Biological Nitrogen Fixation

Biological Nitrogen Fixation

Nitrogen Cycle 1013 gms 4.5 x 1014 gms

Atmospheric pool

Atmospheric N2

Biological N2 fixation

Industrial N2 fixation

Electrical N2 fixation

Denitrification

NH

NO

NO

3

2

3

ChemoDecaying autotrophs biomass Nitrosomonas Nitrobacter

Plant biomass

Animal biomass

Soil pool

Biological pool

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

uninfected and infected cells

Nodules

uninfected cells

Indeterminate nodule of clover; newlyinfected 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

Gene regulation Nodule formation Host recognition

Functions

nodD, nolR Activate/repress transcription nod, nol, noe Enzymatic synthesis of nod factors

Infection thread

exo, lps Synthesis of extracellular 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 cofactors, electron ttransport

Nitrogenase enzyme,

Nod gene expression Rhizobial cell

Nod D proteins Nod D proteins

Flavonoids

+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

Flavonoid signals

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 Leucinerich 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 calmodulindependent protein kinases and other transcription factors have been identified which undertake signaltransduction

Nod-dependent calcium-signaling is restricted to nuclear membranes Cat-ion channels in the nuclear membrane are involved

Lectins, localized in the cell membrane also play a role in binding of rhizobia 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 factorlegume 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

Nucleus

Symbiosome

L bO2 Lb

N H4+

N-assimilation enzymes

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 Location Mol. Wt. (X103 KDa)

Nodulin

Leghemoglobin Infected Cell Cytoplasm

16 Carrier

Oxygen

Sucrose synthase Cytoplasm

100 Carbon Metabolism

Uricase

35

Uninfected Cell

Glutamine Synthetase

Function

Nitrogen assimilation

Infected cell 40 cytoplasm and plastid

Nitrogen assimilation

Flow of metabolites during symbiotic nitrogen fixation

N 2

Sucrose Malate

Glutamine

Bacteroid N2

N H4+ Asparagine

Metabolism of fixed nitrogen Infected cell

Uninfected cell

Bacteroid

Allantoic Acid Allantoic Acid

N

NH3

2 N + Glutamate H4

Glutamine

Plastid

Allantoin

Peroxisome

αKG

Allantoin

Glutamate

CO2

αKG

O 2

Glutamine

OAA

Uric acid IMP

Uric acid

Aspartate

Purine PRPP biosynthesis XMP Xanthine Uric acid

Xanthine

Glutamine

Control of nif gene expression

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

ATP

ADP

Fix-L

Fix-L P

Fix-J

P

nifA P

NifA

Fix-J

e-/ H+

e-/ H+ E + 1H

E 0

e-/ H+ E 2

H

E

H H

NH

e-/ H+

3

3

H+ H

N

2

2

N

H

2

E7-

H

E3 (H+)N2 2 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

Fd

4Fe-4S red.

4Fe-4S red. 2ATP

Dinitrogenase

ox

Dinitrogenase reductase Fd red

4Fe-4S ox.

8e

P Cluster 2X 4Fe-4S

Fe-Mo Cofactor Mo-7Fe-9S

4Fe-4S ox. 2ATP

N2 +

2NH3 +

FeMoCo

S

Histidine S S

Mo

Homocitrate

Fe

S

Fe Fe

S

Fe N

N S

S

Fe Fe

S

S Fe3MoS

Fe

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

Fe-P

e

T

Mo-Fe-P

n

T

e e

Fe-P

T T

Mo-Fe-P

n

D D

Fe-P T

N

2Pi

e

2

D D

Mo-Fe-P

e n+

N H4+

T

Fe-P e

D D

Mo-Fe-P

n+