Nitrogen fixing and genes and their genetic manipulation
Nif genes:-
1. Introduction:-
> Nif means:-
Ni = Nitrogen
f = fixation
> These are genes encoding enzymes involved in the fixation of atmospheric nitrogen.
> Nif genes also encode a number of regulatory proteins involved in nitrogen fixation.
> The nif genes are found in both free-living and symbiotic nitrogen-fixing bacteria.
> The primary enzyme encoded by the nif genes is the nitrogenase.
2. Regulation of Nif genes:- In most bacteria, regulation is done by NifA protein.
i. When there is not enough fixed nitrogen, NtrC triggers NifA expression, and NifA activates the nif genes.
ii. When there is not enough fixed nitrogen, NifL inhibit NifA expression, and nif genes remain inactive.
Reversible ADP-ribosylation:- It is an additional regulation mechanism found in Rhodospirillum rubrum. Reversible ADP-ribosylation of a specific arginine residue in the nitrogenase complex. When reduced nitrogen is present, DraG and DraT catalyze the ribosylation of arginine residue in the nitrogenase. It causes a barrier in the electron transfer flow and thereby inactivates nitrogenase activity.
3. Expression of Nif genes:-
> There are total 20 nif genes.
> nifH, nifD, and nifK:- They encode the nitrogenase subunits.
> nifE, nifN, nifU, nifS, nifV, nifW, nifX, nifB, and nifQ:- They encode proteins involved the assembly and incorporation of Fe and Mo atoms into the nitrogenase subunits.
> nifF and nifJ:- They encode proteins related to electron transfer taking place in the reduction process.
> nifA and nifL:- They are regulatory proteins in charge of regulating the expression of the other nif genes.
Nitrogen fixation:- It is a chemical process by which molecular nitrogen found in the air is converted into ammonia or related nitrogenous compounds.
1. Types of nitrogen fixation:-
a. Physical Nitrogen Fixation
b. Biological Nitrogen Fixation
a. Physical Nitrogen Fixation:-
i. Natural Nitrogen Fixation:- Under the influence of lightning and thunder, N2 and O2 of the air react to form nitric oxide (NO). The nitric oxides are again oxidized with oxygen to form nitrogen peroxide (NO2).
ii. Industrial Nitrogen Fixation:- Ammonia is produced industrially by direct combination of nitrogen with hydrogen (obtained from water) at high temperature and pressure. Later, it is converted into various kinds of fertilizers, such as urea etc.
b. Biological Nitrogen Fixation:- The conversion of atmospheric nitrogen into the nitrogenous compounds by living organisms is called biological nitrogen fixation. Only prokaryotes can fix nitrogen. Nitrogen fixation require anaerobic conditions because oxygen inactivates nitrogenase enzyme.
Hence for obligate anaerobes nitrogen fixation is easy, but in case of facultative anaerobes the nitrogen fixation occurs only in anaerobic conditions. In case of obligate aerobes the oxygen level inside the cell must be kept low for nitrogen fixation.
2. Nitrogen Fixers (Diazotrophs):- Among the earth’s organisms, only some prokaryotes like bacteria and cyanobacteria can fix atmosphere nitrogen. They are called nitrogen fixers or diazotrophs. They fix about 95% of the total global nitrogen fixed annually by natural process.
a. Asymbionts (Free living)
b. Symbionts
a. Asymbionts (Free living):-
i. Bacteria:- They add up to 10-25 kg, of nitrogen/ha/annum.
> Azotobacter (Aerobic, Saprophytic)
> Beijerinckia (Aerobic, Saprophytic)
> Clostridium (Anaerobic, Saprophytic)
> Desulphovibrio (Chemotrophic)
> Rhodopseudomonas (Photoautotrophic)
> Rhodospirillum (Photoautotrophic)
> Chromatium (Photoautotrophic)
ii. Blue Green Algae (Cyanobacteria):- Heterocysts are the special cells that fix nitrogen. They add 20-30 kg Nitrogen/ha/annum.
> Nostoc
> Anabaena
> Aulosira:- A. fertilissima is the most active nitrogen fixer in Rice fields.
> Cylindrospermum:- It is active in sugarcane and maize fields.
> Trichodesmium
b. Symbionts:- Live in close symbiotic association with other plants.
i. Blue Green Algae (Cyanobacteria):-
> Nostoc and Anabaena:- They are common symbionts in lichens, Anthoceros, Azolla and cycad roots.
> Anabaena azollae:- It is found in fronds of Azolla pinnata (a water fern). It is often inoculated to Rice fields for nitrogen fixation.
ii. Bacteria:-
> Rhizobium:- It is aerobic, gram negative nitrogen fixing bacterial symbionts of legume roots. Sesbania rostrata has Rhizobium in root nodules and Aerorhizobium in stem nodules.
> Frankia:- It is symbiont in root nodules of many non-leguminous plants like Casuarina and Alnus.
> Xanthomonas and Mycobacterium:- They occur as symbiont in the leaves of some members of the families Rubiaceae and Myrsinaceae (e.g., Ardisia).
3. Rhizobium Nitrogen Fixation:-
> Rhizobium bacteria:-
i. Free living
ii. Gram negative
iii. Aerobic
iv. Soil bacteria
> Rhizobium becomes anaerobic upon entry into roots.
> Leghaemoglobin (legHb or symbiotic Hb):-
- It is a pink coloured pigment.
- It occurs in the root nodules of leguminous plants.
- It acts as an oxygen scavenger. It provides anaerobic conditions for the nitrogenase enzyme and protects the enzyme from inactivation.
> Two main steps:-
a. Nodule formation
b. Nitrogen fixation
a. Nodule formation:- Root nodule formation is initiated, when the soil contains a low level of nitrogen. Steps of nodulation are:
i. Aggregation:- Roots of legumes secrete flavonoids, which attracts rhizobia towards the root. Rhizobia aggregate around root hairs.
ii. Developmental changes:- Rhizobia secrete nod factors, which causes stimulate many developmental changes:
- Membrane depolarization
- Curling of root hairs
- Cell division in the root cortex
- Intracellular calcium movement
iii. Infection thread:- The nod factor attaches to receptors present on the plasma membrane of the root hairs, which leads to the formation of the infection thread.
iv. Entry:- Infection thread provides the passage to bacteria to enter epidermal cells. Rhizobia then enter cortex cells, each bacterium gets surrounded by a plant-derived membrane known as symbiosome.
v. Nodulation:- Nodule formation is initiated by chemicals produced by rhizobia. It is a result of calcium dependent signal transduction pathway, which triggers biochemical changes leading to cell division and nodule formation. Cytokinin also plays an important role in nodules formation.
vi. Bacteroids:- Within nodules, bacteria get differentiated into bacteroids, which fix nitrogen. The Rhizobia stop dividing, loose cell wall and become nitrogen fixing cells as led bacteroids . Vascular tissues are developed for nodules for exchange of nutrients.
b. Nitrogen fixation:-
- The nodule serves as site for N2 fixation.
- Nodule contains nitrogenase and leghaemoglobin.
- The nitrogenase has 2 components:
i. Molybdoferredoxin (Mo-Fe protein)
ii. Azoferredoxin (Fe-protein).
- The free di-nitrogen first bound to MoFe protein and is not released until completely reduced to ammonia.
- In this process ferredoxin serves as an electron donor to Fe-protein (nitrogenase reductase) which in turn hydrolyzes ATP and reduce MoFe protein, the MoFe protein in Turn reduce the substrate N2. The electrons and ATP are provided by photosynthesis and respiration of the host cells.
- Many intermediates are formed to form ammonia (NH3).
Dinitrogen → Hydrazine → Diamine → Ammonia
- Ammonia (NH3) is immediately protonated at physiological pH to form ammonium ion (NH4+). As NH4+ is toxic to plants, it is rapidly used near the site of generation to synthesize amino acids.
Genetic manipulation of nitrogen fixation:-
> The subunits of nitrogenase from different nitrogen fixing microorganisms can be mixed to produce
functional system
> Genes can be manipulated to improve the fixation of dinitrogen.
> The microorganisms are modified in the host so that they are unable to assimilate the fixed nitrogen until the function of the nitrogenase is over and release NH4+ directly to the plants.
> The nitrogen fixation can be regulated by leguminous plants by:-
i. Reducing the number of root nodule formation
ii. Regulating the carbon flow to the microorganisms.
> The well known nitrogen fixing bacteria K. pneumoniae resembles closely to non-nitrogen fixing bacterium E.coli. The genes of these two species could be transferred and expressed in either of the organisms.
- Nif- mutants of K. pneumoniae that are deficient in fixing nitrogen are located between genes for histidine biosynthesis (his) and shikimik acid uptake (shi A).
- The his and nif regions can be actively transferred from a strain of K. pneumoniae to an E.coli
strain which require histidine.
- E.coli cells that do not require histidine anymore have acquired the ability to fix nitrogen.
- The conjugative plasmid pRDI that picked up the nif and his genes was selected and transferred to other bacterial genome.
- The gene, nifL, that serves as the repressor of nitrogen fixation can be deleted thus allowing constitutive expression of nif promoter mediated by nifA and ntrC (nitrogen regulator) gene products.
- The nitrogenase activity has been observed in E.coli that carries the nif plasmid pRDI which was very
much similar to that of K. pneumoniae strain.
> Agrobacterium tumefaciens, an obligate aerobe not resembling Klebsiella did not result in nitrogen fixing recombinants upon transfer of pRDI to itself.
> Mutants of Azotobacter vinelandii that lack either of the components of nitrogenase regained the
nitrogenase activity when pRDI was transferred to them.
> Nitrogenase can be protected from the inhibitory action of oxygen by a protein leghaemoglobin. The genes encoding these proteins can be isolated and transferred to other nitrogen fixing systems so as to
protect the nitrogenase from oxygen activity.
> Nitrogenase activity is correlated with the hydrogenase activity that evolve hydrogen hence it
requires more energy. The energy can be saved if the evolved hydrogen is further reduced to water
releasing electrons. Many nitrogen fixing bacteria possess ‘uptake hydrogenases’ which consists of two subunits HupS and HupL and it is advantageous to introduce it together with the nif genes into hosts that do not possess uptake hydrogenase system.