GENETIC ENGINEERING OF NITROGEN FIXATION

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GENETIC ENGINEERING OF NITROGEN FIXATION

• REPRESENTED BY
• NIVEDITA CHOUDHARY
• B.Sc. 2nd year

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Nitrogen Fixation-

• Nitrogen fixation is the process by which
  nitrogen is taken from atmosphere and converted
  into nitrogen compounds.
• Biological nitrogen fixation was discovered by
  the Dutch microbiologist Martinus Beigerinck.
• Natural processes fix about 1901012 g yr-1 of
  nitrogen through the following processes-

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Types of nitrogen fixation

• Lightning
• 2.Photochemical Reaction
• 3.Industrial Nitrogen Fixation
• 4.Biological Nitrogen Fixation

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Types of Biological Nitrogen Fixation

• Symbiotic Nitrogen Fixation
• Nonsymbiotic Nitrogen Fixation
• Symbiotic Nitrogen Fixation-
• In symbiotic nitrogen fixation the plant and
  bacteria depend on each other.
• Rates of nitrogen fixation are highly variable
  and are dependent on the bacterial strain-legume
  cultivar used, soil and other environmental
  conditions.
• 100kg of nitrogen are reported to be derived from
  symbiotic nitrogen fixation.

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• The highest amount of nitrogen fixed by the
  legumes takes place either the flowering stage or
  during pod fill stage.
• Legume symbioses- eg- rhizobium species
• Associations with Frankia- Frankia is a group
  termed Actinomycetes-filamentous bacteria that
  are noted for their production of air-borne
  spores. They form nitrogen fixing root nodules
  with several woody plants.eg-elder,sea buckthorn.
• Cyanobacterial associations-The photosynthetic
  cyanobacteria often live as free-living organisms
  in pioneer habitats such as desert soils or as
  symbionts with lichens in other pioneer
  habitats.they form nitrgen fixing nodulation.

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Nonsymbiotic nitrogen fixation

• In nonsymbiotic nitrogen fixation the bacteria
  not associated with plant but they fix nitrogen
  into the plant.
• Azotobacter and Azospirillum fix nitrogen into
  the plant by the nonsymbiotic process.
• Azospirillum is not only capable of nitrogen
  fixation but also codes for plant growth hormones
  auxins and cytokinins.
• These bacteria are the free living bacteria that
  fix nitrogen.

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Mechanism of Nitrogen fixation

• The biological process of nitrogen fixation is
  catalyzed by the nitrogenase enzyme, an enzyme
  complex containing the nitrogenase reductase and
  dinitrogenase.
• Nif genes forming a gene cluster of 24 kb
  nucleotides which are located between the genes
  encoding for histidine and shikimic acid.
• The cluster is organized in 7 operons i.e.
  transcription units (QB AL FM VSUX NE YKDH J).
  Nif HDKY operon encodes nitrogenase.

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• Nitrogenase contains the two proteins
  molybdoferredoxin and azoferredoxin.
• In most bacteria electrons are passed from
  NAD(P)H or pyruvate to ferredoxin, a FeS protein.
• Azoferredoxin transfers electrons from reduced
  flavodoxin (or ferredoxin) to molybdoferredoxin.
  Molybdoferredoxin is a a2ß2 tetramer.
• The alpha and beta subunits are similar but
  distinct and are encoded by genes nifK and nifD.
  Each tetramer contains 2 Mo and several FeS
  groups.
• Azoferredoxin is a dimer of identical subunits
  encoded by nifH and contains a single Fe4S4 group
  per dimer.

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Nodulation

• legumes release compounds called flavonoids from
  their roots, which trigger the production of nod
  factors by the bacteria. when the nod factor is
  sensed by the root, number of biochemical and
  morphological changes are occur
• 1. cell division is triggered in the root to
  create the nodule.
• 2. the root hair growth is redirected to wind
  around the bacteria multiple times until it fully
  encapsulates one or more bacteria.
• Legumes and actinorhizal plants regulate gas
  permeability in their nodules, maintaining a
  level of oxygen within the nodule.
• A nodule contains an oxygen-binding heme protein
  called leghemoglobin that gives the nodules a
  pink color.

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Steps involved in nodulation-

• Attachment of bacteria to the roots of higher
  plant-
• 1.Lectin-mediated root hair binding-
• The host-microsymbiont specificity is governed by
  a specific plant protein called lactins involved
  in recognition of compatible symbiont. It is
  supplemented by secretion of specific
  polysaccharides by the symbiont termed as callose
  which helps in attachment of rhizobia on host
  root surface.
• The rhizobial cell that infect clover posses the
  cross reactive antigen(CRA).clover roots absorb
  CRA and infective R.trifolii cells.

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• 2. Recadhesin -mediated root hair
  binding-
• A Ca2 binding bacterial protein called
  rhicadhesin also appears to be involved in
  bacterial attachment to legume root hairs.
• Calcium ions are not involved in binding of
  rhicadhesin to the root surface. calcium ions
  appears to be involved in anchoring rhicadhesin
  to the rhizobial cell surface.
• An R.l. bv. Trifolii-adhering protein,called
  RapA1,and R.l.bv.viciae rhicadhesin both are
  secreted proteins that bind calcium, bind at
  bacterial cell poles and to root hairs, and
  mediate calcium dependent agglutination.

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Gene involved in nitrogen fixation-

• Nod genes- plant root produce various type of
  flavonoids that stimulates the release of Nod
  factor by Rhizobium.
• The eight nod genes present in the Rhizobium
  bacteria. These nod genes are nod A,B,C,D,E,F,I,
  and nod J.
• nodD genes regulates the transcription of other
  nod genes.
• Nif gene nif gene isolate from
  k.pneumoniae.these nif genes are nif
  J,H,D,K,T,Y,E,N,X, U,S,V,W,Z,M,F, L, A, B, and
  nifQ.
• Nif A and nif L gene regulate the other nif
  genes.

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Phytohormones that involve in nodule formation

• Two processes infection and nodule
  organogenesis-occur simultaneously during root
  nodule formation.
• During the infection process rhizobia that are
  attached to the root hairs release Nod factors
  that induce a pronounced curling of the root hair
  cells.
• The next step is formation of the infection
  thread, an internal tubular extension of the
  plasma membrane that is produced by the fusion of
  Golgi-derived membrane vesicles at the site of
  infection.
• Ethylene is synthesized in the region of the
  pericycle, diffuses into the cortex, and blocks
  cell division opposite the phloem poles of the
  root.

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Alternative Nitrgen fixation system
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Genetic engineering of Rhizobium

• The nif and nod gene of the fast growing
  Rhizobium bacteria are located on plasmid, called
  symbiotic plasmid.
• The rhizobium bacteria have nod gene cluster for
  nodulation.
• These nod genes are nodD,A,B,C,E,F,I, and nodJ .
• The nod I gene encodes a proteins that may be
  involved in membrane transport.
• The nod E gene may help to prevent nodulation of
  legumes other than the normal host for a
  particular Rhizobium species.

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• The last of these nod genes, nod D,has been shown
  to be regulatory, controlling the transcription
  both of itself and of the other nod genes in the
  cluster.
• The plant release some flavonoids that stimulates
  the release of Nod factor by Rhizobium.

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Genetic engineering of nitrogen fixation

• Nif genes are involved in nitrogen fixation that
  are isolate from k.pneumoniae and transfer into
  E.coli.
• The Nif gene cluster required for maturation
  and regulation of the nitrogenase enzyme.
• These Nif genes are nif J,H,D,K,T,Y,E,N,X,
  U,S,V,W,Z,M,F, L, A, B,Q.
• NifL and nifA regulate the expression of all the
  other genes that synthesized the nitrogenase
  enzyme.
• The RNA polymerase bind to the Nif gene promoter
  for the initiation of transcription.

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• The ntrA gene codes for a protein called a sigma
  factor, that confers on RNA polymerase the
  ability to recognize the nif gene promoter.
• The ntrA gene and three additional
  genes-glnA,ntrB,and ntrC- are the part of a
  global system that regulates many aaspects of
  nitrogen metabolism in bacteria.

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Regulation of nif gene-
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Engineering of nif gene-

• The nif gene cluster is introduced in the
  chloroplast genome through genetic engineering.
• The nif gene does not express without ntrA gene
  because ntr gene code the sigma protein that
  required for the recognition of nif gene
  promoter.

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Methods of nif gene transformation into
chloroplast genome-

• Three methods developed for introducing
  chloroplast vector back into the organelle
  genome-
• By using Ti plasmid the gene is transferred into
  plant.
• A two step transformation in which a plasmid with
  both nucleus and chloroplast specific markers is
  first transformed into the cytoplasm and, having
  established replication within the organism,
  selection is made for the chloroplast specific
  marker on the plasmid which could only be
  expressed in the plastid.
• 3. Microinjection method to interduce cloned
  genes directly into chloroplast. By these
  processes the nif gene transferred into
  plants. 
   

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