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.

 Enzyme

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Metabolism 

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Metabolism is the sum of all chemical transformations taking place in a cell or organism.

It occurs through a series of enzyme-catalyzed reactions that constitute metabolic pathway.

In this multistep sequence, the product of one step serves as the substrate for the next step. These metabolic intermediates are called metabolites.

Multiple metabolic pathways remain interconnected to form a meaningful well-coordinated network that determines the overall functioning of the cell.

Complexity of metabolic pathways as depicted in KEGG: Kyoto Encyclopedia of Genes and Genomes

 

             

Catabolism

• The degradative phase of metabolism 
• Organic nutrient molecules (carbohydrates, fats, and proteins) are converted into smaller, simpler end products (such as lactic acid, CO2, NH3) 
• Release energy:
• A part is conserved in the form of ATP and reduced electron carriers (NADH, NADPH, and FADH2)
• The rest is lost as heat

Anabolism

• The biosynthetic phase of metabolism
• Small, simple precursors are built up into larger and more complex molecules (lipids, polysaccharides, proteins, and nucleic acids)
• Require input of energy (phosphoryl group transfer potential of ATP and reducing power of NADH, NADPH, and FADH2)

Some pathways can be either anabolic or catabolic, depending on the energy conditions in the cell. They are referred to as amphibolic pathways.

Example: Citric acid cycle

Metabolic pathways may linear, branched or cyclic.

Catabolic pathways are convergent and anabolic pathways divergent.

• Converging catabolism: convert several starting materials into a single product
• Diverging anabolism: yielding multiple useful end products from a single precursor
• Cyclic pathway: one starting component is regenerated in a series of reactions that converts another starting component into a product

Metabolic processes are regulated in three principal ways:

• by controlling the accessibility of substrates
• by controlling the amounts of enzymes
• by controlling their catalytic activities

Controlling the accessibility of substrates

• At low substrate concentration (near or below Km), rate of reaction depends on substrate concentration.
• Controlling the flux of substrates also regulates metabolism. 
• The transfer of substrates from one compartment of a cell to another (e.g., from the cytosol to mitochondria) can serve as a control point.
• Compartmentalization segregates opposed reactions. For example, fatty acid oxidation takes place in mitochondria, whereas fatty acid synthesis takes place in the cytosol. 

Controlling the amounts of enzymes

• The amount of a particular enzyme depends on:
• its rate of synthesis
• its rate of degradation
• The level of most enzymes is adjusted primarily by changing the rate of transcription of the genes encoding them.

Controlling their catalytic activities

The catalytic activity of enzymes is controlled by: 

• Reversible allosteric control: Key enzymes in many biosynthetic pathways is allosterically inhibited by a metabolic intermediate or coenzyme or the  ultimate product of the pathway. 
• Reversible covalent modification: Activation/deactivation of enzymes by phosphorylation/deposphorylation of particular residues.
• Hormones-mediated reversible modification of key enzymes: Hormones or growth factors coordinate metabolic relations between different tissues, often by regulating the reversible modification of key enzymes.

Classification of the Bryophytes (Goffinet, Buck and Shaw; 2008)

Mosses display a wide range of structural diversity from which relationships and lineages can be inferred. Historically, bryophyte systematics has focused on peristome teeth complexity and sex organ distribution to define taxonomic units.

Modern classifications reflect systematic concepts proposed by Fleischer (1920) and Brotherus (1924, 1925), wherein the peristomate mosses were classified as follows:

• Nematodontous
• Arthrodontous

Based on the position of perichaetia, arthrodontous mosses were sub-divided into:

• Acrocarpous 
• Pleurocarpous

Based on the architecture of the outer ring of peristome teeth, arthrodontous mosses were sub-divided into:

• Haplolepideous
• Diplolepideous

Sphagnum, Andreaea, Andreaeobryum, and Takakia represent additional groupings distinguished by the presence of a pseudopodium and the mode of sporangial dehiscence.

The classification of the Bryophyta is undergoing constant revisions, particularly in the light of phylogenetic inferences. The classification proposed by Goffinet, Buck, and Shaw (2008) builds on those presented by Buck & Goffinet (2000) and Goffinet & Buck (2004). Their classification is outlined below:

BRYOPHYTA

        SUPERCLASS I

                CLASS TAKAKIOPSIDA: Leaves divided into terete filaments; capsules dehiscent by a single longitudinal spiral slit; stomata lacking. Example: Takakia

Takakia sp.

        SUPERCLASS II

                        CLASS SPHAGNOPSIDA: Branches usually in fascicles; leaves composed of a network of chlorophyllose and hyaline cells; setae lacking; capsules elevated on a pseudopodium; stomata lacking. Example: Sphagnum

Sphagnum

        SUPERCLASS III

                CLASS ANDREAEOPSIDA: Plants on acidic rocks, generally autoicous; cauline central strand absent; calyptrae small; capsules valvate, with four valves attached at apex; seta absent, pseudopodium present; stomata lacking. Example: Andreaea

Andreaea

                CLASS ANDREAEOBRYOPSIDA: Plants on calcareous rocks, dioicous; cauline central strand lacking; calyptrae large and covering whole capsule; capsules valvate, apex eroding and valves free when old; stomata lacking; seta present. Example: Andreaeobryum

Andreaeobryum

        SUPERCLASS V

         CLASS OEDIPODIOPSIDA: Leaves unicostate; calyptrae cucullate; capsule symmetric and erect, neck very long; stomata lacking; capsules gymnostomous. Example: Oedipodium

Oedipodium

                    CLASS POLYTRICHOPSIDA: Plants typically robust, dioicous; cauline central strand present; stems typically rhizomatous; costa broad, with adaxial chlorophyllose lamellae; peristome nematodontous, mostly of (16)32–64 teeth. Example: Polytrichum

Polytrichum

                    CLASS TETRAPHIDOPSIDA: Leaves unicostate; calyptrae small conic; capsule symmetric and erect, neck short; peristome nematodontous, of four erect teeth. Example: Tetraphis

Tetraphis

                       CLASS BRYOPSIDA: Plants small to robust; leaves costate or not, typically lacking lamellae; capsules operculate; peristome at least partially arthrodontous. Example: Bryum

Bryum

Features of the classification system:

• The rank of superclass is adopted to unite all arthrodontous mosses in one taxon (i.e. Superclass V).
• Although this system of classification aimed at accepting only monophyletic taxa, given the limited number of ranks available, paraphyletic taxa were incorporated (e.g. Bryanae). 
• Effort was made to resolve the relationships among genera of the Hypnanae.
• Due to the lack of sequence data and availability of information of many taxa at regional level only, the authors have been unable to expand their classification system to a global scale.                                        

Further reading: 

Buck WR, Shaw AJ, Goffinet B. Morphology, anatomy, and classification of the Bryophyta. In: Bryophyte Biology: Second Edition, ed. B. Goffinet & A. J. Shaw. Cambridge University Press; 2008. p. 55–138.