Chemotrophic Anaerobic Respirers: Acetogens, Methanogens, & SRB

Steve Zinder MBL June 18, 2013 Acetogens

• Many can grow on H2/CO2 : 4H2 + 2CO2 ---> CH3COOH + 2H2O • CO2 reduction to acetate is a form of CO2 fixation. • Many acetogens growing on H2/CO2 are autotrophs – Autotrophic methanogenic use a form of the acetogenic pathway to fix CO2 • Most acetogens use a variety of organic compounds • Sometimes also called homoacetogens to differentiate them from other acetate producers • I once suggested calling them acetosynthetic bacteria (acetosynths) - Did not catch on Some acetogens

All are Firmicutes except the two marked with an asterisk, which are in the Spirochetes phylum The Wood-Ljungdahl acetyl CoA pathway

• Harland Wood (1907-1991) – Spent much of his career at Case- Western U – Published 96 papers between the ages of 70 and 84 • Lars Ljungdahl (still alive) – Spent much of his career at University of Georgia • Separate branches for the methyl carbon and the carboxyl carbon of acetate - CH3COOH • The methyl branch involves two well- known one-carbon carriers, tetrahydrofolate and corrinoids Tetrahydrofolate (THF) and vitamin B12

5 10

Pterin ring para-amino- Glutamate benzoic acid

Methyl vitamin B12

Found in Bacteria and Archaea The methyl branch of the acetogenic pathway

CO2 2H Formate Dehydrogenase Formate Formyl-tetrahydrofolate ATP synthetase (ligase) H2O ADP

Formyl tetrahydrofolate 2H

H2O 2H CH -THF Methyl tetrahydrofolate Methyl 3 transferase CH3-Co-Prot Methyl corrinoid protein acetyl-CoA synthetase (CODH/ACS)

CODH

CO2 +2H <--> CO + H2O

CO + CH3 bind here?

CO Ni channel Methyl branch Acetogenic pathway 2H FDH

FTHFS ATP

H2O ADP Carboxyl/carbonyl 2H branch

H2O 2H CO2 2H CH3-THF MT CODH/ACDS H2O CH3-Co-Prot pyruvate biosynthesis HS-CoA CODH/ACDS

PTA AK

Pi HS-CoA ADP ATP Methanogens

CO2-reducers- carboxytrophs? Organisms

4H2 + CO2 ---> CH4 + 2H2O Nearly all

4HCOOH + CO2 ---> CH4 + 2H2O + 4CO2 Most

2CH3CH2OH + CO2 ---> CH4 + 2CH3COOH Some Also short-chain alcohols other than methanol Methylotrophs

4CH3-OH ---> 3CH4 + CO2 + 2H2O Most Msarcinales

4(CH3)3-N + 6H2O ---> 9CH4 + 3CO2 + 4NH3 Most Msarcinales Also dimethyl and monomethyl amines

2CH3-S-CH3 + 2H2O ---> 3CH4 + CO2 + 2H2S Some Msarcinales

Also CH3-SH Acetotrophs

CH3-COOH ---> CH4 + CO2 Methanosarcina and The - methanogens and relatives Anaerobic Heterotroph

Methanobacteriales

SRB AerHet

AerHet

Methanomicrobiales

Methanocellales

Methanosarcinales

CO -reducers-C Methanopyrales 2 Methylotrophs-M Methanobacteriales • All have a "Gram-positive" pseudomurein cell wall similar to G+ Bacteria • Found in soils and GI tracts • Genome of M. thermoautotrophicus is 1.75 Mb

Methanobacterium bryantii Methanothermobacter Methanobrevibacter thermoautotrophicus ruminantium

From: Zeikus, Bacteriol. Rev. 41:514 (1977) Methanopyrales • The only known is Methanopyrus kandleri, originally isolated from an undersea hydrothermal system near Volcano, Italy. • It is a rod with a pseudomurein cell wall, an optimum growth temperature near 98o C and a maximum near 110o C (122o C under pressure) • Its genome is 1.7 Mb, and phylogenetic analyses of genes other than 16S rRNA indicate Methanopyrus is a member of the Methanobacteriales • M. kandleri genome is 1.7 Mb

Fluorescence micrograph of M. kandleri illuminated to

stimulate F420 fluorescence Methanococcales

• All known members are marine CO2-reducing irregular cocci with protein S-layer cell walls o • M. jannaschii (topt = 83 C) genome is only 1.66 Mb (first archaeal genome) despite being an autotroph with a 25 min td

Methanocaldococcus jannaschii, phase contrast and EM showing tufts of flagella.

EMs of thermolithotrophicus showing irregular morphology and S-layer cell wall Methanomicrobiales • All have protein S-layer cell walls found in freshwater habitats and soil • Methanospirillum hungatei has a protein sheath and spacer regions between cells • The M hungatei genome is 3.5 Mb, with many genes associated with motility, chemotaxis and signal transduction • M. boonei is an acidiphile isolated from a peat bog by S. Bräuer

Methanospirillum hungatei Methanogenium cariaci Methanoregula boonei

EMs of rod and coccus forms Motility in Methanospirillum Methanocellales • In 16S rRNA gene surveys of Archaea in rice paddies, an uncultured group related to Mmicrobiales and Msarcinales was dominant • Called Rice Cluster I (RC-I) by a German group • A Japanese group finally got it to grow in coculture with a

propionate oxidizer (see later) and isolated an H2/CO2 and formate using rod called Methanocella paludicola • Its genome contains several genes, like those for using sulfate as a sulfur source, that are common in aerobes • Oxygen emanates from rice roots IJSEM 58:929 (2008) Methanomassiliicoccus

• In many 16S rRNA surveys of Archaea in anaerobic habitats, including animal GI tracts, sequences were found related to the Thermoplasma group – aerobes that grow at low pH and lack a cell wall • A group from Marseille (L- Massilia) enriched and isolated a methanogen from human feces that was in this group

• It grew only on H2 plus methanol but not on H2/CO2 or anything else had an unusual cell wall, and grew at neutral pH

IJSEM 62:1902 (2012) Methanosarcinales • The only group that carries out methanogenic reactions other than CO2 reduction • Most are obligate methylotrophs using methanol, methylamines, and sometimes methyl sulfides • The obligate methylotrophs are marine or halophiles – Methylamines are breakdown products of osmoprotectants like glycine-betaine and trimethylamine N-oxide • Methanosarcina and Methanosaeta are the only genera that can use acetate (acetotrophic or acetoclastic methanogens)

CH3 N  CH3-S-CH3 CH3 CH3 Gycine-betaine Trimethylamine- Trimethylamine Dimethyl sulfide Methylotrophs

• All grow only on methylated compounds • All have protein S-layer cell wall

Phase contrast and EM of Methanolobus zinderi. From: Int. J. System. Evol. Microbiol. 59: 1064 (2009) Methanosarcina- the generalist • Sarcina, L, packet • By far the most versatile methanogen , with all strains using methyl compounds and acetate and some using H2/CO2 (not formate) • The M. acetivorans genome is 5.7 Mb, the largest known in the Archaea Freshwater Methanosarcina cultures form clumps with a thick acidic polysaccharide layer

Phase contrast Phase contrast EMs of M thermophila showing micrograph of an M. micrograph of M. chaotically dispersed division planes barkeri strain that forms thermophila forming large and thick polysaccharide layer. small clumps phase-bright clumps. Marker bars are 1 and 0.1 µm. From: Marker bar = 5 µm. From Zinder et al. Int. J. Syst. Bacteriol. Zinder and Mah, AEM 35:522. 38:996 (1979 Salinity adaptation in Methanosarcina

• Growth of M. barkeri (freshwater, top) and M. acetivorans (marine, bottom) • 0.05 mM NaCl (left) or 0.4 (right) M NaCl. • From Sowers et al. AEM 59:3832 (1993) Methanosaeta, the acetate specialist

• Saeta, L. bristle, thrix, L. hair • Grows very slowly • Methanosaeta spp. can use acetate down to 5 µM while Msarcina cannot use it below 1 mM • The M. thermophila genome is 1.88 Mb Methanosaeta morphology

M thermophila culture showing filaments with squared ends. The refractile granules (right) are gas vesicles which can be collapsed by pressure (left).

Negative stain EM of M. thermophila showing a cell (PC) inside a sheath with gas vesicles (GV) visible. From Zinder et al., Arch. Microbiol. 146:315 (1987) Acetate thresholds

Methanosarcina Methanosaeta Min and Zinder AEM 55:488, 1989 Competition based on rates

Methanosaeta Competition for acetate The Wal-Mart model

Methanosaeta The H2/CO2 pathway CO2 H Fd 2 red MF Methanofuran – C1 carrier

Fdox H2O Formyl-MF dehydrogenase Methanopterin – THMpt similar to folates MF

2H 2 Heterodisulfide F420red reductase 2H H2O F420red H2

CH3-THMpt HS-CoM Methyltransferase CoB-SH CoM-S-S-CoB THMpt CH OH 3 CH -S-CoM 3 CH4 Methylcoenzyme M methylreductase Two cofactors to know about

Cofactor F 420 • Structure resembles flavins but lower O/R potential

• F420 is reduced using Coenzyme M: electrons from H2 Mercaptoethane • Oxidized form is fluorescent sulfonate: the world's Oxidized (Excitation max 420 nm, smallest cofactor 2e– + H+ Fluorescence max at 480 -360 mv nm). • Many methanogens have enough F420 to allow them to autofluoresce under a Methyl-coenzyme M fluorescence microscope A thioether methane precursor Reduced

Fluorescence micrograph of M. kandleri illuminated to Bromoethane sulfonate (BES) stimulate F420 fluorescence An inhibitory CoM analogue Sulfate reducers • Use oxidized forms of sulfur including sulfate, sulfite, and thiosulfate as terminal electron acceptors forming H2S • Generally considered anaerobes, although some have limited ability to use O2 • Were studied by Beijerinck, who first described a culture of Desulfovibrio • Are particularly important in marine habitats (sulfate ≈28 mM in seawater) versus freshwater (~1 mM • Usually use fermentation products as electron donors such as H2, lactate, ethanol, acetate, other fatty acids, and some use aromatic compounds and hydrocarbons Metabolic modes in sulfate reducers • Incomplete oxidation (e.g. Desulfovibrio): - 2- + - 2lactate + SO4 + 2H ---> 2acetate + 2CO2 + H2S ∆G°' = -159 kJ/rxn

• Complete oxidation to CO2 2- + acetate + SO4 + 3 H ---> 2CO2 + H2S ∆G°' = -36 kJ/rxn

• Lithotrophic growth on H2 (sometimes acetate needed as C source) 2- + 4 H2 + SO4 + 2H ---> H2S + 4H2O ∆G°'= -151.9 kJ/rxn • Disproportionation of thiosulfate 2- 2- S2O3 + H2O ----> SO4 + H2S ∆G°' = -27.6 kJ /rxn • Disproportionation of sulfite 2- + 2- 4SO3 + 2H ----> 3SO4 + H2S ∆G°' = -241 kJ/rxn Classic genera enriched with lactate and sulfate

Desulfovibrio Desulfovibrio gigas Desulfotomaculum desulfuricans nigrificans Gram-negative spirals Gram-negative staining endospore forming rods Fritz Widdel made things complicated

Now at: Marine Institute at Bremen, Germany

Desulfotomaculum acetoxidans The first acetate-oxidizing SRB Some of Fritz’s cultures

Desulfobacter Desulfobulbus postgatei propionicus

Desulfococcus Desulfosarcina multivorans variabilis

Desulfonema Desulfonema limicola magnum Delta and epsilon Proteobacteria

Aerobic cannibals

So and Fe3+ reducers

Aerobic cannibals

S-reducer Microaerophiles Now Bacteriovorax Aerobic cannibals Sulfate reducers in Firmicutes

Sulfite reducer

“Gram-negative” Firmicutes

Also Thermodesulfovibrio in the phylum Nitrospirae And Thermodesulfobacterium in it’s own phylum in the Bacteria

Also, Archaeoglobus fulgidus in the Euryarchaeota Anaerobic respiration: competition for H2 Idealized time courses for uptake of H2 10

8 1 Pa (Pascal) = 1 Nt/m2 ≈ 10-5 atm (10 ppm)

6 (Pa)

2 H 4 Methanospirillum

~3 Pa 2 Desulfovibrio ~1 Pa 0 Time

With limiting substrate, Desulfovibrio will take H2 to levels too low for Methanospirillum to use. Effect of H2 on methanogenesis Effect of H2 partial pressure on ∆G of electron-accepting reactions +50 Acetogenesis

0 ≈ Minimum threshold -50

Sulfate G' (kJ/rxn) G' ∆ Reduction -100 Methanogenesis

-150 - - NO3 /NO2 -7 -6 -5 -4 -3 -2 -1 0 ∆G = 0 at ~10-28 atm10 10 10 10 10 10 10 10 H Partial Pressure (atm) ≈ 2 x 10-6 molecules/L 2 Measured thresholds for H2 uptake by anaerobes

From: Arch Microbiol. 149:350, 1988 The good old days….

Sci. Amer. 7/04 p 78