Biological Nitrogen Fixation

 

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Biological Nitrogen Fixation

Nitrogen fixation is the process of converting atmospheric nitrogen (N2​) into ammonia (NH3​), a form that can be used by living organisms. When this process is carried out by microorganisms, it's known as biological nitrogen fixation. It is the primary way that nitrogen enters the biogeochemical nitrogen cycle. This process is exclusive to prokaryotic organisms, which are often called nitrogen fixers. Nitrogen fixers can be either free-living or live in a symbiotic relationship with a host organism.

Examples of nitrogen fixers:

Symbiotic Nitrogen Fixation
Host Plant	N-fixing symbionts
Leguminous: legumes, Parasponia	Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium, Sinorhizobium
Actinorrhizal: alder (tree), Ceanothus (shrub), Casuarina (tree), Datisca (shrub)	Frankia
Gunnera	Nostoc
Azolla (water fern)	Anabaena
Sugarcane	Acetobacter
Miscanthus	Azospirillum
Free-Living Nitrogen Fixation
Type	N-fixing genera
Cyanobacteria (blue-green algae)	Anabaena, Calothrix, Nostoc
Other bacteria
- Aerobic	Azospirillum, Azotobacter, Beijerinckia, Derxia
- Facultative	Bacillus, Klebsiella
- Anaerobic	Clostridium, Methanococcus (archaebacterium)
- Nonphotosynthetic	Clostridium, Methanococcus (archaebacterium)
- Photosynthetic	Rhodospirillum

Association between host plant and rhizobia

Plant host	Rhizobial symbiont
Parasponia (a nonlegume)	Bradyrhizobium spp.
Soybean (Glycine max)	Bradyrhizobium japonicum (slow-growing type); Sinorhizobium fredii (fast-growing type)
Alfalfa (Medicago sativa)	Sinorhizobium meliloti
Sesbania (aquatic)	Azorhizobium (forms both root and stem nodules)
Bean (Phaseolus)	Rhizobium leguminosarum bv. phaseoli, R. tropici, R. etli
Clover (Trifolium)	Rhizobium leguminosarum bv. trifolii
Pea (Pisum sativum)	Rhizobium leguminosarum bv. vicicae
Aeschynomene (aquatic)	Photosynthetic Bradyrhizobium clade

The Nitrogenase Enzyme Complex

The enzyme complex nitrogenase is responsible for biological nitrogen fixation. This enzyme facilitates the high-energy exchange of electrons required for the reduction of N2​ to NH3​.

Components of Nitrogenase

The nitrogenase enzyme complex consists of two main components:

• Fe protein: This is the smaller of the two components and is made of two identical subunits. Each subunit contains an iron-sulfur cluster (4Fe and 4S2−) that participates in redox reactions. The Fe protein is highly sensitive to oxygen and is irreversibly inactivated within 30-45 seconds in its presence.

• MoFe protein: This component has four subunits with a total molecular mass of 180 to 235 kDa. Each subunit has two molybdenum-iron-sulfur (Mo-Fe-S) clusters. The MoFe protein is also inactivated by oxygen, but its half-life in air is about 10 minutes.

Mechanism

In the overall reaction, ferredoxin acts as an electron donor to the Fe protein. The Fe protein, in turn, hydrolyzes ATP and reduces the MoFe protein. The MoFe protein can then react with N2​ and H+ to produce ammonia and hydrogen gas. It is an exergonic reaction

Microanaerobic condition is necessary for nitrogen fixation

Because oxygen is a strong electron acceptor that can damage and inactivate nitrogenase, nitrogen fixation must occur under anaerobic (oxygen-free) or microanaerobic (low-oxygen) conditions.

• Natural anaerobic environments: Some nitrogen-fixing bacteria, like certain cyanobacteria, function in naturally anaerobic conditions such as flooded fields.

• Specialized cells: Cyanobacteria like Anabaena create anaerobic conditions in specialized cells called heterocysts. These cells lack photosystem II, preventing them from generating oxygen.

• High respiration rates: Aerobic nitrogen-fixing bacteria like Azotobacter maintain a low oxygen concentration by having high levels of respiration, which consumes oxygen.

• Temporal separation: Some organisms like Gloeothece fix nitrogen at night when respiration lowers oxygen levels and carry out oxygen-producing photosynthesis during the day.

• Nodule regulation: In symbiotic relationships, plants create a low-oxygen environment within nodules. These nodules contain leghemoglobins which are oxygen-binding proteins that give the nodules a heme-pink color. Leghemoglobins have a high affinity for oxygen and increase its transport to the respiring symbiotic bacteria, effectively reducing the free oxygen concentration to a level (20 to 40 nM) that won't inactivate nitrogenase but is sufficient for respiration.

Symbiotic Nitrogen Fixation

Symbiotic nitrogen fixers usually live in specialized structures called nodules. In Gunnera, nodules are pre-existing stem glands that develop independently of the symbiont. In legumes and actinorhizal plants, the symbionts induce the plant to form root nodules. In grasses, symbionts don't produce root nodules. Instead, they anchor to the root surfaces or live as endophytes inside the apoplasts.

Nodule formation

The symbiotic relationship between legume host plants and rhizobia bacteria is a well-studied example of biological nitrogen fixation. This is not an obligatory relationship; it's established under nitrogen-limited conditions through an elaborate exchange of signals between the plant and the bacterium.

Rhizobia migrates towards the host root hair in response to chemical attractants

1. Colonization of the Rhizosphere

The first step is the migration of rhizobia toward the plant roots. This is a chemotactic response triggered by chemical attractants, such as flavonoids and betaines, secreted by the roots.

Establishing symbiosis requires exchange of signals

2. Nodule Formation

The process of nodule formation involves a complex molecular dialogue:

• Signal Exchange: The plant and rhizobia exchange signals. The plant has nodulin genes, and the rhizobia have nodulation (nod) genes.

• Nod Genes and their functions: 

  Common Nod Genes: These genes are found in all rhizobial strains and are responsible for synthesizing the basic structure of the Nod factors. The key common nod genes and their functions are:

  • nodA: Catalyzes the addition of a fatty acyl chain to the Nod factor.

  • nodB: Acts as a chitin-oligosaccharide deacetylase, removing an acetyl group from the terminal sugar.

  • nodC: Is a chitin-oligosaccharide synthase that links N-acetyl-d-glucosamine monomers to form the chitin backbone of the Nod factor.

  Host-Specific Nod Genes: These genes differ among rhizobial species and play a vital role in determining which plant species a particular rhizobial strain can infect. They modify the basic Nod factor structure synthesized by the common nod genes.

  • nodP, nodQ, nodH, nodF, nodE, and nodL: These genes are involved in modifying the fatty acyl chain or adding specific groups to the sugar moieties of the chitin backbone. 

  Regulatory NodD Gene: The flavonoids from the plant activate the rhizobial NodD protein. NodD is constitutively expressed and its activation induces the transcription of other nod genes.

• Nod Factor Synthesis: The activated nod genes code for proteins that synthesize Nod factors, which are lipochitin oligosaccharide signal molecules. The specific composition of these Nod factors, determined by host-specific nod genes, dictates which plant species can be infected.

• Plant Perception: The plant's root hairs have protein kinase receptors with LysM domains that bind to the Nod factors. This binding induces calcium ion oscillations in the root epidermal cells, activating a complex signaling pathway called the symbiotic pathway that leads to changes in gene expression and nodule formation.

3. Infection and Nodule Organogenesis

The physical process of infection and nodule development proceeds as follows:

• Root Hair Curling: Nod factors cause the root hair to curl, trapping the rhizobia within the coils.

• Infection Thread Formation: The root hair cell wall degrades at the point of infection. An infection thread, an internal tubular extension of the plasma membrane, forms at this site by the fusion of Golgi-derived vesicles. The rhizobia proliferate and are encased within this thread.

    

Root hair curling and infection thread formation

• Nodule Primordium: The cortical cells of the plant root dedifferentiate and begin dividing to form a nodule primordium, which is the precursor to the nodule.

• Rhizobia Release: The infection thread elongates through the root hair and cortical cells toward the nodule primordium. When it reaches the primordium, its tip fuses with the host cell's plasma membrane, releasing the bacteria into the cytoplasm. The bacteria are now surrounded by a host-derived membrane, forming an organelle-like structure called a symbiosome.

Rhizobia release

• Bacteroid Differentiation: Inside the symbiosome, the bacteria divide and then differentiate into nitrogen-fixing forms called bacteroids.

• Vascular System Development: The nodule develops a vascular system to facilitate the exchange of fixed nitrogen (from the bacteroids) for nutrients (from the plant).

Nif and Fix genes

Nitrogen fixation is regulated by specific genes called nitrogen fixation (nif) genes and fixation (fix) genes. These genes encode for proteins that are essential for symbiotic nitrogen fixation in plants and for nitrogen fixation in free-living bacteria.

Nif Genes

The nif genes are the core genes required for nitrogen fixation. They are exclusively found in prokaryotes, such as bacteria and archaea. The products of these genes are directly involved in the nitrogen fixation process, with an important example being the subunits of the nitrogenase enzyme complex. This complex is responsible for catalyzing the reduction of N2​ to NH3​. The nif genes are present in both free-living and symbiotic nitrogen-fixing species.

Fix Genes

The fix genes, or fixation genes, are specifically required for the successful establishment of a functional nitrogen-fixing nodule in a symbiotic relationship. They are found in symbiotic bacteria but have not been identified in free-living nitrogen-fixing bacteria. The products of fix genes include regulatory proteins that monitor and control oxygen levels within the bacteroids (the differentiated form of rhizobia inside a nodule).

Regulation of Nif and Fix Genes

The expression of nif and fix genes is tightly regulated, primarily by oxygen levels. The symbiotic process requires anaerobic or microanaerobic conditions because the nitrogenase enzyme is irreversibly inactivated by oxygen.

• FixL and FixJ: A key regulatory mechanism involves the FixL and FixJ proteins. FixL is a hemoprotein kinase that senses oxygen levels. Under low oxygen conditions, FixL acts as a kinase on FixJ. FixJ, in turn, regulates the expression of other transcriptional regulators, namely NifA and FixK.

• NifA and FixK: NifA is an activator for the transcription of nif genes and some fix genes.

• FixK is another regulator, which may be involved in oxygen sensing.

Outcome: This regulatory cascade ensures that nif gene transcription is activated only when oxygen levels are low, allowing the oxygen-sensitive nitrogenase to function properly. This is crucial for maintaining the symbiotic relationship and successful nitrogen fixation within the nodule.