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Bioteknologi Penambatan Nitrogen Simbiotik

nitrogenase
N2 NH3
Greensulfur Bacteroides
Prokaryotes Spirochetes
Deinococci

Green, Chlamydiae
nonsulfur
Thermotoga Gram positive

Cyano
bacteria
Rhizobium
a Bradyrhizobium
Sinorhizobium
Agrobacterium
b Azospirillum
d
Desulfoivbrio g Herbaspirillum
E. coli
Klebsiella
Azotobacter
Proteobacteria
b
Ralstonia

Burkholderia

Rickettsia
Devosia
Azorhizobium
Sinorhizobium
Methylobacterium
Bradyrhizobium
Proteobacteria Nitrobacter, Afipia

Mesorhizobium,

a
Aminobacter, Phyllobacterium
Bartonella

Brucella
Sinorhizobium
Ensifer
Rhizobium
Agrobacterium

-Rhizobial symbioses have evolved ~10 times


-Nested parasites & non-symbionts Sawada et al. 2003
Biological nitrogen fixation:

nitrogenase
N2 + 8 flavodoxin- + 8H+ + 16 MgATP2- + 18 H2O
2NH4+ + 2OH- + 8 flavodoxin + 16 MgADP- + 16H2PO4- + H2

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
Ammonia assimilatory cycle:
How nitrogen enters biological pathways Amino acids
proteins
purines
Pathway 1 pyrimidines

+ GS
NH4 + glutamate + ATP glutamine + ADP + Pi

GOGAT
glutamate a-ketoglutarate + glutamine

Pathway 2
GDH
NH4+ + a-ketoglutarate glutamate

Amino acids
proteins
A growing population must eat!

•Combined nitrogen is the most common limiting nutrient in agriculture

•Estimated that 90% of population will live in tropical and subtropical areas
where (protein-rich) plant sources contribute 80% of total caloric intake.

•In 1910 humans consumed 10% of total carbon fixed by photosynthesis, by


2030 it is predicted that 80% will be used by humans.
Why chemical fertilizers aren’t the answer

Consumes 1.4%
of total fossil
fuels annually

•Production of nitrogenous fertilizers has “plateaued” in recent years


because of high costs and pollution

•Estimated 90% of applied fertilizers never reach roots and contaminate


groundwater
Rhizobium-legume symbioses

Host plant Bacterial symbiont

Alfalfa Rhizobium meliloti


Clover Rhizobium trifolii
Soybean Bradyrhizobium japonicum
Beans Rhizobium phaseoli
Pea Rhizobium 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


legume

Fixed nitrogen Fixed carbon


(ammonia) (malate, sucrose)

rhizobia
Obvious signs of nodulation by common rhizobial species

MEDICAGO LOTUS
(alfalfa) (birdsfoot trefoil)
Pea Plant

R. leguminosarum
nodules

Pink color is leghaemoglobin a protein


that carries oxygen to the bacteroids
Physiology of a legume nodule
Physiology of a legume nodule
• Rhizobia engulfed into
nodule cells
• Differentiate into
bacteroids

© Simms
Very early events in the Rhizobium-legume symbiosis

Flavonoids rhizosphere Nod-factor


nod-gene
inducers
Sinorhizobium meliloti

chromosome
NodD
nod-gene inducers
from alfalfa roots plasmid
(specificity)

pSym
activated NodD
positively regulates
nod genes

nod genes
Nod factor NodM
biosynthesis

NodC
Nod factor R-group
“decorations”
determine host
specificity
NodB

Nod Factor: a
lipooligosaccharide
Rhizobium Attachment and infection

Nod factor
(specificity)
Invasion through infection tube
Flavonoids
(specificity)

Bacteroid Nitrogen
differentiation fixation

Formation of
nodule primordia
From Hirsch, 1992.
New Phyto. 122, 211-237
• Signals early in infection
– Complex handshaking between legume root
and rhizobium

Correct
signal

Incorrect
signal
Rhizobium encoding GFP from jellyfish as a marker

Infection thread

(From Quaedvlieg et al. Plant Mol. Biol. 37: 715-727, 1998)


Bacteria divide as they traverse infection thread
Nodule development

Enlargement of the
nodule, nitrogen
fixation and
exchange of
nutrients
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
Inoculation of a mutated Sinorhizobium strain does
not transfer fixed N to the plant

wild-type mutant Genes & Development


11:1194, 1997
wt glnB10

6 days

7 days

wt glnBP5
Genes & Development
11:1194, 1997
Azorhizobium caulinodans

on

Sesbania
Actinorhizal symbioses
Frankia in alder root nodules
Frankia vesicles

Frankia root nodules


Spores &
hyphae
Current approaches to improving biological
nitrogen fixation

1 Enhancing survival of nodule forming bacterium by improving


competitiveness of inoculant strains

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?
Symbiotic plasmid of Rhizobium etli

Víctor González et al. Genome Biology 2003 4(6):R36


plasmid 42d
M. loti MAFF303099
plasmid NGR234a
M. loti MAFF303099
B. japonicum
S. meliloti pSymA

The nodulation genes nodABCDIJ are represented in blue


The nitrogen-fixation genes nifHDKNEXAB, fixABCX and fdxBN are
represented in yellow

Víctor González et al. Genome Biology 2003 4(6):R36


Effect of nifH overexpression on
nitrogen fixation and plant growth

Growth response of P. vulgaris plants (45 dpi) inoculated with R. etli strains in
the greenhouse. Images: 1, Noninoculated nonfertilized; 2, inoculated with
CFN42 (wt); 3, inoculated with HP55 (nifHcDK); 4, noninoculated fertilized with
10 mM KNO3–2 mMNH4NO3.
The End