Archaea The latest articles from Hindawi Publishing Corporation © 2016 , Hindawi Publishing Corporation . All rights reserved. The Proteome and Lipidome of Thermococcus kodakarensis across the Stationary Phase Wed, 04 May 2016 08:58:10 +0000 The majority of cells in nature probably exist in a stationary-phase-like state, due to nutrient limitation in most environments. Studies on bacteria and yeast reveal morphological and physiological changes throughout the stationary phase, which lead to an increased ability to survive prolonged nutrient limitation. However, there is little information on archaeal stationary phase responses. We investigated protein- and lipid-level changes in Thermococcus kodakarensis with extended time in the stationary phase. Adaptations to time in stationary phase included increased proportion of membrane lipids with a tetraether backbone, synthesis of proteins that ensure translational fidelity, specific regulation of ABC transporters (upregulation of some, downregulation of others), and upregulation of proteins involved in coenzyme production. Given that the biological mechanism of tetraether synthesis is unknown, we also considered whether any of the protein-level changes in T. kodakarensis might shed light on the production of tetraether lipids across the same period. A putative carbon-nitrogen hydrolase, a TldE (a protease in Escherichia coli) homologue, and a membrane bound hydrogenase complex subunit were candidates for possible involvement in tetraether-related reactions, while upregulation of adenosylcobalamin synthesis proteins might lend support to a possible radical mechanism as a trigger for tetraether synthesis. Emma J. Gagen, Marcos Y. Yoshinaga, Franka Garcia Prado, Kai-Uwe Hinrichs, and Michael Thomm Copyright © 2016 Emma J. Gagen et al. All rights reserved. Characterization of the ATP-Dependent Lon-Like Protease in Methanobrevibacter smithii Thu, 28 Apr 2016 13:48:52 +0000 The Lon protease is highly evolutionarily conserved. However, little is known about Lon in the context of gut microbial communities. A gene encoding a Lon-like protease (Lon-like-Ms) was identified and characterized from Methanobrevibacter smithii, the predominant archaeon in the human gut ecosystem. Phylogenetic and sequence analyses showed that Lon-like-Ms and its homologs are newly identified members of the Lon family. A recombinant form of the enzyme was purified by affinity chromatography, and its catalytic properties were examined. Recombinant Lon-like-Ms exhibited ATPase activity and cleavage activity toward fluorogenic peptides and casein. The peptidase activity of Lon-like-Ms relied strictly on Mg2+ (or other divalent cations) and ATP. These results highlight a new type of Lon-like protease that differs from its bacterial counterpart. Jihua Pei, Jianfang Yan, and Yi Jiang Copyright © 2016 Jihua Pei et al. All rights reserved. Methanogen Diversity in Indigenous and Introduced Ruminant Species on the Tibetan Plateau Thu, 28 Apr 2016 12:19:24 +0000 Host factors are regarded as important in shaping the archaeal community in the rumen but few controlled studies have been performed to demonstrate this across host species under the same environmental conditions. A study was designed to investigate the structure of the methanogen community in the rumen of two indigenous (yak and Tibetan sheep) and two introduced domestic ruminant (cattle and crossbred sheep) species raised and fed under similar conditions on the high altitude Tibetan Plateau. The methylotrophic Methanomassiliicoccaceae was the predominant archaeal group in all animals even though Methanobrevibacter are usually present in greater abundance in ruminants globally. Furthermore, within the Methanomassiliicoccaceae family members from Mmc. group 10 and Mmc. group 4 were dominant in Tibetan Plateau ruminants compared to Mmc. group 12 found to be highest in other ruminants studied. Small ruminants presented the highest number of sequences that belonged to Methanomassiliicoccaceae compared to the larger ruminants. Although the methanogen community structure was different among the ruminant species, there were striking similarities between the animals in this environment. This indicates that factors such as the extreme environmental conditions and diet on the Tibetan Plateau might have a greater impact on rumen methanogen community compared to host differences. Xiao Dan Huang, Gonzalo Martinez-Fernandez, Jagadish Padmanabha, Ruijun Long, Stuart E. Denman, and Christopher S. McSweeney Copyright © 2016 Xiao Dan Huang et al. All rights reserved. Discovery and Characterization of Iron Sulfide and Polyphosphate Bodies Coexisting in Archaeoglobus fulgidus Cells Tue, 19 Apr 2016 13:03:46 +0000 Inorganic storage granules have long been recognized in bacterial and eukaryotic cells but were only recently identified in archaeal cells. Here, we report the cellular organization and chemical compositions of storage granules in the Euryarchaeon, Archaeoglobus fulgidus strain VC16, a hyperthermophilic, anaerobic, and sulfate-reducing microorganism. Dense granules were apparent in A. fulgidus cells imaged by cryo electron microscopy (cryoEM) but not so by negative stain electron microscopy. Cryo electron tomography (cryoET) revealed that each cell contains one to several dense granules located near the cell membrane. Energy dispersive X-ray (EDX) spectroscopy and scanning transmission electron microscopy (STEM) show that, surprisingly, each cell contains not just one but often two types of granules with different elemental compositions. One type, named iron sulfide body (ISB), is composed mainly of the elements iron and sulfur plus copper; and the other one, called polyphosphate body (PPB), is composed of phosphorus and oxygen plus magnesium, calcium, and aluminum. PPBs are likely used for energy storage and/or metal sequestration/detoxification. ISBs could result from the reduction of sulfate to sulfide via anaerobic energy harvesting pathways and may be associated with energy and/or metal storage or detoxification. The exceptional ability of these archaeal cells to sequester different elements may have novel bioengineering applications. Daniel B. Toso, Muhammad Mohsin Javed, Elizabeth Czornyj, Robert P. Gunsalus, and Z. Hong Zhou Copyright © 2016 Daniel B. Toso et al. All rights reserved. Archaeal Community Changes Associated with Cultivation of Amazon Forest Soil with Oil Palm Wed, 24 Feb 2016 10:41:57 +0000 This study compared soil archaeal communities of the Amazon forest with that of an adjacent area under oil palm cultivation by 16S ribosomal RNA gene pyrosequencing. Species richness and diversity were greater in native forest soil than in the oil palm-cultivated area, and 130 OTUs (13.7%) were shared between these areas. Among the classified sequences, Thaumarchaeota were predominant in the native forest, whereas Euryarchaeota were predominant in the oil palm-cultivated area. Archaeal species diversity was 1.7 times higher in the native forest soil, according to the Simpson diversity index, and the Chao1 index showed that richness was five times higher in the native forest soil. A phylogenetic tree of unclassified Thaumarchaeota sequences showed that most of the OTUs belong to Miscellaneous Crenarchaeotic Group. Several archaeal genera involved in nutrient cycling (e.g., methanogens and ammonia oxidizers) were identified in both areas, but significant differences were found in the relative abundances of Candidatus Nitrososphaera and unclassified Soil Crenarchaeotic Group (prevalent in the native forest) and Candidatus Nitrosotalea and unclassified Terrestrial Group (prevalent in the oil palm-cultivated area). More studies are needed to culture some of these Archaea in the laboratory so that their metabolism and physiology can be studied. Daiva Domenech Tupinambá, Maurício Egídio Cantão, Ohana Yonara Assis Costa, Jessica Carvalho Bergmann, Ricardo Henrique Kruger, Cynthia Maria Kyaw, Cristine Chaves Barreto, and Betania Ferraz Quirino Copyright © 2016 Daiva Domenech Tupinambá et al. All rights reserved. The Vertical Distribution of Sediment Archaeal Community in the “Black Bloom” Disturbing Zhushan Bay of Lake Taihu Sun, 17 Jan 2016 13:49:58 +0000 Using the Illumina sequencing technology, we investigated the vertical distribution of archaeal community in the sediment of Zhushan Bay of Lake Taihu, where the black bloom frequently occurred in summer. Overall, the Miscellaneous Crenarchaeotal Group (MCG), Deep Sea Hydrothermal Vent Group 6 (DHVEG-6), and Methanobacterium dominated the archaeal community. However, we observed significant difference in composition of archaeal community among different depths of the sediment. DHVEG-6 dominated in the surface layer (0–3 cm) sediment. Methanobacterium was the dominating archaeal taxa in the L2 (3–6 cm) and L3 (6–10) sediment. MCG was most abundant in the L4 (10–15 cm) and L5 (15–20 cm) sediment. Besides, DHVEG-6 was significantly affected by the concentration of total phosphorus (TP). And loss on ignition (LOI) was an important environmental factor for Methanobacterium. As the typical archaeal taxa in the surface layer sediment, DHVEG-6 and Methanobacterium might be more adapted to abundant substrate supply from cyanobacterial blooms and take active part in the biomass transformation. We propose that DHVEG-6 and Methanobacterium could be the key archaeal taxa correlated with the “black bloom” formation in Zhushan Bay. Xianfang Fan and Peng Xing Copyright © 2016 Xianfang Fan and Peng Xing. All rights reserved. Characterization of Fatty Acids in Crenarchaeota by GC-MS and NMR Thu, 31 Dec 2015 09:21:43 +0000 Lipids composed of condensed isoprenyl units connected to glycerol backbones by ether linkages are a distinguishing feature of Archaea. Data suggesting that fatty acids with linear hydrocarbon chains are present in some Archaea have been available for decades. However, lack of genomic and biochemical evidence for the metabolic machinery required to synthesize and degrade fatty acids has left the field unclear on this potentially significant biochemical aspect. Because lipids are energy currency and cell signaling molecules, their presence in Archaea is significant for understanding archaeal biology. A recent large-scale bioinformatics analysis reignited the debate as to the importance of fatty acids in Archaea by presenting genetic evidence for the presence of enzymes required for anabolic and catabolic fatty acid metabolism across the archaeal domain. Here, we present direct biochemical evidence from gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) spectroscopy for the presence of fatty acids in two members of the Crenarchaeota, Sulfolobus solfataricus and Ignicoccus hospitalis. This is the first report providing biochemical data for the existence of fatty acids in these Crenarchaeota, opening new discussions on energy balance and the potential for the discovery of new thermostable enzymes for industry. Timothy Hamerly, Brian Tripet, Louie Wurch, Robert L. Hettich, Mircea Podar, Brian Bothner, and Valérie Copié Copyright © 2015 Timothy Hamerly et al. All rights reserved. Sequence, Structure, and Binding Analysis of Cyclodextrinase (TK1770) from T. kodakarensis (KOD1) Using an In Silico Approach Thu, 24 Dec 2015 16:02:07 +0000 Thermostable cyclodextrinase (Tk1770 CDase) from hyperthermophilic archaeon Thermococcus kodakarensis (KOD1) hydrolyzes cyclodextrins into linear dextrins. The sequence of Tk1770 CDase retrieved from UniProt was aligned with sequences of sixteen CD hydrolyzing enzymes and a phylogenetic tree was constructed using Bayesian inference. The homology model of Tk1770 CDase was constructed and optimized with Modeller v9.14 program. The model was validated with ProSA server and PROCHECK analysis. Four conserved regions and the catalytic triad consisting of Asp411, Glu437, and Asp502 of GH13 family were identified in catalytic site. Also an additional fifth conserved region downstream to the fourth region was also identified. The structure of Tk1770 CDase consists of an additional N′-domain and a helix-loop-helix motif that is conserved in all archaeal CD hydrolyzing enzymes. The N′-domain contains an extended loop region that forms a part of catalytic domain and plays an important role in stability and substrate binding. The docking of substrate into catalytic site revealed the interactions with different conserved residues involved in substrate binding and formation of enzyme-substrate complex. Ramzan Ali and Muhammad Imtiaz Shafiq Copyright © 2015 Ramzan Ali and Muhammad Imtiaz Shafiq. All rights reserved. Molecular Analysis of Methanogen Richness in Landfill and Marshland Targeting 16S rDNA Sequences Tue, 13 Oct 2015 11:25:38 +0000 Methanogens, a key contributor in global carbon cycling, methane emission, and alternative energy production, generate methane gas via anaerobic digestion of organic matter. The methane emission potential depends upon methanogenic diversity and activity. Since they are anaerobes and difficult to isolate and culture, their diversity present in the landfill sites of Delhi and marshlands of Southern Assam, India, was analyzed using molecular techniques like 16S rDNA sequencing, DGGE, and qPCR. The sequencing results indicated the presence of methanogens belonging to the seventh order and also the order Methanomicrobiales in the Ghazipur and Bhalsawa landfill sites of Delhi. Sequences, related to the phyla Crenarchaeota (thermophilic) and Thaumarchaeota (mesophilic), were detected from marshland sites of Southern Assam, India. Jaccard analysis of DGGE gel using Gel2K showed three main clusters depending on the number and similarity of band patterns. The copy number analysis of hydrogenotrophic methanogens using qPCR indicates higher abundance in landfill sites of Delhi as compared to the marshlands of Southern Assam. The knowledge about “methanogenic archaea composition” and “abundance” in the contrasting ecosystems like “landfill” and “marshland” may reorient our understanding of the Archaea inhabitants. This study could shed light on the relationship between methane-dynamics and the global warming process. Shailendra Yadav, Sharbadeb Kundu, Sankar K. Ghosh, and S. S. Maitra Copyright © 2015 Shailendra Yadav et al. All rights reserved. Biotechnological Uses of Archaeal Proteins Mon, 12 Oct 2015 13:49:37 +0000 Frédéric Pecorari, Vickery L. Arcus, and Juergen Wiegel Copyright © 2015 Frédéric Pecorari et al. All rights reserved. Production and Application of a Soluble Hydrogenase from Pyrococcus furiosus Mon, 12 Oct 2015 08:23:33 +0000 Hydrogen gas is a potential renewable alternative energy carrier that could be used in the future to help supplement humanity’s growing energy needs. Unfortunately, current industrial methods for hydrogen production are expensive or environmentally unfriendly. In recent years research has focused on biological mechanisms for hydrogen production and specifically on hydrogenases, the enzyme responsible for catalyzing the reduction of protons to generate hydrogen. In particular, a better understanding of this enzyme might allow us to generate hydrogen that does not use expensive metals, such as platinum, as catalysts. The soluble hydrogenase I (SHI) from the hyperthermophile Pyrococcus furiosus, a member of the euryarchaeota, has been studied extensively and used in various biotechnological applications. This review summarizes the strategies used in engineering and characterizing three different forms of SHI and the properties of the recombinant enzymes. SHI has also been used in in vitro systems for hydrogen production and NADPH generation and these systems are also discussed. Chang-Hao Wu, Patrick M. McTernan, Mary E. Walter, and Michael W. W. Adams Copyright © 2015 Chang-Hao Wu et al. All rights reserved. Archaeal MCM Proteins as an Analog for the Eukaryotic Mcm2–7 Helicase to Reveal Essential Features of Structure and Function Sun, 11 Oct 2015 12:21:01 +0000 In eukaryotes, the replicative helicase is the large multisubunit CMG complex consisting of the Mcm2–7 hexameric ring, Cdc45, and the tetrameric GINS complex. The Mcm2–7 ring assembles from six different, related proteins and forms the core of this complex. In archaea, a homologous MCM hexameric ring functions as the replicative helicase at the replication fork. Archaeal MCM proteins form thermostable homohexamers, facilitating their use as models of the eukaryotic Mcm2–7 helicase. Here we review archaeal MCM helicase structure and function and how the archaeal findings relate to the eukaryotic Mcm2–7 ring. Justin M. Miller and Eric J. Enemark Copyright © 2015 Justin M. Miller and Eric J. Enemark. All rights reserved. A Novel Highly Thermostable Multifunctional Beta-Glycosidase from Crenarchaeon Acidilobus saccharovorans Sun, 11 Oct 2015 08:14:24 +0000 We expressed a putative β-galactosidase Asac_1390 from hyperthermophilic crenarchaeon Acidilobus saccharovorans in Escherichia coli and purified the recombinant enzyme. Asac_1390 is composed of 490 amino acid residues and showed high sequence similarity to family 1 glycoside hydrolases from various thermophilic Crenarchaeota. The maximum activity was observed at pH 6.0 and 93°C. The half-life of the enzyme at 90°C was about 7 hours. Asac_1390 displayed high tolerance to glucose and exhibits hydrolytic activity towards cellobiose and various aryl glucosides. The hydrolytic activity with p-nitrophenyl (pNP) substrates followed the order pNP-β-D-galactopyranoside (328 U mg−1), pNP-β-D-glucopyranoside (246 U mg−1), pNP-β-D-xylopyranoside (72 U mg−1), and pNP-β-D-mannopyranoside (28 U mg−1). Thus the enzyme was actually a multifunctional β-glycosidase. Therefore, the utilization of Asac_1390 may contribute to facilitating the efficient degradation of lignocellulosic biomass and help enhance bioconversion processes. Vadim M. Gumerov, Andrey L. Rakitin, Andrey V. Mardanov, and Nikolai V. Ravin Copyright © 2015 Vadim M. Gumerov et al. All rights reserved. From Structure-Function Analyses to Protein Engineering for Practical Applications of DNA Ligase Mon, 05 Oct 2015 07:20:42 +0000 DNA ligases are indispensable in all living cells and ubiquitous in all organs. DNA ligases are broadly utilized in molecular biology research fields, such as genetic engineering and DNA sequencing technologies. Here we review the utilization of DNA ligases in a variety of in vitro gene manipulations, developed over the past several decades. During this period, fewer protein engineering attempts for DNA ligases have been made, as compared to those for DNA polymerases. We summarize the recent progress in the elucidation of the DNA ligation mechanisms obtained from the tertiary structures solved thus far, in each step of the ligation reaction scheme. We also present some examples of engineered DNA ligases, developed from the viewpoint of their three-dimensional structures. Maiko Tanabe, Yoshizumi Ishino, and Hirokazu Nishida Copyright © 2015 Maiko Tanabe et al. All rights reserved. Untapped Resources: Biotechnological Potential of Peptides and Secondary Metabolites in Archaea Sun, 04 Oct 2015 09:22:45 +0000 Archaea are an understudied domain of life often found in “extreme” environments in terms of temperature, salinity, and a range of other factors. Archaeal proteins, such as a wide range of enzymes, have adapted to function under these extreme conditions, providing biotechnology with interesting activities to exploit. In addition to producing structural and enzymatic proteins, archaea also produce a range of small peptide molecules (such as archaeocins) and other novel secondary metabolites such as those putatively involved in cell communication (acyl homoserine lactones), which can be exploited for biotechnological purposes. Due to the wide array of metabolites produced there is a great deal of biotechnological potential from antimicrobials such as diketopiperazines and archaeocins, as well as roles in the cosmetics and food industry. In this review we will discuss the diversity of small molecules, both peptide and nonpeptide, produced by archaea and their potential biotechnological applications. James C. Charlesworth and Brendan P. Burns Copyright © 2015 James C. Charlesworth and Brendan P. Burns. All rights reserved. Archaeal Nucleic Acid Ligases and Their Potential in Biotechnology Thu, 01 Oct 2015 08:29:06 +0000 With their ability to catalyse the formation of phosphodiester linkages, DNA ligases and RNA ligases are essential tools for many protocols in molecular biology and biotechnology. Currently, the nucleic acid ligases from bacteriophage T4 are used extensively in these protocols. In this review, we argue that the nucleic acid ligases from Archaea represent a largely untapped pool of enzymes with diverse and potentially favourable properties for new and emerging biotechnological applications. We summarise the current state of knowledge on archaeal DNA and RNA ligases, which makes apparent the relative scarcity of information on in vitro activities that are of most relevance to biotechnologists (such as the ability to join blunt- or cohesive-ended, double-stranded DNA fragments). We highlight the existing biotechnological applications of archaeal DNA ligases and RNA ligases. Finally, we draw attention to recent experiments in which protein engineering was used to modify the activities of the DNA ligase from Pyrococcus furiosus and the RNA ligase from Methanothermobacter thermautotrophicus, thus demonstrating the potential for further work in this area. Cecilia R. Chambers and Wayne M. Patrick Copyright © 2015 Cecilia R. Chambers and Wayne M. Patrick. All rights reserved. Archaeal Enzymes and Applications in Industrial Biocatalysts Thu, 01 Oct 2015 06:26:36 +0000 Archaeal enzymes are playing an important role in industrial biotechnology. Many representatives of organisms living in “extreme” conditions, the so-called Extremophiles, belong to the archaeal kingdom of life. This paper will review studies carried by the Exeter group and others regarding archaeal enzymes that have important applications in commercial biocatalysis. Some of these biocatalysts are already being used in large scale industrial processes for the production of optically pure drug intermediates and amino acids and their analogues. Other enzymes have been characterised at laboratory scale regarding their substrate specificity and properties for potential industrial application. The increasing availability of DNA sequences from new archaeal species and metagenomes will provide a continuing resource to identify new enzymes of commercial interest using both bioinformatics and screening approaches. Jennifer A. Littlechild Copyright © 2015 Jennifer A. Littlechild. All rights reserved. Biochemical Characterisation of Phage Pseudomurein Endoisopeptidases PeiW and PeiP Using Synthetic Peptides Mon, 21 Sep 2015 06:41:41 +0000 Pseudomurein endoisopeptidases cause lysis of the cell walls of methanogens by cleaving the isopeptide bond Ala-ε-Lys in the peptide chain of pseudomurein. PeiW and PeiP are two thermostable pseudomurein endoisopeptidases encoded by phage ΨM100 of Methanothermobacter wolfei and phages ΨM1 and ΨM2 of Methanothermobacter marburgensis, respectively. A continuous assay using synthetic peptide substrates was developed and used in the biochemical characterisation of recombinant PeiW and PeiP. The advantages of these synthetic peptide substrates over natural substrates are sensitivity, high purity, and characterisation and the fact that they are more easily obtained than natural substrates. In the presence of a reducing agent, purified PeiW and PeiP each showed similar activity under aerobic and anaerobic conditions. Both enzymes required a divalent metal for activity and showed greater thermostability in the presence of Ca2+. PeiW and PeiP involve a cysteine residue in catalysis and have a monomeric native conformation. The kinetic parameters, and , were determined, and the ε-isopeptide bond between alanine and lysine was confirmed as the bond lysed by these enzymes in pseudomurein. The new assay may have wider applications for the general study of peptidases and the identification of specific methanogens susceptible to lysis by specific pseudomurein endoisopeptidases. Linley R. Schofield, Amy K. Beattie, Catherine M. Tootill, Debjit Dey, and Ron S. Ronimus Copyright © 2015 Linley R. Schofield et al. All rights reserved. Ecology and Distribution of Thaumarchaea in the Deep Hypolimnion of Lake Maggiore Tue, 25 Aug 2015 11:19:08 +0000 Ammonia-oxidizing Archaea (AOA) play an important role in the oxidation of ammonia in terrestrial, marine, and geothermal habitats, as confirmed by a number of studies specifically focused on those environments. Much less is known about the ecological role of AOA in freshwaters. In order to reach a high resolution at the Thaumarchaea community level, the probe MGI-535 was specifically designed for this study and applied to fluorescence in situ hybridization and catalyzed reporter deposition (CARD-FISH) analysis. We then applied it to a fine analysis of diversity and relative abundance of AOA in the deepest layers of the oligotrophic Lake Maggiore, confirming previous published results of AOA presence, but showing differences in abundance and distribution within the water column without significant seasonal trends with respect to Bacteria. Furthermore, phylogenetic analysis of AOA clone libraries from deep lake water and from a lake tributary, River Maggia, suggested the riverine origin of AOA of the deep hypolimnion of the lake. Manuela Coci, Nina Odermatt, Michaela M. Salcher, Jakob Pernthaler, and Gianluca Corno Copyright © 2015 Manuela Coci et al. All rights reserved. Assessment of the Carbon Monoxide Metabolism of the Hyperthermophilic Sulfate-Reducing Archaeon Archaeoglobus fulgidus VC-16 by Comparative Transcriptome Analyses Thu, 06 Aug 2015 16:23:54 +0000 The hyperthermophilic, sulfate-reducing archaeon, Archaeoglobus fulgidus, utilizes CO as an energy source and it is resistant to the toxic effects of high CO concentrations. Herein, transcription profiles were obtained from A. fulgidus during growth with CO and sulfate or thiosulfate, or without an electron acceptor. This provided a basis for a model of the CO metabolism of A. fulgidus. The model suggests proton translocation by “Mitchell-type” loops facilitated by Fqo catalyzing a :menaquinone oxidoreductase reaction, as the major mode of energy conservation, rather than formate or H2 cycling during respiratory growth. The bifunctional CODH (cdhAB-2) is predicted to play an ubiquitous role in the metabolism of CO, and a novel nitrate reductase-associated respiratory complex was induced specifically in the presence of sulfate. A potential role of this complex in relation to and APS reduction is discussed. Multiple membrane-bound heterodisulfide reductase (DsrMK) could promote both energy-conserving and non-energy-conserving menaquinol oxidation. Finally, the FqoF subunit may catalyze a :F420 oxidoreductase reaction. In the absence of electron acceptor, downregulation of F420H2 dependent steps of the acetyl-CoA pathway is linked to transient formate generation. Overall, carboxidotrophic growth seems as an intrinsic capacity of A. fulgidus with little need for novel resistance or respiratory complexes. William P. Hocking, Irene Roalkvam, Carina Magnussen, Runar Stokke, and Ida H. Steen Copyright © 2015 William P. Hocking et al. All rights reserved. Diversity and Distribution of Archaea in the Mangrove Sediment of Sundarbans Thu, 06 Aug 2015 06:57:28 +0000 Mangroves are among the most diverse and productive coastal ecosystems in the tropical and subtropical regions. Environmental conditions particular to this biome make mangroves hotspots for microbial diversity, and the resident microbial communities play essential roles in maintenance of the ecosystem. Recently, there has been increasing interest to understand the composition and contribution of microorganisms in mangroves. In the present study, we have analyzed the diversity and distribution of archaea in the tropical mangrove sediments of Sundarbans using 16S rRNA gene amplicon sequencing. The extraction of DNA from sediment samples and the direct application of 16S rRNA gene amplicon sequencing resulted in approximately 142 Mb of data from three distinct mangrove areas (Godkhali, Bonnie camp, and Dhulibhashani). The taxonomic analysis revealed the dominance of phyla Euryarchaeota and Thaumarchaeota (Marine Group I) within our dataset. The distribution of different archaeal taxa and respective statistical analysis (SIMPER, NMDS) revealed a clear community shift along the sampling stations. The sampling stations (Godkhali and Bonnie camp) with history of higher hydrocarbon/oil pollution showed different archaeal community pattern (dominated by haloarchaea) compared to station (Dhulibhashani) with nearly pristine environment (dominated by methanogens). It is indicated that sediment archaeal community patterns were influenced by environmental conditions. Anish Bhattacharyya, Niladri Shekhar Majumder, Pijush Basak, Shayantan Mukherji, Debojyoti Roy, Sudip Nag, Anwesha Haldar, Dhrubajyoti Chattopadhyay, Suparna Mitra, Maitree Bhattacharyya, and Abhrajyoti Ghosh Copyright © 2015 Anish Bhattacharyya et al. All rights reserved. Understanding DNA Repair in Hyperthermophilic Archaea: Persistent Gaps and Other Reasons to Focus on the Fork Thu, 04 Jun 2015 09:27:59 +0000 Although hyperthermophilic archaea arguably have a great need for efficient DNA repair, they lack members of several DNA repair protein families broadly conserved among bacteria and eukaryotes. Conversely, the putative DNA repair genes that do occur in these archaea often do not generate the expected phenotype when deleted. The prospect that hyperthermophilic archaea have some unique strategies for coping with DNA damage and replication errors has intellectual and technological appeal, but resolving this question will require alternative coping mechanisms to be proposed and tested experimentally. This review evaluates a combination of four enigmatic properties that distinguishes the hyperthermophilic archaea from all other organisms: DNA polymerase stalling at dU, apparent lack of conventional NER, lack of MutSL homologs, and apparent essentiality of homologous recombination proteins. Hypothetical damage-coping strategies that could explain this set of properties may provide new starting points for efforts to define how archaea differ from conventional models of DNA repair and replication fidelity. Dennis W. Grogan Copyright © 2015 Dennis W. Grogan. All rights reserved. Isolation and Molecular Identification of Auxotrophic Mutants to Develop a Genetic Manipulation System for the Haloarchaeon Natrinema sp. J7-2 Thu, 21 May 2015 09:49:30 +0000 Our understanding of the genus Natrinema is presently limited due to the lack of available genetic tools. Auxotrophic markers have been widely used to construct genetic systems in bacteria and eukaryotes and in some archaeal species. Here, we isolated four auxotrophic mutants of Natrinema sp. J7-2, via 1-methyl-3-nitro-1-nitroso-guanidin mutagenesis, and designated them as J7-2-1, J7-2-22, J7-2-26, and J7-2-52, respectively. The mutant phenotypes were determined to be auxotrophic for leucine (J7-2-1), arginine (J7-2-22 and J7-2-52), and lysine (J7-2-26). The complete genome and the biosynthetic pathways of amino acids in J7-2 identified that the auxotrophic phenotype of three mutants was due to gene mutations in leuB (J7-2-1), dapD (J7-2-26), and argC (J7-2-52). These auxotrophic phenotypes were employed as selectable makers to establish a transformation method. The transformation efficiencies were determined to be approximately 103 transformants per g DNA. And strains J7-2-1 and J7-2-26 were transformed into prototrophic strains with the wild type genomic DNA, amplified fragments of the corresponding genes, or the integrative plasmids carrying the corresponding genes. Additionally, exogenous genes, bgaH or amyH gene, were expressed successfully in J7-2-1. Thus, we have developed a genetic manipulation system for the Natrinema genus based on the isolated auxotrophic mutants of Natrinema sp. J7-2. Jie Lv, Shuai Wang, Yuchen Wang, Yuping Huang, and Xiangdong Chen Copyright © 2015 Jie Lv et al. All rights reserved. Archaeal Communities in a Heterogeneous Hypersaline-Alkaline Soil Wed, 13 May 2015 06:44:39 +0000 In this study the archaeal communities in extreme saline-alkaline soils of the former lake Texcoco, Mexico, with electrolytic conductivities (EC) ranging from 0.7 to 157.2 dS/m and pH from 8.5 to 10.5 were explored. Archaeal communities in the 0.7 dS/m pH 8.5 soil had the lowest alpha diversity values and were dominated by a limited number of phylotypes belonging to the mesophilic Candidatus Nitrososphaera. Diversity and species richness were higher in the soils with EC between 9.0 and 157.2 dS/m. The majority of OTUs detected in the hypersaline soil were members of the Halobacteriaceae family. Novel phylogenetic branches in the Halobacteriales class were detected in the soil, and more abundantly in soil with the higher pH (10.5), indicating that unknown and uncharacterized Archaea can be found in this soil. Thirteen different genera of the Halobacteriaceae family were identified and were distributed differently between the soils. Halobiforma, Halostagnicola, Haloterrigena, and Natronomonas were found in all soil samples. Methanogenic archaea were found only in soil with pH between 10.0 and 10.3. Retrieved methanogenic archaea belonged to the Methanosarcinales and Methanomicrobiales orders. The comparison of the archaeal community structures considering phylogenetic information (UniFrac distances) clearly clustered the communities by pH. Yendi E. Navarro-Noya, César Valenzuela-Encinas, Alonso Sandoval-Yuriar, Norma G. Jiménez-Bueno, Rodolfo Marsch, and Luc Dendooven Copyright © 2015 Yendi E. Navarro-Noya et al. 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.