Hydrogenases of Methanogens
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A Web Tool for Hydrogenase Classification and Analysis Dan Søndergaard1, Christian N
www.nature.com/scientificreports OPEN HydDB: A web tool for hydrogenase classification and analysis Dan Søndergaard1, Christian N. S. Pedersen1 & Chris Greening2,3 H2 metabolism is proposed to be the most ancient and diverse mechanism of energy-conservation. The Received: 24 June 2016 metalloenzymes mediating this metabolism, hydrogenases, are encoded by over 60 microbial phyla Accepted: 09 September 2016 and are present in all major ecosystems. We developed a classification system and web tool, HydDB, Published: 27 September 2016 for the structural and functional analysis of these enzymes. We show that hydrogenase function can be predicted by primary sequence alone using an expanded classification scheme (comprising 29 [NiFe], 8 [FeFe], and 1 [Fe] hydrogenase classes) that defines 11 new classes with distinct biological functions. Using this scheme, we built a web tool that rapidly and reliably classifies hydrogenase primary sequences using a combination of k-nearest neighbors’ algorithms and CDD referencing. Demonstrating its capacity, the tool reliably predicted hydrogenase content and function in 12 newly-sequenced bacteria, archaea, and eukaryotes. HydDB provides the capacity to browse the amino acid sequences of 3248 annotated hydrogenase catalytic subunits and also contains a detailed repository of physiological, biochemical, and structural information about the 38 hydrogenase classes defined here. The database and classifier are freely and publicly available at http://services.birc.au.dk/hyddb/ Microorganisms conserve energy by metabolizing H2. Oxidation of this high-energy fuel yields electrons that can be used for respiration and carbon-fixation. This diffusible gas is also produced in diverse fermentation and 1 anaerobic respiratory processes . H2 metabolism contributes to the growth and survival of microorganisms across the three domains of life, including chemotrophs and phototrophs, lithotrophs and heterotrophs, aerobes and 1,2 anaerobes, mesophiles and extremophiles alike . -
Direct Charging of Trnacua with Pyrrolysine in Vitro and in Vivo
letters to nature .............................................................. gene product (see Supplementary Fig. S1). The tRNA pool extracted from Methanosarcina acetivorans or tRNACUA transcribed in vitro Direct charging of tRNACUA with was used in charging experiments. Charged and uncharged tRNA species were separated by electrophoresis in a denaturing acid-urea pyrrolysine in vitro and in vivo 10,11 polyacrylamide gel and tRNACUA was specifically detected by northern blotting with an oligonucleotide probe. The oligonucleo- Sherry K. Blight1*, Ross C. Larue1*, Anirban Mahapatra1*, tide complementary to tRNA could hybridize to a tRNA in the David G. Longstaff1, Edward Chang1, Gang Zhao2†, Patrick T. Kang4, CUA Kari B. Green-Church5, Michael K. Chan2,3,4 & Joseph A. Krzycki1,4 pool of tRNAs isolated from wild-type M. acetivorans but not to the tRNA pool from a pylT deletion mutant of M. acetivorans (A.M., 1Department of Microbiology, 484 West 12th Avenue, 2Department of Chemistry, A. Patel, J. Soares, R.L. and J.A.K., unpublished observations). 3 100 West 18th Avenue, Department of Biochemistry, 484 West 12th Avenue, Both tRNACUA and aminoacyl-tRNACUA were detectable in the The Ohio State University, Columbus, Ohio 43210, USA isolated cellular tRNA pool (Fig. 1). Alkaline hydrolysis deacylated 4Ohio State University Biochemistry Program, 484 West 12th Avenue, The Ohio the cellular charged species, but subsequent incubation with pyrro- State University, Columbus, Ohio 43210, USA lysine, ATP and PylS-His6 resulted in maximal conversion of 50% of 5CCIC/Mass Spectrometry and Proteomics Facility, The Ohio State University, deacylated tRNACUA to a species that migrated with the same 116 W 19th Ave, Columbus, Ohio 43210, USA electrophoretic mobility as the aminoacyl-tRNACUA present in the * These authors contributed equally to this work. -
Annotation Guidelines for Experimental Procedures
Annotation Guidelines for Experimental Procedures Developed By Mohammed Alliheedi Robert Mercer Version 1 April 14th, 2018 1- Introduction and background information What is rhetorical move? A rhetorical move can be defined as a text fragment that conveys a distinct communicative goal, in other words, a sentence that implies an author’s specific purpose to readers. What are the types of rhetorical moves? There are several types of rhetorical moves. However, we are interested in 4 rhetorical moves that are common in the method section of a scientific article that follows the Introduction Methods Results and Discussion (IMRaD) structure. 1- Description of a method: It is concerned with a sentence(s) that describes experimental events (e.g., “Beads with bound proteins were washed six times (for 10 min under rotation at 4°C) with pulldown buffer and proteins harvested in SDS-sample buffer, separated by SDS-PAGE, and analyzed by autoradiography.” (Ester & Uetz, 2008)). 2- Appeal to authority: It is concerned with a sentence(s) that discusses the use of standard methods, protocols, and procedures. There are two types of this move: - A reference to a well-established “standard” method (e.g., the use of a method like “PCR” or “electrophoresis”). - A reference to a method that was previously described in the literature (e.g., “Protein was determined using fluorescamine assay [41].” (Larsen, Frandesn and Treiman, 2001)). 3- Source of materials: It is concerned with a sentence(s) that lists the source of biological materials that are used in the experiment (e.g., “All microalgal strains used in this study are available at the Elizabeth Aidar Microalgae Culture Collection, Department of Marine Biology, Federal Fluminense University, Brazil.” (Larsen, Frandesn and Treiman, 2001)). -
Supplemental Methods
Supplemental Methods: Sample Collection Duplicate surface samples were collected from the Amazon River plume aboard the R/V Knorr in June 2010 (4 52.71’N, 51 21.59’W) during a period of high river discharge. The collection site (Station 10, 4° 52.71’N, 51° 21.59’W; S = 21.0; T = 29.6°C), located ~ 500 Km to the north of the Amazon River mouth, was characterized by the presence of coastal diatoms in the top 8 m of the water column. Sampling was conducted between 0700 and 0900 local time by gently impeller pumping (modified Rule 1800 submersible sump pump) surface water through 10 m of tygon tubing (3 cm) to the ship's deck where it then flowed through a 156 µm mesh into 20 L carboys. In the lab, cells were partitioned into two size fractions by sequential filtration (using a Masterflex peristaltic pump) of the pre-filtered seawater through a 2.0 µm pore-size, 142 mm diameter polycarbonate (PCTE) membrane filter (Sterlitech Corporation, Kent, CWA) and a 0.22 µm pore-size, 142 mm diameter Supor membrane filter (Pall, Port Washington, NY). Metagenomic and non-selective metatranscriptomic analyses were conducted on both pore-size filters; poly(A)-selected (eukaryote-dominated) metatranscriptomic analyses were conducted only on the larger pore-size filter (2.0 µm pore-size). All filters were immediately submerged in RNAlater (Applied Biosystems, Austin, TX) in sterile 50 mL conical tubes, incubated at room temperature overnight and then stored at -80oC until extraction. Filtration and stabilization of each sample was completed within 30 min of water collection. -
Sequencing, Assembly, and Annotation of the Kaistella Koreensis Genome and Comparison to Closely Related Organisms
Sequencing, Assembly, and Annotation of the Kaistella koreensis Genome and Comparison to Closely Related Organisms Presented to the faculty of Lycoming College in partial fulfillment of the requirements for Departmental Honors in Biology by Timothy Hostelley Lycoming College April 22, 2013 Approved by: (Signature) (Signature) (Signature) (Signature) Abstract Advances in DNA sequencing technology have made DNA sequencing cheaper and more efficient. As a result, there has been an enormous increase in the number of genomes being sequenced. The sequence data can be assembled into complete genomes and annotated in order to reveal information about the organism’s physiology. In this study the DNA of the bacterium Kaistella koreensis was sequenced and assembled into 578 contigs, these contigs were then uploaded to Rapid Annotation Using Subsystems Technology for annotation in order to compute the Average Nucleotide Identity between K. koreensis and closely related organisms in order to dispute the reclassification of K. koreensis as Chryseobacterium koreense. Phenotypic tests including Biolog GenII, API ZYM, and Fatty Acid Methyl Ester analysis were also done in order to supplement the ambiguous results of the ANI. The results of these tests reveal a number of significant differences between K. koreensis and its closest related neighbors that suggests that K. koreensis does not belong in the Chryseobacterium genus or the closely related Lycomia genus. Instead, K. koreensis should be reclassified back to its original classification in the Kaistella genus. This would dispute the proposal made by Kämpfer et al. to reclassify Kaistella koreensis into the Chryseobacterium genus. 1 Introduction DNA sequencing has rapidly evolved from the earliest sequencing efforts using only whole genome shotgun-cloning based sequencing of the 1990’s, to the further advances in Sanger sequencing in the early 2000’s. -
Characterization of Methanosarcina Barkeri MST and 227, Methanosarcina Mazei S-6T, and Methanosarcina Vacuolata Z-76IT GLORIA M
INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Apr. 1991, p. 267-274 Vol. 41, No. 2 0020-7713/91/020267-08$02.OO/O Copyright 0 1991, International Union of Microbiological Societies Characterization of Methanosarcina barkeri MST and 227, Methanosarcina mazei S-6T, and Methanosarcina vacuolata Z-76IT GLORIA M. MAESTROJUAN' AND DAVID R. BOONE172* Departments of Environmental Science and Engineering' and Chemical and Biological Science,2 Oregon Graduate Institute, 19600 N.W. von Neumann Drive, Beaverton, Oregon 97006-1999 Members of the genus Methanosarcina are recognized as major aceticlastic methanogens, and several species which thrive in low-salt, pH-neutral culture medium at mesophilic temperatures have been described. However, the environmental conditions which support the fastest growth of these species (Methanosarcina barkeri MST [T = type strain] and 227, Methanosarcina mazei S-6T, and Methanosarcina vacuolata Z-761T) have not been reported previously. Although the members of the genus Methanosarcina are widely assumed to grow best at pH values near neutrality, we found that some strains prefer acidic pH values. M. vacuolata and the two strains of M. barkeri which we tested were acidophilic when they were grown on H, plus methanol, growing most rapidly at pH 5 and growing at pH values as low as 4.3. M. mazei grew best at pH values near neutrality. We found that all of the strains tested grew most rapidly at 37 to 42°C on all of the growth substrates which we tested. None of the strains was strongly halophilic, although the growth of some strains was slightly stimulated by small amounts of added NaCI. -
A Web Tool for Hydrogenase Classification and Analysis
bioRxiv preprint doi: https://doi.org/10.1101/061994; this version posted September 16, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 HydDB: A web tool for hydrogenase 2 classification and analysis 3 Dan Søndergaarda, Christian N. S. Pedersena, Chris Greeningb, c* 4 5 a Aarhus University, Bioinformatics Research Centre, C.F. Møllers Allé 8, Aarhus 6 DK-8000, Denmark 7 b The Commonwealth Scientific and Industrial Research Organisation, Land and 8 Water Flagship, Clunies Ross Street, Acton, ACT 2060, Australia 9 c Monash University, School of Biological Sciences, Clayton, VIC 2800, Australia 10 11 Correspondence: 12 Dr Chris Greening ([email protected]), Monash University, School of 13 Biological Sciences, Clayton, VIC 2800, Australia 14 Dan Søndergaard ([email protected]), Aarhus University, Bioinformatics Research 15 Centre, C.F. Møllers Allé 8, Aarhus DK-8000, Denmark 1 bioRxiv preprint doi: https://doi.org/10.1101/061994; this version posted September 16, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 16 Abstract 17 H2 metabolism is proposed to be the most ancient and diverse mechanism of 18 energy-conservation. The metalloenzymes mediating this metabolism, 19 hydrogenases, are encoded by over 60 microbial phyla and are present in all major 20 ecosystems. -
The Archaeal Concept and the World It Lives In: a Retrospective
Carl R. Woese (center) with His Majesty Carl XVI Gustaf of Sweden and Queen Silvia on the occassion of his receiving the 2003 Crafoord Prize, given by the Royal Swedish Academy of Sciences. Photo credit: Royal Swedish Academy of Sciences. Photosynthesis Research 80: 361–372, 2004. 363 © 2004 Kluwer Academic Publishers. Printed in the Netherlands. Personal perspective The archaeal concept and the world it lives in: a retrospective Carl R. Woese Department of Microbiology, University of Illinois at Urbana-Champaign, B103 Chemical and Life Sciences Laboratory, 601 South Goodwin Ave, Urbana, IL 61801-3709, USA (e-mail: [email protected]; fax: +1-217-244-6697) Received 9 July 2003; accepted in revised form 30 August 2003 Key words: archaea, evolution, genomics, molecular phylogeny, phylogenetic reconstruction, ribosomal RNA Abstract The present retrospective concerns the discovery and development of the archaea, the so-called ‘third form of life’ that no one anticipated and many did not, and still do not want. In its birth pangs, which the archaea had a plenty, the concept encountered biology unmasked; for it ran up against some of the key struts in the 20th century biological edifice. Consequently, the history of the development of the archaeal concept provides an excellent window on certain of the weaknesses in the 20th century biology paradigm, weaknesses that have now led that paradigm to a conceptual dead end. On the other hand, the archaeal concept has also provided us one of the pillars on which a new holistic paradigm for biology can be built. So, it would seem of value to retrace some of the twists and turns in the history of the development of the archaeal concept. -
Characterization of Methanosarcina Mazei JL01 Isolated from Holocene
Proceedings Characterization of Methanosarcina mazei JL01 Isolated from Holocene Arctic Permafrost and Study of the Archaeon Cooperation with Bacterium Sphaerochaeta associata GLS2T † Viktoriia Oshurkova 1,*, Olga Troshina 1, Vladimir Trubitsyn 1, Yana Ryzhmanova 1, Olga Bochkareva 2 and Viktoria Shcherbakova 1 1 Skryabin Institute of Biochemistry and Physiology of Microorganisms, Federal Research Center Pushchino Center for Biological Research of the Russian Academy of Sciences, prospect Nauki 5, Pushchino, 142290 Moscow, Russia; [email protected] (O.T.); [email protected] (V.T.); [email protected] (Y.R.); [email protected] (V.S.) 2 Institute of Science and Technology (IST Austria), Am Campus 1, 3400 Klosterneuburg, Austria; [email protected] * Correspondence: [email protected] † Presented at the 1st International Electronic Conference on Microbiology, 2–30 November 2020; Available online: https://ecm2020.sciforum.net/. Published: 18 December 2020 Abstract: A mesophilic methanogenic culture, designated JL01, was isolated from Holocene permafrost in the Russian Arctic. After long-term extensive cultivation at 15 °C, it turned out to be a tied binary culture of archaeal (JL01) and bacterial (Sphaerochaeta associata GLS2) strains. Strain JL01 was a strict anaerobe and grew on methanol, acetate, and methylamines as energy and carbon sources. Cells were irregular coccoid, non-motile, non-spore-forming, and Gram-stain-positive. Optimum conditions for growth were 24–28 °C, pH 6.8–7.3, and 0.075–0.1 M NaCl. Phylogenetic tree reconstructions based on 16S rRNA and concatenated alignment of broadly conserved protein- coding genes revealed 16S rRNA’s close relation to Methanosarcina mazei S-6T (similarity 99.5%). -
Reducing the Genetic Code Induces Massive Rearrangement of the Proteome
Reducing the genetic code induces massive rearrangement of the proteome Patrick O’Donoghuea,b, Laure Pratc, Martin Kucklickd, Johannes G. Schäferc, Katharina Riedele, Jesse Rinehartf,g, Dieter Söllc,h,1, and Ilka U. Heinemanna,1 Departments of aBiochemistry and bChemistry, The University of Western Ontario, London, ON N6A 5C1, Canada; Departments of cMolecular Biophysics and Biochemistry, fCellular and Molecular Physiology, and hChemistry, and gSystems Biology Institute, Yale University, New Haven, CT 06520; dDepartment of Microbiology, Technical University of Braunschweig, Braunschweig 38106, Germany; and eDivision of Microbial Physiology and Molecular Biology, University of Greifswald, Greifswald 17487, Germany Contributed by Dieter Söll, October 22, 2014 (sent for review September 29, 2014; reviewed by John A. Leigh) Expanding the genetic code is an important aim of synthetic Opening codons by reducing the genetic code is highly biology, but some organisms developed naturally expanded ge- promising, but it is unknown how removing 1 amino acid from netic codes long ago over the course of evolution. Less than 1% of the genetic code might impact the proteome or cellular viability. all sequenced genomes encode an operon that reassigns the stop Many genetic code variations are found in nature (15), including codon UAG to pyrrolysine (Pyl), a genetic code variant that results stop or sense codon reassignments, codon recoding, and natural from the biosynthesis of Pyl-tRNAPyl. To understand the selective code expansion (16). Pyrrolysine (Pyl) is a rare example of nat- advantage of genetically encoding more than 20 amino acids, we ural genetic code expansion. Evidence for genetically encoded constructed a markerless tRNAPyl deletion strain of Methanosarcina Pyl is found in <1% of all sequenced genomes (17). -
[Nife]-Hydrogenase Ingmar Bürstela,B, Elisabeth Siebertb, Stefan Frielingsdorfa,B, Ingo Zebgerb, Bärbel Friedricha, and Oliver Lenza,B,1
CO synthesized from the central one-carbon pool as source for the iron carbonyl in O2-tolerant [NiFe]-hydrogenase Ingmar Bürstela,b, Elisabeth Siebertb, Stefan Frielingsdorfa,b, Ingo Zebgerb, Bärbel Friedricha, and Oliver Lenza,b,1 aDepartment of Biology, Microbiology, Humboldt-Universität zu Berlin, 10115 Berlin, Germany; and bDepartment of Chemistry, Biophysical Chemistry, Technische Universität Berlin, 10623 Berlin, Germany Edited by Harry B. Gray, California Institute of Technology, Pasadena, CA, and approved November 8, 2016 (received for review September 1, 2016) Hydrogenases are nature’s key catalysts involved in both microbial of the HypD and HypC proteins acts as scaffold for the assembly of consumption and production of molecular hydrogen. H2 exhibits a the Fe(CN)2(CO) entity of the active site (7, 8). The HypF and − strongly bonded, almost inert electron pair and requires transition HypE proteins deliver the CN ligands, which are synthesized metals for activation. Consequently, all hydrogenases are metal- from carbamoyl phosphate (9). Incorporation of the nickel is fa- loenzymes that contain at least one iron atom in the catalytic center. cilitated by the HypB and HypA proteins (10). However, source For appropriate interaction with H2, the iron moiety demands for a and synthesis of the active site CO ligand remained elusive. sophisticated coordination environment that cannot be provided Maturation studies on the O2-tolerant, energy-generating just by standard amino acids. This dilemma has been overcome by [NiFe]-hydrogenases in the facultative H2-oxidizing bacterium the introduction of unprecedented chemistry—that is, by ligating Ralstonia eutropha H16 indicate that at least two different meta- the iron with carbon monoxide (CO) and cyanide (or equivalent) bolic sources exist for CO ligand synthesis (11). -
Composition of the Coenzyme F420-Dependent Formate Dehydrogenase from Methanobacterium Formicicum NEIL L
JOURNAL OF BACTERIOLOGY, Feb. 1986, p. 405-411 Vol. 165, No. 2 0021-9193/86/020405-07$02.00/0 Copyright (C 1986, American Society for Microbiology Composition of the Coenzyme F420-Dependent Formate Dehydrogenase from Methanobacterium formicicum NEIL L. SCHAUERt AND JAMES G. FERRY* Department of Anaerobic Microbiology, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061 Received 16 August 1985/Accepted 19 November 1985 The coenzyme F420-dependent formate dehydrogenase from Methanobacterium formicicum was purified to electrophoretic homogeneity by anoxic procedures which included the addition o(f azide, flavin adenine dinucleotide (FAD), glycerol, and 2-mercaptoethanol to all buffer solutions to stabilize iictivity. The enzyme contains, in approximate miolar ratios, 1 FAD molecule and 1 molybdenum, 2 zinc, 21 to 24 iron, and 25 to 29 Downloaded from inorganic sulfur atoms. Denaturation of the enzyme released a molybdopterin cofactor. The enzyme has a molecular weight of 177,000 and consists of one each of two different subunits, giving the composition olol. The molecular weight of the a-subunit is 85,000, and that of the ,-subunit is 53,000. The UV-visible spectrum is typical of nonheme iron-sulfur flavoprotein. Reduction of the enzyme facilitated dissociation of FAD, and the FAD-depleted enzyme was unable to reduce coenzyme F420. Preincubation of the FAD-depleted enzyme with FAD restored coenzyme F420-dependent activity. The methanogenic bacteria are phylogenetically distant Cells were harvested in the late log phase at an optical density from eubacteria and eucaryotes (10). Consistent with this of 3.0 to 4.5 (550 nm, 1-cm light path).