Getting an N Fix

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Getting an N Fix

• Energetics
• N?N
• Haber-Bosch (100-200 atm, 400-500C, 8,000 kcal
  kg-1 N)
• Nitrogenase (4,000 kcal kg-1 N)

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Biological Nitrogen Fixation
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Pea Plant
R. leguminosarum nodules
Pink color is leghaemoglobin a protein that
carries oxygen to the bacteroids
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Rhizobium-legume symbioses
Host plant Bacterial symbiont Alfalfa Rhizob
ium meliloti Clover Rhizobium
trifolii Soybean Bradyrhizobium
japonicum Beans Rhizobium phaseoli Pea Rhizo
bium leguminosarum Sesbania Azorhizobium
caulinodans Complete listing can be found at at
http//cmgm.stanford.edu/mbarnett/rhiz.htm Both
plant and bacterial factors determine specificity
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Some nitrogen fixing organisms

• Free living aerobic bacteria
• Azotobacter
• Beijerinckia
• Klebsiella
• Cyanobacteria (lichens)
• Free living anaerobic bacteria
• Clostridium
• Desulfovibrio
• Purple sulphur bacteria
• Purple non-sulphur bacteria
• Green sulphur bacteria

• Free living associative bacteria
• Azospirillum
• Symbionts
• Rhizobium (legumes)
• Frankia (alden trees)

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legume
Fixed nitrogen (ammonia)
Fixed carbon (malate, sucrose)
rhizobia
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Obvious signs of nodulation by common rhizobial
species
MEDICAGO (alfalfa)
LOTUS (birdsfoot trefoil)
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Very early events in the Rhizobium-legume
symbiosis
Flavonoids nod-gene inducers
rhizosphere
Nod-factor
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Formation of a Root Nodule
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The Nodulation Process

• Chemical recognition of roots and Rhizobium
• Root hair curling
• Formation of infection thread
• Invasion of roots by Rhizobia
• Cortical cell divisions and formation of nodule
  tissue
• Bacteria fix nitrogen which is transferred to
  plant cells in exchange for fixed carbon

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The Colonization Process

• Signaling
• Rhizobia sense flavonoid compounds release by
  roots
• specific species sense particular flavonoids
  specific to a plant
• Rhizobia move by use of flagella propelling cell
  through soil water
• Rhizobia produce lipo-oligosaccharides or nod
  factors
• these initiate root hair deformation and curling
  and the division of cortical cells in the root at
  very low concentrations (lt 10-9 M soil solution).
  

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Role of Root Exudates

• General
• Amino sugars, sugars
• Specific
• Flavones (luteolin), isoflavones (genistein),
  flavanones, chalcones
• Inducers/repressors of nod genes
• Vary by plant species
• Responsiveness varies by rhizobia species

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Genetics of Nodulation

• Legume plants secrete specific flavonoids, which
  are detected by interaction with bacterial NodD
  proteins.

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The Biology of Symbiotic Development
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Sinorhizobium meliloti
chromosome
NodD
nod-gene inducers from alfalfa roots (specificity)
plasmid
pSym
activated NodD positively regulates nod genes
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• Signals early in infection
• Complex handshaking between legume root and
  rhizobium

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nod Gene Expression
Common nod genes
Nod factorLCO (lipo-chitin oligosaccharide)
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Role of Root Exudates

• General
• Amino sugars, sugars
• Specific
• Flavones (luteolin), isoflavones (genistein),
  flavanones, chalcones
• Inducers/repressors of nod genes
• Vary by plant species
• Responsiveness varies by rhizobia species

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The Colonization Process

• Infection Thread
• Protein called recadhesin and polysaccharides
  from Rhizobia and lectins from plants interact to
  adhere the bacterium to the root hair
• curling of the root hair and hydrolysis of root
  epidermis
• Rhizobia move down centre of the root hair toward
  the root cortex
• plant produces tube called an infection thread
• in the cortex Rhizobia enter enclosed area within
  a plant-derived peribacteroid membrane.
• membrane protect the rhizobia from plant defense
  responses.

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Nod factor biosynthesis
Nod factor R-group decorations determine host
specificity
Nod Factor a lipooligosaccharide
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LysM receptor-like kinases mediate plant
recognition of symbiotic bacteria
Taken from a review Parniske Downie. Locks,
keys and symbioses. Nature 425 569-570.
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Attachment and infection
Rhizobium
Nod factor (specificity)
Invasion through infection tube
Flavonoids (specificity)
Nitrogen fixation
Bacteroid differentiation
Formation of nodule primordia
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Nodule development
Enlargement of the nodule, nitrogen fixation and
exchange of nutrients
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Rhizobium encoding GFP from jellyfish as a marker
Infection thread
(From Quaedvlieg et al. Plant Mol. Biol. 37
715-727, 1998)
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Bacteria divide as they traverse infection thread
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Physiology of a legume nodule
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6 days
7 days
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Rhizobium Root Nodules
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Non-symbiotic nitrogen fixation
Aquatic
Cyanobacteria Anabaena Nostoc
Terrestrial and rhizosphere-associated
Azospirillum Azotobacter Acetobacter Klebsiella Cl
ostridium
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A nitrogen-fixing fern
-Co
Co
The aquatic fern Azolla is the only fern that can
fix nitrogen. It does so by virtue of a symbiotic
association with a cyanobacterium (Anabaena
azollae).
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Another cyanobacterium on the palm Welfia regia
in an epiphyllic relationship
It is believed that these bacteria transfer some
of fixed N to the plants through the leaf
surfaces
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Nitrogen Fixation

• All nitrogen fixing bacteria use highly conserved
  enzyme complex called Nitrogenase
• Nitrogenase is composed of of two subunits an
  iron-sulfur protein and a molybdenum-iron-sulfur
  protein
• Aerobic organisms face special challenges to
  nitrogen fixation because nitrogenase is
  inactivated when oxygen reacts with the iron
  component of the proteins

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Nitrogenase

• All nitrogen fixing bacteria use highly conserved
  enzyme complex called Nitrogenase
• Nitrogenase is composed of of two subunits an
  iron-sulfur protein and a molybdenum-iron-sulfur
  protein
• Aerobic organisms face special challenges to
  nitrogen fixation because nitrogenase is
  inactivated when oxygen reacts with the iron
  component of the proteins

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Nitrogenase Complex

• Two protein components nitrogenase reductase and
  nitrogenase
• Nitrogenase reductase is a 60 kD homodimer with a
  single 4Fe-4S cluster
• Very oxygen-sensitive
• Binds MgATP
• 4ATP required per pair of electrons transferred
• Reduction of N2 to 2NH3 H2 requires 4 pairs of
  electrons, so 16 ATP are consumed per N2

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Nitrogenase

• A 220 kD heterotetramer
• Each molecule of enzyme contains 2 Mo, 32 Fe, 30
  equivalents of acid-labile sulfide (FeS clusters,
  etc)
• Four 4Fe-4S clusters plus two FeMoCo, an
  iron-molybdenum cofactor
• Nitrogenase is slow - 12 e- pairs per second,
  i.e., only three molecules of N2 per second

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Biological N fixation is energetically expensive,
16 ATP/N2.
Note that Molybdenum is a cofactor
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Nitrogen fixation genes are repressed by oxygen
O2 inactivates nitrogenase
Bacterial membranes
FixLI
FixJ
Heme oxidized FixL inactive
O2
nifA
nif
nif
fix
nif regulon
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Exchange of nutrients during Rhizobium-legume
symbiosis
Malate to bacteria
nitrogen- fixing bacteroid containing Rhizobium
TCA
NH4 to plant
ATP
ADPPi
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Assimilation of Nitrogen by the Host

• Indeterminate nodules
• produce ammonia
• exported to the host there by converted to
  glutamine, glutamate, and aspartate to
  asparagines
• Asparagine is then exported to the shoot
• Determinate nodules
• export xanthine (a purine) formed from glutamate
  and aspartate
• Xanthine is converted in the host to ureides,
  allantoin and allantoic acid to be exported
  throughout the plant.

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Ammonia assimilatory cycle How nitrogen enters
biological pathways
Amino acids proteins purines pyrimidines
Pathway 1
GS
NH4
glutamate
glutamine
ATP
ADP Pi

Pathway 2
GDH
NH4
?-ketoglutarate
glutamate

Amino acids proteins
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Alfalfa Nodule
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Current approaches to improving biological
nitrogen fixation

• Enhancing survival of nodule forming bacterium by
  improving competitiveness of inoculant strains
• Extend host range of crops, which can benefit
  from biological nitrogen fixation
• Engineer microbes with high nitrogen fixing
  capacity

What experiments would you propose if you were to
follow each of these approaches?
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Activation of nif promoters by NifA A mechanism
similar to RNAP(?54) activation by NtrC
?54
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Rhizobiums bad brother Agrobacterium tumefaciens
Crown gall on rose
and on grapevine
Opines are an Agrobacterium-specific C- source to
feed future generations
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Chatt-Type Mechanism for Single Site Catalysis
N2 6H 6e- ? 2NH3
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Some nitrogen fixing organisms
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Rhizobiums bad brother Agrobacterium tumefaciens
Crown gall on rose
and on grapevine
Opines are an Agrobacterium-specific C- source to
feed future generations
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END
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Key metabolic step is conversion of fixed ammonia
to organic N
N assimilation is reliant on a steady supply of C
!!
Fig. 8.7