Fismik Metabolisme Nitrogen - ordinary words · Microbial Physiology- Nisa RM 4 The nitrogen cycle...

Microbial Physiology- Nisa RM 1

Nitrogen Metabolism

Nisa Rachmania MubarikMajor Microbiology Department of Biology, IPB


Nitrogen is constantly cycled in the environment, both within organisms and between organisms (free amino acids in the environment are rapidly taken up by bacteria).

Nitrogen Cycle

Microbial Physiology- Nisa RM 3http://www.biology.ed.ac.uk/research/groups/jdeacon/microbes/nitrogen.htm (13-9-08)

The total fixed nitrogen will be locked up in the biomass or in the dead remains of organisms (shown collectively as "organic matter").

Nitrogen Cycle


The nitrogen cycle consists of five main processes:1. Fixation. 2N2 + 3H2 → 2NH3. This is an extremely specialised process, and is

performed by a number of diazotrophs, such as Anabaena (a cyanobacterium), and Rhizobium (the symbiotic bacterium found in legume root nodules).

2. Nitrification. This is the oxidation of ammonia to oxyanions. The initial oxidation to nitrite, 2NH3 + e− + 3O2 → 2NO2− + 2 H2O + 2H+, is performed by bacteria such as Nitrosomonas. Nitrite is less toxic than ammonia, but is still toxic; high levels of nitrite can kill many aquatic organisms. Oxidation to nitrate, 2NO2− + O2 →2NO3− is performed by Nitrobacter. The end product here, nitrate (NO3-), is even less toxic than nitrite, and can be used by many plants as a nitrogen source.

3. Denitrification. Some bacteria, such as Pseudomonas, are able to use nitrate as a terminal electron acceptor in respiration: 2NO3

− + 12H+ + 10e− + N2 + 6H2O. 4. Assimilation. Plants assimilate nitrogen in the form of nitrate and (rarely)

ammonium. The nitrate is first reduced to ammonium, then combined into organic form, generally via glutamate. Animals generally assimilate nitrogen by simply breaking protein down into amino acids and rearranging them.

5. Decay (ammonification) and excretion. When plants and animals decay, putrefying bacteria produce ammonia from the proteins they contain. Animals also produce breakdown products such as ammonia, urea, allantoin and uric acid from excess dietary nitrogen. These compounds are also targets of ammonification by bacteria.


Nitrogen is essential to amino acid (protein), base (DNA) and tetrapyrrole(heme) synthesis. It is very difficult to convert gaseous dinitrogen to biologically available forms (ammonia, etc). It takes 930 kJ mol−1 to break the very strong N≡N bond.

∆G0 (−16 kJ mol−1), the activation energy is so huge, that this spontaneous reaction occurs at a completely negligible rate.


Mechanism of Biological Nitrogen Fixation

Biological nitrogen fixation can be represented by the following equation, in which two moles of ammonia are produced from one mole of nitrogen gas, at the expense of 16 moles of ATP and a supply of electrons and protons (hydrogen ions):

N2 + 8H+ + 8e- + 16 ATP = 2NH3 + H2 + 16ADP + 16 PiThis reaction is performed exclusively by prokaryotes using an enzyme complex termed nitrogenase.

http://www.biology.ed.ac.uk/research/groups/jdeacon/microbes/nitrogen.htm


The nitrogenase reaction itself is:N2 + 6e− + 6H+ + 12ATP → 2NH3 + 12ADP + 12PiThis uses a lot of ATP!

Like Rubisco, nitrogenase is prone to an additional hydrogenase reaction:N2 + 8e− + 8H+ + 16ATP → 2NH3 + 16ADP + 16Pi +H2This wastes a certain amount of ATP, but is unavoidable, as nitrogenase just isn't specific enough.


This enzyme consists of two proteins - an iron protein and a molybdenum-iron protein. The reactions occur while N2 is bound to the nitrogenaseenzyme complex. The Fe protein is first reduced by electrons donated by ferredoxin. Then the reduced Fe protein binds ATP and reduces the molybdenum-iron protein, which donates electrons to N2, producing HN=NH. In two further cycles of this process (each requiring electrons donated by ferredoxin) HN=NH is reduced to H2N-NH2, and this in turn is reduced to 2NH3.

Complex Nitrogenase


Pathway for converting (fixing)

atmospheric nitrogen, N2, into organic nitrogen, NH3.

http://www.cartage.org.lb/en/themes/sciences/botanicalsciences/PlantHormones/PlantHormones/PlantHormones.htm


Nitrogenase, showing iron, molybdenum and ADP cofactors.

Nitrogenase is the enzyme that reduces nitrogen to ammonia. It has two components, a reductase, which is a homodimer containing Fe4S4 clusters, which supplies electrons to nitrogenase from pyruvate; and the nitrogenaseproper, which is a heterotetramer containing Fe4S4 and a molybdenum-FeScluster, which supplies electrons and protons to nitrogen.

Microbial Physiology- Nisa RM 11Myoglobin and leghaemoglobin are only 10% homologous, but have extremely similar tertiary structures.

Nitrogenase is very readily destroyed by oxygen within minutes (t½ = 10 min in air). Legumes reduce [O2] in the vicinity of Rhizobium using leghaemoglobin (Km for O2 = 20 nM, 6 times smaller than haemoglobin). Leghaemoglobin is very similar in shape to animal myoglobin, but has a quite different primary structure. An alternative strategy is that of the cyanobacterium Anabaena, which differentiates into heterocysts with an oxygen-impermeable cell wall.


A point of special interest is that the nitrogenase enzyme complex is highly sensitive to oxygen. It is inactivated when exposed to oxygen, because this reacts with the iron component of the proteins.

It could be a major problem for the aerobic species such as cyanobacteria(which generate oxygen during photosynthesis) and the free-living aerobic bacteria of soils, such as Azotobacter and Beijerinckia. These organisms have various methods to overcome the problem.

Like other nitrogenases, Azotobacter nitrogenase is oxygen-sensitive, but it is believed that the extremely high respiration rate of Azotobacter(possibly the highest of any living organism) soaks up free oxygen within the cells and protects the nitrogenase by maintaining a very low level of oxygen in their cells.

Azotobacter species also produce copious amounts of extracellularpolysaccharide (as do Rhizobium species in culture- Exopolysaccharides). By maintaining water within the polysaccharide slime layer, these bacteria can limit the diffusion rate of oxygen to the cells.


In the symbiotic nitrogen-fixing organisms such as Rhizobium, the root nodules can contain oxygen-scavenging molecules such as leghaemoglobin, which shows as a pink colour when the active nitrogen-fixing nodules of legume roots are cut open. Leghaemoglobin may regulate the supply of oxygen to the nodule tissues in the same way as haemoglobin regulates the supply of oxygen to mammalian tissues.

Some of the cyanobacteria have yet another mechanism for protecting nitrogenase: nitrogen fixation occurs in special cells (heterocysts) which possess only photosystem I (used to generate ATP by light-mediated reactions) whereas the other cells have both photosystem I and photosystem II (which generates oxygen when light energy is used to split water to supply H2 for synthesis of organic compounds).


Nitrification

The term nitrification refers to the conversion of ammonium to nitrate This is brought about by the nitrifying bacteria, which are specialised to gain their energy by oxidising ammonium, while using CO2 as their source of carbon to synthesise organic compounds. Organisms of this sort are termed chemoautotrophs - they gain their energy by chemical oxidations (chemo-) and they are autotrophs (self-feeders) because they do not depend on pre-formed organic matter.

The nitrifying bacteria are found in most soils and waters of moderate pH, but are not active in highly acidic soils. They almost always are found as mixed-species communities (termed consortia). The accumulation of nitrite inhibits Nitrosomonas, so it depends on Nitrobacter to convert this to nitrate, whereas Nitrobacterdepends on Nitrosomonas to generate nitrite.

Nitrosomonas europaea(phase contrast microscopy 2,200X).

Nitrobacter


Energy generation in Nitrosomonas. Only two enzymes, ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO) are involved in the oxidation of ammonia to nitrite. http://dwb.unl.edu/Teacher/NSF/C11/C11Links/www.bact.wisc.edu/microtextbook/metabolism/lithotrophs.html

The reduction of ammonia by nitrifying bacteria (Nitrosomonas)NH3 + 1 1/2 O2 HNO2 + H2O

Nitrification


Nitrogen uptake and conversion by various soil bacteria

Images from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com)


Denitrification

Denitrification refers to the process in which nitrate is converted to gaseous compounds (nitric oxide, nitrous oxide and N2) by microorganisms.

The nitrogen cycle is completed by denitrifying bacteria, which convert nitrates back to nitrogen gas. They include bacteria like Pseudomonas, Alkaligenesand Bacillus which can use nitrate as terminal electron acceptor for respiration.

2NO3− + 12H+ + 10e− → N2 + 6H2O

Denitrifying organisms are characterised by (1) a supply of oxidisable organic matter, and (2) absence of oxygen but availability of reducible nitrogen sources. A mixture of gaseous nitrogen products is often produced because of the stepwise use of nitrate, nitrite, nitric oxide and nitrous oxide as electron acceptors in anaerobic respiration.

These two processes can be a problem for farmers: fertilisers are often based on ammonium salts, which bind well to negatively-charged soil particles. However, activity of nitrifying bacteria oxidise the ammonium to nitrate, which is much more readily leached from soil. Denitrifying bacteria worsen this problem by exhaling even this nitrate back into the air as (di)nitrogen gas.


Ammonification

The biochemical process whereby ammoniacal nitrogen is released from nitrogen-containing organic compounds. Soil bacteria decompose organic nitrogen forms in soil to the ammonium form. This process is referred to as ammonification.

The amount of nitrogen released for plant uptake by this process is most directly related to the organic matter content. The initial breakdown of a urea fertilizer may also be termed as an ammonification process.

In the plantAlanine (an amino acid) + deaminating enzyme --------> ammonia + pyruvicacid, or in the soilRNH2 (Organic N) + heterotrophic (ammonifying) bacteria ---------> NH3(ammonia) + R.

In soils NH3 is rapidly converted to NH4+ when hydrogen ions are plentiful (pH< 7.5)


Nitrogen - Fixing BacteriaEnzymes that fix nitrogen are widely distributed in bacteria.Free Living N fixers --(they fix N2 on their own). Free living nitrogen fixers that generate ammonia for their own use (e.g. bacteria living in soil but not associated with a root) (30 % of all N2 fixed in world)

Azotobacter (aerobic, Gram negative). AzospirillumKlebsiella (facultatively anaerobic). Clostridium (anaerobic, Gram positive).

Photosynthetic bacterium free living Anabaena (Cyanobacteria, some)Anaerobic:RhodospirillumPurple sulphur bacteriaPurple non-sulphur bacteriaGreen sulphur bacteria

Symbiotic N fixers are associated with plants and provide the plant with nitrogen in exchange for the plant's carbon and a protected home (70 % of all N2 fixed in world) Rhizobium + Fabaceae (old names Leguminosae). Rhizobium gets sugars and anaerobic conditions, and the legume gets fixed nitrogen.Frankia (actinomycete, Gram positive)+ Alnus (alder tree).Anabaena + Azolla (water fern).


Azospirillum is a highly efficient species of non-symbiotic bacteria capable of fixing atmospheric nitrogen and making it available to the plant. Azospirillum is found in loose association with roots, and can fix Nitrogen even under microaerophyllic conditions.

Azospirillum species have been shown to fix nitrogen when growing in the root zone (rhizosphere) or tropical grasses, and even of maize plants in field conditions. In both cases the bacteria grow at the expense of sugars and other nutrients that leak from the roots. However, these bacteria can make only a small contribution to the nitrogen nutrition of the plant, because nitrogen-fixation is an energy-expensive process, and large amounts of organic nutrients are not continuously available to microbes in the rhizosphere. This limitation may not apply to the bacteria that live in root nodules or other intimate symbiotic associations with plants.


Azotobacter species can fix nitrogen in the rhizosphere of several plants. There are around six species in the genus, some of which are motile by means of peritrichous flagella, others are not. They are typically polymorphic, i.e. of different sizes and shapes. Their size of the cells ranges from 2-10 µm long and 1-2 µm wide. Old cells tend to form thick-walled, optically refractile cysts, which have capsules consisting largely of alginates and other polysaccharides, enhancing their resistance to heat, desiccation and adverse environmental conditions. However, these cysts cannot withstand the extreme temperatures which bacterial endospores can. Under favourable environmental conditions, the cysts germinate and grow as vegetative cells.

http://www.microbiologybytes.com/video/Azotobacter.html


Cyanobacteria (blue green algae) are single or multi-cell photosynthetic organisms. Most of them can assimilate atmospheric nitrogen under reduced oxygen pressure. Some can do nitrogen fixation under ordinary oxygen pressure. and most of them have large cells, called heterocyst, specialized for nitrogen fixation. Cyanobacteria are either free-living or in symbiosis with plants.

Symbiotic cyanobacteria provide ammonia to the host plants, and the frequency of heterocysts is higher than in free-living ones. Some plants among moss, ferns, and cycads have these symbiotic cyanobacteria. In angiosperms, only Gunnera are such plants.

Anabaena


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 also form symbiotic associations with other organisms such as the water fern Azolla and cycads.

Azolla is useful as a "soybean plant in rice field", because it can assimilate atmospheric nitrogen gas owing to the nitrogen fixation by cyanobacterialiving in the cavities located at the lower side of upper (dorsal) lobes of leaf.

All Azolla species live in symbiosis with a blue green alga Anabaena azollaewhich is able to fixate sufficient nitrogen for both itself and its host plant. In exchange Azolla provides the Anabaena with a protected environment and a fixed source of carbon.

Azolla can, therefore, grow on the water deficient in nitrogen compounds, and is high in nitrogen and protein. It fixes nitrogen as high as 3-5 kg N per ha per day under the optimum condition.


A symbiotic association of cyanobacteriawith cycads

The first image shows a pot-grown plant. The second image shows a close-up of the soil surface in this pot. Short, club-shaped, branching roots have grown into the aerial environment.

These aerial roots contain a nitrogen-fixing cyanobacterialsymbiont.

Anabaena azollae

A symbiotic association of cyanobacteria with Azolla

Azolla pinnata


Symbiotic N fixers

It has been estimated that nitrogen fixation in the nodules of clover roots or other leguminous plants may consume as much as 20% of the total photosynthate.Nodule TypesTwo basic types of root nodules are produced by legumes. One type is ephemeral and lasts days or a few weeks. This is called a determinate structure. It has a short, predestined life-span. Consequently, new nodules are being formed as the root grows in the soil and others are being lost on older parts of the root system. Soybean nodules are like this. The nodule is a spherical elaboration of the ground tissue system in the root cortex and has a specialized anatomy. The second nodule type is illustrated by Clover. In this case the nodule has an apical meristem which functions for many months. It is called Indeterminate in that meristematic activity is theoretically unlimited. These are elongate compared to the determinate nodules. They aretumescent, (swollen). The apical meristem continuously produces new cells which become infected with bacteria from older cells. These nodules have a much more extensive vascular system which surrounds the nitrogen-fixing parenchyma that occupy the center of the nodule. This central location is not a coincidence.


Legume/Rhizobium Nodules are Red. This is due to the production of Leghaemoglobin(Leg = Legume) which sequesters oxygen.

This helps to create a low oxygen environment.

http://www.botany.hawaii.edu/faculty/webb/bot410/Roots/RootSymbioses.htm


Sections through Soybean Determinate Nodules

Note: the extent of cells infected with N-fixing bacteria. The sclerenchyma that helps reduce the oxygen levels inside the nodule. The many vascular bundleswhich facilitate transport of sugar into the nodule and nitrogenous compounds out of the nodule.

The term bacteroid refers to the growth form of the Bacteria in N-fixing Cells. These are structurally modified compared to free-living bacteria.


The roots of an Austrian winter pea plant (Pisum sativum) with nodules harbouring nitrogen-fixing bacteria (Rhizobium). Root nodules develop as a result of a symbiotic relationship between rhizobial bacteria and the root hairs of the plant. (A) The bacteria recognize the root hairs and begin to divide, (B) entering the root through an infection thread that allows bacteria to enter root cells, (C) which divide to form the nodule.


Acacia koa (koa) is a legume tree. It produces Indeterminate nodules which are similar to clover.

Koa produces adventitious nodules from aerial parts of koa stems. These nodules appear to have a similar anatomy compared to root nodules.

Koa root nodules (above) and adventitious stem nodule (right)

Cross Section of an Infected koaNodule: The dark areas are parenchyma cells that contain N-fixing Bacteria.

http://www.botany.hawaii.edu/faculty/webb/bot410/Roots/RootSymbioses.htm


Long section through an clover nodule: The Apical Meristem is at the upper right. The red cells are the most recently infected cells.

Apical region of an clover nodule. The Meristematic cells are small and stain densely. The newly infected cells are enlarged and isodiametric.


Frankia is a genus of the bacterial group termed actinomycetes - filamentous bacteria that are noted for their production of air-borne spores. Frankia species are slow-growing in culture, and require specialised media, suggesting that they are specialised symbionts. They form nitrogen-fixing root nodules (sometimes called actinorhizae) with several woody plants of different families, such as alder(Alnus species), sea buckthorn (Hippophae rhamnoides, which is common in sand-dune environments) and Casuarina (a Mediterranean tree genus). Alder and the other woody hosts of Frankia are typical pioneer species that invade nutrient-poor soils. These plants probably benefit from the nitrogen-fixing association, while supplying the bacterial symbiont with photosynthetic products.

http://www.biology.ed.ac.uk/research/groups/jdeacon/microbes/nitrogen.htm

Figure A (below) shows a young alder tree (Alnus glutinosa) growing in a plant pot, and Figure B shows part of the root system of this tree, bearing the orange-yellow coloured nodules (arrowheads) containing Frankia.


Actinomycete root nodules from Alder (Alnus): similar nodules may fix more atmospheric nitrogen than legume/rhizobium nodules

Frankia fix nitrogen while living in root nodules on “actinorhizalplants”. Frankia thus can supply most or all of the host plants' nitrogen needs. Consequently, actinorhizal plants colonize and often thrive in soils that are low in combined nitrogen. http://web.uconn.edu/mcbstaff/benson/Frankia/FrankiaHome.htm

Fismik Metabolisme Nitrogen - ordinary words · Microbial Physiology- Nisa RM 4 The nitrogen cycle...

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