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Archaea have histone homologues and chromatin proteins to organize their DNA into a compact form. This allows them to survive in extreme climates. Cren7 is one such chromatin protein conserved in Crenarchaeota. When Cren7 binds to model natural DNA, calf thymus DNA (CTD, 58% AT content) and polynucleotides under adverse solution conditions (high temperature, ionic strength), CD bands at 275-290 nm shift to higher wavelengths indicating structural changes in DNA. It formed a strong complex with CTD and poly(dA-dT)·poly(dA-dT), via a combination of electrostatic and non-electrostatic interactions. A low binding enthalpy indicated that the process was driven by entropy. The interaction was independent of the nature of the anions present in the solution. On studying the variation in protein affinity with salt concentration, it was estimated that the electrostatic interaction at the interface involves 3 pairs of ions at the protein-DNA interface. The affinity and binding site size decreased on changing the pH of the solution (between pH 6 and 8), but temperature did not result in such effects. Cren7 bound to 10 bp of DNA, increasing its flexibility and thermal stability by more than 30 ° C. Increasing the amount of Cren7 produces cooperative structural transitions in DNAs without any similar transition in the protein. These crucial binding parameters, energetics, and structural changes decipher the mystery of Cren7 mediated DNA organization in Crenarchaeota.

Because ammonia-oxidizing archaea (AOA) are ubiquitous and highly abundant in almost all terrestrial soils, they play an important role in soil nitrification. However, the changes in the structure and function of AOA communities and their edaphic drivers in paddy soils under different fertilization and irrigation regimes remain unclear. In this study, we investigated AOA abundance, diversity and activity in acid paddy soils by a field experiment. Results indicated that the highest potential ammonia oxidation (PAO) (0.011 μg NO 2 -  -N g -1 d.w.day -1 ) was found in T 2 (optimal irrigation and fertilization)-treated soils, whereas the lowest PAO (0.004 μg NO 2 -  -N g -1 d.w.day -1 ) in T 0 (traditional irrigation)- treated soils. Compared with the T 0 -treated soil, the T 2 treatment significantly (P < 0.05) increased AOA abundances. Furthermore, the abundance of AOA was significantly (P < 0.01) positively correlated with pH, soil organic carbon (SOC), and PAO. Meanwhile, pH and SOC content were significantly (P < 0.05) higher in the T 2 -treated soil than those in the T 1 (traditional irrigation and fertilization)- treated soil. In addition, these two edaphic factors further influenced the AOA community composition. The AOA phylum Crenarchaeota was mainly found in the T 2 -treated soils. Phylogenetic analysis revealed that most of the identified OTUs of AOA were mainly affiliated with Crenarchaeota. Furthermore, the T 2 treatment had higher rice yield than the T 0 and T 1 treatments. Together, our findings confirm that T 2 might ameliorate soil chemical properties, regulate the AOA community structure, increase the AOA abundance, enhance PAO and consequently maintain rice yields in the present study., (© 2021. The Author(s).)

The autotrophic 3-hydroxypropionate/4-hydroxybutyrate (HP/HB) cycle functions in thermoacidophilic, (micro)aerobic, hydrogen-oxidizing Crenarchaeota of the order Sulfolobales as well as in mesophilic, aerobic, ammonia-oxidizing Thaumarchaeota. Notably, the HP/HB cycle evolved independently in these two archaeal lineages, and crenarchaeal and thaumarchaeal versions differ regarding their enzyme properties and phylogeny. These differences result in altered energetic efficiencies between the variants. Compared to the crenarchaeal HP/HB cycle, the thaumarchaeal variant saves two ATP equivalents per turn, rendering it the most energy-efficient aerobic pathway for carbon fixation. Characteristically, the HP/HB cycle includes two enoyl coenzyme A (CoA) hydratase reactions: the 3-hydroxypropionyl-CoA dehydratase reaction and the crotonyl-CoA hydratase reaction. In this study, we show that both reactions are catalyzed in the aforementioned archaeal groups by a promiscuous 3-hydroxypropionyl-CoA dehydratase/crotonyl-CoA hydratase (Msed&#95;2001 in crenarchaeon Metallosphaera sedula and Nmar&#95;1308 in thaumarchaeon Nitrosopumilus maritimus ). Although these two enzymes are homologous, they are closely related to bacterial enoyl-CoA hydratases and were retrieved independently from the same enzyme pool by the ancestors of Crenarchaeota and Thaumarchaeota , despite the existence of multiple alternatives. This striking similarity in the emergence of enzymes involved in inorganic carbon fixation from two independently evolved pathways highlights that convergent evolution of autotrophy could be much more widespread than anticipated. IMPORTANCE Inorganic carbon fixation is the most important biosynthetic process on Earth and the oldest type of metabolism. The autotrophic HP/HB cycle functions in Crenarchaeota of the order Sulfolobales and in ammonia-oxidizing Archaea of the phylum Thaumarchaeota that are highly abundant in marine, terrestrial, and geothermal environments. Bioinformatic prediction of the autotrophic potential of microorganisms or microbial communities requires identification of enzymes involved in autotrophy. However, many microorganisms possess several isoenzymes that may potentially catalyze the reactions of the cycle. Here, we studied the enzymes catalyzing 3-hydroxypropionyl-CoA dehydration and crotonyl-CoA hydration in Nitrosopumilus maritimus ( Thaumarchaeota ) as well as in Metallosphaera sedula ( Crenarchaeota ). We showed that both reactions were catalyzed by homologous promiscuous enzymes, which evolved independently from each other from their bacterial homologs. Furthermore, the HP/HB cycle is of applied value, and knowledge of its enzymes is necessary to transfer them to a heterologous host for synthesis of various value-added products., (Copyright © 2021 Liu et al.)

Members of the phylum Bathyarchaeota and the class Thermoplasmata are widespread in marine and freshwater sediments where they have been recognized as key players in the carbon cycle. Here, we tested the responsiveness of archaeal communities on settled plant debris and sediment from a karstic lake to different organic carbon amendments (amino acids, plant-derived carbohydrates, and aromatics) using a lab-scale microcosm. Changes in the composition and abundance of sediment and biofilm archaeal communities in both DNA and RNA fractions were assessed by 16S rRNA gene amplicon sequencing and qPCR, respectively, after 7 and 30 days of incubation. Archaeal communities showed compositional changes in terms of alpha and beta diversity in relation to the type of carbon source (amino acids vs. plant-derived compounds), the nucleic acid fraction (DNA vs. RNA), and the incubation time (7 vs. 30 days). Distinct groups within the Bathyarchaeota (Bathy-15 and Bathy-6) and the Thermoplasmata (MBG-D) differently reacted to carbon supplements as deduced from the analysis of RNA libraries. Whereas Bathyarchaeota in biofilms showed a long-term positive response to humic acids, their counterparts in the sediment were mainly stimulated by the addition of tryptophan, suggesting the presence of different subpopulations in both habitats. Overall, our work presents an in vitro assessment of the versatility of archaea inhabiting freshwater sediments towards organic carbon and introduces settled leaf litter as a new habitat for the Bathyarchaeota and the Thermoplasmata., Competing Interests: The authors have declared that no competing interests exist.

Spent mushroom substrate (SMS) is the residue of edible mushroom production occurring in huge amounts. The SMS residue can be digested for biogas production in the mesophilic anaerobic digestion. In the present study, performance of batch thermophilic anaerobic digestion (TAD) of SMS was investigated as well as the interconnected microbial population structure changes. The analyzed batch TAD process lasted for 12 days with the cumulative methane yields of 177.69 mL/g volatile solid (VS). Hydrolytic activities of soluble sugar, crude protein, and crude fat in SMS were conducted mainly in the initial phase, accompanied by the excessive accumulation of volatile fatty acids and low methane yield. Biogas production increased dramatically from days 4 to 6. The degradation rates of cellulose and hemicellulose were 47.53 and 55.08%, respectively. The high-throughput sequencing of 16S rRNA gene amplicons revealed that Proteobacteria (56.7%-62.8%) was the dominant phylum in different fermentative stages, which was highly specific compared with other anaerobic processes of lignocellulosic materials reported in the literature. Crenarchaeota was abundant in the archaea. The most dominant genera of archaea were retrieved as Methanothermobacter and Methanobacterium, but the latter decreased sharply with time. This study shows that TAD is a feasible method to handle the waste SMS.

Crenarchaeal chromatin protein Cren7 binds double-stranded DNA in the minor groove, introducing a sharp single-step DNA kink. The side chain of Leu28, a residue conserved among all Cren7 homologs, intercalates into the kinked DNA step. In the present study, we replaced Leu28 with a residue containing a hydrophobic side chain of different sizes (i.e. L28A, L28V, L28I, L28M and L28F). Both the stability of the Cren7-DNA complex and the ability of Cren7 to constrain DNA supercoils correlated well with the size of the intercalated side chain. Structural analysis shows that L28A induces a kink (∼43°), nearly as sharp as that produced by wild-type Cren7 (∼48°), in the bound DNA fragment despite the lack of side chain intercalation. In another duplex DNA fragment, L28F inserts a large hydrophobic side chain deep into the DNA step, but introduces a smaller kink (∼39°) than that formed by the wild-type protein (∼50°). Mutation of Leu28 into methionine yields two protein conformers differing in loop β3-β4 orientation, DNA-binding surface and DNA geometry in the protein-DNA structure. Our results indicate that side chain intercalation is not directly responsible for DNA kinking or bending by Cren7, but plays a critical role in the stabilization of the Cren7-DNA complex. In addition, the flexibility of loop β3-β4 in Cren7, as revealed in the crystal structure of L28M-DNA, may serve a role in the modulation of chromosomal organization and function in the cell., (© 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

Reversible protein phosphorylation is the main mechanism of signal transduction that enables cells to rapidly respond to environmental changes by controlling the functional properties of proteins in response to external stimuli. However, whereas signal transduction is well studied in Eukaryotes and Bacteria, the knowledge in Archaea is still rather scarce. Archaea are special with regard to protein phosphorylation, due to the fact that the two best studied phyla, the Euryarchaeota and Crenarchaeaota, seem to exhibit fundamental differences in regulatory systems. Euryarchaeota (e.g. halophiles, methanogens, thermophiles), like Bacteria and Eukaryotes, rely on bacterial-type two-component signal transduction systems (phosphorylation on His and Asp), as well as on the protein phosphorylation on Ser, Thr and Tyr by Hanks-type protein kinases. Instead, Crenarchaeota (e.g. acidophiles and (hyper)thermophiles) only depend on Hanks-type protein phosphorylation. In this review, the current knowledge of reversible protein phosphorylation in Archaea is presented. It combines results from identified phosphoproteins, biochemical characterization of protein kinases and protein phosphatases as well as target enzymes and first insights into archaeal signal transduction by biochemical, genetic and polyomic studies., (© FEMS 2016.)

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

Biological features can be inferred, based on genomic data, for many microbial lineages that remain uncultured. However, cultivation is important for characterizing an organism's physiology and testing its genome-encoded potential. Here we use single-cell genomics to infer cultivation conditions for the isolation of an ectosymbiotic Nanoarchaeota ('Nanopusillus acidilobi') and its host (Acidilobus, a crenarchaeote) from a terrestrial geothermal environment. The cells of 'Nanopusillus' are among the smallest known cellular organisms (100-300 nm). They appear to have a complete genetic information processing machinery, but lack almost all primary biosynthetic functions as well as respiration and ATP synthesis. Genomic and proteomic comparison with its distant relative, the marine Nanoarchaeum equitans illustrate an ancient, common evolutionary history of adaptation of the Nanoarchaeota to ectosymbiosis, so far unique among the Archaea.

Thermoacidophilic sulfate reduction remains a poorly studied process, which was investigated in the present work. Radioisotope analysis with 35S-Iabeled sulfate was used to determine the rates of dissimilatory sulfate reduction in acidic thermal springs of Kamchatka, Russia. Sulfate reduction rates were found to vary from 0.054 to 12.9 nmol S04/(cm3 day). The Neftyanaya ploshchadka spring (Uzon caldera, 60'C, pH 4.2) and Oreshek spring (Mutnovskii volcano, 91'C, pH 3.5) exhibited the highest activity of sulfate-reducing prokaryotes. Stable enrich- ment'cultures reducing sulfate at pH and temperature values close to'the environmental ones were obtained from these springs. Analysis of the 16S rRNA gene sequences revealed that'a chemolithoautotrophic bacterium Ther- modesufobium sp. 3127-1 was responsible for sulfate reduction in the enrichment from the Oil Site spring. A chemoorganoheterotrophic archaeon Vulcanisaeta sp. 3102-1 (phylum Crenarchaeota) was identified in the en- richment from Oreshek spring. Thus, dissimilatory sulfate reduction under thermoacidophilic conditions was demonstrated and the agents responsible for this process were revealed.

Microorganisms are usually studied either in highly complex natural communities or in isolation as monoclonal model populations that we manage to grow in the laboratory. Here, we uncover the biology of some of the most common and yet-uncultured bacteria in freshwater environments using a mixed culture from Lake Grosse Fuchskuhle. From a single shotgun metagenome of a freshwater mixed culture of low complexity, we recovered four high-quality metagenome-assembled genomes (MAGs) for metabolic reconstruction. This analysis revealed the metabolic interconnectedness and niche partitioning of these naturally dominant bacteria. In particular, vitamin- and amino acid biosynthetic pathways were distributed unequally with a member of Crenarchaeota most likely being the sole producer of vitamin B12 in the mixed culture. Using coverage-based partitioning of the genes recovered from a single MAG intrapopulation metabolic complementarity was revealed pointing to 'social' interactions for the common good of populations dominating freshwater plankton. As such, our MAGs highlight the power of mixed cultures to extract naturally occurring 'interactomes' and to overcome our inability to isolate and grow the microbes dominating in nature., (© 2015 John Wiley & Sons Ltd.)

Stable isotope probing (SIP) of deoxyribonucleic acid (DNA) was used to identify microbes incorporating (13) C-labeled acetate in sulfate-reducing sediment from Aarhus Bay, Denmark. Sediment was incubated in medium containing 10 mM sulfate and different (13) C-acetate (10, 1, 0.1 mM) concentrations. The resultant changes in microbial community composition were monitored in total and SIP-fractionated DNA during long-term incubations. Chemical analyses demonstrated metabolic activity in all sediment slurries, with sulfate-reducing activity largely determined by initial acetate concentrations. Sequencing of 16S rRNA gene PCR amplicons showed that the incubations shifted the bacterial but not the archaeal community composition. After 3 months of incubation, only sediment slurries incubated with 10 mM (13) C-acetate showed detectable (13) C-DNA labeling. Based on 16S rRNA and dsrB gene PCR amplicon sequencing, the (13) C-labeled DNA pool was dominated by a single type of sulfate reducer representing a novel genus in the family Desulfobacteraceae. In addition, members of the uncultivated Crenarchaeotal group C3 were enriched in the (13) C-labeled DNA. Our results were reproducible across biological replicate experiments and provide new information about the identities of uncultured acetate-consuming bacteria and archaea in marine sediments., (© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The anoxic sediments of the White Oak River estuary comprise a distinctive sulfate-methane transition zone (SMTZ) and natural enrichment of the archaea affiliated with the Miscellaneous Crenarchaeotal Group (MCG). Archaeal biphytanes were generally depleted in (13) C, with δ(13) C values being less than -35‰, indicative of production by active sedimentary archaeal populations. Multivariate analysis of the downcore distributions of 63 lipid biomarkers identified three major groups of lipids that were enriched in the surface, SMTZ or subsurface depths. Intact polar lipids with phosphatidylglycerol headgroups and glycerol dibiphytanyl glycerol tetraethers containing one, two or three cyclopentane rings were enriched at the base of the SMTZ and likely represent the accumulated product of a small but active ANME-1 community. The recently identified butanetriol dibiphytanyl glycerol tetraethers (BDGT), which increased relatively to other lipids with depth, were correlated with the relative abundance of MCG in archaeal 16S rRNA clone libraries, and were (13) C depleted throughout the depth profile, suggesting BDGT lipids as putative biomarkers of an MCG community that may either be autotrophic or feeding on (13) C-depleted organic substrates transported by porewater., (© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Unlabelled: Autotrophic microorganisms are able to utilize carbon dioxide as their only carbon source, or, alternatively, many of them can grow heterotrophically on organics. Different variants of autotrophic pathways have been identified in various lineages of the phylum Crenarchaeota. Aerobic members of the order Sulfolobales utilize the hydroxypropionate-hydroxybutyrate cycle (HHC) to fix inorganic carbon, whereas anaerobic Thermoproteales use the dicarboxylate-hydroxybutyrate cycle (DHC). Knowledge of transcriptional regulation of autotrophic pathways in Archaea is limited. We applied a comparative genomics approach to predict novel autotrophic regulons in the Crenarchaeota. We report identification of two novel DNA motifs associated with the autotrophic pathway genes in the Sulfolobales (HHC box) and Thermoproteales (DHC box). Based on genome context evidence, the HHC box regulon was attributed to a novel transcription factor from the TrmB family named HhcR. Orthologs of HhcR are present in all Sulfolobales genomes but were not found in other lineages. A predicted HHC box regulatory motif was confirmed by in vitro binding assays with the recombinant HhcR protein from Metallosphaera yellowstonensis. For the DHC box regulon, we assigned a different potential regulator, named DhcR, which is restricted to the order Thermoproteales. DhcR in Thermoproteus neutrophilus (Tneu&#95;0751) was previously identified as a DNA-binding protein with high affinity for the promoter regions of two autotrophic operons. The global HhcR and DhcR regulons reconstructed by comparative genomics were reconciled with available omics data in Metallosphaera and Thermoproteus spp. The identified regulons constitute two novel mechanisms for transcriptional control of autotrophic pathways in the Crenarchaeota., Importance: Little is known about transcriptional regulation of carbon dioxide fixation pathways in Archaea. We previously applied the comparative genomics approach for reconstruction of DtxR family regulons in diverse lineages of Archaea. Here, we utilize similar computational approaches to identify novel regulatory motifs for genes that are autotrophically induced in microorganisms from two lineages of Crenarchaeota and to reconstruct the respective regulons. The predicted novel regulons in archaeal genomes control the majority of autotrophic pathway genes and also other carbon and energy metabolism genes. The HhcR regulon was experimentally validated by DNA-binding assays in Metallosphaera spp. Novel regulons described for the first time in this work provide a basis for understanding the mechanisms of transcriptional regulation of autotrophic pathways in Archaea., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

16S rRNA Crenarchaeota and Thermoplasmata sequences retrieved from 22 anaerobic digesters were analysed. 4.8 and 0.53 % of archaeal sequences were simultaneously affiliated to these lineages. A core of 2 operational taxonomic units (OTUs) representing 0.6 to -33.6 % of all archaeal sequences were defined for the Crenarchaeotes and identified to already known but not yet cultivable organisms in almost half of the digesters sampled. For the Thermoplasmata, apparently less abundant with 0.7 to -4.7 % of the archaeal sequences, 3 OTUs were identified. We showed here that Crenarchaeotes coexist with methanogens and are particularly abundant when Arch I lineage (also called WSA2 by Hugenholtz) is dominant in digesters. Moreover, Thermoplasmata were detected when Crenarchaeota were present. Interactions between methanogens, Crenarchaeotea and Thermoplamata were thus discussed.

Based on the transient exposure of Chesapeake Bay sediments to hydrocarbons and the metabolic versatility of known anaerobic alkane-degrading microorganisms, it was hypothesized that distinct Bay sediment communities, governed by geochemical gradients, would have intrinsic alkane-utilizing potential under sulfate-reducing and/or methanogenic conditions. Sediment cores were collected along a transect of the Bay. Community DNA was interrogated via pyrosequencing of 16S rRNA genes, PCR of anaerobic hydrocarbon activation genes, and qPCR of 16S rRNA genes and genes involved in sulfate reduction/methanogenesis. Site sediments were used to establish microcosms amended with n-hexadecane under sulfate-reducing and methanogenic conditions. Sequencing of 16S rRNA genes indicated that sediments associated with hypoxic water columns contained significantly greater proportions of Bacteria and Archaea consistent with syntrophic degradation of organic matter and methanogenesis compared to less reduced sediments. Microbial taxa frequently associated with hydrocarbon-degrading communities were found throughout the Bay, and the genetic potential for hydrocarbon metabolism was demonstrated via the detection of benzyl-(bssA) and alkylsuccinate synthase (assA) genes. Although microcosm studies did not indicate sulfidogenic alkane degradation, the data suggested that methanogenic conversion of alkanes was occurring. These findings highlight the potential role that anaerobic microorganisms could play in the bioremediation of hydrocarbons in the Bay., (© FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Anaerobic thermophilic archaea of the genera Thermogladius and Desulfurococcus capable of a- and P3-keratin decomposition were isolated from hot springs of Kamchatka and Kunashir Island. For two of them (strains 2355k and 3008g), the presence of high-molecular mass, cell-bound endopeptidases active against nonhydrolyzed and partially hydrolyzed proteins at high values of temperature and pH was shown. Capacity for β-keratin decomposition was also found in collection strains (type strains of Desulfurococcus amylolyticus subsp. amylolyticus, D. mucosus subsp. mobilis, and D. fermentans).

Production of gaseous hydrocarbons by the microbial community of the Posolsky Bank methane seep bottom sediments (Southern Baikal) was studied at 4°C. Formation of both methane and a heavier gas- eous hydrocarbon, ethane, was detected in enrichment cultures. The highest methane concentrations (6.15 and 4.51 mmol L(-1)) were revealed in enrichments from the sediments from 55-cm depth incubated with-so- dium acetate and H2/CO2 gas mixture, respectively. A decrease in activity of aceticlastic methanogensand a decrease in methane concentration produced by hydrogenotrophic archaea occurred with depth. The highest concentration of ethane was revealed in enrichments from the microbial community of the layer close to gas hydrates (75 cm) incubated with CO2 as a substrate. According to analysis of the 16S rRNA gene fragments from the clone library, these enrichments were found to contain members of the phylum Crenarchaeota form- ing a separate cluster with members of the class Thermoprotei. The phylum Euryarchaeota was represented by nucleotide sequences of the organisms homologous to members of the orders Methanococcales, Methanosa- rcinales, and Thermoplasmatales.

How Arctic climate change might translate into alterations of biogeochemical cycles of carbon (C) and nitrogen (N) with respect to inorganic and organic N utilization is not well understood. This study combined 15N uptake rate measurements for ammonium, nitrate, and urea with 15N- and 13C-based DNA stable-isotope probing (SIP). The objective was to identify active bacterial and archeal plankton and their role in N and C uptake during the Arctic summer and winter seasons. We hypothesized that bacteria and archaea would successfully compete for nitrate and urea during the Arctic winter but not during the summer, when phytoplankton dominate the uptake of these nitrogen sources. Samples were collected at a coastal station near Barrow, AK, during August and January. During both seasons, ammonium uptake rates were greater than those for nitrate or urea, and nitrate uptake rates remained lower than those for ammonium or urea. SIP experiments indicated a strong seasonal shift of bacterial and archaeal N utilization from ammonium during the summer to urea during the winter but did not support a similar seasonal pattern of nitrate utilization. Analysis of 16S rRNA gene sequences obtained from each SIP fraction implicated marine group I Crenarchaeota (MGIC) as well as Betaproteobacteria, Firmicutes, SAR11, and SAR324 in N uptake from urea during the winter. Similarly, 13C SIP data suggested dark carbon fixation for MGIC, as well as for several proteobacterial lineages and the Firmicutes. These data are consistent with urea-fueled nitrification by polar archaea and bacteria, which may be advantageous under dark conditions., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Mesophilic Crenarchaeota (also known as Thaumarchaeota) are ubiquitous and abundant in marine habitats. However, very little is known about their metabolic function in situ. In this study, salt marsh sediments from New Jersey were screened via stable isotope probing (SIP) for heterotrophy by amending with a single (13)C-labeled compound (acetate, glycine or urea) or a complex (13)C-biopolymer (lipids, proteins or growth medium (ISOGRO)). SIP incubations were done at two substrate concentrations (30-150 μM; 2-10 mg ml(-1)), and (13)C-labeled DNA was analyzed by terminal restriction fragment length polymorphism (TRFLP) analysis of 16S rRNA genes. To test for autotrophy, an amendment with (13)C-bicarbonate was also performed. Our SIP analyses indicate salt marsh crenarchaea are heterotrophic, double within 2-3 days and often compete with heterotrophic bacteria for the same organic substrates. A clone library of (13)C-amplicons was screened to find matches to the (13)C-TRFLP peaks, with seven members of the Miscellaneous Crenarchaeal Group and seven members from the Marine Group 1.a Crenarchaeota being discerned. Some of these crenarchaea displayed a preference for particular carbon sources, whereas others incorporated nearly every (13)C-substrate provided. The data suggest salt marshes may be an excellent model system for studying crenarchaeal metabolic capabilities and can provide information on the competition between crenarchaea and other microbial groups to improve our understanding of microbial ecology.

The complete genome of the obligately anaerobic crenarchaeote Fervidicoccus fontis Kam940(T), a terrestrial hot spring inhabitant with a growth optimum of 65-70 °C, has been sequenced and analyzed. The small 1.3-Mb genome encodes several extracellular proteases and no other extracellular hydrolases. No complete pathways of carbohydrate catabolism were found. Genes coding for enzymes necessary for amino acid transamination and further oxidative decarboxylation are present. The genome encodes no mechanisms of acyl-CoA and acetyl-CoA oxidation. Two [NiFe]-hydrogenases are encoded: a membrane-bound energy-converting hydrogenase and a cytoplasmic one. The ATP-synthase is H(+)-dependent as inferred from the amino acid sequence of the membrane rotor subunit. On the whole, genome analysis shows F. fontis to be a peptidolytic heterotroph with a restricted biosynthetic potential, which is in accordance with its phenotypic properties. The analysis of phylogenetic markers and of the distribution of best blastp hits of F. fontis proteins in the available genomes of Crenarchaeota supports distinct phylogenetic position of the order Fervidicoccales as a separate lineage adjoining the heterogeneous order Desulfurococcales. In addition, certain F. fontis genomic features correlate with its adaptation to temperatures of 60-80 °C, which are lower than temperatures preferred by Desulfurococcales.

High-temperature (>70°C) ecosystems in Yellowstone National Park (YNP) provide an unparalleled opportunity to study chemotrophic archaea and their role in microbial community structure and function under highly constrained geochemical conditions. Acidilobus spp. (order Desulfurococcales) comprise one of the dominant phylotypes in hypoxic geothermal sulfur sediment and Fe(III)-oxide environments along with members of the Thermoproteales and Sulfolobales. Consequently, the primary goals of the current study were to analyze and compare replicate de novo sequence assemblies of Acidilobus-like populations from four different mildly acidic (pH 3.3 to 6.1) high-temperature (72°C to 82°C) environments and to identify metabolic pathways and/or protein-encoding genes that provide a detailed foundation of the potential functional role of these populations in situ. De novo assemblies of the highly similar Acidilobus-like populations (>99% 16S rRNA gene identity) represent near-complete consensus genomes based on an inventory of single-copy genes, deduced metabolic potential, and assembly statistics generated across sites. Functional analysis of coding sequences and confirmation of gene transcription by Acidilobus-like populations provide evidence that they are primarily chemoorganoheterotrophs, generating acetyl coenzyme A (acetyl-CoA) via the degradation of carbohydrates, lipids, and proteins, and auxotrophic with respect to several external vitamins, cofactors, and metabolites. No obvious pathways or protein-encoding genes responsible for the dissimilatory reduction of sulfur were identified. The presence of a formate dehydrogenase (Fdh) and other protein-encoding genes involved in mixed-acid fermentation supports the hypothesis that Acidilobus spp. function as degraders of complex organic constituents in high-temperature, mildly acidic, hypoxic geothermal systems.

Like bacteria, archaea predominately exist as biofilms in nature. However, the environmental cues and the molecular mechanisms driving archaeal biofilm development are not characterized. Here we provide data suggesting that the transcriptional regulators belonging to the Lrs14-like protein family constitute a key regulatory factor during Sulfolobus biofilm development. Among the six lrs14-like genes encoded by Sulfolobus acidocaldarius, the deletion of three led to markedly altered biofilm phenotypes. Although Δsaci1223 and Δsaci1242 deletion mutants were impaired in biofilm formation, the Δsaci0446 deletion strain exhibited a highly increased extracellular polymeric substance (EPS) production, leading to a robust biofilm structure. Moreover, although the expression of the adhesive pili (aap) genes was upregulated, the genes of the motility structure, the archaellum (fla), were downregulated rendering the Δsaci0446 strain non-motile. Gel shift assays confirmed that Saci0446 bound to the promoter regions of fla and aap thus controlling the expression of both cell surface structures. In addition, genetic epistasis analysis using Δsaci0446 as background strain identified a gene cluster involved in the EPS biosynthetic pathway of S. acidocaldarius. These results provide insights into both the molecular mechanisms that govern biofilm formation in Crenarchaea and the functionality of the Lrs14-like proteins, an archaea-specific class of transcriptional regulators.

The coupled nitrification-denitrification process plays a pivotal role in cycling and removal of nitrogen in aquatic ecosystems. In the present study, the communities of ammonia oxidizers and denitrifiers in the sediments of 2 basins (Guozhenghu Basin and Tuanhu Basin) of a large urban eutrophic lake (Lake Donghu) were determined using the ammonia monooxygenase subunit A (amoA) gene and the nitrite reductase gene. At all sites of this study, the archaeal amoA gene predominated over the bacterial amoA gene, whereas the functional gene for denitrification nirK gene far outnumbered the nirS gene. Spatially, compared with the Tuanhu Basin, the Guozhenghu Basin showed a significantly greater abundance of the archaeal amoA gene but less abundance of the nirK and nirS genes, while there was no significant difference of bacterial amoA gene copy numbers between the 2 basins. Unlike the archaeal amoA gene, the nirK gene showed a significant difference in community structure between the 2 basins. Archaeal amoA diversity was limited to the water-sediment cluster of Crenarchaeota, in sharp contrast with nirK for which 22 distinct operational taxonomic units were found. Accumulation of organic substances were found to be positively related to nirK and nirS gene copy numbers but negatively related to archaeal amoA gene copy numbers, whereas the abundance of the bacterial amoA gene was related to ammonia concentration.

The thermoacidophile and obligate elemental sulfur (S(8)(0))-reducing anaerobe Acidilobus sulfurireducens 18D70 does not associate with bulk solid-phase sulfur during S(8)(0)-dependent batch culture growth. Cyclic voltammetry indicated the production of hydrogen sulfide (H(2)S) as well as polysulfides after 1 day of batch growth of the organism at pH 3.0 and 81°C. The production of polysulfide is likely due to the abiotic reaction between S(8)(0) and the biologically produced H(2)S, as evinced by a rapid cessation of polysulfide formation when the growth temperature was decreased, inhibiting the biological production of sulfide. After an additional 5 days of growth, nanoparticulate S(8)(0) was detected in the cultivation medium, a result of the hydrolysis of polysulfides in acidic medium. To examine whether soluble polysulfides and/or nanoparticulate S(8)(0) can serve as terminal electron acceptors (TEA) supporting the growth of A. sulfurireducens, total sulfide concentration and cell density were monitored in batch cultures with S(8)(0) provided as a solid phase in the medium or with S(8)(0) sequestered in dialysis tubing. The rates of sulfide production in 7-day-old cultures with S(8)(0) sequestered in dialysis tubing with pore sizes of 12 to 14 kDa and 6 to 8 kDa were 55% and 22%, respectively, of that of cultures with S(8)(0) provided as a solid phase in the medium. These results indicate that the TEA existed in a range of particle sizes that affected its ability to diffuse through dialysis tubing of different pore sizes. Dynamic light scattering revealed that S(8)(0) particles generated through polysulfide rapidly grew in size, a rate which was influenced by the pH of the medium and the presence of organic carbon. Thus, S(8)(0) particles formed through abiological hydrolysis of polysulfide under acidic conditions appeared to serve as a growth-promoting TEA for A. sulfurireducens.

Constructed wetlands are used for biological treatment of wastewater from agricultural lands carrying pollutants such as nitrates. Nitrogen removal in wetlands occurs from direct assimilation by plants and through microbial nitrification and denitrification. We investigated the spatial distribution of N-cycling microbial communities and genes involved in nitrification and denitrification in constructed wetland sediments receiving irrigation water. We used quantitative real-time PCR (qPCR) to characterize microbial communities. Geostatistical variance analysis was used to relate them with vegetation cover and biogeochemical sediment properties. The spatial distribution of the N-cycling microbial communities of sediments was heterogeneous and complex. Total communities of bacteria and crenarchaea showed different spatial distributions. Analysis of autocorrelation patterns through semivariance indicated a tendency towards a patchy distribution over scales around 10 m for genes involved in the nitrification and denitrification processes. In contrast, biogeochemical sediment properties showed diverse spatial distributions. While almost no patchiness was found for pH and moisture, patchiness at scales between 8 and 10 m was detected for carbon, nitrate and ammonia. Denitrification variables showed spatial autocorrelation at scales comparable to genes. However, denitrifying enzyme activity and potential N(2)O production showed a common spatial pattern, different from that of the N(2)O/(N(2)O + N(2))., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

A metaproteomic survey of surface coastal waters near Palmer Station on the Antarctic Peninsula, West Antarctica, was performed, revealing marked differences in the functional capacity of summer and winter communities of bacterioplankton. Proteins from Flavobacteria were more abundant in the summer metaproteome, whereas winter was characterized by proteins from ammonia-oxidizing Marine Group I Crenarchaeota. Proteins prevalent in both seasons were from SAR11 and Rhodobacterales clades of Alphaproteobacteria, as well as many lineages of Gammaproteobacteria. The metaproteome data were used to elucidate the main metabolic and energy generation pathways and transport processes occurring at the microbial level in each season. In summer, autotrophic carbon assimilation appears to be driven by oxygenic photoautotrophy, consistent with high light availability and intensity. In contrast, during the dark polar winter, the metaproteome supported the occurrence of chemolithoautotrophy via the 3-hydroxypropionate/4-hydroxybutyrate cycle and the reverse tricarboxylic acid cycle of ammonia-oxidizing archaea and nitrite-oxidizing bacteria, respectively. Proteins involved in nitrification were also detected in the metaproteome. Taurine appears to be an important source of carbon and nitrogen for heterotrophs (especially SAR11), with transporters and enzymes for taurine uptake and degradation abundant in the metaproteome. Divergent heterotrophic strategies for Alphaproteobacteria and Flavobacteria were indicated by the metaproteome data, with Alphaproteobacteria capturing (by high-affinity transport) and processing labile solutes, and Flavobacteria expressing outer membrane receptors for particle adhesion to facilitate the exploitation of non-labile substrates. TonB-dependent receptors from Gammaproteobacteria and Flavobacteria (particularly in summer) were abundant, indicating that scavenging of substrates was likely an important strategy for these clades of Southern Ocean bacteria. This study provides the first insight into differences in functional processes occurring between summer and winter microbial communities in coastal Antarctic waters, and particularly highlights the important role that 'dark' carbon fixation has in winter.

Despite the knowledge on anaerobic degradation of hydrocarbons and signature metabolites in the oil reservoirs, little is known about the functioning microbes and the related biochemical pathways involved, especially about the methanogenic communities. In the present study, a methanogenic consortium enriched from high-temperature oil reservoir production water and incubated at 55 °C with a mixture of long chain n-alkanes (C(15)-C(20)) as the sole carbon and energy sources was characterized. Biodegradation of n-alkanes was observed as methane production in the alkanes-amended methanogenic enrichment reached 141.47 μmol above the controls after 749 days of incubation, corresponding to 17 % of the theoretical total. GC-MS analysis confirmed the presence of putative downstream metabolites probably from the anaerobic biodegradation of n-alkanes and indicating an incomplete conversion of the n-alkanes to methane. Enrichment cultures taken at different incubation times were subjected to microbial community analysis. Both 16S rRNA gene clone libraries and DGGE profiles showed that alkanes-degrading community was dynamic during incubation. The dominant bacterial species in the enrichment cultures were affiliated with Firmicutes members clustering with thermophilic syntrophic bacteria of the genera Moorella sp. and Gelria sp. Other represented within the bacterial community were members of the Leptospiraceae, Thermodesulfobiaceae, Thermotogaceae, Chloroflexi, Bacteroidetes and Candidate Division OP1. The archaeal community was predominantly represented by members of the phyla Crenarchaeota and Euryarchaeota. Corresponding sequences within the Euryarchaeota were associated with methanogens clustering with orders Methanomicrobiales, Methanosarcinales and Methanobacteriales. On the other hand, PCR amplification for detection of functional genes encoding the alkylsuccinate synthase α-subunit (assA) was positive in the enrichment cultures. Moreover, the appearance of a new assA gene sequence identified in day 749 supported the establishment of a functioning microbial species in the enrichment. Our results indicate that n-alkanes are converted to methane slowly by a microbial community enriched from oilfield production water and fumarate addition is most likely the initial activation step of n-alkanes degradation under thermophilic methanogenic conditions.

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

The lipid cores from Ignisphaera aggregans, a hyperthermophilic Crenarchaeon recently isolated from New Zealand hot springs, have been profiled by liquid chromatography-tandem mass spectrometry. The distribution revealed includes relatively high proportions of monoalkyl (also known as H-shaped) tetraether cores which have previously been implicated as kingdom-specific biomarkers for the Euryarchaeota. Such high expression of monoalkyl tetraether lipids is unusual in the archaeal domain and may indicate that formation of these components is an adaptive mechanism that allows I. aggregans to regulate membrane behaviour at high temperatures. The observed dialkyl tetraether and monoalkyl tetraether lipid distributions are similar but not fully concordant, showing differences in the average number of incorporated rings. The similarity supports a biosynthetic route to the ring-containing dialkyl and monoalkyl tetraether lipids via a dialkyl tetraether core containing zero rings, or a closely related structural relative, as an intermediate. Currently, however, the precise nature of the biosynthetic route to these lipids cannot be deduced.

Mesophilic Crenarchaeota have recently been thought to be significant contributors to nitrogen (N) and carbon (C) cycling. In this study, we examined the vertical distribution of ammonia-oxidizing Crenarchaeota at offshore site in Southern Tyrrhenian Sea. The median value of the crenachaeal cell to amoA gene ratio was close to one suggesting that virtually all deep-sea Crenarchaeota possess the capacity to oxidize ammonia. Crenarchaea-specific genes, nirK and ureC, for nitrite reductase and urease were identified and their affiliation demonstrated the presence of 'deep-sea' clades distinct from 'shallow' representatives. Measured deep-sea dark CO(2) fixation estimates were comparable to the median value of photosynthetic biomass production calculated for this area of Tyrrhenian Sea, pointing to the significance of this process in the C cycle of aphotic marine ecosystems. To elucidate the pivotal organisms in this process, we targeted known marine crenarchaeal autotrophy-related genes, coding for acetyl-CoA carboxylase (accA) and 4-hydroxybutyryl-CoA dehydratase (4-hbd). As in case of nirK and ureC, these genes are grouped with deep-sea sequences being distantly related to those retrieved from the epipelagic zone. To pair the molecular data with specific functional attributes we performed [(14)C]HCO(3) incorporation experiments followed by analyses of radiolabeled proteins using shotgun proteomics approach. More than 100 oligopeptides were attributed to 40 marine crenarchaeal-specific proteins that are involved in 10 different metabolic processes, including autotrophy. Obtained results provided a clear proof of chemolithoautotrophic physiology of bathypelagic crenarchaeota and indicated that this numerically predominant group of microorganisms facilitate a hitherto unrecognized sink for inorganic C of a global importance.

Marine Crenarchaeota are among the most abundant groups of prokaryotes in the ocean and recent reports suggest that they oxidize ammonia as an energy source and inorganic carbon as carbon source, while other studies indicate that Crenarchaeota use organic carbon and hence, live heterotrophically. We used catalysed reporter deposition fluorescence in situ hybridization (CARD-FISH) to determine the crenarchaeal and bacterial contribution to total prokaryotic abundance in the (sub)tropical Atlantic. Bacteria contributed ~ 50% to total prokaryotes throughout the water column. Marine Crenarchaeota Group I (MCGI) accounted for ~ 5% of the prokaryotes in subsurface waters (100 m depth) and between 10 and 20% in the oxygen minimum layer (250-500 m depth) and deep waters (North East Atlantic Deep Water). The fraction of both MCGI and Bacteria fixing inorganic carbon, determined by combining microautoradiography with CARD-FISH (MICRO-CARD-FISH), decreased with depth, ranging from ~ 30% in the oxygen minimum zone to < 10% in the intermediate waters (Mediterranean Sea Outflow Water, Antarctic Intermediate Water). In the deeper water masses, however, MCGI were not taking up inorganic carbon. Using quantitative MICRO-CARD-FISH to determine autotrophy activity on a single cell level revealed that MCGI are incorporating inorganic carbon (0.002-0.1 fmol C cell⁻¹ day⁻¹) at a significantly lower rate than Bacteria (0.01-0.6 fmol C cell⁻¹ day⁻¹). Hence, it appears that MCGI contribute substantially less to autotrophy than Bacteria. Taking the stoichiometry of nitrification together with our findings suggests that MCGI might not dominate the ammonia oxidation step in the mesopelagic waters of the ocean to that extent as the reported dominance of archaeal over bacterial amoA would suggest., (© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Quantitative PCR (qPCR) analysis revealed elevated relative abundance (1.8% of prokaryotes) of marine group 1 Crenarchaeota (MG1C) in two samples of southeastern US coastal bacterioplankton, collected in August 2008, compared with samples collected from the same site at different times (mean 0.026%). We analyzed the MG1C sequences in metatranscriptomes from these samples to gain an insight into the metabolism of MG1C population growing in the environment, and for comparison with ammonia-oxidizing bacteria (AOB) in the same samples. Assemblies revealed low diversity within sequences assigned to most individual MG1C open reading frames (ORFs) and high homology with 'Candidatus Nitrosopumilus maritimus' strain SCM1 genome sequences. Reads assigned to ORFs for ammonia uptake and oxidation accounted for 37% of all MG1C transcripts. We did not recover any reads for Nmar&#95;1354-Nmar&#95;1357, proposed to encode components of an alternative, nitroxyl-based ammonia oxidation pathway; however, reads from Nmar&#95;1259 and Nmar&#95;1667, annotated as encoding a multicopper oxidase with homology to nirK, were abundant. Reads assigned to two homologous ORFs (Nmar&#95;1201 and Nmar&#95;1547), annotated as hypothetical proteins were also abundant, suggesting that their unknown function is important to MG1C. Superoxide dismutase and peroxiredoxin-like transcripts were more abundant in the MG1C transcript pool than in the complete metatranscriptome, suggesting an enhanced response to oxidative stress by the MG1C population. qPCR indicated low AOB abundance (0.0010% of prokaryotes), and we found no transcripts related to ammonia oxidation and only one RuBisCO transcript among the transcripts assigned to AOB, suggesting they were not responding to the same environmental cues as the MG1C population.

The objective of this study is to investigate the responses of methanogen populations to poultry waste addition by comparing the archaeal microbial populations in continuous anaerobic digesters with or without the addition of poultry waste as a co-substrate. Poultry waste was characterized as an organic/nitrogen-rich substrate for anaerobic digestion. Supplementing dilute dairy waste with poultry waste for anaerobic co-digestion to increase organic loading rate by 50% resulted in improved biogas production. Elevated ammonia derived from poultry waste did not lead to process inhibition at the organic loadings tested, demonstrating the feasibility of the anaerobic co-digestion of dairy and poultry wastes for improved treatment efficiency. The stability of the anaerobic co-digestion process was linked to the robust archaeal microbial community, which remained mostly unchanged in community structure following increases in organic loading and ammonia levels. Surprisingly, Crenarchaeota archaeal populations, instead of the Euryarchaeota methanogens, dominated the archaeal communities in the anaerobic digesters. The ecological functions of these abundant non-methanogen archaeal populations in anaerobic digestion remain to be identified., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Nitrification is the process that converts ammonium to nitrate and thus links the regeneration of organic nitrogen to fixed nitrogen loss by denitrification. The first step, oxidation of ammonia to nitrite, is performed by a phylogenetically restricted group of proteobacteria (ammonia-oxidizing bacteria, AOB) and Crenarchaea (ammonia-oxidizing archaea, AOA). The second step is restricted to nitrite-oxidizing bacteria (NOB) as far as currently known. All three groups are assumed to be autotrophic and obligately aerobic, but the true extent of autotrophy and potential anaerobic pathways in these organisms is currently under investigation. Here, we describe methods for the measurement of nitrification rates in the marine environment, with a focus on seawater systems and stable isotopic tracer methods. The methods vary in analytical requirements but share the need for incubations, which must be optimized for different environments with different substrate concentrations. Recent advances in mass spectrometry now make it possible to minimize incubation artifacts and to achieve greatly improved sensitivity., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Cyclization in glycerol dibiphytanyl glycerol tetraethers (GDGTs) results in internal cyclopentane moieties which are believed to confer thermal stability to crenarchaeal membranes. While the average number of rings per GDGT lipid (ring index) is positively correlated with temperature in many temperate environments, poor correlations are often observed in geothermal environments, suggesting that additional parameters may influence GDGT core lipid composition in these systems. However, the physical and chemical parameters likely to influence GDGT cyclization which are often difficult to decouple in geothermal systems, making it challenging to assess their influence on lipid composition. In the present study, the influence of temperature (range 65-81°C), pH (range 3.0-5.0), and ionic strength (range 10.1-55.7 mM) on GDGT core lipid composition was examined in the hyperthermoacidophile Acidilobus sulfurireducens, a crenarchaeon originally isolated from a geothermal spring in Yellowstone National Park, Wyoming. When cultivated under defined laboratory conditions, the composition of individual and total GDGTs varied significantly with temperature and to a lesser extent with the pH of the growth medium. Ionic strength over the range of values tested did not influence GDGT composition. The GDGT core lipid ring index was positively correlated with temperature and negatively correlated with pH, suggesting that A. sulfurireducens responds to increasing temperature and acidity by increasing the number of cyclopentyl rings in GDGT core membrane lipids.

Our knowledge concerning the metabolic potentials of as yet to be cultured microorganisms has increased tremendously with the advance of sequencing technologies and the consequent discoveries of novel genes. On the other hand, it is often difficult to reliably assign a particular gene to a phylogenetic clade, because these sequences are usually found on genomic fragments that carry no direct marker of cell identity, such as rRNA genes. Therefore, the aim of the present study was to develop geneFISH - a protocol for linking gene presence with cell identity in environmental samples, the signals of which can be visualized at a single cell level. This protocol combines rRNA-targeted catalysed reporter deposition - fluorescence in situ hybridization and in situ gene detection. To test the protocol, it was applied to seawater samples from the Benguela upwelling system. For gene detection, a polynucleotide probe mix was used, which was designed based on crenarchaeotal amoA clone libraries prepared from each seawater sample. Each probe in the mix was selected to bind to targets with up to 5% mismatches. To determine the hybridization parameters, the T(m) of probes, targets and hybrids was estimated based on theoretical calculations and in vitro measurements. It was shown that at least 30%, but potentially the majority of the Crenarchaeota present in these samples harboured the amoA gene and were therefore likely to be catalysing the oxidation of ammonia., (© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Four stratified basins in Lake Kivu (Rwanda-Democratic Republic of the Congo) were sampled in March 2007 to investigate the abundance, distribution, and potential biogeochemical role of planktonic archaea. We used fluorescence in situ hybridization with catalyzed-reported deposition microscopic counts (CARD-FISH), denaturing gradient gel electrophoresis (DGGE) fingerprinting, and quantitative PCR (qPCR) of signature genes for ammonia-oxidizing archaea (16S rRNA for marine Crenarchaeota group 1.1a [MCG1] and ammonia monooxygenase subunit A [amoA]). Abundance of archaea ranged from 1 to 4.5% of total DAPI (4',6-diamidino-2-phenylindole) counts with maximal concentrations at the oxic-anoxic transition zone (∼50-m depth). Phylogenetic analysis of the archaeal planktonic community revealed a higher level of richness of crenarchaeal 16S rRNA gene sequences (21 of the 28 operational taxonomic units [OTUs] identified [75%]) over euryarchaeotal ones (7 OTUs). Sequences affiliated with the kingdom Euryarchaeota were mainly recovered from the anoxic water compartment and mostly grouped into methanogenic lineages (Methanosarcinales and Methanocellales). In turn, crenarchaeal phylotypes were recovered throughout the sampled epipelagic waters (0- to 100-m depth), with clear phylogenetic segregation along the transition from oxic to anoxic water masses. Thus, whereas in the anoxic hypolimnion crenarchaeotal OTUs were mainly assigned to the miscellaneous crenarchaeotic group, the OTUs from the oxic-anoxic transition and above belonged to Crenarchaeota groups 1.1a and 1.1b, two lineages containing most of the ammonia-oxidizing representatives known so far. The concomitant vertical distribution of both nitrite and nitrate maxima and the copy numbers of both MCG1 16S rRNA and amoA genes suggest the potential implication of Crenarchaeota in nitrification processes occurring in the epilimnetic waters of the lake.

Marine Crenarchaeota, ubiquitous and abundant organisms in the oceans worldwide, remain metabolically uncharacterized, largely due to their low cultivability. Identification of candidate genes for bicarbonate fixation pathway in the Cenarchaeum symbiosum A was an initial step in understanding the physiology and ecology of marine Crenarchaeota. Recent cultivation and genome sequencing of obligate chemoautotrophic Nitrosopumilus maritimus SCM1 were a major breakthrough towards understanding of their functioning and provide a valuable model for experimental validation of genomic data. Here we present the identification of multiple key components of 3-hydroxipropionate/4-hydroxybutyrate cycle, the fifth pathway in carbon fixation, found in data sets of environmental sequences representing uncultivated superficial and bathypelagic Crenarchaeota from Sargasso sea (GOS data set) and KM3 (Mediterranean Sea) and ALOHA (Atlantic ocean) stations. These organisms are likely to use acetyl-CoA/propionyl-CoA carboxylase(s) as CO₂-fixing enzyme(s) to form succinyl-CoA, from which one molecule of acetyl-CoA is regenerated via 4-hydroxybutyrate cleavage and another acetyl-CoA to be the pathway product. The genetic distinctiveness and matching sympatric abundance imply that marine crenarchaeal genotypes from the three different geographic sites share similar ecophysiological properties, and therefore may represent fundamental units of marine ecosystem functioning. To couple results of sequence comparison with the dark ocean primary production, dissolved inorganic carbon fixation rates were measured at KM3 Station (3000 m depth, Eastern Mediterranean Sea), i.e. at the same site and depth used for metagenomic library construction., (© 2010 The Authors. Journal compilation © 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Globally distributed archaea comprising ammonia oxidizers of moderate terrestrial and marine environments are considered the most abundant archaeal organisms on Earth. Based on 16S rRNA phylogeny, initial assignment of these archaea was to the Crenarchaeota. By contrast, features of the first genome sequence from a member of this group suggested that they belong to a novel phylum, the Thaumarchaeota. Here, we re-investigate the Thaumarchaeota hypothesis by including two newly available genomes, that of the marine ammonia oxidizer Nitrosopumilus maritimus and that of Nitrososphaera gargensis, a representative of another evolutionary lineage within this group predominantly detected in terrestrial environments. Phylogenetic studies based on r-proteins and other core genes, as well as comparative genomics, confirm the assignment of these organisms to a separate phylum and reveal a Thaumarchaeota-specific set of core informational processing genes, as well as potentially ancestral features of the archaea., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Marine Crenarchaeota are among the most abundant microbial groups in the ocean, and although relatively little is currently known about their biogeochemical roles in marine ecosystems, recognition that Crenarchaeota posses ammonia monooxygenase (amoA) genes and may act as ammonia-oxidizing archaea (AOA) offers another means of probing the ecology of these microorganisms. Here we use a time series approach combining quantification of archaeal and bacterial ammonia oxidizers with bacterial community fingerprints and biogeochemistry, to explore the population and community ecology of nitrification. At multiple depths (150, 500 and 890 m) in the Southern California Bight sampled monthly from 2003 to 2006, AOA were enumerated via quantitative PCR of archaeal amoA and marine group 1 Crenarchaeota 16S rRNA genes. Based on amoA genes, AOA were highly variable in time - a consistent feature of marine Crenarchaeota- however, average values were similar at different depths and ranged from 2.20 to 2.76 x 10(4) amoA copies ml(-1). Archaeal amoA genes were correlated with Crenarchaeota 16S rRNA genes (r(2) = 0.79) and the slope of this relationship was 1.02, demonstrating that the majority of marine group 1 Crenarchaeota present over the dates and depths sampled possessed amoA. Two AOA clades were specifically quantified and compared with betaproteobacterial ammonia-oxidizing bacteria (beta-AOB) amoA genes at 150 m; these AOA groups were found to strongly co-vary in time (r(2) = 0.70, P < 0.001) whereas AOA : beta-AOB ratios ranged from 13 to 5630. Increases in the AOA : beta-AOB ratio correlated with the accumulation of nitrite (r(2) = 0.87, P < 0.001), and may be indicative of differences in substrate affinities and activities leading to periodic decoupling between ammonia and nitrite oxidation. These data capture a dynamic nitrogen cycle in which multiple microbial groups appear to be active participants.

Nitrification and denitrification are key steps in nitrogen (N) cycling. The coupling of these processes, which affects the flow of N in ecosystems, requires close interaction of nitrifying and denitrifying microorganisms, both spatially and temporally. The diversity, temporal and spatial variations in the microbial communities affecting these processes was examined, in relation to N cycling, across 12 sites in the Fitzroy river estuary, which is a turbid subtropical estuary in central Queensland. The estuary is a major source of nutrients discharged to the Great Barrier Reef near-shore zone. Measurement of nitrogen fluxes showed an active denitrifying community during all sampling months. Archaeal ammonia monooxygenase (amoA of AOA, functional marker for nitrification) was significantly more abundant than Betaproteobacterial (beta-AOB) amoA. Nitrite reductase genes, functional markers for denitrification, were dominated by nirS and not nirK types at all sites during the year. AOA communities were dominated by the soil/sediment cluster of Crenarchaeota, with sequences found in estuarine sediment, marine and terrestrial environments, whereas nirS sequences were significantly more diverse (where operational taxonomic units were defined at both the threshold of 5% and 15% sequence similarity) and were closely related to sequences originating from estuarine sediments. Terminal-restriction fragment length polymorphism (T-RFLP) analysis revealed that AOA population compositions varied spatially along the estuary, whereas nirS populations changed temporally. Statistical analysis of individual T-RF dominance suggested that salinity and C:N were associated with the community succession of AOA, whereas the nirS-type denitrifier communities were related to salinity and chlorophyll-alpha in the Fitzroy river estuary.

Anaerobic methanotrophs help regulate Earth's climate and may have been an important part of the microbial ecosystem on the early Earth. The anaerobic oxidation of methane (AOM) is often thought of as a sulfate-dependent process, despite the fact that other electron acceptors are more energetically favorable. Here, we show that microorganisms from marine methane-seep sediment in the Eel River Basin in California are capable of using manganese (birnessite) and iron (ferrihydrite) to oxidize methane, revealing that marine AOM is coupled, either directly or indirectly, to a larger variety of oxidants than previously thought. Large amounts of manganese and iron are provided to oceans from rivers, indicating that manganese- and iron-dependent AOM have the potential to be globally important.

Ancestral trait reconstruction was used to identify the relative ancestry of metabolic and physiological traits in the archaeal domain of life. First, well-resolved phylogenetic trees were inferred with multiple gene sequences obtained from whole genome sequences. Next, metabolic and physiological traits were coded into characters, and ancestral state reconstruction was used to identify ancient and derived traits. Traits inferred to be ancient included sulfur reduction, methanogenesis, and hydrogen oxidation. By using the articulation of the "oxygen age constraint," several other traits were inferred to have arisen at or after 2.32 Ga: aerobic respiration, nitrate reduction, sulfate reduction, thiosulfate reduction, sulfur oxidation, and sulfide oxidation. Complex organic metabolism appeared to be nearly as ancient as autotrophy. Hyperthermophily was ancestral, while hyperacidophily and extreme halophily likely arose after 2.32 Ga. The ancestral euryarchaeote was inferred to have been a hyperthermophilic marine methanogen that lived in a deep-sea hydrothermal vent. In contrast, the ancestral crenarchaeote was most likely a hyperthermophilic sulfur reducer that lived in a slightly acidic terrestrial environment, perhaps a fumarole. Cross-colonization of these habitats may not have occurred until after 2.32 Ga, which suggests that both archaeal lineages exhibited niche specialization on early Earth for a protracted period of time.

Marine Crenarchaeota represent an abundant component of the oceanic microbiota that play an important role in the global nitrogen cycle. Here we report the association of the colonial ascidian Cystodytes dellechiajei with putative ammonia-oxidizing Crenarchaeota that could actively be involved in nitrification inside the animal tissue. As shown by 16S rRNA gene analysis, the ascidian-associated Crenarchaeota were phylogenetically related to Nitrosopumilus maritimus, the first marine archaeon isolated in pure culture that grows chemolithoautotrophically oxidizing ammonia to nitrite aerobically. Catalysed reporter deposition (CARD)-FISH revealed that the Crenarchaeota were specifically located inside the tunic tissue of the colony, where moreover the expression of amoA gene was detected. The amoA gene encodes the alpha-subunit of ammonia monooxygenase, which is involved in the first step of nitrification, the oxidation of ammonia to nitrite. Sequencing of amoA gene showed that they were phylogenetically related to amoA genes of N. maritimus and other putative ammonia-oxidizing marine Crenarchaeota. In order to track the suspected nitrification activity inside the ascidian colony under in vivo conditions, microsensor profiles were measured through the tunic tissue. Net NO(x) production was detected in the tunic layer 1200-1800 microm with rates of 58-90 nmol cm(-3) h(-1). Oxygen and pH microsensor profiles showed that the layer of net NO(x) production coincided with O(2) concentrations of 103-116 microM and pH value of 5.2. Together, molecular and microsensor data indicate that Crenarchaeota could oxidize ammonia to nitrite aerobically, and thus be involved in nitrification inside the ascidian tissue.

The potential for nitrification in the Mediterranean sponge Aplysina aerophoba was assessed using a combined physiological and molecular approach. Nitrate excretion rates in whole sponges reached values of up to 344 nmol g(-1) dry weight (wt) h(-1) (unstimulated) and 1325 nmol g(-1) dry wt h(-1) (stimulated). Addition of nitrapyrin, a nitrification-specific inhibitor, effectively inhibited nitrate excretion. Ammonium was taken up by sponges in spring and excreted in fall, the sponges thus serving as either an ammonium sink or ammonium source. Nitrosospira cluster 1 and Crenarchaeota group I.1A 16S rRNA and amoA genes were recovered from A. aerophoba and other sponges from different world's oceans. The archaeal 16S rRNA genes formed a sponge-specific subcluster, indicating that their representatives are members of the stable microbial community of sponges. On the other hand, clustering was not evident for Nitrosospira rRNA genes which is consistent with their presence in sediment and seawater samples. The presence of both Nitrosospira cluster 1 and crenarchaeal group 1 phylotypes in sponge tissue was confirmed using fluorescently labelled 16S rRNA gene probes. This study contributes to an ongoing effort to link microbial diversity with metabolic functions in the phylogenetically diverse, elusive and so far uncultivated microbial communities of marine sponges.

Previous results from a 16S rRNA gene library analysis showed high diversity within the prokaryotic community of a subterranean radioactive thermal spring, the "Franz-Josef-Quelle" (FJQ) in Bad Gastein, Austria, as well as evidence for ammonia oxidation by crenarchaeota. This study reports further characterization of the community by denaturing gradient gel electrophoresis (DGGE) analysis, fluorescence in situ hybridization (FISH), and semiquantitative nitrification measurements. DGGE bands from three types of samples (filtered water, biofilms on glass slides, and naturally grown biofilms), including samples collected at two distinct times (January 2005 and July 2006), were analyzed. The archaeal community consisted mainly of Crenarchaeota of the soil-subsurface-freshwater group (group 1.1b) and showed a higher diversity than in the previous 16S rRNA gene library analysis, as was also found for crenarchaeal amoA genes. No bacterial amoA genes were detected. FISH analysis of biofilms indicated the presence of archaeal cells with an abundance of 5.3% (+/-4.5%) in the total 4',6-diamidino-2-phenylindole (DAPI)-stained community. Microcosm experiments of several weeks in duration showed a decline of ammonium that correlated with an increase of nitrite, the presence of crenarchaeal amoA genes, and the absence of bacterial amoA genes. The data suggested that only ammonia-oxidizing archaea (AOA) perform the first step of nitrification in this 45 degrees C environment. The crenarchaeal amoA gene sequences grouped within a novel cluster of amoA sequences from the database, originating from geothermally influenced environments, for which we propose the designation "thermal spring" cluster and which may be older than most AOA from soils on earth.