•ɴɪᴛʀᴏɢᴇɴ ғɪxᴀᴛɪᴏɴ ᴀɴᴅ ᴄʏᴄʟᴇ-ɪɴᴛʀᴏᴅᴜᴄᴛᴏʀʏ sᴇʟᴇᴄᴛᴇᴅ ᴀsᴘᴇᴄᴛs ɪɴ ᴍɪᴄʀᴏʙɪᴀʟ,ᴘʟᴀɴᴛ & ᴀɴɪᴍᴀʟ ʙɪᴏᴄʜᴇᴍɪsᴛʀʏ

•ɴɪᴛʀᴏɢᴇɴ ғɪxᴀᴛɪᴏɴ ᴀɴᴅ ᴄʏᴄʟᴇ-ɪɴᴛʀᴏᴅᴜᴄᴛᴏʀʏ sᴇʟᴇᴄᴛᴇᴅ ᴀsᴘᴇᴄᴛs ɪɴ ᴍɪᴄʀᴏʙɪᴀʟ,ᴘʟᴀɴᴛ & ᴀɴɪᴍᴀʟ ʙɪᴏᴄʜᴇᴍɪsᴛʀʏ

-ʙɪᴏᴄʜᴇᴍɪsᴛʀʏ

1. Objectives

2. Understand how nitrogen is mobilized in the environment via the nitrogen cycle.

3. Explain the different modes of nitrogen fixation

4. Explain the process of biological nitrogen fixation and appreciate its importance.

5. Understand the underlying biochemistry and genetics of biological nitrogen fixation.

• Nitrogen cycle :-

Nitrogen is present in many forms in the biosphere. For the most part, this large reservoir of nitrogen is not directly available to living organisms. Acquisition of nitrogen from the atmosphere requires the breaking of an exceptionally stable triple covalent bond between two nitrogen atoms (N N) to produce ammonia (NH3) or nitrate (NO3– ). These reactions, known as nitrogen fixation, can be accomplished by both industrial and natural processes. Nitrogen fixation is the process of conversion of atmospheric N2 (nitrogen)to NH3 (ammonia). It plays an important role in the mobilization of nitrogen in the ecosystem. Its importance stems from the fact that despite its high concentration in the atmosphere, most of the living organisms are unable to directly utilize free nitrogen for synthesis of nitrogenous biomolecules viz. amino acids, purines, pyrimidines and others. Hence there is a need to convert atmospheric nitrogen in utilizable form, a process termed as nitrogen fixation. Once fixed in ammonium or nitrate, nitrogen enters a biogeochemical cycle and passes through several organic or inorganic forms before it eventually returns to molecular nitrogen (Figure 1). The ammonium (NH4 + ) and nitrate (NO3 –) ions are generated through fixation or released through decomposition of soil organic matter.

2. Concept Map

Concept Map-Nitrogen Fixation.
Concept Map-Nitrogen Fixation.
Schematic representation of nitrogen cycle
Schematic representation of nitrogen cycle

• Importance of nitrogen in the living cell:-

Nitrogen is an essential constituent of cell amino acids and nucleic acids. However, the higher organisms are unable to directly utilize the atmospheric form.

This nitrogen is fixed in various forms such as nitrous acid, nitric acid and ammonia in the soil from where the higher organisms take up nitrogen for their protein and nucleic acid anabolisms.

The deficiency of nitrogen manifests in the form of growth inhibition.

• Abiotic nitrogen fixation:-

The nitrogen atoms in the nitrogen molecule are held together by means of triple bonded nitrogen atoms.

This confers exceptional stability to the nitrogen molecule and is responsible for the inert nature of nitrogen.

This accounts for the high activation energy of the chemical reaction required to convert nitrogen to ammonia by Haber-Bosch process.

This process is the starting point for the manufacture of many industrial and agricultural chemicals.

Nitrogen fixation by Haber-Bosch process
Nitrogen fixation by Haber-Bosch process

Lightning converts water vapour and oxygen into highly reactive hydroxyl free radicals, free hydrogen atoms, and free oxygen atoms that attack molecular nitrogen (N2) to form nitric acid (HNO3).

This nitric acid subsequently falls to Earth with rain.

Photochemical reactions between gaseous nitric oxide (NO) and ozone (O3) that produce nitric acid (HNO3) also account for nitrogen fixation by abiotic means.

Biological nitrogen fixation :-

It remains the single most important method of nitrogen fixation in nature meeting most of the nitrogen demand of plants.

This process is confined to a select few species of free-living and symbiotic bacteria and archae in association with plants. These organisms assimilate gaseous nitrogen (N2) as nitrogen source and are known as diazotrophs.

Nitrogen fixing free-living microorganisms
Nitrogen fixing free-living microorganisms
Symbiotic nitrogen fixing bacteria
Symbiotic nitrogen fixing bacteria

What makes the nitrogen fixers unique ?

The nitrogen fixing bacteria are endowed with special nitrogen fixing apparatus which include the both genetic as well as the biochemical components actively involved in the reduction of nitrogen to ammonia. Additionally, they are equipped with a mechanism to preserve anoxic conditions to protect the above molecular apparatus from oxidation.

• The nif genes, encode the nitrogenase enzyme responsible for converting N2 to NH3. Expression of these genes is therefore a key requirement for nitrogen fixation.

• The nitrogenase enzyme is very sensitive to oxygen and is prone to oxygen inhibition. Hence, the maintenance of anoxic conditions for the activity of the nitrogenase enzyme becomes mandatory.

• The symbiotic nitrogen fixers create an anerobic environment by means of leghaemoglobin in the root nodules of the symbiont plant host. This is a molecule analogous to the haemoglobin in animals that transports oxygen to respiring tissues in animals. Leghaemoglobin, however has ten times more affinity for oxygen than human haemoglobin β chain.

• The free-living nitrogen fixers do so by different mechanisms, Azotobacter, for instance, maintains reduced oxygen conditions through their high levels of respiration. Gloeotheca, evolves O2 photosynthetically during the day and fix nitrogen during the night. Facultative aerobes and anaerobes generally fix nitrogen under anaerobic conditions. For the photosynthetic anaerobe such as Rhodospirillum and non-photosynthetic anaerobe such as Clostridium, oxygen does not pose a threat owing to their anaerobic habit.

• Presence of reduced ferredoxin or reduced flavodoxins that act as electron acceptors are critical for the reduction reaction of N2 to NH3.

• The process of biological nitrogen fixation, together with the subsequent assimilation of NH3 into an amino acid, is highly energy intensive, consumes about 16 -24 ATPs per nitrogen. Hence the organism involved in nitrogen fixation should have an abundant supply of ATP molecules.

• Mo6+ is present in nitrogenase and nitrate reductase and Fe is present in the nitrogenase enzyme structure. The symbiotic microorganisms obtain these minerals from their plant partners, while the free living ones most probably obtain them from soil.

• Presence of nitrate and ammonia in the cell’s environment is detrimental to nitrogen fixation.

Symbiotic nitrogen fixation :-

Root nodules of legumes, actinorrhiza and Gunnera are the dwelling sites of the symbiotic nitrogen-fixing bacteria.

The formation of these root nodules themselves may be induced by the symbiotic bacteria or may be stem glands that develop independently of the symbiotic bacteria.

Besides, the occurrence of root nodules is not even mandatory for the purpose of symbiotic nitrogen fixation. Some grasses are known to be symbiotically associate with nitrogen fixing bacteria that mainly colonize the root surface.

The nodules are pink in colour due to the presence of leghaemoglobin, a molecule analogous to the haemoglobin in animals that transports oxygen to respiring tissues in animals. Leghaemoglobin, however has ten times more affinity for oxygen than human haemoglobin β chain.

• Root nodules on a legume root
• Root nodules on a legume root

• Establishment of symbiosis :-

There is no obligatory relationship between rhizobia and legumes, as each is capable of survival without the other. However, under nitrogen-limited conditions, the symbionts seek out one another through an elaborate exchange of chemical signals. The signalling is followed by infection process and the development of nodules. All these processes are driven by underlying genetic mechanisms.

Genetics of nitrogen fixation :-

Nod and nod genes

· Genes specific to nodules in plants are called nodulin (Nod) genes

· Genes that participate in nodule formation in rhizobia are called nodulation (nod) genes

The nod genes are classified as common nod genes or host-specific nod genes. The common nod genes are nodA, nodB, and nodC. On the other hand,nodP, nodQ, and nodH; or nodF, nodE, and nodL arehost-specific. nodD generegulates the function of other nod genes, it is constitutively expressed as the protein NodD.

• Chemotaxis of Rhizobia

The first stage in the formation of the symbiotic relationship between the nitrogen-fixing bacteria and their host is migration of the bacteria toward the roots of the host plant. This migration is a chemotactic response mediated mainly by flavonoids and betaines, secreted by the roots. Following chemotaxis, rhizobia attach to the root hairs and release Nod factors. These attractants activate the rhizobial NodD protein, which then induces the expression other nod genes involved in the synthesis of Nod factors (Table 3). Nod factors are lipochitooligosaccharides (LCOs) essentially glycolipids, with 3-5 units of N-acetyl glucosamine molecules. The terminal residue is attached to a 16-18 C long fatty acyl side chain at the C2 position. The functional group substitutions on the oligosaccharide moieties are variable and result in a great diversity among Nod factors. The response of legumes is specific to the Nod factor and is mediated by lectin receptors present on root hairs. Nod factors activate these lectins, increasing their hydrolysis of phosphoanhydride bonds of nucleoside di- and triphosphates. This lectin activation directs rhizobia to anchor to the cell walls of the root hair. The role of these molecules in the initiation of root nodule formation have been summarized in Figure.

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•  Role of nod genes in Rhizobium sp.
• Role of nod genes in Rhizobium sp.
• Biochemistry of rhizobial chemotaxis
• Biochemistry of rhizobial chemotaxis

Infection of plant cells by rhizobia :-

Following the chemotactic movement of rhizobia towards the plant cells, infection of the plant cells by the

rhizobia occurs in the following stages.

a) Formation of infection thread: The Nod factors induce a curling of the root hair cells which enclose the rhizobia. The cell wall of the root hair degrades in these regions allowing the bacterial cells direct access to the plant plasma membrane. The plasma membrane then invaginates and fuses with Golgi derived membrane vesicles to form an infection thread at the site of infection.

b) Generation of nodule primordia: Deeper into the root cortex, near the xylem, cortical cells dedifferentiate and start dividing, forming a distinct area within the cortex, called a nodule primordium, from which the nodule will develop. The nucleoside uridine and ethylene control cell division in the root primordia.

c) Release of bacterial cells into root primordia: The infection thread filled with proliferating rhizobia elongates through the root hair and cortical cell layers, in the direction of the nodule primordium.

When the infection thread reaches specialized cells within the nodule, its tip fuses with the plasma membrane of the host cell, releasing bacterial cells that are packaged in a membrane derived from the host cell plasma membrane.

d) Branching of the infection thread: Inside the nodule, the infection thread branches, enabling the bacteria to infect many cells.

e) Formation of bacteroids: At first, the bacteria continue to divide, and the surrounding membrane increases in surface area to accommodate this growth by fusing with smaller vesicles. Soon thereafter, upon an undetermined signal from the plant, the bacteria stop dividing and begin to enlarge and to differentiate into nitrogen-fixing endosymbiotic organelles called bacteroids. The membrane surrounding the bacteroids is called the peribacteroid membrane.

f) Development of vascular system of the nodule: The nodule develops 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 stages in the development of the root nodule have been depicted in Figure.

• Steps in the nodule formation in legumes
• Steps in the nodule formation in legumes

Biochemistry of nitrogen fixation :-

The overall reaction of BNF may be represented as follows:

N2 + 8e + H+ +16 ATP ‡ 2NH3 + H2+ 16 ADP + 16 Pi

The reduction of N2 to 2NH3 is coupled to the reduction of two protons to evolve H2. This reaction is catalysed by the nitrogenase enzyme complex.

• Nitrogenase enzyme structure :-

This enzyme consists of the MoFe protein (component I) and the Fe protein (component II) (Figure 8). The MoFe protein has four subunits, with a total molecular mass of 180 to 235 kDa, depending on the species. This subunit has the dinitrogenase activity. The Fe protein has the dinitrogen reductase activity and is the smaller of the two components and has two identical subunits of 30 to 72 kDa each, depending on the organism. Each subunit contains an iron–sulfur cluster (4 Fe and 4 S2– ) that participates in the redox reactions involved in the conversion of N2 to NH3. Each subunit has two Mo–Fe–S clusters. Both the subunits are required for the reduction of N2 to NH3 and are susceptible to irreversible inactivation by O2.

Activation of nitrogenase enzyme :-

For activation, dinitrogenase reductase requires ATP which gets hydrolysed to ADP and inorganic phosphate. Only such activated dinitrogenase can mediate the transfer of one electron at a time from reduced ferredoxin or flavodoxin to dinitrogenase which contains the sites for its substrate. Such repeated one electron transfer processes result in building up of pool of electrons in dinitrogenase which are finally used to reduce nitrogenase substrates.In the overall nitrogen reduction reaction, ferredoxin serves as an electron donor to the Fe protein, which in turn hydrolyzes ATP and reduces the MoFe protein. The MoFe protein then can reduce numerous substrates such as hydrogen cyanide, azide or acetylene, although under natural conditions it reacts only with N2 and H+ .

• Nitrogenase enzyme complex
• Nitrogenase enzyme complex

Factors influencing nitrogenase activity :-

The availability of Molybdenum in the soil is necessary for the formation of the nitrogenase enzyme. The hydrogen produced during the process of nitrogen fixation has an inhibitory effect on nitrogenase. To prevent this, the nitrogen fixing organisms also produce a membrane bound enzyme called uptake hydrogenase, in order to consume the hydrogen produced during nitrogenase activity. Oxygen is a strong inhibitor of nitrogenase activity. The problem of oxygen is countered by various mechanisms in different nitrogen fixers. The anaerobic nitrogen fixers, for instance, take care of this problem by their very anaerobic mode of nutrition. The aerobic bacteria, such as Azotobacter, protect their nitrogenase by Fe-S redox protein and by adjusting the rate of aerobic respiration such that anaerobic environment is created within the cell engaged in N2-fixation. Klebsiella pneumoniae overcomes the oxygen problem by repressing the production of nitrogenase enzyme in presence of oxygen. Cyanobacteria like Nostoc and Anabaena have heterocysts as the exclusive sites of nitrogen fixation under aerobic growth conditions. They lack photosystem II activity which results in loss of oxygen production during photosynthesis. Besides, the heterocysts also have a glycolipid layer laid in the wall of the heterocyst which prevent oxygen entry from the external environment. Leghaemoglobin formed in Rhizobium acts as the reservoir of oxygen for the bacteroid for aerobic respiration as well as to bind and quench the diffusing oxygen within the nodule.

Mechanisms to tackle the oxygen problem:-

The problem of oxygen inhibition of nitrogenase enzyme is a crucial one and various mechanisms are adopted to overcome this problem. These include anaerobic respiration, modified respiration rate, regulation of nitrogenase gene expression and the presence of specially modified cells known as heterocysts in the nitrogen fixing species. Leghaemoglobin is yet another mechanism of preventing the damage due to oxygen. Let us look at each of these mechanisms in more detail.

Leghaemoglobin :-

To solve the oxygen-toxicity problem, the bacteria in root nodules are bathed in a solution of the oxygen binding heme protein leghaemoglobin, produced by the plant for which the heme may be contributed by the bacteria. Leghaemoglobin binds all available oxygen so that oxygen cannot interfere with nitrogen fixation efficiently delivers the oxygen to the bacterial electron-transfer system. Leghaemoglobin formed in Rhizobium.

acts as the reservoir of oxygen for the bacteroid for aerobic respiration as well as to bind and quench the diffusing oxygen within the nodule. In the part a) of the figure is the artificially colored electron micrograph of a thin section through a pea root nodule. Symbiotic nitrogen-fixing bacteria, or bacteroids are seen as red structures inside the nodule cells, surrounded by the peribacteroid membrane which is blue.

Heterocysts :-

The heterocysts are specialized nitrogen fixing cells of filamentous cyanobacteria such as Nostoc and Anabaena. They are exclusive sites of nitrogen fixation. They fix nitrogen using the nitrogenase enzyme. They create a microanaerobic environment for dealing with the oxygen problem by various mechanisms such as by synthesizing three additional cell walls. One of these cell walls is a glycolipid cell wall. These cell walls act as a hydrophobic barrier to oxygen. Secondly the photosystem II which is responsible for the liberation of oxygen is absent in them. The heterocyst cells selectively degrade their photosystem II by means of specific proteases. Further, the glycolytic enzymes are also overexpressed in the heterocyst cells. This increases the rate of glycolysis to meet the increased energy demand of nitrogen fixation. Cyanobacteria like Nostoc use oxygen scavenging proteins such as cyanoglobin (GlbN) for oxygen scavenging and thus creating an anaerobic microenvironment for nitrogen fixation to occur. This protein is specifically localized to the peripheral membrane of the heterocyst. One in about 9-15 cells in the cyanobacteria filament, develops into a heterocyst. In the later stages of the heterocyst development, there is accumulation of the polypeptide known as cyanophycin. This is important for the storage of the fixed nitrogen in the heterocyst as well as to prevent the diffusion of oxygen from the adjacent vegetative cells.

Klebsiella pneumoniae overcomes the oxygen problem by repressing the production of nitrogenase enzyme in presence of oxygen. Since efficient operation of nitrogenase requires considerable amounts of ATP and reducing power, synthesis and activity of this enzyme in response to environmental stimuli are subject to strict regulatory controls at transcriptional and post-translational levels. In photosynthetic bacteria, the physiological regulation of nitrogenase activity involves inactivation by covalent modification of the Fe protein of nitrogenase. This inactivation can be reversed by an activating factor which is an extrinsic membrane protein. The primary inhibitor or repressor of nitrogenase is oxygen which interferes with biological nitrogen fixation at different levels. At genetic level, oxygen represses the nitrogenase synthesis by down regulation of the expression of nif genes. Furthermore, due to its oxidative properties oxygen causes irreversible damage to the enzyme by degrading the Fe-S cofactors. Dinitrogenase reductase the iron protein is more sensitive to inactivation by Oxygen than dinitrogenase the Molybdenum Iron protein. The nitrogenase activity of the cells might be restored partially or completely by reestablishing anaerobic conditions after respiring the traces of oxygen present in the culture. Furthermore, once the anaerobic conditions are set, new nitrogenase enzymes will be synthesized. Ammonium is another inhibitor of the nitrogenase. It represses both the synthesis and activity of nitrogenase enzyme. NtrB/C and GlnB/K are the two-component systems which sense the fixed nitrogen status of the cell and accordingly regulate the nif operon and nifA gene. The gene product of nifA which is a member of enhancer binding proteins (EBPs) is a transcriptional activator of nif structural genes in bacteria and thereby regulates the activity of nif genes. The presence of ammonium ion in the culture medium causes a decrease in the expression levels of nif operon and nifA gene and results in a decrease in hydrogen production. At the post-translational level, the nitrogenase enzyme activity is reversibly inactivated in a way that an ADP ribose group from NAD+ is attached to an arginine residue in one subunit of the homodimeric NifH protein resulting in NifH inactivation (switch-off). This process is catalysed by dinitrogenase reductase ADP ribosyltransferase (DraT). However, this inactivation is reversible such that when the added ammonium is exhausted by cellular metabolism, the ADP-ribose group is removed by dinitrogenase reductase activating glycohydrolase (DraG) leading to NifH activation (switch-on). However, nitrogenase biosynthesis is strongly stimulated by light. It was observed that nitrogenase represents up to 40% of the cytoplasmic proteins in Rb. capsulatus under high illumination. It is also worth noting that a diurnal pattern of illumination (one involving alternating periods of light and dark) rather than continuous light results in a more stable nitrogenase activity.

Anaerobic respiration :-

Free-living nitrogen fixing bacteria are both aerobic, facultative or anaerobic. Anaerobic nitrogen fixing bacteria may be either facultative anaerobes or obligate anaerobes. Facultative organisms, which are able to grow under both aerobic and anaerobic conditions, generally fix nitrogen only under anaerobic conditions. The anaerobic nitrogen fixers take care of the oxygen problem by their very anaerobic mode of nutrition. For them oxygen does not pose a problem, because it is absent in their habitat. These anaerobic organisms can be either photosynthetic Rhodospirillum for example, or non-photosynthetic Clostridium, for example. Anaerobic respiration is a form of respiration using electron acceptors other than oxygen. These include sulphate, nitrate, sulphur or fumarate are used. These terminal electron acceptors have smaller reduction potentials than O2, meaning that less energy is released per oxidized molecule. Anaerobic respiration therefore does not pose any problem of oxygen inhibition to the nitrogenase enzyme in general energetically less efficient than aerobic respiration. These nitrogen fixing species include, Clostridium acetobutylicum,Clostridium beijerinckii, Rhodospirillum rubrum, Enterobacter agglomerans and Enterobacter oryzae.

High respiration rate :-

The aerobic bacteria, such as Azotobacter, protect their nitrogenase by adjusting the rate of aerobic respiration such that anaerobic environment is created within the cell engaged in nitrogen fixation.Azotobacter spp. have the highest metabolic rate of any organism. They play a vital role in any ecosystem by making nitrogen available to all organisms. The cysts of Azotobacter are spherical and consist of a highly vacuolar central body a two-layer shell. The inner intine and an outer exine. The cyst helps in surviving extreme conditions.

• Azotobacter cyst
• Azotobacter cyst

Summary

In this lecture we learnt about:

· Nitrogen cycle in the environment

· Abiotic and biotic modes of nitrogen fixation

· Biological nitrogen fixation in the Rhizobium-legume system

· Genetic and biochemical basis of BNF

· Factors that affect nitrogenase enzyme function

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