Nitrogen, why is it so important?

1
Nitrogen, why is it so important?
Name Symbol Percent by Volume
Nitrogen N2 78.084
                                                                                                                                                                                                                                                                                                                                                                                                                                         
Oxygen O2 20.9476
                                                                                                                                                                                                                                                                                                                                                                                                                                         
Argon Ar 0.934
                                                                                                                                                                                                                                                                                                                                                                                                                                         
Carbon Dioxide CO2 0.0314
                                                                                                                                                                                                                                                                                                                                                                                                                                         
Neon Ne 0.001818
                                                                                                                                                                                                                                                                                                                                                                                                                                         
Methane CH4 0.0002
                                                                                                                                                                                                                                                                                                                                                                                                                                         
Helium He 0.000524
                                                                                                                                                                                                                                                                                                                                                                                                                                         
Krypton Kr 0.000114
                                                                                                                                                                                                                                                                                                                                                                                                                                         
Hydrogen H2 0.00005
                                                                                                                                                                                                                                                                                                                                                                                                                                         
Xenon Xe 0.0000087
NN
air
NN
NN
NN
SourceCRC Handbook of Chemistry and Physicsby
David R. Lide, Editor-in-Chief1997 Edition
2
Excess Nitrogen

• Nitrogen Fixation
• Atmospheric dinitrogen(N2) ? Ammonia(NH3 )
• Nitrogen-fixers bacteria, free-living and
  symbiotic

http//dragon.seowon.ac.kr/bioedu/bio/ohp/t-294.j
pg
3
The Big Picture
N2 8H 8e- 16 ATP 2NH3 H2 16ADP 16
Pi
4
Nitrogen-Fixing Bacteria
5
Nitrogen Fixation Mechanism
6
Nitrogen Dependence Cryptobiotic Crust
http//helios.bto.ed.ac.uk/bto/microbes/nitrogen.h
tm
7
How they know how to fix nitrogen
8
Nitrogenase
9
Nitrogenase Enzyme Complex
Fe Protein
Fe-Mo Protein
a
b
a
b
http//www.tau.ac.il/ecology/virtau/2tali/The20N
itrogenase20complex.htm
10
Fe-Mo Protein
Regulation
Fe Protein
b
a
a
b
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Activation of NifA
Fe-Mo Protein
Regulation
Fe Protein
b
a
a
b
NtrC-RNA polymerase
12
Electron Transport
Fe-Mo Protein
Regulation
Fe Protein
b
a
a
b
ATP
Ferredoxin
13
Electron Transport
Fe-Mo Protein
Regulation
Reduced Fe Protein
b
a
a
b
ATP
Ferredoxin
14
Electron Transport
Reduction of
Fe-Mo Protein
Regulation
Reduced Fe Protein
b
a
a
b
ATP
Ferredoxin
15
Electrons Donated to N2
Electron Transport
Reduction of
Fe-Mo Protein
Regulation
Reduced Fe Protein
b
a
a
b
ATP
Ferredoxin
16
Electrons Donated to N2
Formation of NH3
Electron Transport
Reduction of
Fe-Mo Protein
Regulation
Reduced Fe Protein
b
a
a
b
ATP
Ferredoxin
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(No Transcript)
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Fe-Mo Protein
Regulation
Fe Protein
b
a
a
b
19
Activation of NifL
Fe-Mo Protein
Regulation
Fe Protein
b
a
a
b
Oxygen Presence
20
Activation of NifL
Suppression of NifA
Fe-Mo Protein
Regulation
Fe Protein
b
a
a
b
Oxygen Presence
21
Electron Transport
Fe-Mo Protein
Regulation
Fe Protein
b
a
a
b
Ferredoxin
22
Electron Transport
Fe-Mo Protein
Regulation
Fe Protein
b
a
a
b
Ferredoxin
23
Electron Transport
Fe-Mo Protein
Regulation
Fe Protein
b
a
a
b
Order Importance?
Ferredoxin
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Is there a correlation among the position of nif
genes among nitrogen-fixers?
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Methods

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

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Examination of Synteny

• Nif genes in Trichodesmium similar to
  Crocosphaera?
• Examine orthologs of specific genes related to NIF

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Genes Described By Nif Trichodesmium/Crocosphaera

• (("Tn10-like transposase Bacteroides uniformis"
  "NifU Trichodesmium sp. IMS101" "hypothetical
  protein Trichodesmium erythraeum IMS101
  gi20138873spO34106NIFH_TRIS1 Nitro"
  "hypothetical protein Trichodesmium erythraeum
  IMS101 gi9081910gbAAF82641.1 NifX Tricho"
  "hypothetical protein Trichodesmium erythraeum
  IMS101 gi20139219spQ9KJL4NIFW_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
  gi1709285spP52965NIFJ_SYNY3 Putativ" "Unknown
  Vibrio vulnificus CMCP6 gi27361669gbAAO10576.
  1AE016804_86 Unknown Vibrio vulnificus" "NifT
  protein Nostoc sp. PCC 7120 gi25532906pirAF1
  982 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
  gi2183304gbAAC35194.1 NifE" "Nitrogenase
  iron-molybdenum cofactor biosynthesis protein
  nifN gi2183305gbAAC35195.1 NifN" "NifX
  Cyanothece sp. PCC 8801" "Nitrogenase
  stabilizing/protective protein nifW
  gi2183307gbAAC35197.1 NifW Cyanothece sp"
  "unknown protein Synechocystis sp. PCC 6803
  gi1652359dbjBAA17282.1 ORF_IDslr2030unknow"
  "light-independent protochlorophyllide reductase
  iron protein subunit ChlL Synechocystis sp."
  "putative NifU-like protein Synechococcus sp.
  PCC 7942"))

But what does this mean?
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Are the Nif Genes in Different Order?
Crocosphaera(CWAT)
NifH
Trichodesmium (TERY)
NifH
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Are the Nif Genes in Different Order?
Crocosphaera(CWAT)
NifH
Trichodesmium (TERY)
NifH
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Genes Downstream
Crocosphaera(CWAT)
MOFE
NifH
Trichodesmium (TERY)
NifH
MOFE
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Genes Upstream
Crocosphaera(CWAT)
NifH
Trichodesmium (TERY)
NifH
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Synteny Among Crocosphaera and Trichodesmium
Similar Functionally and Geographically
Crocosphaera(CWAT)
NifH
Trichodesmium (TERY)
NifH
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Synteny Among Crocosphaera and Trichodesmium
Crocosphaera(CWAT)
MOFE
NifH
Trichodesmium (TERY)
NifH
MOFE
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Synteny Among Crocosphaera and Trichodesmium
Crocosphaera(CWAT)
Alpha chain only
MOFE
NifH
Trichodesmium (TERY)
NifH
MOFE
Alpha and beta chain
36
Synteny Among Crocosphaera and Trichodesmium
Makes senseneed e- for transport
Crocosphaera(CWAT)
Alpha chain only
Hypothetical Protein
MOFE
NifH
Trichodesmium (TERY)
NifH
MOFE
Hemolysin activation/secretion protein
Alpha and beta chain
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Synteny Among Crocosphaera and Trichodesmium
Similar Function?
Crocosphaera(CWAT)
Alpha chain only
Hypothetical Protein
MOFE
NifH
Trichodesmium (TERY)
NifH
MOFE
Hemolysin activation/secretion protein
Alpha and beta chain
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In Reality, Synteny Exists!
Crocosphaera(CWAT)
Alpha chain only
Hypothetical Protein
MOFE
NifH
Trichodesmium (TERY)
NifH
MOFE
Hemolysin activation/secretion protein
Alpha and beta chain
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Regulators
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Gene Conservation Among Other N-Fixers
NifA
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Gene Conservation
NifA
Ferrodoxin
43
Gene Conservation
NifA Transcriptional Activator
NifA
Ferrodoxin
44
Other Regulators?
A
L
45
Electron Transport
Fe-Mo Protein
Regulation
Fe Protein
b
a
a
b
Ferredoxin
46
Electron Transport
Fe-Mo Protein
Regulation
Fe Protein
b
a
a
b
Ferredoxin
H
D
A
K
L
47
Mutational Regulation
H
D
A
K
L
48
Mutational Regulation
H
D
A
K
L
49
Electron Transport
Fe-Mo Protein
Regulation
Fe Protein
b
a
a
b
Ferredoxin
H
D
A
K
L
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Similarities and Differences of Nif in
Nitrogen-fixing bacteria?

• Functional conservation between nif-genes of
  nitrogen fixers

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A Special Thanks.

• Dr. Jeff Elhai
• Dr. Jonathan Zehr