Methanogenic Archaeaand Consortia

Methanogenic Archaeaand Consortia

Kevin R. Sowers

University of Maryland Biotechnology Institute

Kevin R. Sowers

University of Maryland Biotechnology InstituteExtremophile Research: Theory & Techniques 2003Extremophile Research: Theory & Techniques 2003

Hyperthermophiles

Methanopyrus

Methanococcus

Methanothermus

Methanobacterium

Methanosarcina

MethanoplanusMethanospirillum

Crenarchaeota Euryarchaeota

Archaea

MethanolobusMethanohalophilus

Methanosaeta

Methanobrevibacter

Halophiles

EukaryaBacteria

Alessandro VoltaAlessandro Volta

Volta ExperimentVolta Experiment

Historical OverviewHistorical Overview

• 1776 Volta described “combustible air”• 1806 Henry lamp gas = methane• 1869 Becamp methanogenesis biological• 1936 Barker “isolated” M. omelianskii • 1947 Kluyver first true isolates• 1950 Hungate anaerobic technique• 1977 Woese described “archaebacteria”

• 1776 Volta described “combustible air”• 1806 Henry lamp gas = methane• 1869 Becamp methanogenesis biological• 1936 Barker “isolated” M. omelianskii • 1947 Kluyver first true isolates• 1950 Hungate anaerobic technique• 1977 Woese described “archaebacteria”

Phylogeny of MethanogensPhylogeny of MethanogensMethanococcalesMethanococcales

MethanobacterialesMethanobacteriales

MethanosarcinalesMethanosarcinales

MethanopyralesMethanopyrales

Boone et al., 1993

MethanomicrobialesMethanomicrobiales

Substrates Used by Orders ofMethanogenic Archaea

Substrates Used by Orders ofMethanogenic Archaea

• Methanobacteriales H2,HCOOH,H2/Me, 2P,2B,CP• Methanococcales H2,HCOOH• Methanomicrobiales H2,HCOOH,E,P,2P,2B,CP• Methanopyrales H2

• Methanosarcinales H2,CH3COOH,CH3OH,MMA, DMA,TMA,DMS

• Methanobacteriales H2,HCOOH,H2/Me, 2P,2B,CP• Methanococcales H2,HCOOH• Methanomicrobiales H2,HCOOH,E,P,2P,2B,CP• Methanopyrales H2

• Methanosarcinales H2,CH3COOH,CH3OH,MMA, DMA,TMA,DMS

CharacteristicsCharacteristics

• psychrophiles (-2.5 OC) - thermophiles (110 OC)• acidophiles (pH 5) - alkaliphiles (pH 10.3)• non-halophiles (<0.1 M) - halophiles (5.1 M)• autotrophs (H2 -CO2 ) - methylotrophs (TMA, DMS)

• psychrophiles (-2.5 OC) - thermophiles (110 OC)• acidophiles (pH 5) - alkaliphiles (pH 10.3)• non-halophiles (<0.1 M) - halophiles (5.1 M)• autotrophs (H2 -CO2 ) - methylotrophs (TMA, DMS)

O2

REDUCTANT

Methanococcus jannaschiiMethanococcus jannaschii

Morphology coccusDiameter 1.0 µmMotility YespH optimum/range 6.0/5.2-7.6Temp optimum 85 C/48-94 CSubstrates H2 /formateGrowth factors NoneNaCl optimum/range 0.4M/0.1-0.85MMol % G+C 31

Morphology coccusDiameter 1.0 µmMotility YespH optimum/range 6.0/5.2-7.6Temp optimum 85 C/48-94 CSubstrates H2 /formateGrowth factors NoneNaCl optimum/range 0.4M/0.1-0.85MMol % G+C 31

Methanococcoides burtoniiMethanococcoides burtonii

Morphology coccusDiameter 1.2 µmMotility YespH optimum/range 7.7/6.8-8.2Temp optimum 23.4 C/-2.5-30 CSubstrates MeOH/methylaminesGrowth factors NoneNaCl optimum/range 0.2M/0.2-0.5MMol % G+C 39.6

Morphology coccusDiameter 1.2 µmMotility YespH optimum/range 7.7/6.8-8.2Temp optimum 23.4 C/-2.5-30 CSubstrates MeOH/methylaminesGrowth factors NoneNaCl optimum/range 0.2M/0.2-0.5MMol % G+C 39.6

Morphological DiversityMorphological Diversity

Sprott and Beveridge, 1993

Cell EnvelopesCell Envelopes

• Methanobacteriaceae pseudomurein• Methanothermaceae pseudomurein/glycoprotein• Methanosarcina methanochondroitin/glycoprotein• Methanococcales protein• Methanomicrobium mobile protein• Methanogenium marisnigri protein• Methanogenium marisnigri glycoprotein• Methanoplanus limicola glycoprotein• Methanosaeta concilii glycoprotein/sheath• Methanosprillum hungatei glycoprotein/sheath

• Methanobacteriaceae pseudomurein• Methanothermaceae pseudomurein/glycoprotein• Methanosarcina methanochondroitin/glycoprotein• Methanococcales protein• Methanomicrobium mobile protein• Methanogenium marisnigri protein• Methanogenium marisnigri glycoprotein• Methanoplanus limicola glycoprotein• Methanosaeta concilii glycoprotein/sheath• Methanosprillum hungatei glycoprotein/sheath

PseudomureinPseudomurein

Kandler, 1982

Thin-section EM micrograph ofMethanobacterium thermoautotrophicum

Thin-section EM micrograph ofMethanobacterium thermoautotrophicum

Sprott and Beveridge, 1993

MethanochondroitinMethanochondroitin

Kreisl and Kandler, 1986

Thin-section EM Micrograph ofMethanosarcina thermophila

Thin-section EM Micrograph ofMethanosarcina thermophila

Freeze-etched EM Micrograph ofMethanocorpusculum sinense

Freeze-etched EM Micrograph ofMethanocorpusculum sinense

Sprott and Beveridge, 1993

Methanogenic EnvironmentsMethanogenic Environments

Wolfe, 1996

Anaerobic Digestor / Freshwater SedimentsAnaerobic Digestor / Freshwater Sediments

COMPLEX POLYMERS(proteins, polysaccharides, etc.)

FERMENTATIVEBACTERIA

MONOMERS/POLYMERS(sugars, amino acids, peptides)

FATTY ACIDSPRIMARY ALCOHOLS

AROMATICS

GROUP I

H2-PRODUCING/ CO2 -REDUCINGACETOGENIC BACTERIA HYDROGEN

FORMATE, CO2

ACETATE

GROUP II

METHANOGENICARCHAEA

CH4, CO2

GROUP III

Net Effect of Interspecies Hydrogen Exchange

Net Effect of Interspecies Hydrogen Exchange

Zinder, 1993

Microstructural Analysis of UASB Granule

Microstructural Analysis of UASB Granule

Fang et al., 1994, 1995

Marine SedimentsMarine SedimentsCOMPLEX POLYMERS

(proteins, polysaccharides, etc.)

METHYLAMINOMETHYLTHIOOSMOLYTES

FERMENTATIVEBACTERIA

MONOMERS/POLYMERS(sugars, amino acids, peptides)

FATTY ACIDSPRIMARY ALCOHOLS

AROMATICSMETHYLAMINES

METHYL SULFIDES

GROUP I

H2-PRODUCINGACETOGENIC BACTERIA

SULFIDOGENIC BACTERIAHYDROGEN

CO2

ACETATE

GROUP II

METHANOGENIC ARCHAEASULFIDOGENIC BACTERIA

H2S, CH4, CO2

CH4, CO2

GROUP III

Animal RumenAnimal Rumen

COMPLEX POLYMERS(proteins, polysaccharides, etc.)

FERMENTATIVEBACTERIA

MONOMERS/POLYMERS(sugars, amino acids, peptides)

FATTY ACIDSPRIMARY ALCOHOLS

AROMATICS

HYDROGENCO2

ACETATE

GROUP I

METHANOGENIC ARCHAEACH4, CO2

GROUP III

RUMINANT

Hydrothermal VentsHydrothermal Vents

METHANOGENIC ARCHAEAGROUP III

HYDROGENCO2

CH4, CO2

Deep Subsurface Microbial ProcessesDeep Subsurface Microbial Processes

Kaiser, 1995

Methods - Hungate TechniqueMethods - Hungate Technique

Methods - Anaerobic Glove BoxMethods - Anaerobic Glove BoxChamberChamber

Air lockAir lock

A B C

Methods - Anaerobic Plating JarMethods - Anaerobic Plating Jar

Methods - Anaerobic Scale-UpMethods - Anaerobic Scale-Up

Genomic AnalysisGenomic Analysis

ArchaeaArchaea

Bacterial characteristicsBacterial characteristics Eukaryal characteristicsEukaryal characteristics

•Small molecule biosynthesis•Intermediary metabolism•Transport•Nitrogen fixation•Regulatory functions

•Small molecule biosynthesis•Intermediary metabolism•Transport•Nitrogen fixation•Regulatory functions

•DNA metabolism•Transcription•Translation

•DNA metabolism•Transcription•Translation

Transcription ProteinsTranscription Proteins

Genotypic Characteristics Undetected in Phenotype

Genotypic Characteristics Undetected in Phenotype

• multiple methyltransferases• flagellin/chemotaxis gene clusters• cytochrome d oxidase• 3 nitrogenases (Mo/Fe,V/Fe,Fe)• unique 2 component reg system

• multiple methyltransferases• flagellin/chemotaxis gene clusters• cytochrome d oxidase• 3 nitrogenases (Mo/Fe,V/Fe,Fe)• unique 2 component reg system

Pyrodictium

Desulfurococcus

SulfolobusThermofilum

Thermoproteus

Methanopyrus

Thermococcus

Methanococcus

Methanothermus

Methanobacterium

Archaeoglobus

Methanosarcina

MethanoplanusMethanospirillum

Halococcus

Halobacterium

Crenarchaeota Euryarchaeota

Archaea

Haloferax

MethanolobusMethanohalophilus

Methanosaeta

Pyrococcus

Pyrobaculum

Methanobrevibacter

Haloarcula

Halorubrum

NatronococcusNatronobacterium

Transformation

Transduction

Conjugation

EukaryaBacteria

Gene Transfer SystemsGene Transfer Systems

Sowers and Schreier, 1999

Viruses & Virus-like ParticlesViruses & Virus-like Particles

• ψΜ1 M. thermoautotrophicum lytic• ΦF3 M. thermoautotrophicum lytic• ΦF3 M. thermoautotrophicum lytic• PG M. smithii lytic• VLP M. voltae temperate

• ψΜ1 M. thermoautotrophicum lytic• ΦF3 M. thermoautotrophicum lytic• ΦF3 M. thermoautotrophicum lytic• PG M. smithii lytic• VLP M. voltae temperate

Restriction-Modification SystemsRestriction-Modification Systems

• MaeI, II, III Methanococcus. aeolicus endonuclease

• MthF1, T1, Z1 Methanobacterium endonuclease/thermoautotropicum methylase

• MvnI Methanococcus vanielii endonuclease

• MwoI Methanobacterium wolfei endonuclease/methylase

• PstI Methanococcus maripaludis endonuclease

• MaeI, II, III Methanococcus. aeolicus endonuclease

• MthF1, T1, Z1 Methanobacterium endonuclease/thermoautotropicum methylase

• MvnI Methanococcus vanielii endonuclease

• MwoI Methanobacterium wolfei endonuclease/methylase

• PstI Methanococcus maripaludis endonuclease

Plasmid DNAPlasmid DNA

• pT3 Methanobacterium sp. 7.3 kb Unknown function • pME2001 Methanobacterium 4.4 kb Unknown function

thermoautotropicum• pFV1,2,3 Methanobacterium 13.5,11,11 kb Restriction/

thermoautotropicum methylation• pURB500 Methanococcus sp 8.7 kb Unknown function • pURB900 Methanococcus sp. AG86 20 kb Unknown function• pURB800 Methanococcus jannaschii 64 kb Unknown function• pURB801 Methanococcus jannaschii 18 kb Unknown function• pC2A Methanosarcina acetivorans 5.4 kb Recombinase/

replication initiation• pMP1 Methanolobus vulcanii 6.9 kb Unknown function

• pT3 Methanobacterium sp. 7.3 kb Unknown function • pME2001 Methanobacterium 4.4 kb Unknown function

thermoautotropicum• pFV1,2,3 Methanobacterium 13.5,11,11 kb Restriction/

thermoautotropicum methylation• pURB500 Methanococcus sp 8.7 kb Unknown function • pURB900 Methanococcus sp. AG86 20 kb Unknown function• pURB800 Methanococcus jannaschii 64 kb Unknown function• pURB801 Methanococcus jannaschii 18 kb Unknown function• pC2A Methanosarcina acetivorans 5.4 kb Recombinase/

replication initiation• pMP1 Methanolobus vulcanii 6.9 kb Unknown function

00

pC2ApC2A5467bp5467bp

ssrAssrA

repArepA

orf1orf1

orf2orf2

Metcalfe et al, 1997Metcalfe et al, 1997

M. Acetivorans Native PlasmidM. Acetivorans Native Plasmid

Selection CassetteSelection Cassette

Hybrid Shuttle VectorHybrid Shuttle Vector

PWM3158745bp

EcoRV (183) BamHI (187) XhoI (265) Ecl136II (233)EcoICRI (233)

SstI (235)SacI (235)Asp718I (240)Acc65I (240)KpnI (244)SphI (253)ClaI (256)

lacZ

pC2A

mcr promoter

pac

mcr terminator

oriRK6

bla

trpA terminator

Metcalf et al., 1997Metcalf et al., 1997

Shuttle Vector Hybrid ConstructsShuttle Vector Hybrid Constructs

• pJK series Integration vector Methanosarcina spp.• pWM series Autonomous vector Methanosarcina spp.• pWAY1/2 Integration vector Methanosarcina

mazei• pMIP1 Integration vector Methanococcus voltae• pKAS Integration vector Methanococcus

maripaludis• pDLT44 Autonomous vector Methanococcus

maripaludis

• pJK series Integration vector Methanosarcina spp.• pWM series Autonomous vector Methanosarcina spp.• pWAY1/2 Integration vector Methanosarcina

mazei• pMIP1 Integration vector Methanococcus voltae• pKAS Integration vector Methanococcus

maripaludis• pDLT44 Autonomous vector Methanococcus

maripaludis

Transformation Protocols Transformation Protocols

• Natural transformation (M. voltae) < 7 x 10 2 /µg• Electroporation (M. voltae) < 7 x 10 2 /µg • PEG (M. maripaludis) < 2 x 10 7 /µg• Liposome (Methanosarcina spp.) < 2 x 10 9 /µg

• Natural transformation (M. voltae) < 7 x 10 2 /µg• Electroporation (M. voltae) < 7 x 10 2 /µg • PEG (M. maripaludis) < 2 x 10 7 /µg• Liposome (Methanosarcina spp.) < 2 x 10 9 /µg

Gene DisruptionGene Disruption

• Directed: Integration gene disruption

• Random: Ethylmethanesulfonate and Transposon mutagenesis

• Directed: Integration gene disruption

• Random: Ethylmethanesulfonate and Transposon mutagenesis

pac casette

pMIPbla

Gernhardt et al., 1990Gernhardt et al., 1990

HisA’HisA’

HisA

Methanococcus voltaeMethanococcus voltae

Directed Gene DisruptionDirected Gene Disruption

Random Gene DisruptionRandom Gene Disruption

Zhang et al., 2000

Reporter Vector for Methanosarcina acetivoransReporter Vector for Methanosarcina acetivorans

Sowers et al.

1

pKJ10412994bp

lacZ

cdhP

C2A

mcrP

pac

mcrToriR6K

bla

Current Limitations• Limited genetic selection markers• DNA restriction/modification systems • DNA repair/recombination mutants

Current Limitations• Limited genetic selection markers• DNA restriction/modification systems • DNA repair/recombination mutants

Current Capabilities• Genomes sequenced • Integration/autonomous plasmid vectors • Transposon mutagenesis• Reporter plasmids

Current Capabilities• Genomes sequenced • Integration/autonomous plasmid vectors • Transposon mutagenesis• Reporter plasmids

Technological ApplicationsTechnological Applications

• Human/animal waste treatment• Energy production • Bioremediation• Thermostable enzymes• Thermostable lipids• Synthesis of other unique compounds

• Human/animal waste treatment• Energy production • Bioremediation• Thermostable enzymes• Thermostable lipids• Synthesis of other unique compounds