Archaea The latest articles from Hindawi Publishing Corporation © 2015 , Hindawi Publishing Corporation . All rights reserved. Corrigendum to “Unique Characteristics of the Pyrrolysine System in the 7th Order of Methanogens: Implications for the Evolution of a Genetic Code Expansion Cassette” Mon, 02 Feb 2015 13:41:00 +0000 Guillaume Borrel, Nadia Gaci, Pierre Peyret, Paul W. O’Toole, Simonetta Gribaldo, and Jean-François Brugère Copyright © 2015 Guillaume Borrel et al. All rights reserved. De Novo Sequences of Haloquadratum walsbyi from Lake Tyrrell, Australia, Reveal a Variable Genomic Landscape Sun, 01 Feb 2015 08:05:46 +0000 Hypersaline systems near salt saturation levels represent an extreme environment, in which organisms grow and survive near the limits of life. One of the abundant members of the microbial communities in hypersaline systems is the square archaeon, Haloquadratum walsbyi. Utilizing a short-read metagenome from Lake Tyrrell, a hypersaline ecosystem in Victoria, Australia, we performed a comparative genomic analysis of H. walsbyi to better understand the extent of variation between strains/subspecies. Results revealed that previously isolated strains/subspecies do not fully describe the complete repertoire of the genomic landscape present in H. walsbyi. Rearrangements, insertions, and deletions were observed for the Lake Tyrrell derived Haloquadratum genomes and were supported by environmental de novo sequences, including shifts in the dominant genomic landscape of the two most abundant strains. Analysis pertaining to halomucins indicated that homologs for this large protein are not a feature common for all species of Haloquadratum. Further, we analyzed ATP-binding cassette transporters (ABC-type transporters) for evidence of niche partitioning between different strains/subspecies. We were able to identify unique and variable transporter subunits from all five genomes analyzed and the de novo environmental sequences, suggesting that differences in nutrient and carbon source acquisition may play a role in maintaining distinct strains/subspecies. Benjamin J. Tully, Joanne B. Emerson, Karen Andrade, Jochen J. Brocks, Eric E. Allen, Jillian F. Banfield, and Karla B. Heidelberg Copyright © 2015 Benjamin J. Tully et al. All rights reserved. Adaptation, Ecology, and Evolution of the Halophilic Stromatolite Archaeon Halococcus hamelinensis Inferred through Genome Analyses Thu, 29 Jan 2015 12:31:23 +0000 Halococcus hamelinensis was the first archaeon isolated from stromatolites. These geomicrobial ecosystems are thought to be some of the earliest known on Earth, yet, despite their evolutionary significance, the role of Archaea in these systems is still not well understood. Detailed here is the genome sequencing and analysis of an archaeon isolated from stromatolites. The genome of H. hamelinensis consisted of 3,133,046 base pairs with an average G+C content of 60.08% and contained 3,150 predicted coding sequences or ORFs, 2,196 (68.67%) of which were protein-coding genes with functional assignments and 954 (29.83%) of which were of unknown function. Codon usage of the H. hamelinensis genome was consistent with a highly acidic proteome, a major adaptive mechanism towards high salinity. Amino acid transport and metabolism, inorganic ion transport and metabolism, energy production and conversion, ribosomal structure, and unknown function COG genes were overrepresented. The genome of H. hamelinensis also revealed characteristics reflecting its survival in its extreme environment, including putative genes/pathways involved in osmoprotection, oxidative stress response, and UV damage repair. Finally, genome analyses indicated the presence of putative transposases as well as positive matches of genes of H. hamelinensis against various genomes of Bacteria, Archaea, and viruses, suggesting the potential for horizontal gene transfer. Reema K. Gudhka, Brett A. Neilan, and Brendan P. Burns Copyright © 2015 Reema K. Gudhka et al. All rights reserved. Recombinant Cyclodextrinase from Thermococcus kodakarensis KOD1: Expression, Purification, and Enzymatic Characterization Mon, 26 Jan 2015 07:54:02 +0000 A gene encoding a cyclodextrinase from Thermococcus kodakarensis KOD1 (CDase-Tk) was identified and characterized. The gene encodes a protein of 656 amino acid residues with a molecular mass of 76.4 kDa harboring four conserved regions found in all members of the α-amylase family. A recombinant form of the enzyme was purified by ion-exchange chromatography, and its catalytic properties were examined. The enzyme was active in a broad range of pH conditions (pHs 4.0–10.0), with an optimal pH of 7.5 and a temperature optimum of 65°C. The purified enzyme preferred to hydrolyze β-cyclodextrin (CD) but not α- or γ-CD, soluble starch, or pullulan. The final product from β-CD was glucose. The and values were 3.13 ± 0.47 U mg−1 and 2.94 ± 0.16 mg mL−1 for β-CD. The unique characteristics of CDase-Tk with a low catalytic temperature and substrate specificity are discussed, and the starch utilization pathway in a broad range of temperatures is also proposed. Ying Sun, Xiaomin Lv, Zhengqun Li, Jiaqiang Wang, Baolei Jia, and Jinliang Liu Copyright © 2015 Ying Sun et al. All rights reserved. Diversity and Distribution of Archaea Community along a Stratigraphic Permafrost Profile from Qinghai-Tibetan Plateau, China Tue, 25 Nov 2014 11:46:21 +0000 Accompanying the thawing permafrost expected to result from the climate change, microbial decomposition of the massive amounts of frozen organic carbon stored in permafrost is a potential emission source of greenhouse gases, possibly leading to positive feedbacks to the greenhouse effect. In this study, the community composition of archaea in stratigraphic soils from an alpine permafrost of Qinghai-Tibetan Plateau was investigated. Phylogenic analysis of 16S rRNA sequences revealed that the community was predominantly constituted by Crenarchaeota and Euryarchaeota. The active layer contained a proportion of Crenarchaeota at 51.2%, with the proportion of Euryarchaeota at 48.8%, whereas the permafrost contained 41.2% Crenarchaeota and 58.8% Euryarchaeota, based on 16S rRNA gene sequence analysis. OTU1 and OTU11, affiliated to Group 1.3b/MCG-A within Crenarchaeota and the unclassified group within Euryarchaeota, respectively, were widely distributed in all sediment layers. However, OTU5 affiliated to Group 1.3b/MCG-A was primarily distributed in the active layers. Sequence analysis of the DGGE bands from the 16S rRNAs of methanogenic archaea showed that the majority of methanogens belonged to Methanosarcinales and Methanomicrobiales affiliated to Euryarchaeota and the uncultured ZC-I cluster affiliated to Methanosarcinales distributed in all the depths along the permafrost profile, which indicated a dominant group of methanogens occurring in the cold ecosystems. Shiping Wei, Hongpeng Cui, Hao He, Fei Hu, Xin Su, and Youhai Zhu Copyright © 2014 Shiping Wei et al. All rights reserved. Children Living near a Sanitary Landfill Have Increased Breath Methane and Methanobrevibacter smithii in Their Intestinal Microbiota Mon, 13 Oct 2014 07:43:58 +0000 This study evaluated the breath CH4 excretion and concentration of M. smithii in intestinal microbiota of schoolchildren from 2 slums. One hundred and eleven children from a slum near a sanitary landfill, 35 children of a slum located away from the sanitary landfill, and 32 children from a high socioeconomic level school were included in the study. Real-time PCR was performed to quantify the M. smithii nifH gene and it was present in the microbiota of all the participating children, with higher concentrations in those who lived in the slum near the landfill ( CFU/g of feces), comparing with the children from the slum away from the landfill ( CFU/g of feces) and those from the high socioeconomic level group ( CFU/g of feces). The prevalence of children who present breath methane was 53% in the slum near the landfill, 31% in the slum further away from the landfill and, 22% in the high socioeconomic level group. To live near a landfill is associated with higher concentrations of M. smithii in intestinal microbiota, comparing with those who live away from the landfill, regardless of their socioeconomics conditions. Humberto Bezerra de Araujo Filho, Mirian Silva Carmo-Rodrigues, Carolina Santos Mello, Lígia Cristina Fonseca Lahoz Melli, Soraia Tahan, Antonio Carlos Campos Pignatari, and Mauro Batista de Morais Copyright © 2014 Humberto Bezerra de Araujo Filho et al. All rights reserved. A Phylogenomic Census of Molecular Functions Identifies Modern Thermophilic Archaea as the Most Ancient Form of Cellular Life Sun, 31 Aug 2014 06:21:58 +0000 The origins of diversified life remain mysterious despite considerable efforts devoted to untangling the roots of the universal tree of life. Here we reconstructed phylogenies that described the evolution of molecular functions and the evolution of species directly from a genomic census of gene ontology (GO) definitions. We sampled 249 free-living genomes spanning organisms in the three superkingdoms of life, Archaea, Bacteria, and Eukarya, and used the abundance of GO terms as molecular characters to produce rooted phylogenetic trees. Results revealed an early thermophilic origin of Archaea that was followed by genome reduction events in microbial superkingdoms. Eukaryal genomes displayed extraordinary functional diversity and were enriched with hundreds of novel molecular activities not detected in the akaryotic microbial cells. Remarkably, the majority of these novel functions appeared quite late in evolution, synchronized with the diversification of the eukaryal superkingdom. The distribution of GO terms in superkingdoms confirms that Archaea appears to be the simplest and most ancient form of cellular life, while Eukarya is the most diverse and recent. Arshan Nasir, Kyung Mo Kim, and Gustavo Caetano-Anollés Copyright © 2014 Arshan Nasir et al. All rights reserved. Seasonal Effects in a Lake Sediment Archaeal Community of the Brazilian Savanna Sun, 20 Jul 2014 11:32:43 +0000 The Cerrado is a biome that corresponds to 24% of Brazil’s territory. Only recently microbial communities of this biome have been investigated. Here we describe for the first time the diversity of archaeal communities from freshwater lake sediments of the Cerrado in the dry season and in the transition period between the dry and rainy seasons, when the first rains occur. Gene libraries were constructed, using Archaea-specific primers for the 16S rRNA and amoA genes. Analysis revealed marked differences between the archaeal communities found in the two seasons. I.1a and I.1c Thaumarchaeota were found in greater numbers in the transition period, while MCG Archaea was dominant on the dry season. Methanogens were only found in the dry season. Analysis of 16S rRNA sequences revealed lower diversity on the transition period. We detected archaeal amoA sequences in both seasons, but there were more OTUs during the dry season. These sequences were within the same cluster as Nitrosotalea devanaterra’s amoA gene. The principal coordinate analysis (PCoA) test revealed significant differences between samples from different seasons. These results provide information on archaeal diversity in freshwater lake sediments of the Cerrado and indicates that rain is likely a factor that impacts these communities. Thiago Rodrigues, Elisa Catão, Mercedes M. C. Bustamante, Betania F. Quirino, Ricardo H. Kruger, and Cynthia M. Kyaw Copyright © 2014 Thiago Rodrigues et al. All rights reserved. The Origin and Evolution of the Archaeal Domain Wed, 04 Jun 2014 11:19:24 +0000 Gustavo Caetano-Anollés and Kyung Mo Kim Copyright © 2014 Gustavo Caetano-Anollés and Kyung Mo Kim. All rights reserved. Archaea: The First Domain of Diversified Life Mon, 02 Jun 2014 09:05:15 +0000 The study of the origin of diversified life has been plagued by technical and conceptual difficulties, controversy, and apriorism. It is now popularly accepted that the universal tree of life is rooted in the akaryotes and that Archaea and Eukarya are sister groups to each other. However, evolutionary studies have overwhelmingly focused on nucleic acid and protein sequences, which partially fulfill only two of the three main steps of phylogenetic analysis, formulation of realistic evolutionary models, and optimization of tree reconstruction. In the absence of character polarization, that is, the ability to identify ancestral and derived character states, any statement about the rooting of the tree of life should be considered suspect. Here we show that macromolecular structure and a new phylogenetic framework of analysis that focuses on the parts of biological systems instead of the whole provide both deep and reliable phylogenetic signal and enable us to put forth hypotheses of origin. We review over a decade of phylogenomic studies, which mine information in a genomic census of millions of encoded proteins and RNAs. We show how the use of process models of molecular accumulation that comply with Weston’s generality criterion supports a consistent phylogenomic scenario in which the origin of diversified life can be traced back to the early history of Archaea. Gustavo Caetano-Anollés, Arshan Nasir, Kaiyue Zhou, Derek Caetano-Anollés, Jay E. Mittenthal, Feng-Jie Sun, and Kyung Mo Kim Copyright © 2014 Gustavo Caetano-Anollés et al. All rights reserved. The Bifunctional Pyruvate Decarboxylase/Pyruvate Ferredoxin Oxidoreductase from Thermococcus guaymasensis Thu, 29 May 2014 12:58:16 +0000 The hyperthermophilic archaeon Thermococcus guaymasensis produces ethanol as a metabolic end product, and an alcohol dehydrogenase (ADH) catalyzing the reduction of acetaldehyde to ethanol has been purified and characterized. However, the enzyme catalyzing the formation of acetaldehyde has not been identified. In this study an enzyme catalyzing the production of acetaldehyde from pyruvate was purified and characterized from T. guaymasensis under strictly anaerobic conditions. The enzyme had both pyruvate decarboxylase (PDC) and pyruvate ferredoxin oxidoreductase (POR) activities. It was oxygen sensitive, and the optimal temperatures were 85°C and >95°C for the PDC and POR activities, respectively. The purified enzyme had activities of  U mg−1 and  U mg−1, with optimal pH-values of 9.5 and 8.4 for each activity, respectively. Coenzyme A was essential for both activities, although it did not serve as a substrate for the former. Enzyme kinetic parameters were determined separately for each activity. The purified enzyme was a heterotetramer. The sequences of the genes encoding the subunits of the bifunctional PDC/POR were determined. It is predicted that all hyperthermophilic β-keto acids ferredoxin oxidoreductases are bifunctional, catalyzing the activities of nonoxidative and oxidative decarboxylation of the corresponding β-keto acids. Mohammad S. Eram, Erica Oduaran, and Kesen Ma Copyright © 2014 Mohammad S. Eram et al. All rights reserved. Genome-Wide miRNA Seeds Prediction in Archaea Wed, 14 May 2014 09:11:39 +0000 Growing evidence indicates that miRNA genes exist in the archaeal genome, though the functional role of such noncoding RNA remains unclear. Here, we integrated the phylogenetic information of available archaeal genomes to predict miRNA seeds (typically defined as the 2–8 nucleotides of mature miRNAs) on the genomic scale. Finally, we found 2649 candidate seeds with significant conservation signal. Eleven of 29 unique seeds from previous study support our result ( value ), which demonstrates that the pipeline is suitable to predict experimentally detectable miRNA seeds. The statistical significance of the overlap between the detected archaeal seeds and known eukaryotic seeds shows that the miRNA may evolve before the divergence of these two domains of cellular life. In addition, miRNA targets are enriched for genes involved in transcriptional regulation, which is consistent with the situation in eukaryote. Our research will enhance the regulatory network analysis in Archaea. Shengqin Wang, Yuming Xu, and Zuhong Lu Copyright © 2014 Shengqin Wang et al. All rights reserved. An Antimethanogenic Nutritional Intervention in Early Life of Ruminants Modifies Ruminal Colonization by Archaea Sun, 06 Apr 2014 12:10:52 +0000 The aim of this work was to study whether feeding a methanogen inhibitor from birth of goat kids and their does has an impact on the archaeal population colonizing the rumen and to what extent the impact persists later in life. Sixteen goats giving birth to two kids were used. Eight does were treated (D+) with bromochloromethane after giving birth and over 2 months. The other 8 goats were not treated (D−). One kid per doe in both groups was treated with bromochloromethane (k+) for 3 months while the other was untreated (k−), resulting in four experimental groups: D+/k+, D+/k−, D−/k+, and D−/k−. Rumen samples were collected from kids at weaning and 1 and 4 months after (3 and 6 months after birth) and from does at the end of the treating period (2 months). Pyrosequencing analyses showed a modified archaeal community composition colonizing the rumen of kids, although such effect did not persist entirely 4 months after; however, some less abundant groups remained different in treated and control animals. The different response on the archaeal community composition observed between offspring and adult goats suggests that the competition occurring in the developing rumen to occupy different niches offer potential for intervention. Leticia Abecia, Kate E. Waddams, Gonzalo Martínez-Fernandez, A. Ignacio Martín-García, Eva Ramos-Morales, C. Jamie Newbold, and David R. Yáñez-Ruiz Copyright © 2014 Leticia Abecia et al. All rights reserved. Diversity of the DNA Replication System in the Archaea Domain Wed, 26 Mar 2014 09:21:14 +0000 The precise and timely duplication of the genome is essential for cellular life. It is achieved by DNA replication, a complex process that is conserved among the three domains of life. Even though the cellular structure of archaea closely resembles that of bacteria, the information processing machinery of archaea is evolutionarily more closely related to the eukaryotic system, especially for the proteins involved in the DNA replication process. While the general DNA replication mechanism is conserved among the different domains of life, modifications in functionality and in some of the specialized replication proteins are observed. Indeed, Archaea possess specific features unique to this domain. Moreover, even though the general pattern of the replicative system is the same in all archaea, a great deal of variation exists between specific groups. Felipe Sarmiento, Feng Long, Isaac Cann, and William B. Whitman Copyright © 2014 Felipe Sarmiento et al. All rights reserved. Phylogenetic Diversity of Archaea and the Archaeal Ammonia Monooxygenase Gene in Uranium Mining-Impacted Locations in Bulgaria Tue, 11 Mar 2014 00:00:00 +0000 Uranium mining and milling activities adversely affect the microbial populations of impacted sites. The negative effects of uranium on soil bacteria and fungi are well studied, but little is known about the effects of radionuclides and heavy metals on archaea. The composition and diversity of archaeal communities inhabiting the waste pile of the Sliven uranium mine and the soil of the Buhovo uranium mine were investigated using 16S rRNA gene retrieval. A total of 355 archaeal clones were selected, and their 16S rDNA inserts were analysed by restriction fragment length polymorphism (RFLP) discriminating 14 different RFLP types. All evaluated archaeal 16S rRNA gene sequences belong to the 1.1b/Nitrososphaera cluster of Crenarchaeota. The composition of the archaeal community is distinct for each site of interest and dependent on environmental characteristics, including pollution levels. Since the members of 1.1b/Nitrososphaera cluster have been implicated in the nitrogen cycle, the archaeal communities from these sites were probed for the presence of the ammonia monooxygenase gene (amoA). Our data indicate that amoA gene sequences are distributed in a similar manner as in Crenarchaeota, suggesting that archaeal nitrification processes in uranium mining-impacted locations are under the control of the same key factors controlling archaeal diversity. Galina Radeva, Anelia Kenarova, Velina Bachvarova, Katrin Flemming, Ivan Popov, Dimitar Vassilev, and Sonja Selenska-Pobell Copyright © 2014 Galina Radeva et al. All rights reserved. Towards a Computational Model of a Methane Producing Archaeum Tue, 04 Mar 2014 09:58:31 +0000 Progress towards a complete model of the methanogenic archaeum Methanosarcina acetivorans is reported. We characterized size distribution of the cells using differential interference contrast microscopy, finding them to be ellipsoidal with mean length and width of 2.9 μm and 2.3 μm, respectively, when grown on methanol and 30% smaller when grown on acetate. We used the single molecule pull down (SiMPull) technique to measure average copy number of the Mcr complex and ribosomes. A kinetic model for the methanogenesis pathways based on biochemical studies and recent metabolic reconstructions for several related methanogens is presented. In this model, 26 reactions in the methanogenesis pathways are coupled to a cell mass production reaction that updates enzyme concentrations. RNA expression data (RNA-seq) measured for cell cultures grown on acetate and methanol is used to estimate relative protein production per mole of ATP consumed. The model captures the experimentally observed methane production rates for cells growing on methanol and is most sensitive to the number of methyl-coenzyme-M reductase (Mcr) and methyl-tetrahydromethanopterin:coenzyme-M methyltransferase (Mtr) proteins. A draft transcriptional regulation network based on known interactions is proposed which we intend to integrate with the kinetic model to allow dynamic regulation. Joseph R. Peterson, Piyush Labhsetwar, Jeremy R. Ellermeier, Petra R. A. Kohler, Ankur Jain, Taekjip Ha, William W. Metcalf, and Zaida Luthey-Schulten Copyright © 2014 Joseph R. Peterson et al. All rights reserved. Archaeal Genome Guardians Give Insights into Eukaryotic DNA Replication and Damage Response Proteins Thu, 20 Feb 2014 10:17:10 +0000 As the third domain of life, archaea, like the eukarya and bacteria, must have robust DNA replication and repair complexes to ensure genome fidelity. Archaea moreover display a breadth of unique habitats and characteristics, and structural biologists increasingly appreciate these features. As archaea include extremophiles that can withstand diverse environmental stresses, they provide fundamental systems for understanding enzymes and pathways critical to genome integrity and stress responses. Such archaeal extremophiles provide critical data on the periodic table for life as well as on the biochemical, geochemical, and physical limitations to adaptive strategies allowing organisms to thrive under environmental stress relevant to determining the boundaries for life as we know it. Specifically, archaeal enzyme structures have informed the architecture and mechanisms of key DNA repair proteins and complexes. With added abilities to temperature-trap flexible complexes and reveal core domains of transient and dynamic complexes, these structures provide insights into mechanisms of maintaining genome integrity despite extreme environmental stress. The DNA damage response protein structures noted in this review therefore inform the basis for genome integrity in the face of environmental stress, with implications for all domains of life as well as for biomanufacturing, astrobiology, and medicine. David S. Shin, Ashley J. Pratt, and John A. Tainer Copyright © 2014 David S. Shin et al. All rights reserved. Characterization of Ten Heterotetrameric NDP-Dependent Acyl-CoA Synthetases of the Hyperthermophilic Archaeon Pyrococcus furiosus Tue, 11 Feb 2014 07:00:46 +0000 The hyperthermophilic archaeon Pyrococcus furiosus grows by fermenting peptides and carbohydrates to organic acids. In the terminal step, acyl-CoA synthetase (ACS) isoenzymes convert acyl-CoA derivatives to the corresponding acid and conserve energy in the form of ATP. ACS1 and ACS2 were previously purified from P. furiosus and have structures but the genome contains genes encoding three additional -subunits. The ten possible combinations of and genes were expressed in E. coli and each resulted in stable and active isoenzymes. The -subunit of each isoenzyme determined CoA-based substrate specificity and between them they accounted for the CoA derivatives of fourteen amino acids. The -subunit determined preference for adenine or guanine nucleotides. The GTP-generating isoenzymes are proposed to play a role in gluconeogenesis by producing GTP for GTP-dependent phosphoenolpyruvate carboxykinase and for other GTP-dependent processes. Transcriptional and proteomic data showed that all ten isoenzymes are constitutively expressed indicating that both ATP and GTP are generated from the metabolism of most of the amino acids. A phylogenetic analysis showed that the ACSs of P. furiosus and other members of the Thermococcales are evolutionarily distinct from those found throughout the rest of biology, including those of other hyperthermophilic archaea. Joseph W. Scott, Farris L. Poole, and Michael W. W. Adams Copyright © 2014 Joseph W. Scott et al. All rights reserved. Comparative Analysis of the Methanogen Diversity in Horse and Pony by Using mcrA Gene and Archaeal 16S rRNA Gene Clone Libraries Thu, 30 Jan 2014 08:09:52 +0000 Comparative analysis of methanogen compositions in the feces of horse and pony was carried out by constructing the α-subunit of methyl coenzyme-M reductase (mcrA) gene and 16S ribosomal RNA gene (16S rRNA) clone libraries. The mcrA clone library analysis indicated that Methanomicrobiales was predominant in both horse and pony. Furthermore, most of the clones of the 16S rRNA gene library showed that Methanomicrobiales was also predominant in horse and pony, but the LIBSHUFF analysis showed that the horse and pony libraries were significantly different (). Most of operational taxonomic units (OTUs) showed low similarity to the identified methanogens in both the mcrA and the 16S rRNA clone libraries. The results suggest that horse and pony harbor unidentified and novel methanogens in their hindgut. The methanogen population was higher in horse than in pony; however, the anaerobic fungal population was similar in horse and pony. The methanogen diversity was different between two breeds of Equus caballus. Khin-Ohnmar Lwin and Hiroki Matsui Copyright © 2014 Khin-Ohnmar Lwin and Hiroki Matsui. All rights reserved. Unique Characteristics of the Pyrrolysine System in the 7th Order of Methanogens: Implications for the Evolution of a Genetic Code Expansion Cassette Mon, 27 Jan 2014 07:17:14 +0000 Pyrrolysine (Pyl), the 22nd proteogenic amino acid, was restricted until recently to few organisms. Its translational use necessitates the presence of enzymes for synthesizing it from lysine, a dedicated amber stop codon suppressor tRNA, and a specific amino-acyl tRNA synthetase. The three genomes of the recently proposed Thermoplasmata-related 7th order of methanogens contain the complete genetic set for Pyl synthesis and its translational use. Here, we have analyzed the genomic features of the Pyl-coding system in these three genomes with those previously known from Bacteria and Archaea and analyzed the phylogeny of each component. This shows unique peculiarities, notably an amber    with an imperfect anticodon stem and a shortened synthetase. Phylogenetic analysis indicates that a Pyl-coding system was present in the ancestor of the seventh order of methanogens and appears more closely related to Bacteria than to Methanosarcinaceae, suggesting the involvement of lateral gene transfer in the spreading of pyrrolysine between the two prokaryotic domains. We propose that the Pyl-coding system likely emerged once in Archaea, in a hydrogenotrophic and methanol-H2-dependent methylotrophic methanogen. The close relationship between methanogenesis and the Pyl system provides a possible example of expansion of a still evolving genetic code, shaped by metabolic requirements. Guillaume Borrel, Nadia Gaci, Pierre Peyret, Paul W. O'Toole, Simonetta Gribaldo, and Jean-François Brugère Copyright © 2014 Guillaume Borrel et al. All rights reserved. The Alternative Route to Heme in the Methanogenic Archaeon Methanosarcina barkeri Thu, 23 Jan 2014 13:14:47 +0000 In living organisms heme is formed from the common precursor uroporphyrinogen III by either one of two substantially different pathways. In contrast to eukaryotes and most bacteria which employ the so-called “classical” heme biosynthesis pathway, the archaea use an alternative route. In this pathway, heme is formed from uroporphyrinogen III via the intermediates precorrin-2, sirohydrochlorin, siroheme, 12,18-didecarboxysiroheme, and iron-coproporphyrin III. In this study the heme biosynthesis proteins AhbAB, AhbC, and AhbD from Methanosarcina barkeri were functionally characterized. Using an in vivo enzyme activity assay it was shown that AhbA and AhbB (Mbar_A1459 and Mbar_A1460) together catalyze the conversion of siroheme into 12,18-didecarboxysiroheme. The two proteins form a heterodimeric complex which might be subject to feedback regulation by the pathway end-product heme. Further, AhbC (Mbar_A1793) was shown to catalyze the formation of iron-coproporphyrin III in vivo. Finally, recombinant AhbD (Mbar_A1458) was produced in E. coli and purified indicating that this protein most likely contains two [4Fe-4S] clusters. Using an in vitro enzyme activity assay it was demonstrated that AhbD catalyzes the conversion of iron-coproporphyrin III into heme. Melanie Kühner, Kristin Haufschildt, Alexander Neumann, Sonja Storbeck, Judith Streif, and Gunhild Layer Copyright © 2014 Melanie Kühner et al. All rights reserved. Development of β-Lactamase as a Tool for Monitoring Conditional Gene Expression by a Tetracycline-Riboswitch in Methanosarcina acetivorans Mon, 20 Jan 2014 06:35:15 +0000 The use of reporter gene fusions to assess cellular processes such as protein targeting and regulation of transcription or translation is established technology in archaeal, bacterial, and eukaryal genetics. Fluorescent proteins or enzymes resulting in chromogenic substrate turnover, like β-galactosidase, have been particularly useful for microscopic and screening purposes. However, application of such methodology is of limited use for strictly anaerobic organisms due to the requirement of molecular oxygen for chromophore formation or color development. We have developed β-lactamase from Escherichia coli (encoded by bla) in conjunction with the chromogenic substrate nitrocefin into a reporter system usable under anaerobic conditions for the methanogenic archaeon Methanosarcina acetivorans. By using a signal peptide of a putative flagellin from M. acetivorans and different catabolic promoters, we could demonstrate growth substrate-dependent secretion of β-lactamase, facilitating its use in colony screening on agar plates. Furthermore, a series of fusions comprised of a constitutive promoter and sequences encoding variants of the synthetic tetracycline-responsive riboswitch (tc-RS) was created to characterize its influence on translation initiation in M. acetivorans. One tc-RS variant resulted in more than 11-fold tetracycline-dependent regulation of bla expression, which is in the range of regulation by naturally occurring riboswitches. Thus, tc-RS fusions represent the first solely cis-active, that is, factor-independent system for controlled gene expression in Archaea. Shemsi Demolli, Miriam M. Geist, Julia E. Weigand, Nicole Matschiavelli, Beatrix Suess, and Michael Rother Copyright © 2014 Shemsi Demolli et al. All rights reserved. Early Methanogenic Colonisation in the Faeces of Meishan and Yorkshire Piglets as Determined by Pyrosequencing Analysis Mon, 06 Jan 2014 16:28:05 +0000 Gut methanogenic archaea of monogastric animals are considered to be related to energy metabolism and adipose deposition of the host; however, information on their development in young piglets is limited. Thus, to investigate early methanogenic colonisation in the faeces of Meishan and Yorkshire piglets, faecal samples were collected from piglets at 1, 3, 7, and 14 days after birth and used to analyse the methanogenic community with 16S rRNA gene pyrosequencing. Results showed that the diversity of the methanogenic community in the faeces of neonatal piglets decreased from one to 14 days of age, as the total methanogen populations increased. The age of piglets, but not the breed, significantly affected the diversity of the methanogenic community which was dominated by the genus Methanobrevibacter. From the ages of one to 14 days, the abundance of M. smithii-related operational taxonomic units (OTUs) increased significantly, while the abundances of M. thaueri- and M. millerae-related OTUs decreased significantly. The substitution of M. smithii for M. thaueri/M. millerae was faster in Yorkshire piglets than in Meishan piglets. These results suggest that the early establishment of microbiota in neonatal piglets is accompanied by dramatic changes in the methanogenic community, and that the changes vary among pigs of different genotypes. Yong Su, Gaorui Bian, Zhigang Zhu, Hauke Smidt, and Weiyun Zhu Copyright © 2014 Yong Su et al. All rights reserved. Comparative Analysis of Proteomes and Functionomes Provides Insights into Origins of Cellular Diversification Tue, 31 Dec 2013 14:30:25 +0000 Reconstructing the evolutionary history of modern species is a difficult problem complicated by the conceptual and technical limitations of phylogenetic tree building methods. Here, we propose a comparative proteomic and functionomic inferential framework for genome evolution that allows resolving the tripartite division of cells and sketching their history. Evolutionary inferences were derived from the spread of conserved molecular features, such as molecular structures and functions, in the proteomes and functionomes of contemporary organisms. Patterns of use and reuse of these traits yielded significant insights into the origins of cellular diversification. Results uncovered an unprecedented strong evolutionary association between Bacteria and Eukarya while revealing marked evolutionary reductive tendencies in the archaeal genomic repertoires. The effects of nonvertical evolutionary processes (e.g., HGT, convergent evolution) were found to be limited while reductive evolution and molecular innovation appeared to be prevalent during the evolution of cells. Our study revealed a strong vertical trace in the history of proteins and associated molecular functions, which was reliably recovered using the comparative genomics approach. The trace supported the existence of a stem line of descent and the very early appearance of Archaea as a diversified superkingdom, but failed to uncover a hidden canonical pattern in which Bacteria was the first superkingdom to deploy superkingdom-specific structures and functions. Arshan Nasir and Gustavo Caetano-Anollés Copyright © 2013 Arshan Nasir and Gustavo Caetano-Anollés. All rights reserved. The Potent In Vitro Skin Permeation of Archaeosome Made from Lipids Extracted of Sulfolobus acidocaldarius Thu, 26 Dec 2013 11:20:40 +0000 Archaeosomes are a new generation of liposomes that exhibit higher stabilities under different conditions, such as high temperatures, alkaline or acidic pH, and presence of bile salts in comparison with liposomes, and can be used in biotechnology including drug, gene, and vaccine delivery. The objective of this study was to prepare archaeosomes using lipid extracted from Sulfolobus acidocaldarius and evaluate their physicochemical properties. The lipids were extracted from S. acidocaldarius and assayed by High Performance Thin-Layer Chromatography (HPTLC). Archaeosomes were prepared using film method and methylene blue was used as drug model. They were characterized for their vesicle size and Differential Scanning Calorimetry (DSC) was used to investigate changes in their thermal behavior. The released amount of methylene blue was determined using a dialysis membrane and rat skin. HPTLC analysis of the extracted lipids showed that glycerol ether may be the major lipid with more than 78 percent probability. Results of particle size determination showed a mean size of 158.33 nm and the results of DSC indicated the possible interaction of methylene blue with lipids during the preparation of archaeosome. The addition of cholesterol significantly improved the encapsulation of methylene blue in the archaeosome so that the encapsulation efficiency was 61.66 ± 2.88%. The result of in vitro skin permeation showed that methylene blue could pass through skin model according to Peppas model and there was about 41.66% release after 6 h, whereas no release was observed through dialysis membrane. According to the results of the study, it is concluded that archaeosome may be successfully used as drug delivery system. Eskandar Moghimipour, Mohammad Kargar, Zahra Ramezani, and Somayeh Handali Copyright © 2013 Eskandar Moghimipour et al. All rights reserved. Archaeal Production of Polyhydroxyalkanoate (PHA) Co- and Terpolyesters from Biodiesel Industry-Derived By-Products Thu, 19 Dec 2013 14:54:11 +0000 The archaeon Haloferax mediterranei was selected for production of PHA co- and terpolyesters using inexpensive crude glycerol phase (CGP) from biodiesel production as carbon source. CGP was assessed by comparison with the application of pure glycerol. Applying pure glycerol, a copolyester with a molar fraction of 3-hydroxybutyrate (3HB) of 0.90 mol/mol and 3-hydroxyvalerate (3HV) of 0.10 mol/mol, was produced at a volumetric productivity of 0.12 g/Lh and an intracellular PHA content of 75.4 wt.-% in the sum of biomass protein plus PHA. Application of CGP resulted in the same polyester composition and volumetric productivity, indicating the feasibility of applying CGP as feedstock. Analysis of molar mass distribution revealed a weight average molar mass of 150 kDa and polydispersity of 2.1 for pure glycerol and 253 kDa and 2.7 for CGP, respectively; melting temperatures ranged between 130 and 140°C in both setups. Supplying γ-butyrolactone as 4-hydroxybutyrate (4HB) precursor resulted in a poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyvalerate-co-4-hydroxybutyrate] (PHBHV4HB) terpolyester containing 3HV (0.12 mol/mol) and 4HB (0.05 mol/mol) in the poly[(R)-3-hydroxybutyrate] (PHB) matrix; in addition, this process runs without sterilization of the bioreactor. The terpolyester displayed reduced melting (melting endotherms at 122 and 137°C) and glass transition temperature (2.5°C), increased molar mass (391 kDa), and a polydispersity similar to the copolyesters. Carmen Hermann-Krauss, Martin Koller, Alexander Muhr, Hubert Fasl, Franz Stelzer, and Gerhart Braunegg Copyright © 2013 Carmen Hermann-Krauss et al. All rights reserved. Close Encounters of the Third Domain: The Emerging Genomic View of Archaeal Diversity and Evolution Tue, 19 Nov 2013 09:34:36 +0000 The Archaea represent the so-called Third Domain of life, which has evolved in parallel with the Bacteria and which is implicated to have played a pivotal role in the emergence of the eukaryotic domain of life. Recent progress in genomic sequencing technologies and cultivation-independent methods has started to unearth a plethora of data of novel, uncultivated archaeal lineages. Here, we review how the availability of such genomic data has revealed several important insights into the diversity, ecological relevance, metabolic capacity, and the origin and evolution of the archaeal domain of life. Anja Spang, Joran Martijn, Jimmy H. Saw, Anders E. Lind, Lionel Guy, and Thijs J. G. Ettema Copyright © 2013 Anja Spang et al. All rights reserved. The Common Ancestor of Archaea and Eukarya Was Not an Archaeon Sun, 17 Nov 2013 10:43:39 +0000 It is often assumed that eukarya originated from archaea. This view has been recently supported by phylogenetic analyses in which eukarya are nested within archaea. Here, I argue that these analyses are not reliable, and I critically discuss archaeal ancestor scenarios, as well as fusion scenarios for the origin of eukaryotes. Based on recognized evolutionary trends toward reduction in archaea and toward complexity in eukarya, I suggest that their last common ancestor was more complex than modern archaea but simpler than modern eukaryotes (the bug in-between scenario). I propose that the ancestors of archaea (and bacteria) escaped protoeukaryotic predators by invading high temperature biotopes, triggering their reductive evolution toward the “prokaryotic” phenotype (the thermoreduction hypothesis). Intriguingly, whereas archaea and eukarya share many basic features at the molecular level, the archaeal mobilome resembles more the bacterial than the eukaryotic one. I suggest that selection of different parts of the ancestral virosphere at the onset of the three domains played a critical role in shaping their respective biology. Eukarya probably evolved toward complexity with the help of retroviruses and large DNA viruses, whereas similar selection pressure (thermoreduction) could explain why the archaeal and bacterial mobilomes somehow resemble each other. Patrick Forterre Copyright © 2013 Patrick Forterre. All rights reserved. A Single-Culture Bioprocess of Methanothermobacter thermautotrophicus to Upgrade Digester Biogas by CO2-to-CH4 Conversion with H2 Tue, 01 Oct 2013 08:04:16 +0000 We optimized and tested a postbioprocessing step with a single-culture archaeon to upgrade biogas (i.e., increase methane content) from anaerobic digesters via conversion of CO2 into CH4 by feeding H2 gas. We optimized a culture of the thermophilic methanogen Methanothermobacter thermautotrophicus using: (1) a synthetic H2/CO2 mixture; (2) the same mixture with pressurization; (3) a synthetic biogas with different CH4 contents and H2; and (4) an industrial, untreated biogas and H2. A laboratory culture with a robust growth (dry weight of 6.4–7.4 g/L; OD600 of 13.6–15.4), a volumetric methane production rate of 21 L/L culture-day, and a H2 conversion efficiency of 89% was moved to an industrial anaerobic digester facility, where it was restarted and fed untreated biogas with a methane content of ~70% at a rate such that CO2 was in excess of the stoichiometric requirements in relation to H2. Over an 8-day operating period, the dry weight of the culture initially decreased slightly before stabilizing at an elevated level of ~8 g/L to achieve a volumetric methane production rate of 21 L/L culture-day and a H2 conversion efficiency of 62%. While some microbial contamination of the culture was observed via microscopy, it did not affect the methane production rate of the culture. Matthew R. Martin, Jeffrey J. Fornero, Rebecca Stark, Laurens Mets, and Largus T. Angenent Copyright © 2013 Matthew R. Martin et al. All rights reserved. Comparative Analysis of Barophily-Related Amino Acid Content in Protein Domains of Pyrococcus abyssi and Pyrococcus furiosus Thu, 26 Sep 2013 13:38:53 +0000 Amino acid substitution patterns between the nonbarophilic Pyrococcus furiosus and its barophilic relative P. abyssi confirm that hydrostatic pressure asymmetry indices reflect the extent to which amino acids are preferred by barophilic archaeal organisms. Substitution patterns in entire protein sequences, shared protein domains defined at fold superfamily level, domains in homologous sequence pairs, and domains of very ancient and very recent origin now provide further clues about the environment that led to the genetic code and diversified life. The pyrococcal proteomes are very similar and share a very early ancestor. Relative amino acid abundance analyses showed that biases in the use of amino acids are due to their shared fold superfamilies. Within these repertoires, only two of the five amino acids that are preferentially barophilic, aspartic acid and arginine, displayed this preference significantly and consistently across structure and in domains appearing in the ancestor. The more primordial asparagine, lysine and threonine displayed a consistent preference for nonbarophily across structure and in the ancestor. Since barophilic preferences are already evident in ancient domains that are at least ~3 billion year old, we conclude that barophily is a very ancient trait that unfolded concurrently with genetic idiosyncrasies in convergence towards a universal code. Liudmila S. Yafremava, Massimo Di Giulio, and Gustavo Caetano-Anollés Copyright © 2013 Liudmila S. Yafremava et al. All rights reserved.