Microbial communities of thermal environments - possible analogues of early Earth ecosystems?

1. Microbial communities of thermal environments - possible analogues of early Earth ecosystems? E.A. Bonch-Osmolovskaya Winogradsky Institute of Microbiology Russian Academy of Sciences

2. Summary • Archaean biosphere • Thermal habitats • Electron donors and acceptors • Metabolic diversity of thermophilic prokaryotes • Evidence for new metabolic groups • Carbon cycle in thermal ecosystems – is it closed?

3. Summary • Archaean biosphere • Thermal habitats • Electron donors and acceptors • Metabolic diversity of thermophilic prokaryotes • Evidence for new metabolic groups • Carbon cycle in thermal ecosystems – is it closed? Georgy A. Zavarzin 1933-2011

4. Archaean biosphere • -4.0 - -2.5 billion years • Temperature: +70 - +100oC • Anaerobic • Reduced

5. Thermal habitats

6. Thermophiles on the Tree of Life

7. Thermophiles on the Tree of Life

8. Methanogens, sulfur and sulfate reducers CO2 CH4 SO4-2 H2 So H2S

9. Methanogens, sulfur and sulfate reducers

10. Alexander Merkel New methanogens in terrestrial hot springs Geyser Valley, Kamchatka Hot spring 2012 (Т 58˚C, pH 5.7) 108 clones

11. Methanogens, sulfur and sulfate reducers

12. Methanogens, sulfur and sulfate reducers

13. Sulfate reduction: Vulcanisaetamoutnovskia MariaProkofeva Isolated from the hot springs of Moutnovsky Volcano, Kamchatka Nikolai Chernyh Evgeny Frolov Nikolay Pimenov Grows in the temperature range from 59-102oC with the optimum at 83oC and in pH range 3.5-6.5 with the optimum at 5.2

14. Sulfate reduction: Vulcanisaetamoutnovskia Growth Growth V. moutnovskya was found to be able to grow be sulfate reduction Substrates are yeast extract, ethanol and glycerol SO4 SO4 mM Cells, 107/ml H2S H2S Time, hours

15. Sulfate reduction: Vulcanisaetamoutnovskia Crenarchaeal genes encoding sulfate reduction enzymes make a separate cluster, while those of Archaeoglobus are related to bacterial ones

16. Sulfate reduction: Vulcanisaetamoutnovskia

17. H2O CO2 Disproportionation of sulfur compounds H2S CH4 SO4-2 So SO4-2 H2 S2O3-2 So H2S

18. Disproportionation of sulfur compounds Alexander Slobodkin Galina Slobodkina Disproportionation - redox reaction in which compound with an intermediate oxidation state is simultaneously reduced and oxidized to form two different products Electron donor and electron acceptor Inorganic sulfur fermentation Disproportionation of sulfur compounds: sulfite, thiosulfate, elemental sulfur Formation of sulfate and sulfide 4SO32- + H+ = 3SO42- + HS-3:1ΔG°’= -58.9 kJ mol-1 SO32- S2O32- + H2O = SO42- + HS- + H+ 1:1ΔG°’= -22.3 kJ mol-1 S2O32- 4S0 + 4H2O = SO42- + 3HS- + 5H+ 1:3ΔG°’= +10.3 kJ mol-1S0

19. Thermosulfurimonasdismutans Isolated from the hydrothermal chimney of Lau Spreading Center, Pacific Ocean, depth 2060 m Growth in the temperatures range from 50 to 92 oC, opt 74 oC Obligate anaerobe Obligate lithoautotroph Needs Fe(III) for H2S scavenging (growth up to 108 cells/ml Capable to grow with H2 reducing thiosulfate

20. Thermosulfurimonasdismutans New genus in Thermodesulfobacteria

21. ‘Dissulfurimicrobium hydrothermalis’ Sh68 97 Dissulfuribacter thermophilus S69T (JQ414031) 100 Desulfobulbaceae 70 Syntrophaceae 100 59 Desulfobacca acetoxidans DSM 11109T(CP002629) 100 Desulfomonile 100 Syntrophobacteraceae 69 52 DeferrisomacaminiS3R1T (JF802205) 29 Desulfuromonadaceae 100 0.02 New thermophilicDeltaproteobacteriacapable of sulfur disproportionation Uzon Caldera, Kamchatka Lau Spreading Center, Pacific Ocean

22. Genome of Thermosulfurimonasdismutans • Genome size – 2.20 Mb • Carbon metabolism - autotrophic CO2 fixation via reductive acetyl-CoA pathway • Identified genes: CO dehydrogenase/acetyl-CoA synthase, acetyl-CoA synthase subunit, • Acetyl-CoA synthase corrinoid iron-sulfur protein, large subunit; • Acetyl-CoA synthase corrinoid activation protein • NAD-dependent formate dehydrogenase alpha subunit • 5,10-methylenetetrahydrofolate reductase • Carbon monoxide dehydrogenase CooS subunit • Methylenetetrahydrofolate dehydrogenase • Formate--tetrahydrofolate ligase • Hydrogen metabolism – uptake [Ni/Fe] hydrogenase • Identified genes: [Ni/Fe] hydrogenase, group 1, large subunit • [Ni/Fe] hydrogenase, group 1, small subunit • Uptake hydrogenase large subunit • Ni,Fe-hydrogenase I cytochrome b subunit • Hydrogenase maturation protease • [NiFe] hydrogenase metallocenter assembly protein HypC • [NiFe] hydrogenase nickel incorporation protein HypA • [NiFe] hydrogenase nickel incorporation-associated protein HypB • [NiFe] hydrogenase metallocenter assembly protein HypF

23. Genome of Thermosulfurimonas dismutans • Sulfur metabolism – complete pathway of sulfate reduction • Identified genes: • Thiosulfate sulfurtransferase, rhodanase • Dissimilatory sulfite reductase (desulfoviridin), alpha and beta subunits • Tetrathionate reductase subunit A • Sulfite reduction-associated complex DsrMKJOP protein DsrP (= HmeB) • Sulfite reduction-associated complex DsrMKJOP iron-sulfur protein DsrO (=HmeA) • Sulfite reduction-associated complex DsrMKJOP multiheme protein DsrJ (=HmeF) • Sulfite reduction-associated complex DsrMKJOP protein DsrK (=HmeD) • Sulfite reduction-associated complex DsrMKJOP protein DsrM (= HmeC) • Tetrathionate reductase subunit C • Tetrathionate reductase subunit B • Anaerobic dimethyl sulfoxide reductase chain B • Anaerobic dimethyl sulfoxide reductase, A subunit • Polysulphide reductase, NrfD • Adenylylsulfate reductase alpha-subunit • Adenylylsulfate reductase beta-subunit • Sulfate adenylyltransferase, dissimilatory-type • Sulfite reductase, dissimilatory-type gamma subunit • Sulfite reductase alpha subunit • Sulfite reductase beta subunit • Dissimilatory sulfite reductase clustered protein DsrD • Octaheme tetrathionate reductase

24. Anaerobic CO and formate oxidation H2O CO2 H2O H2S CH4 H2 CO SO4-2 So SO4-2 H2 S2O3-2 So H2O CO2 H2S H2 HCOOH

25. Anaerobic CO and formate oxidation CO + H2O = CO2 + H2 (Svetlichny et al., 1991) Alexander Lebedinsky Tatyana Sokolova CO Tatyana Kochetkova Daria Kozhevnikova H2 100% CO: phylogenetically diverse Firmicutes hyperthermophilic archaea of genus Thermococcus 45% CO: hyperthermophilic archaea of genus Thermofilum 5% CO: Thermophilic bacteria of genus Dictyoglomus Growth of Thermococcus barophilus Ch5 on CO

26. Anaerobic CO and formate oxidation cooA cooC cooM cooK cooL cooX cooU cooH hypA cooF cooS Carboxydothermus hydrogenoformans Thermococcus sp. AM4 T. barophilus MPT and Ch5 T. onnurineus cooRa cooF cooS cooC 1/2 cooM cooK cooU+cooH cooX cooL cooRa cooF cooS cooC 1/2 cooM cooU cooH cooY cooL cooK cooX “Thermofilum carboxydotrophus"

27. Anaerobic CO and formate oxidation cooA cooC cooM cooK cooL cooX cooU cooH hypA cooF cooS Carboxydothermus hydrogenoformans Thermococcus sp. AM4 T. barophilus MPT and Ch5 T. onnurineus cooRa cooF cooS cooC 1/2 cooM cooK cooU+cooH cooX cooL T. onnurineus T. gammatolerans T. barophilus Ch5 fdh cooF 1/2 cooM 1/2 cooM 1/2 cooM cooK cooU+cooH cooX cooL h f-tr cooRa cooF cooS cooC 1/2 cooM cooU cooH cooY cooL cooK cooX “Thermofilum carboxydotrophus"

28. Anaerobic CO and formate oxidation The energy of reaction: HCOO- + H2O → HCO3- + H2 ΔG0' = +1.3 kJ/mol was always considered to be insufficient to support microbial growth In our experimental conditions ΔG0‘ varied from -8 to -20 kJ/mol Kim et al., Nature, 2010, 467:352-355

29. Anaerobic CO and formate oxidation Cells Thermococcus sp. able to grow on formate producing hydrogen: T. barophilus T. gammatolerance T. onnurineus three new isolates from different deep-sea hydrothermal areas H2 Formate

30. Anaerobic CO and formate oxidation H2O CO2 H2O H2S CH4 H2 CO SO4-2 So SO4-2 H2 S2O3-2 So H2O CO2 H2S H2 HCOOH

31. Radioisotopic tracing: detection of new metabolic groups Uzon Caldera, Kamchatka In situ incubation Na14CO3 14C-acetate 14C-products 65 70 85 pH 8.5 Nikolay Pimenov pH 3.5 pH 7.0

32. Radioisotopic tracing: detection of new metabolic groups Uzon Caldera, Kamchatka ? In situ incubation Na14CO3 14C-acetate 14C-products 65 70 85 65 70 85 pH 8.5 ? ? ? ? pH 3.5 pH 7.0

33. Anaerobic CO and formate oxidation H2O CO2 H2O H2S CH4 H2 CO SO4-2 So SO4-2 H2 Acetate S2O3-2 So H2O CO2 H2S H2 HCOOH

34. Radioisotopic tracing: detection of new metabolic groups Uzon Caldera, Kamchatka In situ incubation Na14CO3 14C-acetate 14C-products 65 70 85 pH 8.5 ? ? ? ? pH 3.5 pH 7.0

35. Conclusions • Microbial communities of thermal environments contain anaerobic lithoautotrophic microorganisms capable to use electron donors and acceptors of volcanic origin, and to assimilate inorganic carbon in cell material. • C1 compounds of abiogenic origin can also fuel microbial ecosystems; no electron acceptor is required. • Anaerobic thermophiliclithoautotrophs able to disproportionate sulfur compounds are phylogenetically diverse, widely spread and also could act as the primary producers in primary ecosystems of the Archaean Earth. • New anaerobic lithotrophicthermophiles are still to be discovered. • Microbial communities of thermal habitats are able to perform both primary production and complete mineralization of organic matter, thus, closing the carbon cycle in these environments.

36. Kamchatka-1997

37. Acknowledgements: Collaboration: Institute of Volcanology and Seysmology RAS (expeditions) IFREMER, France (expeditions) University of Portland, USA (expeditions) Center «Bioengineering» RAS (sequencing and annotation of genomes) KORDI, Republic of Korea (the genomics of formate-utilizing archaea) Financial support: Programs of RAS Russian Foundation of Basic Research

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