Biological Nitrogen Fixation

Nitrogen Cycle
1013 gms
4.5 x 1014 Atmospheric N2 Atmospheric
gms pool

Biological N2 Industrial N2 Electrical N2 Denitrification

fixation fixation fixation

Soil pool
NH NO NO
3 2 3

Chemo- Decaying Plant

autotrophs biomass biomass Biological
pool
Nitrosomonas
Nitrobacter Animal biomass
 Free-living and symbiotic nitrogen-fixing micro-organisms

Klebsiella, Azotobacter, Clostridium, most are anaerobic or micro-aerophilic

Symbiotic microorganisms
2. Gram-negative bacteria (rhizobia) → Fabaceae (Legume plants)
3. Gram-positive actinomycete (fungi)→Alder, Myrtle, Casuarina (Woody
species)
4. Cyanobacteria → Dicots, Ferns, Cycads

Rhizobium and related bacteria have characteristc host-range

Sinorhizobium melilotii Medicago (alfalfa), Trigonella (fenugreek),

Melilotus (sweet clover)
S. fredii Glycine, Vigna
Rhizobium leguminosarum Vicia, Pisum, Cicer, Phaseolus
Mesorhizobium loti Lotus
Root nodules
 Nodules

In determinate nodules, such as in soybean, bean etc., the nodule meristem is

inactive and all infected cells are at the same stage of differentiatio and infection
 Nodules
uninfected and infected cells

uninfected cells
Indeterminate nodule of clover; newly-
infected cells stain heavily
Senescent cells

In indeterminate nodules, such as in clover, alfalfa, pea, etc., the nodule meristem
is active and cell division constantly takes place at the tip
 Cross-talk between legumes and rhizobia
Rhizobia live in soil and grow heterotrophically in the presence of
organic compounds

Cross-talk between rhizobia and legume roots include flavonoid exudates

from roots inducing the production of Nod factors by the rhizobia. Nod
factors induce rapid changes in the root hair cells, leading to the
formation of infection thread, nodulins, differentiation of rhizobia into
bacteroids, development of nodule and eventually, nitrogen fixation.

Alfalfa produces luteolin and 4-hydroxymethyl chalcone, which induce

Nod gene expression in Sinorhizobium meliloti

Of the thousands of flavonoids produced by plants, only a few are

involved in stimulating nod gene expression, which is extremely specific
 Bacterial genes used in symbiosis

Stage Genes Functions

Gene regulation nodD, nolR Activate/repress

transcription
Nodule formation nod, nol, noe Enzymatic synthesis
Host recognition of nod factors

Infection thread exo, lps Synthesis of extra-

cellular polysaccharides
Differentiation bacA Signal transduction
Bacteroid metabolism dct import of dicarboxylic
acids
Regulation of nitrogen fixL, fixJ, nifA, nifK Response to oxygen,
fixation transcriptional control

Nitrogen fixation nifHDK, other nif Nitrogenase enzyme,

cofactors, electron
ttransport
 Nod gene expression

Rhizobial cell

Nod D proteins Flavonoid

Nod D proteins Flavonoids
signals
+ve and –ve
regulators

P nod gene

nod D

Bacterial genes required for nodule formation – nod

Present as clustered in the chromosome or plasmid or can be
dispersed
Common nod genes: nodA, -B,-C are present in all rhizobia; nodD
is the transcription factor, responsible for their expression
 Nod factors

Nod factors are N-acetylated chitooligosaccharides with a backbone of N-acetyl glucoseamine

Mutants having a different acyl group are defective

in nodulation

Mutants of rhozobia defective in eliciting the calcium-spiking,root-hair curling response,

formation of infection thread, etc. have been mapped to nod genes

Thus, nod factors may have a wide and diverse role to play in the cross-talk between the
symbiotic partners

Oldroyd and Downie, Ann

Rev Pl. Biol. 59: 519-146
(2008)
Nod factors

Fucosyl group

Mutants lacking the

groups cannot infect
root-hairs, although can
establish infection in
cracked epidermis

Arabinosyl group

Oldroyd and Downie, Ann

Rev Pl. Biol. 59: 519-146
(2008)
Nod factors

Arttached by Nod X

Oldroyd and Downie, Ann

Rev Pl. Biol. 59: 519-146
(2008)
 Nod signaling pathway

Receptors have Leucine-

rich repeats and lysine
motifs

Second messenger targets

a potassium channel in
the nuclear membrane

Second messenger may

initiate changes at the
membrane and involve
phospholipases

Calcium- and calmodulin-

dependent protein
kinases and other
transcription factors have
been identified which
undertake signal-
transduction

Lectins, localized in the

cell membrane also play a
Nod-dependent calcium-signaling is restricted to nuclear membranes
role in binding of rhizobia
Cat-ion channels in the nuclear membrane are involved
to the root hairs

Oldroyd and Downie, Ann

Rev Pl. Biol. 59: 519-146
(2008)
Perception of nod factors takes place in the
epidermis

Oldroyd and Downie, Ann

Rev Pl. Biol. 59: 519-146
(2008)

Receptor-like kinases
(LysM) have been identified
as receptors for Nod factors
in the epidermal layers

They are responsible for the

specificity of Nod factor-
legume interaction

Although binding between

the putative receptors and
Nod factor has not yet been
shown
 Formation of infection thread
The response at the epidermal cell layers (leading to bacterial
infection) and that at the cortical cell layers (leading to nodule
morphogenesis) are distinct responses

Infection threads are trans-cellular channels harboring rhizobia with

associated lignification of adjacent cell walls

Transposon tagging in Lotus japonicus has produced mutants (nin),

which show root hair curling but no infection thread

NIN protein encodes a transcriptional factor, which acts as a positive

regulator for both epidermal and the cortical responses

NORK mutant of alfalfa does not show Ca+2spiking

NORK protein encodes a LRR kinase, but does not bind to Nod factors

Various Nod mutants are also deficient in establishing mycorrhizal

associations, indicating a common receptor, upstream of Ca+2 response

Nod factors are extremely potent signaling molecules

Can initiate depolarisation of membranes at a concentration of 10-9M
ENOD gene expression is detectable within 6 hrs. of Nod factor
treatment and cortical cell division in 18-30 hours.

Both nod factors and surface polysaccharides impact the formation of

infection thread

Oldroyd and Downie, Ann

Rev Pl. Biol. 59: 519-146
(2008)
 Nodulins

Symbiosome Nucleus

L
bO2
Lb

N-assimilation
N enzymes
H4+ Ureides
Amides
 Nodulins

Plant proteins expressed only in response to rihizobial infection

Expressed only in the developing and mature nodules

Early nodulins: expressed during nodule morphogenesis

Late nodulins: expressed during the release of rhizobia and fixation of nitrogen

Nodulin Location Mol. Wt. (X103 KDa) Function

Leghemoglobin Infected Cell 16 Oxygen

Cytoplasm Carrier

Sucrose synthase Cytoplasm 100 Carbon

Metabolism

Uricase Uninfected 35 Nitrogen

Cell assimilation

Glutamine Infected cell 40 Nitrogen

Synthetase cytoplasm and plastid assimilation
Flow of metabolites during symbiotic
nitrogen fixation

N
2

Glutamine
Sucrose
Bacteroid
N
Malate
N2 H4+
Asparagine
 Metabolism of fixed nitrogen

Uninfected cell Infected cell

Allantoic Bacteroid
Acid
N NH3
Allantoic
2
Acid
N
+
Glutamate H4
Glutamine Glutamine
Plastid
Allantoin Peroxisome
αKG
Allantoin Glutamate Glutamine
CO2
αKG
O
Uric acid OAA Aspartate
2
Purine
IMP biosynthesis PRPP
XMP Xanthine

Uric acid Uric acid Xanthine

 Control of nif gene expression
ATP ADP

Fix-L Fix-L P
In response to low oxygen
conditions, Fix-L /Fix-J
signaling cascades activates
gene expression of the nif Fix-J P Fix-J
genes
Fix-L is a heme-containing
membrane-anchored nifA P
protein
Phosphorelay cascade is
initiated in the absence of
oxygen NifA
The control is reversible
NifA is the transactivator for
most nif genes

Nif gene promoters

 e-/ H+ e-/ H+ e-/ H+
H
H H+
E E E E
1H
+ H H
0 2 3
H N
2 2
NH
e-/ H+ N H
3
2 E3 (H+)N2 2
E7-
e-/ H+
e-/ H+
e-/ H+

E6= NH E5= N- E4= N-

Theoretical scheme for the reduction of nitrogen on the MoFe
NH
protein of nitrogenase e-/ H+
3
 N2 + 8H+ + 8e- + 16 ATP → 2NH3 + H2 + 16ADP + 16 Pi

Nitrogenase Complex

4Fe-4S
4Fe-4S
red.
red.
Fd 2ATP Dinitrogenase
ox
8e P Cluster Fe-Mo
Dinitrogenase
2X Cofactor
reductase
4Fe-4S Mo-7Fe-9S

Fd 4Fe-4S 4Fe-4S
red ox. ox.
2ATP

N2 + 2NH3 +
 FeMoCo

S
Histidine
S S
Fe Fe
N N
Mo S Fe S
Fe Fe

Homocitrate S
S Fe Fe
S
S
Fe3MoS Fe4S

Molecular model of molybdenum-iron cofactor

Mo can be replaced by V or Fe in some free-living diazotrophs but is
invariably present in all symbiotic bacteria

Heldt
 The Fe-protein cycle

T
Fe-P Mo-Fe-P
e T n

e T
Fe-P Mo-Fe-P
e T n
D
D
N
2Pi
2
D e
Fe-P Mo-Fe-P
e D
T n+
N
T
H4+

D
Fe-P Mo-Fe-P
e D n+