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Plant-associated Proteobacteria (and a few outsiders):the good and the bad
N2 NH3
nitrogenase
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Today’s Topics:
1. Rhizobeacae and other nitrogen-fixing genera2. Nitrogen fixation and why we need it3. Examples of nitrogen-fixing symbioses in plants4. Processes of nodulation4. Processes of nodulation5. Non-symbiotic nitrogen-fixing Proteobacteria6. Cyanobacterial associations7. Crown gall: the selfish doings of Agrobacterium
tumefaciens
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Thermotoga
Green,nonsulfur
Deinococci
Spirochetes
Greensulfur Bacteroides
Chlamydiae
Gram positive
Cyanobacteria
Rhizobium
Prokaryotes
a
b
gd
Proteobacteria
RhizobiumBradyrhizobiumSinorhizobiumAgrobacteriumAzospirillum
E. coliKlebsiellaAzotobacter
HerbaspirillumDesulfoivbrio
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Ecology of nitrogen-fixing bacteria
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N2 + 8 flavodoxin- + 8H+ + 16 MgATP2- + 18 H2O
+ 2OH- + 8 flavodoxin + 16 MgADP- + 16H2PO4- + H2
nitrogenase
Biological nitrogen fixation:
2NH4+ + 2OH + 8 flavodoxin + 16 MgADP + 16H2PO4 + H22NH4
1. Rare, extremely energy consuming conversion because of stability of triply bonded N2
2. Produces fixed N which can be directly assimilated into N containing biomolecules
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Ammonia assimilatory cycle:How nitrogen enters biological pathways Amino acids
proteinspurinespyrimidines
glutamate glutamine+ ATP + ADP + PiGS
+NH4+
GOGAT
Pathway 1
NH4+
Amino acidsproteins
�-ketoglutarate glutamateGDH
+
glutamineglutamate �-ketoglutarateGOGAT
+
Pathway 2
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N2dinitrogen gas
(78% of air)
NH4+
N2Onitrous oxide
Denitrification Nitrogen fixationthe Haber Process
and lightning
The Nitrogen CycleNH4
ammonium
NO2-
nitrite
NO3-
nitrate
BIOSPHEREDenitrification
nitrificationnitrification
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N2dinitrogen gas
(78% of air)
NH4+
N2Onitrous oxide Prokaryotes
assimilation
nitrification
Biological nitrogen fixation
The Nitrogen Cycle
NH4
ammonium
NO2-
nitrite
NO3-
nitrate
Animals
Plants
uptake
consumption
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A growing population must eat!
•In 1910 humans consumed 10% of total carbon fixed by photosynthesis, by 2030 it is predicted that 80% will be used by humans.
•Estimated that 90% of population will live in tropical and subtropical areas where (protein-rich) plant sources contribute 80% of total caloric intake.
•Combined nitrogen is the most common limiting nutrient in agriculture
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Consumes 1.4% of total fossil fuels annually
Why chemical fertilizers aren’t the answer
•Production of nitrogenous fertilizers has “plateaued” in recent years because of high costs and pollution
•Estimated 90% of applied fertilizers never reach roots and contaminategroundwater
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Host plant Bacterial symbiont
Alfalfa Rhizobium melilotiClover Rhizobium trifoliiSoybean Bradyrhizobium japonicumBeans Rhizobium phaseoli
Rhizobium-legume symbioses
Beans Rhizobium phaseoliPea Rhizobium leguminosarumSesbania 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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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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Pea Plant
R. leguminosarumnodules
Pink color is leghaemoglobin a protein that carries oxygen to the bacteroids
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Physiology of a legume nodule
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Very early events in the Rhizobium-legume symbiosis
rhizosphereFlavonoidsnod-geneinducers
Nod-factor
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NodD
pSym
Sinorhizobium meliloti
nod-gene inducers from alfalfa roots
(specificity)
chromosome
plasmid
pSym
nod genes
activated NodD positively regulates
nod genes
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Nod factor biosynthesis
Nod factor R-group “decorations”
determine host
NodM
NodC
Nod Factor: alipooligosaccharide
determine host specificity
NodB
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Rhizobium
Nod factor(specificity)
Invasion through infection tube
Attachment and infection
Flavonoids
From Hirsch, 1992.New Phyto. 122, 211-237
Formation ofnodule primordia
Bacteroiddifferentiation
Nitrogenfixation
Flavonoids(specificity)
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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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Nodule development
Enlargement of the nodule, nitrogen
fixation and exchange of
nutrients
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The Nodulation Process
• Chemical recognition of roots and Rhizobium
• Root hair curling
• Formation of infection thread• 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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Inoculation of a mutated Sinorhizobium strain does not transfer fixed N to the plant
Genes & Development 11:1194, 1997
wild-type mutant
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glnB�10
6 days
wt
glnBP5
7 days
wt
Genes & Development 11:1194, 1997
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Azorhizobium caulinodans
on
Sesbania
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Non-symbiotic nitrogen fixation
CyanobacteriaAnabaenaNostoc
Aquatic:
Terrestrial and rhizosphere-associated:
AzospirillumAzotobacterAcetobacterKlebsiellaClostridium
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Plant-associated nitrogen fixation:the endophytes and epiphytes
Acetobacter diazotropicus
Lives as an endophyte of sugarcane and various other monocots and some dicots
On sugarcane
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-Co +Co
A nitrogen-fixing fern
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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Symptoms of crown gall
http://ohioline.osu.edu/hyg-fact/3000/3054.html
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Agrobacterium tumefaciens “transforms” plant cells
Transgenes produce OPINES, unique amino acid-like molecules,as well as plant hormones
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The End
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1 Enhancing survival of nodule forming bacterium by improving competitiveness of inoculant strains
2 Extend host range of crops, which can benefit from biological
Current approaches to improving biological nitrogen fixation
2 Extend host range of crops, which can benefit from biological nitrogen fixation
3 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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FixLI
O2 inactivates nitrogenase
Bacterial membranes
Nitrogen fixation genes are repressed by oxygen
O2
Heme oxidizedFixL inactive
FixJ
nif regulonnifA nif nif fix
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Rhizobium’s bad brother: Agrobacterium tumefaciens
Crown gall on rose
Opines are an Agrobacterium-specific C- source to feed future generations
Crown gall on rose
and on grapevine
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Rhizobium’s bad brother: Agrobacterium tumefaciens
Crown gall on rose
Opines are an Agrobacterium-specific C- source to feed future generations
Crown gall on rose
and on grapevine
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Malateto bacteria
nitrogen-fixing bacteroid
containing Rhizobium
Exchange of nutrients during Rhizobium-legume symbiosis
NH4+
to plantN2 NH4
+
TCAATP ADP+Pi
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