CARBON FIXATION IN NITRIFIERS

Transcription

1 CARBON FIXATION IN NITRIFIERS Logan Hodgskiss, MSc PhD student Division of Archaea Biology and Ecogenomics Department of Ecogenomics and Systems Biology University of Vienna, Austria October 4 th, 2017

2 Outline Important details of carbon fixation pathways in nitrifiers Ecological significance Functionality of carbon fixation in nitrifiers

3 Nitrification Commamox NH 4 + NO 2 - NO 3 - Ammonium Nitrite Nitrate Ammonia Oxidizing Bacteria (AOB) Nitrite Oxidizing Bacteria (NOB) Ammonia Oxidizing Archaea (AOA)

4 Carbon Fixation CO 2 biomass Multiple carbon fixation pathways Differentiated by:

5 Carbon Fixation CO 2 biomass Multiple carbon fixation pathways Differentiated by: End product (central metabolite)

6 End Products s&cd=&cad=rja&uact=8&ved=0ahukewjylyn709twahwobfa KHdirC0cQjRwIBw&url=http%3A%2F%2Fwww.uky.edu%2F~dh ild%2fbiochem%2f24%2flect24.html&psig=aovvaw3lduxlde ARO8gCPA6mggJn&ust=

7 Carbon Fixation CO 2 biomass Multiple carbon fixation pathways Differentiated by: End product (central metabolite) Electron Donors

8 Electron Donors CO 2 Oxidation state of carbon: +4 biomass Oxidation state of carbon: typically 0

9 Electron Donors CO 2 Oxidation state of carbon: +4 NADPH reduced Ferredoxin biomass Oxidation state of carbon: typically 0 E 0 = -320 mv E mv ***oxygen sensitive*** FFe2S2.svg%2F220px-Fe2S2.svg.png&imgrefurl=https%3A%2F%2Fen.wikipedia.org%2Fwiki%2FFerredoxin&docid=whN_- iil6yv_am&tbnid=prydfekseup5zm%3a&vet=10ahukewjbr- XkhNDWAhVpP5oKHan4BJEQMwhdKAAwAA..i&w=220&h=118&bih=637&biw=1366&q=ferredoxin&ved=0ahUKEwjBr- XkhNDWAhVpP5oKHan4BJEQMwhdKAAwAA&iact=mrc&uact=8

10 Carbon Fixation CO 2 biomass Multiple carbon fixation pathways Differentiated by: End product (central metabolite) Electron Donors Energetic Costs ATP required per molecule produced Protein synthesis requirements

11 Ammonia Oxidizing Bacteria (AOB) Calvin-Benson Cycle (Berg, 2011)

12 Ammonia Oxidizing Bacteria (AOB) Calvin-Benson Cycle Key Enzyme Rubisco (ribulose-1,5- bisphosphate carboxylase/oxygenase) (Berg, 2011)

13 Ammonia Oxidizing Bacteria (AOB) Calvin-Benson Cycle Key Enzyme Rubisco (ribulose-1,5- bisphosphate carboxylase/oxygenase) End Product Glyceraldehyde-3-P (Berg, 2011)

14 Ammonia Oxidizing Bacteria (AOB) Calvin-Benson Cycle * Key Enzyme Rubisco (ribulose-1,5- bisphosphate carboxylase/oxygenase) End Product Glyceraldehyde-3-P Electron Donor NADPH (Berg, 2011)

15 Ammonia Oxidizing Bacteria (AOB) Calvin-Benson Cycle (Berg, 2011) * Key Enzyme Rubisco (ribulose-1,5- bisphosphate carboxylase/oxygenase) End Product Glyceraldehyde-3-P Electron Donor NADPH Costs 9 ATP and 6 NADPH (per glyceraldehyde-3-p) Rubisco side reaction

16 Rubisco %3A%2F%2Fcsls-text.c.u-tokyo.ac.jp%2Factive%2F08_08.html&psig=AOvVaw3j2XrwABAjqAqofXNIK3Nj&ust=

17 Rubisco Both phosphoglycerates incorporated into carbon fixation cycle. Phosphoglycolate cannot be incorporated and must be discarded. %3A%2F%2Fcsls-text.c.u-tokyo.ac.jp%2Factive%2F08_08.html&psig=AOvVaw3j2XrwABAjqAqofXNIK3Nj&ust=

18 Rubisco Both phosphoglycerate s incorporated into carbon fixation cycle. Phosphoglycolate cannot be incorporated and must be discarded. %3A%2F%2Fcsls-text.c.u-tokyo.ac.jp%2Factive%2F08_08.html&psig=AOvVaw3j2XrwABAjqAqofXNIK3Nj&ust=

19 Nitrite Oxidizing Bacteria (NOB) Calvin-Benson Cycle

20 Nitrite Oxidizing Bacteria (NOB) Calvin-Benson Cycle Reductive TCA Cycle (White, Drummond, & Fuqua, 2012)

21 Nitrite Oxidizing Bacteria (NOB) Calvin-Benson Cycle Reductive TCA Cycle Key Enzymes: 2-oxoglutarate synthase Citrate lyase (White, Drummond, & Fuqua, 2012)

22 Nitrite Oxidizing Bacteria (NOB) Calvin-Benson Cycle Reductive TCA Cycle Key Enzymes: 2-oxoglutarte synthase Citrate lyase End product Pyruvate (White, Drummond, & Fuqua, 2012)

23 Nitrite Oxidizing Bacteria (NOB) Calvin-Benson Cycle Reductive TCA Cycle * * * * * Key Enzymes: 2-oxoglutarte synthase Citrate lyase End product Pyruvate Electron Donors NAD(P)H Fd red (White, Drummond, & Fuqua, 2012)

24 Nitrite Oxidizing Bacteria (NOB) Calvin-Benson Cycle Reductive TCA Cycle * * * (White, Drummond, & Fuqua, 2012) * * Key Enzymes: 2-oxoglutarte synthase Citrate lyase End product Pyruvate Electron Donors NADPH Fd red Costs 2 ATP, 2 Fd red, 2 NAD(P)H, FADH 2 (per pyruvate)

25 Ammonia Oxidizing Archaea (AOA) 3-hydroxypropionate/ 4-hydroxybutyrate Cycle (Berg, Kockelkorn, Buckel, & Fuchs, 2007)

26 Ammonia Oxidizing Archaea (AOA) 3-hydroxypropionate/ 4-hydroxybutyrate Cycle Key Enzymes Acetyl-CoA/propionyl- CoA carboxylase (Berg, Kockelkorn, Buckel, & Fuchs, 2007)

27 Ammonia Oxidizing Archaea (AOA) 3-hydroxypropionate/ 4-hydroxybutyrate Cycle Key Enzymes Acetyl-CoA/propionyl- CoA carboxylase End Product Succinyl-CoA (Berg, Kockelkorn, Buckel, & Fuchs, 2007)

28 Ammonia Oxidizing Archaea (AOA) 3-hydroxypropionate/ 4-hydroxybutyrate Cycle * * * (Berg, Kockelkorn, Buckel, & Fuchs, 2007) * * * Key Enzymes Acetyl-CoA/propionyl- CoA carboxylase End Product Succinyl-CoA Electron Donor NAD(P)H

29 Ammonia Oxidizing Archaea (AOA) 3-hydroxypropionate/ 4-hydroxybutyrate Cycle * * * (Berg, Kockelkorn, Buckel, & Fuchs, 2007) * * * Key Enzymes Acetyl-CoA/propionyl- CoA carboxylase End Product Succinyl-CoA Electron Donor NAD(P)H Costs 7 ATP and 9 NAD(P)H (per succinyl-coa)

30 AOB Calvin Cycle 9 ATP/ end product Rubisco side reaction Oxygen tolerant AOA NOB Calvin Cycle 9 ATP/ end product Rubisco side reaction Oxygen tolerant rtca Cycle 2 ATP/ end product Oxygen sensitive Comammox 3-HP/4-HB Cycle 7 ATP/ end product Oxygen tolerant Heat tolerant rtca Cycle 2 ATP/ end product Oxygen sensitive

31 Ecological Significance Ammonia Oxidation AOB Calvin Cycle AOA Expensive in terms of ATP 3-hydroxypropionate/4-hydroxybutyrate More cost effective Stable at high temperatures

32 Ecological Significance Ammonia Oxidation AOB Calvin Cycle AOA Expensive in terms of ATP 3-hydroxypropionate/4-hydroxybutyrate More cost effective Stable at high temperatures Typically associated with HIGH ammonia concentrations. Typically associated with LOW ammonia concentrations. *stability at high temperatures supports the hot origin theory of AOA

33 Ecological Significance Nitrite Oxidation Calvin Cycle Expensive in terms of ATP Resistant to oxygen Reductive TCA Cycle Cheaper in terms of ATP Oxygen sensitive (ferredoxins)

34 Ecological Significance Nitrite Oxidation Calvin Cycle Expensive in terms of ATP Resistant to oxygen Reductive TCA Cycle Cheaper in terms of ATP Oxygen sensitive (ferredoxins) Possibly more adapted to higher nitrite concentrations. More commonly found in micro-aerophilic environments

35 Ecological Significance Nitrite Oxidation Calvin Cycle Expensive in terms of ATP Resistant to oxygen Reductive TCA Cycle Cheaper in terms of ATP Oxygen sensitive (ferredoxins) Possibly more adapted to higher nitrite concentrations. More commonly found in micro-aerophilic environments *Commamox organisms utilize rtca and grow in biofilms.

36 Functionality? Carbon fixation cycles based on genomic data Presence of key enzymes (i.e. Rubisco) Functionality can be tested in wet-lab experiments Protein assays Transcriptomics Proteomics Metabolomics

37 Nitrososphaera viennensis AOA-utilizes ammonia as an energy source Tourna et al. (2011) and Stieglmeier et al. (2014). Stieglmeier et al. (2014)

38 Pyruvate Dependence Could N. viennensis be mixotrophic? Tourna et al. (2010)

39 Pyruvate Dependence Could N. viennensis be mixotrophic? Is pyruvate acting as a scavenger of radical oxygen species (ROS)? Tourna (2010) Kim et al. (2016)

40 Oxidative Stress Kim et al. (2016) N. viennensis is an obligate autotroph

41 2.52 Mb genome 3123 predicted protein-coding genes Proteome of late-exponential phase Recovered 1503 proteins (48%) 733 proteins of core COG families of AOA

42 Kerou et al. (2016)

43 Transcriptomic Experimental Setup Allowed a culture to run out of ammonia Starved for 10 days Re-supplemented ammonia after 10 days Transcriptomics End of starvation period After re-addition of ammonia (recovery)

44 3-HP/4-HB in the AOA Nitrososphaera viennensis (Kerou et al., 2016 (pathway figure only))

45 3-HP/4-HB in the AOA Nitrososphaera viennensis Low High Starved Starved (Kerou et al., 2016 (pathway figure only))

46 3-HP/4-HB in the AOA Nitrososphaera viennensis Low High Starved Recovered Starved Recovered (Kerou et al., 2016 (pathway figure only))

47 Summary Carbon fixation requires the following: A replenishing cycle Electron donors for the reduction of carbon dioxide A direct link to the central carbon metabolism Various carbon fixation cycles in nitrifiers are adapted for organisms and environments Temperature, presence of oxygen, energy requirements, etc. Genomic data is substantially strengthened with physiological tests

48 References Arp, D., Sayavedra-Soto, L., & Hommes, N. (2002). Molecular biology and biochemistry of ammonia oxidation by Nitrosomonas europaea. Archives of Microbiology, 178(4), Berg, I. A. (2011). Ecological aspects of the distribution of different autotrophic CO2 fixation pathways. Applied and Environmental Microbiology, 77(6), Berg, I. A., Kockelkorn, D., Buckel, W., & Fuchs, G. (2007). A 3-Hydroxypropionate/4-Hydroxybutyrate Autotrophic Carbon Dioxide Assimilation Pathway in Archaea. Science, 318(5857). Berg, I. A., Kockelkorn, D., Ramos-Vera, W. H., Say, R. F., Zarzycki, J., Hügler, M., Fuchs, G. (2010). Autotrophic carbon fixation in archaea. Nature Reviews Microbiology, 8(6), Daims, H., Lebedeva, E. V., Pjevac, P., Han, P., Herbold, C., Albertsen, M., Wagner, M. (2015). Complete nitrification by Nitrospira bacteria. Nature, 528(7583), Estelmann, S., Hügler, M., Eisenreich, W., Werner, K., Berg, I. A., Ramos-Vera, W. H., Fuchs, G. (2011). Labeling and enzyme studies of the central carbon metabolism in Metallosphaera sedula. Journal of Bacteriology, 193(5), Hommes, N. G., Sayavedra-Soto, L. A., & Arp, D. J. (2003). Chemolithoorganotrophic growth of Nitrosomonas europaea on fructose. Journal of Bacteriology, 185(23), Kerou, M., Offre, P., Valledor, L., Abby, S. S., Melcher, M., Nagler, M., Schleper, C. (2016). Proteomics and comparative genomics of Nitrososphaera viennensis reveal the core genome and adaptations of archaeal ammonia oxidizers. Proceedings of the National Academy of Sciences, 113(49), E7937 E Kim, J.-G., Park, S.-J., Sinninghe Damsté, J. S., Schouten, S., Rijpstra, W. I. C., Jung, M.-Y., Rhee, S.- K. (2016). Hydrogen peroxide detoxification is a key mechanism for growth of ammonia-oxidizing archaea. Proceedings of the National Academy of Sciences of the United States of America, 113(28),

49 References (continued) Klotz, M. G., Arp, D. J., Chain, P. S. G., El-Sheikh, A. F., Hauser, L. J., Hommes, N. G., Ward, B. B. (2006). Complete genome sequence of the marine, chemolithoautotrophic, ammonia-oxidizing bacterium Nitrosococcus oceani ATCC Applied and Environmental Microbiology, 72(9), Könneke, M., Schubert, D. M., Brown, P. C., Hügler, M., Standfest, S., Schwander, T., Berg, I. A. (2014). Ammonia-oxidizing archaea use the most energy-efficient aerobic pathway for CO2 fixation. Proceedings of the National Academy of Sciences of the United States of America, 111(22), Lücker, S., Nowka, B., Rattei, T., Spieck, E., & Daims, H. (2013). The Genome of Nitrospina gracilis Illuminates the Metabolism and Evolution of the Major Marine Nitrite Oxidizer. Frontiers in Microbiology, 4, Norton, J. M., Klotz, M. G., Stein, L. Y., Arp, D. J., Bottomley, P. J., Chain, P. S. G., Starkenburg, S. R. (2008). Complete genome sequence of Nitrosospira multiformis, an ammonia-oxidizing bacterium from the soil environment. Applied and Environmental Microbiology, 74(11), Stieglmeier, M., Klingl, A., Alves, R. J. E., Rittmann, S. K. M. R., Melcher, M., Leisch, N., & Schleper, C. (2014). Nitrososphaera viennensis gen. nov., sp. nov., an aerobic and mesophilic, ammonia-oxidizing archaeon from soil and a member of the archaeal phylum Thaumarchaeota. International Journal of Systematic and Evolutionary Microbiology, 64(PART 8), Tourna, M., Stieglmeier, M., Spang, A., Könneke, M., Schintlmeister, A., & Urich, T. (2011). Nitrososphaera viennensis, an ammonia oxidizing archaeon from soil. Proc. Natl. Acad. Sci. USA, 108(20), /DCSupplemental. van Kessel, M. A. H. J., Speth, D. R., Albertsen, M., Nielsen, P. H., Op den Camp, H. J. M., Kartal, B., Lücker, S. (2015). Complete nitrification by a single microorganism. Nature. White, D., Drummond, J., & Fuqua, C. (2012). The Physiology and Biochemistry of Prokaryotes. Oxford University Press.