Methanogenic Archaea and Consortia

Home , Methanococcus, Methanogenium, Methanolobus, Methanoplanus, Methanopyrales, Methanopyrus

Kevin R. Sowers

University of Maryland Biotechnology Institute

Extremophile Research: Theory & Techniques 2003

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 H H22,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

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 Unknown Unknown 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 Unknown Unknown 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