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Nitrogen, why is it so important? NameSymbolPercent by Volume Nitrogen N2N2 78.084 % Oxygen O2O2 20.9476 % Argon Ar 0.934 % Carbon Dioxide CO 2 0.0314.

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Presentation on theme: "Nitrogen, why is it so important? NameSymbolPercent by Volume Nitrogen N2N2 78.084 % Oxygen O2O2 20.9476 % Argon Ar 0.934 % Carbon Dioxide CO 2 0.0314."— Presentation transcript:

1 Nitrogen, why is it so important? NameSymbolPercent by Volume Nitrogen N2N % Oxygen O2O % Argon Ar % Carbon Dioxide CO % Neon Ne % Methane CH % Helium He % Krypton Kr % Hydrogen H2H % Xenon Xe % Source: CRC Handbook of Chemistry and Physics by David R. Lide, Editor-in-Chief 1997 Edition

2 Excess Nitrogen Nitrogen Fixation: Nitrogen Fixation: Atmospheric dinitrogen(N 2 )  Ammonia(NH 3 ) Atmospheric dinitrogen(N 2 )  Ammonia(NH 3 ) Nitrogen-fixers – bacteria, free-living and symbiotic Nitrogen-fixers – bacteria, free-living and symbiotic

3 The Big Picture N2 + 8H+ + 8e ATP = 2NH3 + H2 + 16ADP + 16 Pi

4 Nitrogen-Fixing Bacteria

5 Nitrogen Fixation Mechanism

6 Nitrogen Dependence : Cryptobiotic Crust

7 How they know how to fix nitrogen:

8 Nitrogenase

9 Fe Protein Nitrogenase Enzyme Complex Fe-Mo Protein a a b b

10 b a a b Fe Protein Fe-Mo ProteinRegulation

11 b a a b Fe Protein Fe-Mo ProteinRegulation NtrC-RNA polymerase Activation of NifA

12 b a a b Fe Protein Fe-Mo ProteinRegulation Electron Transport Ferredoxin ATP

13 b a a b Reduced Fe Protein Fe-Mo ProteinRegulation Electron Transport Ferredoxin ATP

14 b a a b Reduced Fe Protein Fe-Mo ProteinRegulation Electron Transport Ferredoxin ATP Reduction of

15 b a a b Reduced Fe Protein Fe-Mo ProteinRegulation Electron Transport Ferredoxin ATP Reduction of Electrons Donated to N2

16 b a a b Reduced Fe Protein Fe-Mo ProteinRegulation Electron Transport Ferredoxin ATP Reduction of Electrons Donated to N2Formation of NH3

17

18 b a a b Fe Protein Fe-Mo ProteinRegulation

19 b a a b Fe Protein Fe-Mo ProteinRegulation Oxygen Presence Activation of NifL

20 b a a b Fe Protein Fe-Mo ProteinRegulation Oxygen Presence Activation of NifL Suppression of NifA

21 b a a b Fe Protein Fe-Mo ProteinRegulation Electron Transport Ferredoxin

22 b a a b Fe Protein Fe-Mo ProteinRegulation Electron Transport Ferredoxin

23 b a a b Fe Protein Fe-Mo ProteinRegulation Electron Transport Ferredoxin Order Importance?

24

25 Is there a correlation among the position of nif genes among nitrogen-fixers?

26 Methods: Identify the nif genes involved in nitrogen fixation Identify the nif genes involved in nitrogen fixation Map out the location of specific nif genes in Tery and Cwat Map out the location of specific nif genes in Tery and Cwat Compare geographic proximity of cyanobacteria to other nitrogen fixers. Compare geographic proximity of cyanobacteria to other nitrogen fixers.

27 Examination of Synteny Nif genes in Trichodesmium similar to Crocosphaera? Nif genes in Trichodesmium similar to Crocosphaera? –Examine orthologs of specific genes related to NIF

28 (("Tn10-like transposase [Bacteroides uniformis]" "NifU [Trichodesmium sp. IMS101]" "hypothetical protein [Trichodesmium erythraeum IMS101] gi| |sp|O34106|NIFH_TRIS1 Nitro" "hypothetical protein [Trichodesmium erythraeum IMS101] gi| |gb|AAF | NifX [Tricho" "hypothetical protein [Trichodesmium erythraeum IMS101] gi| |sp|Q9KJL4|NIFW_TRIS1 Nitro" "COG1348: Nitrogenase subunit NifH (ATPase) [Trichodesmium erythraeum IMS101]" "COG1625: Fe-S oxidoreductase, related to NifB/MoaA family [Trichodesmium erythraeum IMS1") ("pyruvate oxidoreductase [Synechocystis sp. PCC 6803] gi| |sp|P52965|NIFJ_SYNY3 Putativ" "Unknown [Vibrio vulnificus CMCP6] gi| |gb|AAO |AE016804_86 Unknown [Vibrio vulnificus" "NifT protein [Nostoc sp. PCC 7120] gi| |pir||AF1982 NifT protein [imported] - Nostoc sp." "NifP [Cyanothece sp. PCC 8801]" "NifS [Cyanothece sp. PCC 8801]" "Nitrogenase iron protein (Nitrogenase component II) (Nitrogenase Fe protein) (Nitrogenas" "Nitrogenase iron-molybdenum cofactor biosynthesis protein nifE gi| |gb|AAC | NifE" "Nitrogenase iron-molybdenum cofactor biosynthesis protein nifN gi| |gb|AAC | NifN" "NifX [Cyanothece sp. PCC 8801]" "Nitrogenase stabilizing/protective protein nifW gi| |gb|AAC | NifW [Cyanothece sp" "unknown protein [Synechocystis sp. PCC 6803] gi| |dbj|BAA | ORF_ID:slr2030~unknow" "light-independent protochlorophyllide reductase iron protein subunit; ChlL [Synechocystis sp." "putative NifU-like protein [Synechococcus sp. PCC 7942]")) (("Tn10-like transposase [Bacteroides uniformis]" "NifU [Trichodesmium sp. IMS101]" "hypothetical protein [Trichodesmium erythraeum IMS101] gi| |sp|O34106|NIFH_TRIS1 Nitro" "hypothetical protein [Trichodesmium erythraeum IMS101] gi| |gb|AAF | NifX [Tricho" "hypothetical protein [Trichodesmium erythraeum IMS101] gi| |sp|Q9KJL4|NIFW_TRIS1 Nitro" "COG1348: Nitrogenase subunit NifH (ATPase) [Trichodesmium erythraeum IMS101]" "COG1625: Fe-S oxidoreductase, related to NifB/MoaA family [Trichodesmium erythraeum IMS1") ("pyruvate oxidoreductase [Synechocystis sp. PCC 6803] gi| |sp|P52965|NIFJ_SYNY3 Putativ" "Unknown [Vibrio vulnificus CMCP6] gi| |gb|AAO |AE016804_86 Unknown [Vibrio vulnificus" "NifT protein [Nostoc sp. PCC 7120] gi| |pir||AF1982 NifT protein [imported] - Nostoc sp." "NifP [Cyanothece sp. PCC 8801]" "NifS [Cyanothece sp. PCC 8801]" "Nitrogenase iron protein (Nitrogenase component II) (Nitrogenase Fe protein) (Nitrogenas" "Nitrogenase iron-molybdenum cofactor biosynthesis protein nifE gi| |gb|AAC | NifE" "Nitrogenase iron-molybdenum cofactor biosynthesis protein nifN gi| |gb|AAC | NifN" "NifX [Cyanothece sp. PCC 8801]" "Nitrogenase stabilizing/protective protein nifW gi| |gb|AAC | NifW [Cyanothece sp" "unknown protein [Synechocystis sp. PCC 6803] gi| |dbj|BAA | ORF_ID:slr2030~unknow" "light-independent protochlorophyllide reductase iron protein subunit; ChlL [Synechocystis sp." "putative NifU-like protein [Synechococcus sp. PCC 7942]")) But what does this mean?

29 NifH Crocosphaera(CWAT) Trichodesmium (TERY) Are the Nif Genes in Different Order?

30 NifH Crocosphaera(CWAT) Trichodesmium (TERY) Are the Nif Genes in Different Order?

31 NifH Crocosphaera(CWAT) Trichodesmium (TERY) MOFE Genes Downstream…

32 NifH Crocosphaera(CWAT) Trichodesmium (TERY) Genes Upstream…

33 NifH Crocosphaera(CWAT) Trichodesmium (TERY) Synteny Among Crocosphaera and Trichodesmium Similar Functionally and Geographically

34 NifH Crocosphaera(CWAT) Trichodesmium (TERY) MOFE Synteny Among Crocosphaera and Trichodesmium

35 NifH Crocosphaera(CWAT) Trichodesmium (TERY) MOFE Synteny Among Crocosphaera and Trichodesmium Alpha chain only Alpha and beta chain

36 NifH Crocosphaera(CWAT) Trichodesmium (TERY) MOFE Hypothetical Protein Hemolysin activation/secretion protein Synteny Among Crocosphaera and Trichodesmium Alpha chain only Alpha and beta chain Makes sense…need e- for transport

37 NifH Crocosphaera(CWAT) Trichodesmium (TERY) MOFE Hypothetical Protein Hemolysin activation/secretion protein Synteny Among Crocosphaera and Trichodesmium Alpha chain only Alpha and beta chain Similar Function?

38 NifH Crocosphaera(CWAT) Trichodesmium (TERY) MOFE Hypothetical Protein Hemolysin activation/secretion protein In Reality, Synteny Exists! Alpha chain only Alpha and beta chain

39

40 Regulators

41 Gene Conservation Among Other N-Fixers NifA

42 Gene Conservation NifA Ferrodoxin

43 Gene Conservation NifA Ferrodoxin NifA Transcriptional Activator

44 L Other Regulators? A

45 b a a b Fe Protein Fe-Mo ProteinRegulation Electron Transport Ferredoxin

46 b a a b Fe Protein Fe-Mo ProteinRegulation Electron Transport Ferredoxin D L A K H

47 Mutational Regulation D L A K H

48 D L A K H

49 b a a b Fe Protein Fe-Mo ProteinRegulation Electron Transport Ferredoxin D L A K H

50 Similarities and Differences of Nif in Nitrogen-fixing bacteria? Functional conservation between nif-genes of nitrogen fixers Functional conservation between nif-genes of nitrogen fixers

51

52 A Special Thanks…. Dr. Jeff Elhai Dr. Jeff Elhai Dr. Jonathan Zehr Dr. Jonathan Zehr


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