Showing papers in "Archaea in 2014"

Unique characteristics of the pyrrolysine system in the 7th order of methanogens: implications for the evolution of a genetic code expansion cassette.

Abstract: 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 tRNA Pyl with an imperfect anticodon stem and a shortened tRNA Pyl 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-H 2-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.

An Antimethanogenic Nutritional Intervention in Early Life of Ruminants Modifies Ruminal Colonization by Archaea

Abstract: 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.

The Alternative Route to Heme in the Methanogenic Archaeon Methanosarcina barkeri

Abstract: 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.

Early Methanogenic Colonisation in the Faeces of Meishan and Yorkshire Piglets as Determined by Pyrosequencing Analysis

Abstract: 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.

Archaea: the first domain of diversified life.

Abstract: 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.

Comparative Analysis of the Methanogen Diversity in Horse and Pony by Using mcrA Gene and Archaeal 16S rRNA Gene Clone Libraries

Abstract: 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 (P < 0.05). 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.

Development of β -lactamase as a tool for monitoring conditional gene expression by a tetracycline-riboswitch in Methanosarcina acetivorans.

Abstract: 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.

A Phylogenomic Census of Molecular Functions Identifies Modern Thermophilic Archaea as the Most Ancient Form of Cellular Life

Abstract: 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.

Diversity and Distribution of Archaea Community along a Stratigraphic Permafrost Profile from Qinghai-Tibetan Plateau, China

Abstract: 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.

Diversity of the DNA replication system in the Archaea domain.

Abstract: 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.

Archaeal genome guardians give insights into eukaryotic DNA replication and damage response proteins.

Abstract: 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.

Children living near a sanitary landfill have increased breath methane and Methanobrevibacter smithii in their intestinal microbiota.

Abstract: 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.

The bifunctional pyruvate decarboxylase/pyruvate ferredoxin oxidoreductase from Thermococcus guaymasensis.

Abstract: 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 3.8 ± 0.22 U mg(-1) and 20.2 ± 1.8 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.

Towards a Computational Model of a Methane Producing Archaeum

Abstract: 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.

Seasonal effects in a lake sediment archaeal community of the Brazilian Savanna.

Abstract: 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.

Characterization of ten heterotetrameric NDP-dependent acyl-CoA synthetases of the hyperthermophilic archaeon Pyrococcus furiosus.

Abstract: 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 α 2 β 2 structures but the genome contains genes encoding three additional α-subunits. The ten possible combinations of α and β genes were expressed in E. coli and each resulted in stable and active α 2 β 2 isoenzymes. The α-subunit of each isoenzyme determined CoA-based substrate specificity and between them they accounted for the CoA derivatives of fourteen amino acids. The β-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.

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

Abstract: 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 11b/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 11b/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

Genome-wide miRNA seeds prediction in Archaea.

Abstract: 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 (P value <0.01), 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.

The Origin and Evolution of the Archaeal Domain

Abstract: With this special issue on the origin and evolution of Archaea we honor and celebrate the life and impactful contributions of Carl Woese (July 15, 1928-December 30, 2012). Carl was born and raised in Syracuse, New York. His undergraduate studies were in Amherst College and his graduate training in Yale. Sol Spiegelman brought him to the University of Illinois at Urbana-Champaign, where he unfolded a brilliant career. Carl was inspired by the originality of his mentor, Ernest C. Pollard, the tradition of biological form of D'arcy Thompson, the charisma of Francis Crick, the evolutionary tempo-mode perspective of G. G. Simpson, and the foresight of Darwin and Wallace. He understood the centrality of evolution in our understanding of biology and championed this perspective as he explored the molecular makeup of the translation machinery. His insightful mind is responsible for the discovery of the archaeal domain and for transforming comparative views of microbial diversity into an overarching evolutionary framework. Archaea constitutes the third domain of life, a remarkable group of akaryotic microbes with unique biochemical and genomic features, some of which resemble those of eukaryotes. Their habitats and lifestyles are very diverse, from extremophiles living in harsh environments to soil and marine mesophiles, from free-living microbes to gut-inhabiting methanogens and symbionts. Carl's work did not only result in the definition of a new “urkingdom,” originally named by him as “archaebacteria,” but his insights prompted an appreciation (and respect) for the incredible microbial diversity of the biosphere. He battled the establishment to make way to a redefined microbiological science that treasured evolutionary thinking and acknowledged the centrality of microbes in the global ecosystems of our planet. He was also a harsh critic of the field of biology in general. He felt our biological views are still governed by reductionistic biases inherited from the genetic and genomic revolutions of last century, which could not identify any important questions left to answer. Furthermore, he strongly felt the biological sciences were devoted and defined by the application side, that is, by focusing on providing “service to society” through bioengineering instead of acting as “society's teacher” of man's place in the universe. A number of unsolved problems that are central to understanding life remain to be answered, and Carl posed some of the basic questions from the very beginning. What were life's origins? How did molecular and organismal complexity unfold? What are the ultimate governing principles of life? He recognized the limitations of the primacy of a genetic, molecular biology and mechanistic outlook that was gene-centered and prompted an exploration of biological complexity and emergence of biological organization within an evolutionary and physics framework. He recognized the importance of the proteinaceous backbone of life and how its design and function is delimited by the genetic code, translation, and its complex regulatory control. In this special issue we bring back some of Carl's basic unanswered questions. While it is becoming clear that the archaeal domain may have an independent evolutionary history, its origin and links to the other two domains of cellular complexity remain contentious, as well as its placement in the tree of life. The question demands urgent attention. Several contributions of this special issue tackle important aspects of the origin, diversity, and evolution of the archaeal domain. A review article by A. Spang et al. comprehensively describes current hypotheses on the relationships of the three domains and evaluates archaeal diversity and evolution using recent genomic data (e.g., metagenomes and single-cell genomes). P. Forterre also evaluates the contemporary scenarios for the origins of the three domains. Archaeal ancestor scenarios and the fusion hypothesis are criticized. Interestingly, he brings the evolutionary role of the virosphere to explain the diversification of the three domains from the last universal common ancestor of life. A. Nasir and G. Caetano-Anolles explore a novel comparative genomic framework that makes the vertical horizontal evolutionary contributions explicit, and G. Caetano-Anolles et al. advance structural phylogenomic analyses of protein and nucleic acid structures and their associated functions. These approaches reveal that Archaea is the most ancient domain, which prompts a careful reevaluation of current phylogenetic methodologies and our understanding of the rooting of the tree of life. D. S. Shin et al. review the robustness of archaeal proteins against extremophilic environments at the protein 3-dimensional structural level. C. J. Reed et al. describe how archaeal species can be adapted into thermophilic, psychrophilic, piezophilic, and halophilic environments by characterizing the biophysical property of archaeal proteins. Both studies emphasize the importance of archaeal structural biology for understanding human biology with medical and industrial impacts. G. Borrel et al. present a bioinformatics analysis of three genomes from a newly identified order of methanogens and find the pyrrolysine (22nd amino acid) coding system. The phylogenetic analysis indicates that this genomic feature is conserved in both archaeal methanogens and bacteria, which can be an example of continuing evolution of the genetic code directed by metabolic requirements. On another front, L. S. Yafremava et al. study amino acid substitution patterns in the protein domains of nonbarophilic and barophilic Pyrococcus species and reveal that barophily is a very ancient trait that unfolded with the early evolution of the genetic code during early adaptation to deep ocean environments. J. R. Peterson et al. use many different state-of-the-art approaches (e.g., SiMPull and RNA-Seq) to quantitatively characterize the methanogenesis pathways and translational machinery of the methanogen Methanosarcina acetivorans. This bioinformatics modeling can be a first step to establish new archaeal model systems, very much as E. coli is used for bacteria. Taken together, articles highlight patterns and processes responsible for archaeal diversity at genetic, genomic, biochemical, physiological, and ecological levels. It is our intention that the work presented here will stimulate further evolutionary thinking, following Carl's pioneering and unorthodox spirit. Gustavo Caetano-Anolles Kyung Mo Kim