial abundance (LMA) nature. We found that the H. fulva core microbiome is dominated by sequences belonging to the orders Nitrosomonadales and Cenarchaeales. Seventy percent of the reads assigned to these phylotypes grouped in a very small number of high-frequency operational taxonomic units, representing niche-specific species Cenarchaeum symbiosum and uncultured Betaproteobacteria HF1, a new eubacterial ribotype variant found in H. fulva. The microbial composition of H. fulva is quite distinct from those reported in sponge species of the same Haliclona genus. We also detected evidence of an excretion/capturing loop between these abundant microorganisms and planktonic microbes by analyzing shifts in seawater planktonic microbial content exposed to healthy sponge specimens maintained in aquaria. Our results suggest that horizontal transmission is very likely the main mechanism for symbionts' acquisition by H. fulva. So far, this is the first shallow water sponge species harboring such a specific and predominant assemblage composed of these eubacterial and archaeal ribotypes. Our data suggests that this symbiotic relationship is very stable over time, indicating that the identified core microbial symbionts may play key roles in the holobiont functioning.

Methylotrophic methanogenic archaea are an integral part of the carbon cycle in various anaerobic environments. Different from methylotrophic bacteria, methylotrophic methanogens assimilate both, the methyl compound and dissolved inorganic carbon. Here, we present DNA- and RNA-stable isotope probing (SIP) methods involving an effective labeling strategy using 13 C-labeled dissolved inorganic carbon (DIC) as carbon source along with methanol as dissimilatory substrate.

Meiobenthic community structure in the northern Gulf of Mexico has been shown to be driven by geographical differences due to inshore-offshore gradients and location relative to river discharge. Samples collected along three transects spanning Mobile Bay, Alabama, showed significant differences in meiobenthic communities east of the bay compared to those sampled from the west. In contrast, analysis of bacterial and archaeal communities from the same sediment samples shows that the inshore-offshore gradient has minimal impact on their community structure. Significant differences in community structure were observed for Bacteria and Archaea between the east and west samples, but there was no difference in richness or diversity. Grouped by sediment type, higher richness was observed in silty samples compared to sandy samples. Significant differences were also observed among sediment types for community structure with bacteria communities in silty samples having more anaerobic sulfate reducers compared to aerobic heterotrophs, which had higher abundances in sandy sediments. This is likely due to increased organic matter in the silty sediments from the overlying river leading to low oxygen habitats. Most archaeal sequences represented poorly characterized high-level taxa, limiting interpretation of their distributions. Overlap between groups based on transect and sediment characteristics made determining which factor is more important in structuring bacterial and archaeal communities difficult. However, both factors are driven by discharge from the Mobile River. Although inshore-offshore gradients do not affect Bacteria or Archaea to the same extent as the meiobenthic communities, all three groups are strongly affected by sediment characteristics.

Quantification of microbes in water systems is essential to industrial practices ranging from drinking water and wastewater treatment to groundwater remediation. While quantification using DNA-based molecular methods is precise, the accuracy is dependent on DNA extraction efficiencies. We show that the DNA yield is strongly impacted by the cell concentration in groundwater samples (r = -0.92, P < 0.0001). This has major implications for industrial applications using quantitative polymerase chain reaction (qPCR) to determine cell concentrations in water, including bioremediation. We propose a simple normalization method using a DNA recovery ratio, calculated with the total cell count and DNA yield. Application of this method to enumeration of bacteria and archaea in groundwater samples targeting phylogenetic markers (16S rRNA) demonstrated an increased goodness of fit after normalization (7.04 vs 0.94 difference in Akaike's information criteria). Furthermore, normalization was applied to qPCR quantification of functional genes and combined with DNA sequencing of archaeal and bacterial 16S rRNA genes to monitor changes in abundance of methanogenic archaea and sulphate-reducing bacteria in groundwater. The integration of qPCR and DNA sequencing with appropriate normalization enables high-throughput quantification of microbial groups using increasingly affordable and accessible techniques. This research has implications for microbial ecology and engineering research as well as industrial practice.

The newly discovered Candidatus 'Methanoperedens nitroreducens' (M. nitroreducens), mediating nitrate-dependent anaerobic oxidation of methane, is an important microorganism in linking carbon and nitrogen cycles. In order to explore the diversity of M. nitroreducens-like archaea in various environmental niches with advanced high-throughput sequencing, new primers based on alpha subunit of methyl-coenzyme M reductase gene were designed. The PCR results demonstrated that the new primers could effectively detect M. nitroreducens-like archaea from an enrichment culture dominated by M. nitroreducens as well as samples collected from a natural freshwater lake and a full-scale wastewater treatment plant (WWTP). By high-throughput sequencing, more than 30,000 M. nitroreducens-like sequences were obtained. Phylogenetic analysis of these sequences along with published sequences showed that M. nitroreducens-like archaea could be divided into three sub-branches (named as Group A, Group B and Group C in this study). Clear geographical difference was observed, with Group A and Group B dominating samples in Queensland (Australia) and in European ecosystems, respectively. Further quantitative PCR revealed that the M. nitroreducens-like archaea were more abundant in WWTP than the freshwater lake. The study provided a large number of sequences for M. nitroreducens-like archaeal communities, thus expanded our understanding on the ecological diversity of M. nitroreducens-like archaea.

Microbial colonization of the intestinal tract of commercial poultry is highly variable, likely due to the fact that poults and chicks are hatched and raised without exposure to adult birds and their microbiota. In industrial poultry production, it is hypothesized that most of the microbiota is obtained through horizontal transmission from the environment and very little by maternal transmission. The initial gut microbiota will therefore differ between flocks and companies based on environmental conditions at the hatchery. Day-old poults were collected from the hatchery of 2 companies at 3 different time points to monitor the initial colonizing microbiota by sequencing amplicons of marker genes for bacteria, lactic acid bacteria (LAB), fungi, and archaea. Bacterial colonizers were distinct by company (pseudo-F 38.7, P ≤ 0.05) with the predominant bacteria at Company A being clostridia, specifically Clostridium celatum group, C. paraputrificum, and C. tertium. Predominant bacteria at Company B were Enterobacteriaceae, belonging to 2 different groups, one that included Escherichia; Shigella and Salmonella and the other Klebsiella; Enterobacter; and others. The predominant LAB at both companies were Enterococcus faecalis and E. gallinarum, confirmed by sequencing the 16S ribosomal RNA (rRNA) gene of colonies picked from lactobacilli agar plate counts. The predominant fungi were Aspergillus niger and Saccharomyces cerevisiae, with Candida sake or Alterneria sp. in some samples of Company A. Archaeal sequences were detected only in a single poult from Company B. The initial gastrointestinal colonizers of poults vary across company and time, signifying a strong environmental effect on microbiota acquisition. There was an indication of maternal effects in certain breeder flocks from Company B. Further work is necessary to determine how this variability affects microbiota succession and impacts growth and production of the birds.

The ubiquity and relevance of extracellular DNA (exDNA) are well-known and increasingly gaining importance in many fields of application such as medicine and environmental microbiology. Although sources and types of exDNA are manifold, ratios of specific DNA-molecules inside and outside of living cells can give reliable information about the activity of entire systems and of specific microbial groups or species. Here, we introduce a method to discriminate between internal (iDNA), as well as bound and free exDNA, and evaluate various DNA fractions and related ratios (ex:iDNA) regarding their applicability to be used as a fast, convenient, and reliable alternative to more tedious RNA-based activity measurements. In order to deal with microbial consortia that can be regulated regarding their activity, we tested and evaluated the proposed method in comparison to sophisticated dehydrogenase- and RNA-based activity measurements with two anaerobic microbial consortia (anaerobic fungi and syntrophic archaea and a microbial rumen consortium) and three levels of resolution (overall activity, total bacteria, methanogenic archaea). Furthermore, we introduce a 28S rRNA gene-specific primer set and qPCR protocol, targeting anaerobic fungi (Neocallimastigomycota). Our findings show that the amount of actively released free exDNA (fDNA) strongly correlates with different activity measurements and is thus suggested to serve as a proxy for microbial activity.

Shotgun metagenomics is a low biased technology for assessing environmental microbial diversity and function. However, the requirement for a sufficient amount of DNA and the contamination of inhibitors in environmental DNA leads to difficulties in constructing a shotgun metagenomic library. We herein examined metagenomic library construction from subnanogram amounts of input environmental DNA from subarctic surface water and deep-sea sediments using two library construction kits: the KAPA Hyper Prep Kit and Nextera XT DNA Library Preparation Kit, with several modifications. The influence of chemical contaminants associated with these environmental DNA samples on library construction was also investigated. Overall, shotgun metagenomic libraries were constructed from 1 pg to 1 ng of input DNA using both kits without harsh library microbial contamination. However, the libraries constructed from 1 pg of input DNA exhibited larger biases in GC contents, k-mers, or small subunit (SSU) rRNA gene compositions than those constructed from 10 pg to 1 ng DNA. The lower limit of input DNA for low biased library construction in this study was 10 pg. Moreover, we revealed that technology-dependent biases (physical fragmentation and linker ligation vs. tagmentation) were larger than those due to the amount of input DNA.

Background: Total community rDNA was used to determine the diversity of bacteria and archaea from water, wet sediment and microbial mats samples of hot springs in the Eastern lowlands of Eritrea. The temperatures of the springs range from 49.5 °C to 100 °C while pH levels varied from 6.97 to 7.54. Akwar and Maiwooi have high carbonate levels. The springs near the seashore, Garbanabra and Gelti, are more saline with higher levels of sodium and chlorides. Elegedi, situated in the Alid volcanic area, has the highest temperature, iron and sulfate concentrations., Results: The five hot springs shared 901 of 4371 OTUs recovered while the three sample types (water, wet sediment and microbial mats) also shared 1429 OTUs. The Chao1 OTU estimate in water sample was significantly higher than the wet sediment and microbial mat samples. As indicated by NMDS, the community samples at genus level showed location specific clustering. Certain genera correlated with temperature, sodium, carbonate, iron, sulfate and ammonium levels in water. The abundant phyla included Proteobacteria (6.2-82.3%), Firmicutes (1.6-63.5%), Deinococcus-Thermus (0.0-19.2%), Planctomycetes (0.0-11.8%), Aquificae (0.0-9.9%), Chlorobi (0.0-22.3%) and Bacteroidetes (2.7-8.4%)., Conclusion: There were significant differences in microbial community structure within the five locations and sample types at OTU level. The occurence of Aquificae, Deinococcus-Thermus, some Cyanobacteria and Crenarchaeota were highly dependent on temperature. The Halobacterium, unclassified Thaumarchaeota, Actinobacteria and Cyanobacteria showed significant correlation with salinity occurring abundantly in Garbanabra and Gelti. Firmicutes and unclassified Rhodocylaceae were higher in the microbial mat samples, while Archaea were prominent in the wet sediment samples.

Salar de Uyuni, situated in the Southwest of the Bolivian Altiplano, is the largest salt flat on Earth. Brines of this athalassohaline hypersaline environment are rich in lithium and boron. Due to the ever- increasing commodity demand, the industrial exploitation of brines for metal recovery from the world's biggest lithium reservoir is likely to increase substantially in the near future. Studies on the composition of halophilic microbial communities in brines of the salar have not been published yet. Here we report for the first time on the prokaryotic diversity of four brine habitats across the salar. The brine is characterized by salinity values between 132 and 177 PSU, slightly acidic to near-neutral pH and lithium and boron concentrations of up to 2.0 and 1.4g/L, respectively. Community analysis was performed after sequencing the V3-V4 region of the 16S rRNA genes employing the Illumina MiSeq technology. The mothur software package was used for sequence processing and data analysis. Metagenomic analysis revealed the occurrence of an exclusively archaeal community comprising 26 halobacterial genera including only recently identified genera like Halapricum, Halorubellus and Salinarchaeum. Despite the high diversity of the halobacteria-dominated community in sample P3 (Shannon-Weaver index H'=3.12 at 3% OTU cutoff) almost 40% of the Halobacteriaceae-assigned sequences could not be classified on the genus level under stringent filtering conditions. Even if the limited taxonomic resolution of the V3-V4 region for halobacteria is considered, it seems likely to discover new, hitherto undescribed genera of the family halobacteriaceae in this particular habitat of Salar de Uyuni in future., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Propionate degradation is a critical step during the conversion of complex organic matter under methanogenic conditions, and it requires a syntrophic cooperation between propionate-oxidizing bacteria and methanogenic archaea. Increasing evidences suggest that interspecies electron transfer for syntrophic metabolism is not limited to the reducing equivalents of hydrogen and formate. This study tested the ability of an electron shuttle to mediate interspecies electron transfer in syntrophic methanogenesis. We found that cysteine supplementation (100, 400, and 800 μM) accelerated CH 4 production from propionate in paddy soil enrichments. Of the concentrations tested, 100 μM cysteine was the most effective at enhancing propionate degradation to CH 4 , and the rates of CH 4 production and propionate degradation were increased by 109 and 79%, respectively, compared with the cysteine-free control incubations. We eliminated the possibility that the stimulatory effect of cysteine on methanogenesis was attributable to the function of cysteine as a methanogenic substrate in the presence of propionate. The potential catalytic effect involved cysteine serving as an electron carrier to mediate interspecies electron transfer in syntrophic propionate oxidization. The redox potential of cystine/cysteine, which is dependent on the concentration, might be more suitable to facilitate interspecies electron transfer between syntrophic partners at a concentration of 100 μM. Pelotomaculum, obligately syntrophic, propionate-oxidizing bacteria, and hydrogenotrophic methanogens of the family Methanobacteriaceae are predominant in cysteine-mediated methanogenic propionate degradation. The stimulatory effect of cysteine on syntrophic methanogenesis offers remarkable potential for improving the performance of anaerobic digestion and conceptually broaden strategies for interspecies electron transfer in syntrophic metabolism.

In the present study, we used culture-independent methods to investigate the diversity of methanogenic archaea and their distribution in five permafrost samples collected from a borehole in the Kolyma River Lowland (north-east of Russia). Total DNA was extracted from methane-containing permafrost samples of different age and amplified by PCR. The resulting DNA fragments were cloned. Phylogenetic analysis of the sequences showed the presence of archaea in all studied samples; 60%-95% of sequences belonged to the Euryarchaeota. Methanogenic archaea were novel representatives of Methanosarcinales, Methanomicrobiales, Methanobacteriales and Methanocellales orders. Bathyarchaeota (Miscellaneous Crenarchaeota Group) representatives were found among nonmethanogenic archaea in all the samples studied. The Thaumarchaeota representatives were not found in the upper sample, whereas Woesearchaeota (formerly DHVEG-6) were found in the three deepest samples. Unexpectedly, the greatest diversity of archaea was observed at a depth of 22.3 m, probably due to the availability of the labile organic carbon and/or due to the migration of the microbial cells during the freezing front towards the bottom., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Lake Magadi and little Magadi are hypersaline, alkaline lakes situated in the southern part of Kenyan Rift Valley. Solutes are supplied mainly by a series of alkaline hot springs with temperatures as high as 86 °C. Previous culture-dependent and culture-independent studies have revealed diverse groups of microorganisms thriving under these conditions. Previous culture independent studies were based on the analysis of 16S rDNA but were done on less saline lakes. For the first time, this study combined illumina sequencing and analysis of amplicons of both total community rDNA and 16S rRNA cDNA to determine the diversity and community structure of bacteria and archaea within 3 hot springs of L. Magadi and little Magadi., Methods: Water, wet sediments and microbial mats were collected from springs in the main lake at a temperature of 45.1 °C and from Little Magadi "Nasikie eng'ida" (temperature of 81 °C and 83.6 °C). Total community DNA and RNA were extracted from samples using phenol-chloroform and Trizol RNA extraction protocols respectively. The 16S rRNA gene variable region (V4 - V7) of the extracted DNA and RNA were amplified and library construction performed following Illumina sequencing protocol. Sequences were analyzed done using QIIME while calculation of Bray-Curtis dissimilarities between datasets, hierarchical clustering, Non Metric Dimensional Scaling (NMDS) redundancy analysis (RDA) and diversity indices were carried out using the R programming language and the Vegan package., Results: Three thousand four hundred twenty-six and one thousand nine hundred thirteen OTUs were recovered from 16S rDNA and 16S rRNA cDNA respectively. Uncultured diversity accounted for 89.35 % 16S rDNA and 87.61 % 16S rRNA cDNA reads. The most abundant phyla in both the 16S rDNA and 16S rRNA cDNA datasets included: Proteobacteria (8.33-50 %), Firmicutes 3.52-28.92 %, Bacteroidetes (3.45-26.44 %), Actinobacteria (0.98-28.57 %) and Euryarchaeota (3.55-34.48 %) in all samples. NMDS analyses of taxonomic composition clustered the taxa into three groups according to sample types (i.e. wet sediments, mats and water samples) with evident overlap of clusters between wet sediments and microbial mats from the three sample types in both DNA and cDNA datasets. The hot spring (45.1 °C) contained less diverse populations compared to those in Little Magadi (81-83 °C)., Conclusion: There were significant differences in microbial community structure at 95 % level of confidence for both total diversity (P value, 0.009) based on 16S rDNA analysis and active microbial diversity (P value, 0.01) based on 16S rRNA cDNA analysis, within the three hot springs. Differences in microbial composition and structure were observed as a function of sample type and temperature, with wet sediments harboring the highest diversity.

Ammonia oxidation is the first and rate-limiting step in nitrification and is dominated by two distinct groups of microorganisms in soil: ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). AOA are often more abundant than AOB and dominate activity in acid soils. The mechanism of ammonia oxidation under acidic conditions has been a long-standing paradox. While high rates of ammonia oxidation are frequently measured in acid soils, cultivated ammonia oxidizers grew only at near-neutral pH when grown in standard laboratory culture. Although a number of mechanisms have been demonstrated to enable neutrophilic AOB growth at low pH in the laboratory, these have not been demonstrated in soil, and the recent cultivation of the obligately acidophilic ammonia oxidizer "Candidatus Nitrosotalea devanaterra" provides a more parsimonious explanation for the observed high rates of activity. Analysis of the sequenced genome, transcriptional activity, and lipid content of "Ca Nitrosotalea devanaterra" reveals that previously proposed mechanisms used by AOB for growth at low pH are not essential for archaeal ammonia oxidation in acidic environments. Instead, the genome indicates that "Ca Nitrosotalea devanaterra" contains genes encoding both a predicted high-affinity substrate acquisition system and potential pH homeostasis mechanisms absent in neutrophilic AOA. Analysis of mRNA revealed that candidate genes encoding the proposed homeostasis mechanisms were all expressed during acidophilic growth, and lipid profiling by high-performance liquid chromatography-mass spectrometry (HPLC-MS) demonstrated that the membrane lipids of "Ca Nitrosotalea devanaterra" were not dominated by crenarchaeol, as found in neutrophilic AOA. This study for the first time describes a genome of an obligately acidophilic ammonia oxidizer and identifies potential mechanisms enabling this unique phenotype for future biochemical characterization., (Copyright © 2016 Lehtovirta-Morley et al.)

The evolutionary interrelationships between the archaeal organisms which comprise the class Halobacteria have proven difficult to elucidate using traditional phylogenetic tools. The class currently contains three orders. However, little is known about the family level relationships within these orders. In this work, we have completed a comprehensive comparative analysis of 129 sequenced genomes from members of the class Halobacteria in order to identify shared molecular characteristics, in the forms of conserved signature insertions/deletions (CSIs) and conserved signature proteins (CSPs), which can provide reliable evidence, independent of phylogenetic trees, that the species from the groups in which they are found are specifically related to each other due to common ancestry. Here we present 20 CSIs and 31 CSPs which are unique characteristics of infra-order level groups of genera within the class Halobacteria. We also present 40 CSIs and 234 CSPs which are characteristic of Haloarcula, Halococcus, Haloferax, or Halorubrum. Importantly, the CSIs and CSPs identified here provide evidence that the order Haloferacales contains two main groups, one consisting of Haloferax and related genera supported by four CSIs and five CSPs and the other consisting of Halorubrum and related genera supported by four CSPs. We have also identified molecular characteristics that suggest that the polyphyletic order Halobacteriales contains at least two large monophyletic clusters of organisms in addition to the polyphyletic members of the order, one cluster consisting of Haloarcula and related genera supported by ten CSIs and nineteen CSPs and the other group consisting of the members of the genus Halococcus supported by nine CSIs and 23 CSPs. We have also produced a highly robust phylogenetic tree based on the concatenated sequences of 766 proteins which provide additional support for the relationships identified by the CSIs and CSPs. On the basis of the phylogenetic analyses and the identified conserved molecular characteristics presented here, we propose a division of the order Haloferacales into two families, an emended family Haloferacaceae and Halorubraceae fam. nov. and a division of the order Halobacteriales into three families, an emended family Halobacteriaceae, Haloarculaceae fam. nov., and Halococcaceae fam. nov.

Hydrogenotrophic methanogens can use gaseous substrates, such as H2 and CO2, in CH4 production. H2 gas is used to reduce CO2. We have successfully operated a hollow-fiber membrane biofilm reactor (Hf-MBfR) for stable and continuous CH4 production from CO2 and H2. CO2 and H2 were diffused into the culture medium through the membrane without bubble formation in the Hf-MBfR, which was operated at pH 4.5-5.5 over 70 days. Focusing on the presence of hydrogenotrophic methanogens, we analyzed the structure of the microbial community in the reactor. Denaturing gradient gel electrophoresis (DGGE) was conducted with bacterial and archaeal 16S rDNA primers. Real-time qPCR was used to track changes in the community composition of methanogens over the course of operation. Finally, the microbial community and its diversity at the time of maximum CH4 production were analyzed by pyrosequencing methods. Genus Methanobacterium, related to hydrogenotrophic methanogens, dominated the microbial community, but acetate consumption by bacteria, such as unclassified Clostridium sp., restricted the development of acetoclastic methanogens in the acidic CH4 production process. The results show that acidic operation of a CH4 production reactor without any pH adjustment inhibited acetogenic growth and enriched the hydrogenotrophic methanogens, decreasing the growth of acetoclastic methanogens.

During the anaerobic digestion of dewatered sludge, polyacrylamide (PAM), a chemical conditioner, can usually be consumed as a carbon and nitrogen source along with other organic matter (e.g., proteins and carbohydrates in the sludge). However, a significant accumulation of acrylamide monomers (AMs) was observed during the PAM biodegradation process. To improve the anaerobic hydrolysis of PAM, especially the amide hydrolysis process, and to avoid the generation of the intermediate product AM, a new strategy is reported herein that uses an initial pH of 9, 200 mg COD/L of PAM and a fermentation time of 17 d. First, response surface methodology (RSM) was applied to optimize PAM removal in the anaerobic digestion of the sludge. The biological hydrolysis of PAM reached 86.64% under the optimal conditions obtained from the RSM. Then, the mechanisms for the optimized parameters that significantly improved the biological hydrolysis of PAM were investigated by the synergistic effect of the main organic compounds in the sludge, the floc size distribution, and the enzymatic activities. Finally, semi-continuous-flow experiments for a microbial community study were investigated based on the determination of key microorganisms involved in the biological hydrolysis of PAM.

Background: Although high-throughput sequencing, such as Illumina-based technologies (e.g. MiSeq), has revolutionized microbial ecology, adaptation of amplicon sequencing for environmental microbial community analysis is challenging due to the problem of low base diversity., Results: A new phasing amplicon sequencing approach (PAS) was developed by shifting sequencing phases among different community samples from both directions via adding various numbers of bases (0-7) as spacers to both forward and reverse primers. Our results first indicated that the PAS method substantially ameliorated the problem of unbalanced base composition. Second, the PAS method substantially improved the sequence read base quality (an average of 10 % higher of bases above Q30). Third, the PAS method effectively increased raw sequence throughput (~15 % more raw reads). In addition, the PAS method significantly increased effective reads (9-47 %) and the effective read sequence length (16-96 more bases) after quality trim at Q30 with window 5. In addition, the PAS method reduced half of the sequencing errors (0.54-1.1 % less). Finally, two-step PCR amplification of the PAS method effectively ameliorated the amplification biases introduced by the long barcoded PCR primers., Conclusion: The developed strategy is robust for 16S rRNA gene amplicon sequencing. In addition, a similar strategy could also be used for sequencing other genes important to ecosystem functional processes.

The haloarchaeon Haloferax volcanii degrades D-xylose and L-arabinose via oxidative pathways to α-ketoglutarate. The genes involved in these pathways are clustered and were transcriptionally upregulated by both D-xylose and L-arabinose suggesting a common regulator. Adjacent to the gene cluster, a putative IclR-like transcriptional regulator, HVO&#95;B0040, was identified. It is shown that HVO&#95;B0040, designated xacR, encodes an activator of both D-xylose and L-arabinose catabolism: in ΔxacR cells, transcripts of genes involved in pentose catabolism could not be detected; transcript formation could be recovered by complementation, indicating XacR dependent transcriptional activation. Upstream activation promoter regions and nucleotide sequences that were essential for XacR-mediated activation of pentose-specific genes were identified by in vivo deletion and scanning mutagenesis. Besides its activator function XacR acted as repressor of its own synthesis: xacR deletion resulted in an increase of xacR promoter activity. A palindromic sequence was identified at the operator site of xacR promoter, and mutation of this sequence also resulted in an increase and thus derepression of xacR promoter activity. It is concluded that the palindromic sequence represents the binding site of XacR as repressor. This is the first report of a transcriptional regulator of pentose catabolism in the domain of archaea., (© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The combined zero-valent iron (ZVI) and upflow anaerobic sludge blanket (UASB) process is established for the treatment of chloronitrobenzenes (ClNBs) wastewater, and the succession of microbial community and its enhanced mechanism are investigated in the study. Results showed that compared with the control UASB (R1), the stable COD removal, ClNBs transformation, and dechlorination occurred in the combined system (R2) when operated at influent COD and 3,4-Dichloronitrobenzene (3,4-DClNB) loading rates of 4200-7700 g m(-3) d(-1) and 6.0-70.0 g m(-3) d(-1), and R2 had the better shock resistance and buffering capacity for the anaerobic acidification. The dechlorination for the intermediate products of p-chloroanaline (p-ClAn) to analine (AN) occurred in R2 reactor after 45 days, whereas it did not occur in R1 after a long-term operation. The novel ZVI-based anaerobic granular sludge (ZVI-AGS) was successfully developed in the combined system, and higher microbial activities including ClNB transformation and H2/CH4 production were achieved simultaneously. The dominant bacteria were closely related to the groups of Megasphaera, Chloroflexi, and Clostridium, and the majority of archaea were correlated with the groups of Methanosarcinalesarchaeon, Methanosaetaconcilii, and Methanothrixsoehngenii, which are capable of reductively dechlorinating PCB, HCB, and TCE in anaerobic niche and EPS secretion., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Quantitating relative (32)P-band intensity in gels is desired, e.g., to study primer-extension kinetics of DNA polymerases (DNAPs). Following imaging, multiple (32)P-bands are often present in lanes. Though individual bands appear by eye to be simple and well-resolved, scanning reveals they are actually skewed-Gaussian in shape and neighboring bands are overlapping, which complicates quantitation, because slower migrating bands often have considerable contributions from the trailing edges of faster migrating bands. A method is described to accurately quantitate adjacent (32)P-bands, which relies on having a standard: a simple skewed-Gaussian curve from an analogous pure, single-component band (e.g., primer alone). This single-component scan/curve is superimposed on its corresponding band in an experimentally determined scan/curve containing multiple bands (e.g., generated in a primer-extension reaction); intensity exceeding the single-component scan/curve is attributed to other components (e.g., insertion products). Relative areas/intensities are determined via pixel analysis, from which relative molarity of components is computed. Common software is used. Commonly used alternative methods (e.g., drawing boxes around bands) are shown to be less accurate. Our method was used to study kinetics of dNTP primer-extension opposite a benzo[a]pyrene-N(2)-dG-adduct with four DNAPs, including Sulfolobus solfataricus Dpo4 and Sulfolobus acidocaldarius Dbh. Vmax/Km is similar for correct dCTP insertion with Dpo4 and Dbh. Compared to Dpo4, Dbh misinsertion is slower for dATP (∼20-fold), dGTP (∼110-fold) and dTTP (∼6-fold), due to decreases in Vmax. These findings provide support that Dbh is in the same Y-Family DNAP class as eukaryotic DNAP κ and bacterial DNAP IV, which accurately bypass N(2)-dG adducts, as well as establish the scan-method described herein as an accurate method to quantitate relative intensity of overlapping bands in a single lane, whether generated from (32)P-signals or by other means (e.g., staining)., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Sampling strategy is important for unbiased analysis of the characteristics of microbial communities in the environment. During field work it is not always possible to analyze fresh samples immediately or store them frozen. Therefore, the effect of short-term storage temperature was investigated on the abundance and composition of bacterial, archaeal and denitrifying communities in environmental samples from two different sampling sites. Oxic forest soil and anoxic pond sediment were investigated by measuring microbial abundance (DNA) and transcriptional activity (RNA). Prior to investigating the effect of storage temperature, samples were immediately analyzed, in order to represent the original situation in the habitat. The effect of storage temperature was then determined after 11 days at different low temperatures (room temperature, 4 °C, −22 °C and −80 °C). Community profiling using terminal restriction fragment length polymorphism (T-RFLP) showed no significant differences between the immediately analyzed reference sample and the samples stored at different incubation temperatures, both for DNA and RNA extracts. The abundance of microbial communities was determined using quantitative PCR and it also revealed a stable community size at all temperatures tested. By contrast, incubation at an elevated temperature (37 °C) resulted in changed bacterial community composition. In conclusion, short-term storage, even at room temperature, did not affect microbial community composition, abundance and transcriptional activity in aerated forest soil and anoxic pond sediment.

Bacteria and archaea are characterized by an amazing metabolic diversity, which allows them to persist in diverse and often extreme habitats. Apart from oxygenic photosynthesis and oxidative phosphorylation, well-studied processes from chloroplasts and mitochondria of plants and animals, prokaryotes utilize various chemo- or lithotrophic modes, such as anoxygenic photosynthesis, iron oxidation and reduction, sulfate reduction, and methanogenesis. Most bioenergetic pathways have a similar general structure, with an electron transport chain composed of protein complexes acting as electron donors and acceptors, as well as a central cytochrome complex, mobile electron carriers, and an ATP synthase. While each pathway has been studied in considerable detail in isolation, not much is known about their relative evolutionary relationships. Wanting to address how this metabolic diversity evolved, we mapped the distribution of nine bioenergetic modes on a phylogenetic tree based on 16S rRNA sequences from 272 species representing the full diversity of prokaryotic lineages. This highlights the patchy distribution of many pathways across different lineages, and suggests either up to 26 independent origins or 17 horizontal gene transfer events. Next, we used comparative genomics and phylogenetic analysis of all subunits of the F0F1 ATP synthase, common to most bacterial lineages regardless of their bioenergetic mode. Our results indicate an ancient origin of this protein complex, and no clustering based on bioenergetic mode, which suggests that no special modifications are needed for the ATP synthase to work with different electron transport chains. Moreover, examination of the ATP synthase genetic locus indicates various gene rearrangements in the different bacterial lineages, ancient duplications of atpI and of the beta subunit of the F0 subcomplex, as well as more recent stochastic lineage-specific and species-specific duplications of all subunits. We discuss the implications of the overall pattern of conservation and flexibility of the F0F1 ATP synthase genetic locus.

In spite of considerable insights into the microbial diversity of marine sponges, quantitative information on microbial abundances and community composition remains scarce. Here, we established qPCR assays for the specific quantification of four bacterial phyla of representative sponge symbionts as well as the kingdoms Eubacteria and Archaea. We could show that the 16S rRNA gene numbers of Archaea, Chloroflexi, and the candidate phylum Poribacteria were 4-6 orders of magnitude higher in high microbial abundance (HMA) than in low microbial abundance (LMA) sponges and that actinobacterial 16S rRNA gene numbers were 1-2 orders higher in HMA over LMA sponges, while those for Cyanobacteria were stable between HMA and LMA sponges. Fluorescence in situ hybridization of Aplysina aerophoba tissue sections confirmed the numerical dominance of Chloroflexi, which was followed by Poribacteria. Archaeal and actinobacterial cells were detected in much lower numbers. By use of fluorescence-activated cell sorting as a primer- and probe-independent approach, the dominance of Chloroflexi, Proteobacteria, and Poribacteria in A. aerophoba was confirmed. Our study provides new quantitative insights into the microbiology of sponges and contributes to a better understanding of the HMA/LMA dichotomy., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

This study investigated the cycling of C and N following application of olive mill wastewater (OMW) at various rates (0, 42, 84, and 168 m(3)/ha). OMW stimulated respiration rate throughout the study period, but an increase in soil organic matter was observed only at the highest rate. Soil phenol content decreased rapidly within 2 weeks following application but neither phenol oxidase and peroxidase activity nor laccase gene copies could explain this response. Soil NH4 (+)-N content increased in response to OMW application rate, while an opposite trend observed for NO3 (-)-N, which attributed to immobilization. This decrease was in accordance with amoA gene copies of archaeal and bacterial ammonia oxidizers in the first days following OMW application. Afterwards, although amoA gene copies and potential nitrification rates recovered to values similar to or higher than those in the non-treated soils, NO3 (-)-N content did not change among the treatments. A corresponding increase in denitrifying gene copies (nirK, nirS, nosZ) during that period indicates that denitrification, stimulated by OMW application rate, was responsible for this effect; a hypothesis consistent with the decrease in total Kjeldahl nitrogen content late in the season. The findings suggest that land application of OMW is a promising practice for OMW management, even at rates approaching the soil water holding capacity.

The fluorinated compounds are becoming a ubiquitous class of environmental contaminants because of their widespread applications, and their fate is a matter of great concern under anaerobic environment. In this work, the biotransformation of five fluoronitrobenzenes (FNBs), i.e., 2-fluoronitrobenzene (2-FNB), 3-fluoronitrobenzene (3-FNB), 4-fluoronitrobenzene (4-FNB), 2,4-difluoronitrobenzene (2,4-DFNB), and 2,3,4-trifluoronitrobenzene (2,3,4-TFNB), under methanogenic conditions had been studied by semicontinuous and batch tests for the first time. In 245 days, the five FNBs except 3-FNB were transformed mainly via nitro-reduction, and the reductive defluorination was not observed. During the biotransformation of 3-FNB, the reductive defluorination occurred after 98 days; however, its product was not aniline. The maximum transformation rates of 2-FNB, 3-FNB, 4-FNB, 2,4-DFNB, and 2,3,4-TFNB were 21.21 ± 1.73, 32.14 ± 2.33, 21.33 ± 2.48, 33.89 ± 6.87, and 10.87 ± 0.84 mg FNB (g VSS h)(-1), respectively. With the increase in the number of the fluorous groups, the transformation rates did not increase. Besides, the microbial communities were characterized by means of denaturing gradient gel electrophoresis (DGGE). Results showed that the predominant archaea were Methanobacterium, Methanosphaerula, Methanofollis, Methanospirillum, Methanolinea, and Methanosaeta; the predominant bacteria were Sphingbacteriales, Flavobacteriales, bacteroidales, Deltaproteobacteria, Desulfovibrionales, Clostridates, and Pseudomonadates. Few bacteria found were with high similarity to dechlorinating microorganisms reported. The results demonstrated that the pathways of FNBs biotransformation were different from those of the chloronitrobenzenes under methanogenic conditions.

DNA replication initiates at defined sites called origins, which serve as binding sites for initiator proteins that recruit the replicative machinery. Origins differ in number and structure across the three domains of life and their properties determine the dynamics of chromosome replication. Bacteria and some archaea replicate from single origins, whereas most archaea and all eukaryotes replicate using multiple origins. Initiation mechanisms that rely on homologous recombination operate in some viruses. Here we show that such mechanisms also operate in archaea. We use deep sequencing to study replication in Haloferax volcanii and identify four chromosomal origins of differing activity. Deletion of individual origins results in perturbed replication dynamics and reduced growth. However, a strain lacking all origins has no apparent defects and grows significantly faster than wild type. Origin-less cells initiate replication at dispersed sites rather than at discrete origins and have an absolute requirement for the recombinase RadA, unlike strains lacking individual origins. Our results demonstrate that homologous recombination alone can efficiently initiate the replication of an entire cellular genome. This raises the question of what purpose replication origins serve and why they have evolved.

Granules are large, self-supporting biofilms that form naturally in high-rate anaerobic treatment systems and are extremely important to reactor functionality. Granules exhibit functional and phylogenetic layering, interesting to both scientists and technologists. Until now, it has only been possible to analyze layering through sectioning and microscopic analysis with fluorescent in situ hybridization, or to analyze the whole granule through DNA extraction and microbial community profiling methods. This means different functional and spatial layers cannot be analyzed separately, including next generation sequencing techniques, such as pyrotag sequencing. In this work, we describe a method to remove microbes selectively from successive spatial layers through hydraulic shearing and demonstrate its application on anaerobic granules of three different types (VFA-, carbohydrate-, protein-fed) in size ranges 0.6-2 mm. Outer layers in particular could be selectively sheared as confirmed by FISH. TRFLP was used as an example bulk DNA method on selectively sheared fractions. A shift in dominant population was found from presumptive acidogens (such as Bacteroidetes and Anaerolinea) in outer layers to syntrophs (such as Syntrophomonas and Geobacter) in inner layers, with progressive changes through the depth. The strength of the shear-bulk molecular method over FISH was that a deeper phylogenetic profile could be obtained, even with TRFLP, and that prior knowledge of the community is not required., (Copyright © 2013 Wiley Periodicals, Inc.)

Archaea are non-bacterial prokaryotes associated with oral microbiota in humans, but their roles in oral pathologies remain controversial. Several studies reported the molecular detection of methanogenic archaea from periodontitis, but the significance of this association has not been confirmed yet. An electronic search was therefore conducted in MEDLINE-Pubmed to identify all papers published in English connecting archaea and periodontal infections. Data analysis of the selected studies showed that five genera of methanogenic archaea have been detected in the subgingival microbiota, Methanobrevibacter oralis being the most frequently detected species in 41% of periodontitis patients and 55% of periodontal pockets compared to 6% of healthy subjects and 5% of periodontally-healthy sites (p < 10(-5) , Chi-squared test). Based on the five determination-criteria proposed by Socransky (association with disease, elimination of the organism, host response, animal pathogenicity and mechanisms of pathogenicity), M. oralis is a periodontal pathogen. The methanogenic archaea load correlating with periodontitis severity further supports the pathogenic role of methanogenic archaea in periodontitis. Therefore, detection and quantification of M. oralis in periodontal pockets could help the laboratory diagnosis and follow-up of periodontitis. Determining the origin, diversity and pathogenesis of archaea in periodontal infections warrants further investigations., (© 2012 APMIS Published by John Wiley & Sons Ltd.)

Motivation: Metagenome analysis requires tools that can estimate the taxonomic abundances in anonymous sequence data over the whole range of biological entities. Because there is usually no prior knowledge about the data composition, not only all domains of life but also viruses have to be included in taxonomic profiling. Such a full-range approach, however, is difficult to realize owing to the limited coverage of available reference data. In particular, archaea and viruses are generally not well represented by current genome databases., Results: We introduce a novel approach to taxonomic profiling of metagenomes that is based on mixture model analysis of protein signatures. Our results on simulated and real data reveal the difficulties of the existing methods when measuring achaeal or viral abundances and show the overall good profiling performance of the protein-based mixture model. As an application example, we provide a large-scale analysis of data from the Human Microbiome Project. This demonstrates the utility of our method as a first instance profiling tool for a fast estimate of the community structure., Availability: http://gobics.de/TaxyPro., Supplementary Information: Supplementary Material is available at Bioinformatics online.

Unlabelled: The objective of this study was to compare the extraction efficiency of commercial DNA kits by evaluating the quantity and purity of DNA extracts obtained from paddy soils. DNA was extracted from three paddy soils using the FastDNA® SPIN kit for soil (FD), the innuSPEED soil DNA kit (INS) and the NucleoSpin® soil kit (NSP). DNA extracts were analysed by agarose gel electrophoresis and UV spectroscopy. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analyses were conducted to evaluate the potential bias of the DNA extractions on fingerprinting techniques. Regarding the quantity and the purity of the extracted DNA, the NSP kit was detected superior to the FD kit, while the INS kit failed to extract detectable amounts of DNA. DGGE fingerprints generated from PCR products (FD, NSP) showed high levels of similarity for the amplified 16S rRNA genes of methanogenic archaea (>95%) and bacteria (up to 100%) in each soil. This study suggested that the recently introduced NSP kit allowed for the adjustment of the lysis buffer composition to the soil of interest and is at least equivalent to the well-established FD kit for the extraction of DNA from paddy soils., Significance and Impact of the Study: The choice of commercial kits (FD, INS, NSP) has been of great importance regarding the quantity and purity of DNA extracted from paddy soils in this study. The composition of the cell lysis buffer represented a key component for successful extractions of DNA from different soils. The possibility of adjusting the lysis buffer to the soil of interest as well as the reproducibility of DGGE banding patterns makes the recently introduced NSP kit a strong competitor to the well-established FD kit for the extraction of DNA from paddy soils., (© 2012 The Society for Applied Microbiology.)

The concentration of CO(2) in the Earth's atmosphere has increased over the last century. Although this increase is unlikely to have direct effects on soil microbial communities, increased atmospheric CO(2) may impact soil ecosystems indirectly through plant responses. This study tested the hypothesis that exposure of plants to elevated CO(2) would impact soil microorganisms responsible for key nitrogen cycling processes, specifically denitrification and nitrification. We grew trembling aspen (Populus tremuloides) trees in outdoor chambers under ambient (360 ppm) or elevated (720 ppm) levels of CO(2) for 5 years and analyzed the microbial communities in the soils below the trees using quantitative polymerase chain reaction and clone library sequencing targeting the nitrite reductase (nirK) and ammonia monooxygenase (amoA) genes. We observed a more than twofold increase in copy numbers of nirK and a decrease in nirK diversity with CO(2) enrichment, with an increased predominance of Bradyrhizobia-like nirK sequences. We suggest that this dramatic increase in nirK-containing bacteria may have contributed to the significant loss of soil N in the CO(2)-treated chambers. Elevated CO(2) also resulted in a significant decrease in copy numbers of bacterial amoA, but no change in archaeal amoA copy numbers. The decrease in abundance of bacterial amoA was likely a result of the loss of soil N in the CO(2)-treated chambers, while the lack of response for archaeal amoA supports the hypothesis that physiological differences in these two groups of ammonia oxidizers may enable them to occupy distinct ecological niches and respond differently to environmental change.

Tundra ecosystem is of importance for its high accumulation of organic carbon and vulnerability to future climate change. Microorganisms play a key role in carbon dynamics of the tundra ecosystem by mineralizing organic carbon. We assessed both ecosystem process rates and community structure of Bacteria, Archaea, and Fungi in different soil layers (surface organic layer and subsurface mineral soil) in an Arctic soil ecosystem located at Spitsbergen, Svalbard during the summer of 2008 by using biochemical and molecular analyses, such as enzymatic assay, terminal restriction fragment length polymorphism (T-RFLP), quantitative polymerase chain reaction (qPCR), and pyrosequencing. Activity of hydrolytic enzymes showed difference according to soil type. For all three microbial communities, the average gene copy number did not significantly differ between soil types. However, archaeal diversities appeared to differ according to soil type, whereas bacterial and fungal diversity indices did not show any variation. Correlation analysis between biogeochemical and microbial parameters exhibited a discriminating pattern according to microbial or soil types. Analysis of the microbial community structure showed that bacterial and archaeal communities have different profiles with unique phylotypes in terms of soil types. Water content and hydrolytic enzymes were found to be related with the structure of bacterial and archaeal communities, whereas soil organic matter (SOM) and total organic carbon (TOC) were related with bacterial communities. The overall results of this study indicate that microbial enzyme activity were generally higher in the organic layer than in mineral soils and that bacterial and archaeal communities differed between the organic layer and mineral soils in the Arctic region. Compared to mineral soil, peat-covered organic layer may represent a hotspot for secondary productivity and nutrient cycling in this ecosystem.

The study of natural archaeal assemblages requires community context, namely, a concurrent assessment of the dynamics of archaeal, bacterial, and viral populations. Here, we use filter size-resolved metagenomic analyses to report the dynamics of 101 archaeal and bacterial OTUs and 140 viral populations across 17 samples collected over different timescales from 2007-2010 from Australian hypersaline Lake Tyrrell (LT). All samples were dominated by Archaea (75-95%). Archaeal, bacterial, and viral populations were found to be dynamic on timescales of months to years, and different viral assemblages were present in planktonic, relative to host-associated (active and provirus) size fractions. Analyses of clustered regularly interspaced short palindromic repeat (CRISPR) regions indicate that both rare and abundant viruses were targeted, primarily by lower abundance hosts. Although very few spacers had hits to the NCBI nr database or to the 140 LT viral populations, 21% had hits to unassembled LT viral concentrate reads. This suggests local adaptation to LT-specific viruses and/or undersampling of haloviral assemblages in public databases, along with successful CRISPR-mediated maintenance of viral populations at abundances low enough to preclude genomic assembly. This is the first metagenomic report evaluating widespread archaeal dynamics at the population level on short timescales in a hypersaline system.

Molecular diversity of protists from bovine rumen fluid incubated with condensed tannins of Leucaena leucocephala hybrid-Rendang at 20 mg/500 mg dry matter (treatment) or without condensed tannins (control) was investigated using 18S rRNA gene library. Clones from the control library were distributed within nine genera, but clones from the condensed tannin treatment clone library were related to only six genera. Diversity estimators such as abundance-based coverage estimation and Chao1 showed significant differences between the two libraries, although no differences were found based on Shannon-Weaver index and Libshuff., (© 2012 The Author(s) Journal of Eukaryotic Microbiology © 2012 International Society of Protistologists.)

In Mediterranean-type grassland ecosystems, the timing of rainfall events controls biogeochemical cycles, as well as the phenology and productivity of plants and animals. Here, we investigate the effect of short-term (days) soil environmental conditions on microbial community structure and composition during a natural wetting and drying cycle. Soil samples were collected from a meadow in Northern California at four time points after the first two rainfall events of the rainy season. We used 16S rRNA microarrays (PhyloChip) to track changes in bacterial and archaeal community composition. Microbial communities at time points 1 and 3 were significantly different than communities at time points 2 and 4. Based on ordination analysis, the available carbon, soil moisture, and temperature explained most of the variation in community structure. For the first time, a complementary and more comprehensive approach using linear regression and generalized logical networks were used to identify linear and nonlinear associations among environmental variables and with the relative abundance of subfamilies. Changes in soil moisture and available carbon were correlated with the relative abundance of many phyla. Only the phylum Actinobacteria showed a lineage-specific relationship to soil moisture but not to carbon or nitrogen. The results indicate that the use of a high taxonomic rank in correlations with nutritional indicators might obscure divergent subfamily-level responses to environmental parameters. An important implication of this research is that there is short-term variation in microbial community composition driven in part by rainfall fluctuation that may not be evident in long-term studies with coarser time resolution.

Ammonia oxidation has been intensively studied for its sensitivity to environmental shifts and stresses. However, acute stress effects on the occurrence and composition of ammonia oxidizing bacteria (AOB) and archaea (AOA) based on expression of related molecular markers in complex soil environments have been to an extent overlooked, particularly concerning transient but commonly occurring environmental changes like soil moisture shifts. The present study investigates the responses of AOB and AOA to moisture shifts and high Zn soil content. AmoA gene copies and transcripts of AOB and AOA along with potential nitrification activity were measured in a soil microcosm approach for investigating the referred environmental shifts. Moisture change from 87 to 50 % of the water holding capacity caused a ~99 % reduction of AOB but not of AOA amoA transcripts that did not change significantly. Increasing applied zinc concentrations resulted in a reduction of potential nitrification rates and negatively affected studied gene expressions of both AOB and AOA, with AOB being more responsive. Both 16 S rRNA and amoA transcripts of AOB had an inverse relation to the applied zinc, indicating a gradual loss in total cell activity. Our results suggest the existence of pronounced differences between AOB and AOA concerning ammonia oxidation activity.

A 16S rDNA sequence-based investigation of methanogenic Archaea in the human stools found Methanobrevibacter smithii in 99.2% and Methanosphaera stadtmanae in 32.6%. The recently described Methanomassiliicoccus luminyensis found by others to be representative of a new order of methanogenic Archaea was found in 4% of stool specimens. The prevalence of M. luminyensis significantly increased with age, contrary to M. smithii and M. stadtmanae., (© 2012 The Authors APMIS © 2012 APMIS.)

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.

Propionate is one of the major intermediary products in the anaerobic decomposition of organic matter in wetlands and paddy fields. Under methanogenic conditions, propionate is decomposed through syntrophic interaction between proton-reducing and propionate-oxidizing bacteria and H(2)-consuming methanogens. Temperature is an important environmental regulator; yet its effect on syntrophic propionate oxidation has been poorly understood. In the present study, we investigated the syntrophic oxidation of propionate in a rice field soil at 15°C and 30°C. [U-(13)C]propionate (99 atom%) was applied to anoxic soil slurries, and the bacteria and archaea assimilating (13)C were traced by DNA-based stable isotope probing. Syntrophobacter spp., Pelotomaculum spp., and Smithella spp. were found significantly incorporating (13)C into their nucleic acids after [(13)C]propionate incubation at 30°C. The activity of Smithella spp. increased in the later stage, and concurrently that of Syntrophomonas spp. increased. Aceticlastic Methanosaetaceae and hydrogenotrophic Methanomicrobiales and Methanocellales acted as methanogenic partners at 30°C. Syntrophic oxidation of propionate also occurred actively at 15°C. Syntrophobacter spp. were significantly labeled with (13)C, whereas Pelotomaculum spp. were less active at this temperature. In addition, Methanomicrobiales, Methanocellales, and Methanosarcinaceae dominated the methanogenic community, while Methanosaetaceae decreased. Collectively, temperature markedly influenced the activity and community structure of syntrophic guilds degrading propionate in the rice field soil. Interestingly, Geobacter spp. and some other anaerobic organisms like Rhodocyclaceae, Acidobacteria, Actinobacteria, and Thermomicrobia probably also assimilated propionate-derived (13)C. The mechanisms for the involvement of these organisms remain unclear.

Analyses of the DNA replication-associated proteins of hyperthermophilic archaea have yielded considerable insight into the structure and biochemical function of these evolutionarily conserved factors. However, little is known about the regulation and progression of DNA replication in the context of archaeal cells. In the current work, we describe the generation of strains of Sulfolobus solfataricus and Sulfolobus acidocaldarius that allow the incorporation of nucleoside analogues during DNA replication. We employ this technology, in conjunction with immunolocalization analyses of replisomes, to investigate the sub-cellular localization of nascent DNA and replisomes. Our data reveal a peripheral localization of replisomes in the cell. Furthermore, while the two replication forks emerging from any one of the three replication origins in the Sulfolobus chromosome remain in close proximity, the three origin loci are separated.

Anaerobic oxidation of methane (AOM) was investigated in hydrothermal sediments of Guaymas Basin based on δ(13)C signatures of CH(4), dissolved inorganic carbon and porewater concentration profiles of CH(4) and sulfate. Cool, warm and hot in-situ temperature regimes (15-20 °C, 30-35 °C and 70-95 °C) were selected from hydrothermal locations in Guaymas Basin to compare AOM geochemistry and 16S ribosomal RNA (rRNA), mcrA and dsrAB genes of the microbial communities. 16S rRNA gene clone libraries from the cool and hot AOM cores yielded similar archaeal types such as Miscellaneous Crenarchaeotal Group, Thermoproteales and anaerobic methane-oxidizing archaea (ANME)-1; some of the ANME-1 archaea formed a separate 16S rRNA lineage that at present seems to be limited to Guaymas Basin. Congruent results were obtained by mcrA gene analysis. The warm AOM core, chemically distinct by lower porewater sulfide concentrations, hosted a different archaeal community dominated by the two deep subsurface archaeal lineages Marine Benthic Group D and Marine Benthic Group B, and by members of the Methanosarcinales including ANME-2 archaea. This distinct composition of the methane-cycling archaeal community in the warm AOM core was confirmed by mcrA gene analysis. Functional genes of sulfate-reducing bacteria and archaea, dsrAB, showed more overlap between all cores, regardless of the core temperature. 16S rRNA gene clone libraries with Euryarchaeota-specific primers detected members of the Archaeoglobus clade in the cool and hot cores. A V6-tag high-throughput sequencing survey generally supported the clone library results while providing high-resolution detail on archaeal and bacterial community structure. These results indicate that AOM and the responsible archaeal communities persist over a wide temperature range.

A reductionist ecological approach of using a model genus was adopted in order to understand how microbial community structure is driven by metabolic properties. The distribution along an estuarine gradient of the highly specialised genus Methanosaeta was investigated and compared to the previously determined distribution of the more metabolically flexible Desulfobulbus. Methanosaeta genotypic distribution along the Colne estuary (Essex, UK) was determined by DNA- and RNA-based denaturing gradient gel electrophoresis and 16S rRNA gene sequence analyses. Methanosaeta distribution was monotonic, with a consistently diverse community and no apparent niche partitioning either in DNA or RNA analyses. This distribution pattern contrasts markedly with the previously described niche partitioning and sympatric differentiation of the model generalist, Desulfobulbus. To explain this difference, it is hypothesised that Methanosaeta's strict metabolic needs limit its adaptation potential, thus populations do not partition into spatially distinct groups and so do not appear to be constrained by gross environmental factors such as salinity. Thus, at least for these two model genera, it appears that metabolic flexibility may be an important factor in spatial distribution and this may be applicable to other microbes.

Aquatic environments harbour large and diverse microbial populations that ensure their functioning and sustainability. In the current context of global change, characterizing microbial diversity has become crucial, and new tools have been developed to overcome the methodological challenges posed by working with microbes in nature. The advent of Sanger sequencing and now next-generation sequencing technologies has enabled the resolution of microbial communities to an unprecedented degree of precision. However, to correctly interpret microbial diversity and its patterns this revolution must also consider conceptual and methodological matters. This review presents advances, gaps and caveats of these recent approaches when considering microorganisms in aquatic ecosystems. We also discuss potentials and limitations of the available methodologies, from water sampling to sequence analysis, and suggest alternative ways to incorporate results in a conceptual and methodological framework. Together, these methods will allow us to gain an unprecedented understanding of microbial diversity in aquatic ecosystems., (© 2011 Blackwell Publishing Ltd.)