Biological Nitrogen Fixation

Dr. Riddhi Datta

Assistant Professor

PG Department of Botany

Barasat Government College

Dr. Riddhi Datta

Biological Nitrogen Fixation

• The process of conversion of atmospheric nitrogen into the biologically acceptable form, i.e. ammonia is called nitrogen fixation.

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• If it occurs via agency of microorganisms, it is referred to as biological nitrogen fixation.

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• The microorganisms involved are called nitrogen fixers.

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• Biological nitrogen serves as the key entry point of molecular nitrogen into the biogeochemical cycle of nitrogen.

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• Exclusively a prokaryotic domain.

Dr. Riddhi Datta

Nitrogen fixers can be free-living or symbiotic

Dr. Riddhi Datta

Association between host plants and rhizobia

Dr. Riddhi Datta

Nitrogen fixation requires anaerobic (or microanaerobic ) conditions

• The principal enzyme responsible for biological nitrogen fixation is nitrogenase.

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• Nitrogenase have sites that facilitate the high-energy exchange of electrons.

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• Oxygen is a strong electron acceptor that damage these sites and irreversibly inactivate nitrogenase.

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• So nitrogen must be fixed under anaerobic conditions.

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• Nitrogen fixers either functions under natural anaerobic conditions or creates an internal, local anaerobic environment (microanaerobic) separated from the oxygen in the atmosphere.

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• In cyanobacteria, anaerobic conditions are created in specialized cells called heterocysts. These cells lack photosystem II so they do not generate oxygen.

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• Some cyanobacteria (non-heterocyst types) can fix nitrogen under anaerobic conditions such as those that occur in flooded fields.

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• Aerobic nitrogen-fixing bacteria maintains a low oxygen concentration (microaerobic conditions) through their high levels of respiration (Ex: Azotobacter) or evolve O₂ photosynthetically during the day and fix nitrogen during the night when respiration lowers oxygen levels (Ex: Gloeothece).

Dr. Riddhi Datta

Symbiotic nitrogen fixation

• Some symbiotic nitrogen-fixing prokaryotes dwell within nodules, the special organs of the plant host that enclose the nitrogen-fixing bacteria.

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• In Gunnera, nodules are preexisting stem glands that develop independently of the symbiont.

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• In legumes and actinorhizal plants, the symbionts induce the plant to form root nodules.

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• In grasses, root nodules are not produced and the symbionts anchor to the root surfaces, around the elongation zone and the root hairs, or live as endophytes inside apoplasts.

Root nodule

Dr. Riddhi Datta

• Legumes and actinorhizal plants regulate gas permeability in their nodules.

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• Oxygen concentration is maintained at 20 to 40 nM within the nodule.

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• These levels can support respiration but are sufficiently low to avoid inactivation of the nitrogenase.

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• Nodules contain oxygen-binding heme proteins called leghemoglobins that gives them a heme-pink color, and are crucial for symbiotic nitrogen fixation.

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• Leghemoglobins have a high affinity for oxygen.

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• They increase the rate of oxygen transport to the respiring symbiotic bacterial cells substantially decreasing the steady-state level of oxygen in infected cells.

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• To continue aerobic respiration under such conditions, the bacteroid uses a specialized electron transport chain in which the terminal oxidase has an affinity for oxygen even higher than that of leghemoglobins

Microaerobic condition within nodule

Dr. Riddhi Datta

• The symbiosis between legumes and rhizobia is not obligatory.

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• Under nitrogen-limited conditions the symbionts seek each other out through an elaborate exchange of signals.

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Colonization of the rhizosphere:

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• The first stage is migration of the bacteria toward the roots of the host plant.
• This migration is a chemotactic response mediated by chemical attractants, especially (iso)flavonoids and betaines, secreted by the roots.

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Nodule Formation

Chemotactic binding of rhizobia bind to an emerging root hair

Dr. Riddhi Datta

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Establishing symbiosis requires an exchange of signals:

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• Plant genes specific to nodules are called nodulin genes.

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• Rhizobial genes for nodule formation are called

nodulation (nod) genes.

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• The nod genes are classified as:
• Common nod genes:
• nodA, nodB, and nodC
• found in all rhizobial strains
• Host-specific nod genes:
• nodP, nodQ, and nodH; or nodF, nodE, and nodL
• differ among rhizobial species and determine the host range

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• Only the regulatory nodD is constitutively expressed as NodD protein regulates the transcription of the other nod genes.
• These attractants activate the rhizobial NodD protein, which then induces transcription of the other nod genes.
• The promoter region of all nod operons, except that of nodD, contains a highly conserved sequence called the nod box.
• Binding of the activated NodD to the nod box induces transcription of the other nod genes.

Dr. Riddhi Datta

• The nod genes activated by NodD code for nodulation proteins, most of which are involved in the biosynthesis of Nod factors.

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• Nod factors are lipochitin oligosaccharide signal molecules.

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• Three of the nod proteins are required for synthesizing this basic structure:
• NodA is an N-acyltransferase that catalyzes the addition of a fatty acyl chain.
• NodB is a chitin-oligosaccharide deacetylase that removes the acetyl group from the terminal non-reducing sugar.
• NodC is a chitin-oligosaccharide synthase that links N-acetyl-d-glucosamine monomers.

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• Host-specific nod genes are involved in the modification of the fatty acyl chain or the addition of groups important in determining host specificity:
• NodE and NodF determine the length and degree of saturation of the fatty acyl chain
• NodL, influence the host specificity of Nod factors through the addition of specific substitutions at the reducing or non-reducing sugar moieties of the chitin backbone.

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• A particular legume host responds to a specific Nod factor

Establishing symbiosis requires an exchange of signals:

Dr. Riddhi Datta

• The legume receptors for Nod factors are protein kinases with extracellular sugar-binding LysM domains (for lysin motif) in the root hairs.

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• Nod factors induces oscillations in the concentrations of free calcium ions in the nuclear regions of root epidermal cells.

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• This activates LysM domains.

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• Recognition of the calcium ion oscillations requires a calcium ion/calmodulin-dependent protein kinase (CaMK) that is associated with a protein of unknown function named CYCLOPS.

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• Once the plant epidermal cell recognizes ongoing calcium ion oscillations, Nod factor–responsive transcriptional regulators directly associate with the promoters of Nod factor–inducible genes.

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• The overall process links Nod factor perception at the plasma membrane to gene expression changes in the nucleus and is called the symbiotic pathway.

Establishing symbiosis requires an exchange of signals:

Dr. Riddhi Datta

Poole et al 2018; Nat Rev Microbiol 16, 291–303

Plant flavonoids

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Production of nod factors (Lipochitooligosaccharides or LCOs)

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Bind a lysine motif (LysM) receptor heterocomplex

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Activates the leucine-rich repeat protein receptor-like kinase (SYMRK)

Triggers symbiosis (SYM) signalling pathway

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calcium oscillations in the nucleus

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calcium/calmodulin-dependent serine/threonine protein kinase (CCAMK)

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Induction of nodule formation

Dr. Riddhi Datta

Formation of infection thread and nodule organogenesis:

• Rhizobia usually infect root hairs by first releasing Nod factors that induce a pronounced curling of the root hair cells.

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• The rhizobia become enclosed in the small compartment formed by the curling.

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• The cell wall of the root hair degrades in these regions, also in response to Nod factors, allowing the bacterial cells direct access to the outer surface of the plant plasma membrane.

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• Next the infection thread (an internal tubular extension of the plasma membrane) is produced by the fusion of Golgi-derived membrane vesicles at the site of infection.

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• The thread grows at its tip by the fusion of secretory vesicles to the end of the tube.

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• The cortical cells dedifferentiate and start dividing, forming a distinct area within the cortex, called a nodule primordium, from which the nodule will develop.

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• The process is modulated by several phytohormones including cytokinin and ethylene.

Root hair exhibits abnormal curling growth, and rhizobia proliferate within the coils

Localized degradation of the root

hair wall leads to infection and formation of the infection thread from Golgi secretory vesicles of root cells

Dr. Riddhi Datta

Release of Rhizobia:

• The infection thread filled with proliferating rhizobia elongates through the root hair and cortical cell layers, in the direction of the nodule primordium.

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• When the infection thread reaches the nodule primordium, its tip fuses with the plasma membrane of a host cell and penetrates into the cytoplasm.

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• Bacterial cells are subsequently released into the cytoplasm, surrounded by the host plasma membrane, resulting in the formation of an organelle called the symbiosome.

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Rhizobia are released into the apoplast

The infection thread reaches the end of the cell, and its membrane fuses with the plasma membrane of the root hair cell

Dr. Riddhi Datta

Release of Rhizobia:

• At first the bacteria within symbiosomes continue to divide, and the surrounding symbiosome membrane (called the peribacteroid membrane) increases in surface area to accommodate this growth by fusing with smaller vesicles.

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• The bacteria then stop dividing and begin to differentiate into nitrogen-fixing bacteroids.

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• The nodule as a whole develops features like a vascular system (which facilitates the exchange of fixed nitrogen produced by the bacteroids for nutrients contributed by the plant) and a layer of cells to exclude O₂ from the root nodule interior.

The infection thread extends and branches until it reaches target cells, where vesicles composed of plant membrane that enclose bacterial cells are released into the cytosol.

Dr. Riddhi Datta

Nitrogenase enzyme complex fixes N₂

• Produces ammonia from molecular nitrogen.

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• The reduction of N₂ to 2 NH₃ is a six-electron transfer reaction.

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• It is coupled to the reduction of two protons to evolve H₂.

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• The nitrogenase enzyme complex catalyzes this reaction.

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Dr. Riddhi Datta

Nitrogenase enzyme complex fixes N₂

• The nitrogenase enzyme complex can be separated into two components—

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• Fe protein
• The smaller of the two components
• has two identical subunits
• Each subunit contains an iron–sulfur cluster (4 Fe and 4 S^2–),
• Participates in the redox reactions that convert N₂ to NH₃
• Irreversibly inactivated by O₂ with half life 30-45 seconds

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• MoFe protein
• Has four subunits,
• Total molecular mass of 180 to 235 kDa
• Each subunit has two Mo–Fe–S clusters.
• Inactivated by O₂ with half life 10 minutes

Dr. Riddhi Datta

Nitrogenase enzyme complex fixes N₂

• In the overall nitrogen reduction reaction, ferredoxin serves as an electron donor to the Fe protein,
• It, in turn hydrolyzes ATP and reduces the MoFe protein.
• The MoFe protein can then reduce numerous substrates, although under natural conditions it reacts only with N₂ and H⁺.
• The production of NH₃ from N₂ and H₂ is an exergonic reaction.
• However, industrial production of NH₃ from N₂ and H₂ is endergonic, requiring a very large energy input because of the activation energy needed to break the triple bond in N₂.

Dr. Riddhi Datta

nif (nitrogen fixation) genes

Gene products are required for symbiotic nitrogen fixation, and for nitrogen fixation in free-living N-fixing species.

Example: subunits of nitrogenase

fix (fixation) genes

Gene products required to successfully establish a functional N-fixing nodule.

No fix homologues have been identified in free-living N-fixing bacteria.

Example: regulatory proteins that monitor and control

oxygen levels within the bacteroids

Dr. Riddhi Datta

• FixL senses the oxygen level and at low oxygen tensions, it acts as a kinase on FixJ
• FixJ regulates expression of two more transcriptional regulators:
• NifA, the upstream activator of nif and some fix genes;
• FixK, the regulator of fixN (another oxgen sensor?)
• FixL is a novel hemoprotein kinase with a complex structure.
• It has an N-terminal membrane-anchoring domain, followed by the heme binding section, and a C-terminal kinase catalytic domain.
• Result:
• Low oxygen tension activates nif gene transcription and permits the oxygen-sensitive nitrogenase to function.