Methanogenic Archaea and Consortia Kevin R. Sowers University of Maryland Biotechnology Institute Extremophile Research: Theory & Techniques 2003 Archaea Crenarchaeota Euryarchaeota Halophiles Methanobacterium Methanobrevibacter Methanoplanus Methanothermus Methanospirillum Methanosarcina Hyperthermophiles Methanolobus Methanohalophilus Methanosaeta Methanococcus Methanopyrus Bacteria Eukarya Alessandro Volta Volta Experiment Historical Overview •• 1776 1776 VoltaVolta described described “combustible“combustible air”air” •• 1806 1806 HenryHenry lamp lamp gasgas == methanemethane •• 1869 1869 Becamp Becamp methanogenesis methanogenesis biologicalbiological •• 1936 1936 BarkerBarker “isolated” “isolated” M.M. omelianskii omelianskii •• 1947 1947 Kluyver Kluyver first first truetrue isolatesisolates •• 1950 1950 Hungate Hungate anaerobic anaerobic techniquetechnique •• 1977 1977 Woese Woese described described “archaebacteria”“archaebacteria” Phylogeny of Methanogens Methanococcales Methanobacteriales Methanosarcinales Methanomicrobiales Methanopyrales Boone et al., 1993 Substrates Used by Orders of Methanogenic Archaea •• Methanobacteriales Methanobacteriales H H22,HCOOH,H,HCOOH,H22/Me,/Me, 2P,2B,CP2P,2B,CP •• Methanococcales Methanococcales H H22,HCOOH,HCOOH •• Methanomicrobiales Methanomicrobiales HH22,HCOOH,E,P,2P,2B,CP,HCOOH,E,P,2P,2B,CP •• Methanopyrales Methanopyrales H H22 •• Methanosarcinales Methanosarcinales H H22,CH,CH33COOH,CH33OH,MMA, DMA,TMA,DMS Characteristics •• psychrophiles psychrophiles (-2.5 (-2.5 OC) - thermophiles (110 OC) •• acidophiles acidophiles (pH (pH 5)5) -- alkaliphiles alkaliphiles (pH (pH 10.3)10.3) •• non-halophiles non-halophiles (<0.1 (<0.1 M)M) -- halophiles halophiles (5.1 (5.1 M)M) •• autotrophs autotrophs (H (H22 -CO-CO22 )) -- methylotrophs methylotrophs (TMA, (TMA, DMS)DMS) REDUCTANT O2 Methanococcus jannaschii Morphology coccus Diameter 1.0 µm Motility Yes pH optimum/range 6.0/5.2-7.6 Temp optimum 85 C/48-94 C Substrates H22 /formate/formate Growth factors None NaCl optimum/range 0.4M/0.1-0.85M Mol % G+C 31 Methanococcoides burtonii Morphology coccus Diameter 1.2 µm Motility Yes pH optimum/range 7.7/6.8-8.2 Temp optimum 23.4 C/-2.5-30 C Substrates MeOH/methylamines Growth factors None NaCl optimum/range 0.2M/0.2-0.5M Mol % G+C 39.6 Morphological Diversity Sprott and Beveridge, 1993 Cell Envelopes •• Methanobacteriaceae pseudomureinpseudomurein •• Methanothermaceae pseudomurein/pseudomurein/glycoproteinglycoprotein •• Methanosarcina methanochondroitin/methanochondroitin/glycoproteinglycoprotein •• Methanococcales proteinprotein •• Methanomicrobium mobile proteinprotein •• Methanogenium marisnigri proteinprotein •• Methanogenium marisnigri glycoproteinglycoprotein •• Methanoplanus limicola glycoproteinglycoprotein •• Methanosaeta concilii glycoprotein/sheathglycoprotein/sheath •• Methanosprillum hungatei glycoprotein/sheathglycoprotein/sheath Pseudomurein Kandler, 1982 Thin-section EM micrograph of Methanobacterium thermoautotrophicum Sprott and Beveridge, 1993 Methanochondroitin Kreisl and Kandler, 1986 Thin-section EM Micrograph of Methanosarcina thermophila Freeze-etched EM Micrograph of Methanocorpusculum sinense Sprott and Beveridge, 1993 Methanogenic Environments Wolfe, 1996 Anaerobic Digestor / Freshwater Sediments COMPLEX POLYMERS (proteins, polysaccharides, etc.) GROUP I MONOMERS/POLYMERS FERMENTATIVE (sugars, amino acids, peptides) BACTERIA FATTY ACIDS PRIMARY ALCOHOLS AROMATICS GROUP II H2-PRODUCING/ CO2 -REDUCING ACETOGENIC BACTERIA HYDROGEN ACETATE FORMATE, CO2 GROUP III METHANOGENIC CH , CO ARCHAEA 4 2 Net Effect of Interspecies Hydrogen Exchange Zinder, 1993 Microstructural Analysis of UASB Granule Fang et al., 1994, 1995 Marine Sediments COMPLEX POLYMERS (proteins, polysaccharides, etc.) GROUP I MONOMERS/POLYMERS METHYLAMINO FERMENTATIVE (sugars, amino acids, peptides) METHYLTHIO BACTERIA OSMOLYTES FATTY ACIDS PRIMARY ALCOHOLS METHYLAMINES AROMATICS METHYL SULFIDES GROUP II H2-PRODUCING ACETOGENIC BACTERIA HYDROGEN ACETATE SULFIDOGENIC BACTERIA CO2 GROUP III METHANOGENIC ARCHAEA H S, CH , CH , CO SULFIDOGENIC BACTERIA 2 4 4 2 CO2 Animal Rumen COMPLEX POLYMERS (proteins, polysaccharides, etc.) MONOMERS/POLYMERS (sugars, amino acids, peptides) GROUP I FERMENTATIVE BACTERIA FATTY ACIDS PRIMARY ALCOHOLS AROMATICS RUMINANT HYDROGEN ACETATE CO2 GROUP III METHANOGENIC ARCHAEA CH4, CO2 Hydrothermal Vents HYDROGEN CO2 GROUP III METHANOGENIC ARCHAEA CH4, CO2 Deep Subsurface Microbial Processes Kaiser, 1995 Methods - Hungate Technique Methods - Anaerobic Glove Box Chamber Air lock Methods - Anaerobic Plating Jar A BC Methods - Anaerobic Scale-Up Genomic Analysis Archaea Bacterial characteristics Eukaryal characteristics ••Small molecule biosynthesis ••DNA metabolism ••IntermediaryIntermediary metabolismmetabolism ••Transcription ••Transport ••Translation ••Nitrogen fixation ••Regulatory functions Transcription Proteins Genotypic Characteristics Undetected inin PhenotypePhenotype •• multiple multiple methyltransferasesmethyltransferases •• flagellin/chemotaxis flagellin/chemotaxis gene gene clustersclusters •• cytochrome cytochrome d oxidase •• 3 3 nitrogenasesnitrogenases (Mo/Fe,V/Fe,Fe)(Mo/Fe,V/Fe,Fe) •• unique unique 22 componentcomponent regreg systemsystem Gene Transfer Systems Archaea Crenarchaeota Euryarchaeota Halobacterium Haloferax Transformation Halorubrum Halococcus Haloarcula Natronococcus Transduction Methanobacterium Natronobacterium Methanobrevibacter Archaeoglobus Conjugation Methanoplanus Thermoproteus Methanothermus Methanospirillum Pyrobaculum Methanosarcina Thermococcus Thermofilum Pyrococcus Methanolobus Sulfolobus Methanohalophilus Methanosaeta Methanococcus Desulfurococcus Methanopyrus Pyrodictium Bacteria Eukarya Sowers and Schreier, 1999 Viruses & Virus-like Particles •• ψΜ1 M. thermoautotrophicum lytic •• ΦF3 M. thermoautotrophicum lytic •• ΦF3 M. thermoautotrophicum lytic •PG•PG M. smithii lytic •VLP•VLP M. voltae temperate Restriction-Modification Systems •• MaeI,I, II,II, IIIIII Methanococcus. aeolicus endonucleaseendonuclease •• MthF1, T1, Z1 Methanobacterium endonuclease/endonuclease/ thermoautotropicumthermoautotropicum methylase •• MvnII Methanococcus vanielii endonucleaseendonuclease •• MwoII Methanobacterium wolfei endonuclease/endonuclease/ methylase •• PstII Methanococcus maripaludis endonucleaseendonuclease Plasmid DNA •• pT3pT3 Methanobacterium sp.sp. 7.3 7.3 kbkb Unknown Unknown functionfunction •• pME2001pME2001 Methanobacterium 4.44.4 kbkb UnknownUnknown functionfunction thermoautotropicumthermoautotropicum •• pFV1,2,3pFV1,2,3 Methanobacterium 13.5,11,1113.5,11,11 kbkb Restriction/ Restriction/ thermoautotropicumthermoautotropicum methylation •• pURB500pURB500 Methanococcus sp 8.78.7 kbkb Unknown Unknown functionfunction •• pURB900pURB900 Methanococcus sp.sp. AG86AG86 20 20 kbkb Unknown Unknown functionfunction •• pURB800pURB800 Methanococcus jannaschii 6464 kbkb UnknownUnknown functionfunction •• pURB801pURB801 Methanococcus jannaschii 1818 kbkb Unknown Unknown functionfunction •• pC2ApC2A Methanosarcina acetivorans 5.45.4 kbkb Recombinase/ Recombinase/ replicationreplication initiationinitiation •• pMP1pMP1 Methanolobus vulcanii 6.96.9 kbkb Unknown Unknown functionfunction M. Acetivorans Native Plasmid orf2 0 orf1 ssrA pC2A 5467bp repArepA Metcalfe etet al,al, 19971997 Selection Cassette Hybrid Shuttle Vector EcoRV (183) BamHI (187) XhoI (265) Ecl136II (233) EcoICRI (233) SstI (235) SacI (235) bla Asp718I (240) trpA terminator lacZ Acc65I (240) oriRK6 KpnI (244) SphI (253) mcr terminator ClaI (256) PWM315 pac 8745bp mcr promoter pC2A Metcalf etet al.,al., 19971997 Shuttle Vector Hybrid Constructs •• pJKpJK series series Integration Integration vectorvector Methanosarcina spp.spp. •• pWMpWM series series Autonomous Autonomous vectorvector Methanosarcina spp.spp. •• pWAY1/2pWAY1/2 Integration Integration vectorvector Methanosarcina mazei •• pMIP1pMIP1 Integration Integration vectorvector Methanococcus voltae •• pKASpKAS Integration Integration vectorvector Methanococcus maripaludis •• pDLT44pDLT44 Autonomous Autonomous vectorvector Methanococcus maripaludis Transformation Protocols •• Natural Natural transformationtransformation (M. (M. voltae) voltae) << 77 xx 101022 //µgg •• Electroporation Electroporation (M.(M. voltae) voltae) << 77 xx 101022 //µgg •• PEG PEG (M.(M. maripaludis) maripaludis) < < 22 xx 101077 //µgg •• Liposome Liposome (Methanosarcina (Methanosarcina spp.)spp.) < < 22 xx 101099 //µgg Gene Disruption •Directed• Directed:: IntegrationIntegration genegene disruptiondisruption •• Random Random:: EthylmethanesulfonateEthylmethanesulfonate andand Transposon mutagenesis Directed Gene Disruption bla pMIP HisA’ pac casette HisA’ HisA Methanococcus voltae Gernhardt etet al.,al., 19901990 Random Gene Disruption Zhang et al., 2000 Reporter Vector for Methanosarcina acetivorans bla oriR6K mcrT 1 lacZ pac mcrP pKJ104 12994bp cdhP C2A Sowers et al. Current Capabilities •• Genomes Genomes sequencedsequenced •• Integration/autonomous Integration/autonomous plasmidplasmid vectorsvectors •• Transposon Transposon mutagenesismutagenesis •• Reporter Reporter plasmidsplasmids Current Limitations •• Limited Limited geneticgenetic selectionselection markersmarkers •• DNA DNA restriction/modificationrestriction/modification systemssystems •• DNA DNA repair/recombinationrepair/recombination mutantsmutants Technological Applications •• Human/animal Human/animal wastewaste treatmenttreatment •• Energy Energy productionproduction •• Bioremediation Bioremediation •• Thermostable Thermostable enzymes enzymes •• Thermostable Thermostable lipidslipids •• Synthesis Synthesis ofof otherother uniqueunique compoundscompounds.