1 9/30/2010 Nitrogen Metabolism Nisa Rachmania Mubarik Major Microbiology Department of Biology, IPB 1212 Microbial Physiology- Nisa RM Nitrogen Cycle

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Nitrogen Metabolism

Nisa Rachmania Mubarik

Major MicrobiologyDepartment of Biology,IPB

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Microbial Physiology- Nisa RM

Nitrogen Cycle

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


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Nitrogen Cycle

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

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


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 tonitrite, 2NH3 + e− + 3O2 → 2NO2− + 2 H2O + 2H+, is performed by bacteria suchas Nitrosomonas. Nitrite is less toxic than ammonia, but is still toxic; high levels ofnitrite 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 beused by many plants as a nitrogen source.

3. Denitrification. Some bacteria, such as Pseudomonas, are able to use nitrate as aterminal 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 organicform, generally via glutamate. Animals generally assimilate nitrogen by simplybreaking protein down into amino acids and rearranging them.

5. Decay (ammonification) and excretion. When plants and animals decay, putrefyingbacteria produce ammonia from the proteins they contain. Animals also producebreakdown products such as ammonia, urea, allantoin and uric acid from excessdietary nitrogen. These compounds are also targets of ammonification bybacteria.

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

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

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Mechanism of Biological Nitrogen Fixation

Biological nitrogen fixation can be represented by the following equation, inwhich two moles of ammonia are produced from one mole of nitrogen gas, atthe 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 enzymecomplex termed nitrogenase.

http://www.biology.ed.ac.uk/research/groups/jdeacon/microbes/nitrogen.htmMicrobial Physiology- Nisa RM

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

Like Rubisco, nitrogenase is prone to an additionalhydrogenase reaction:N2 + 8e− + 8H+ + 16ATP → 2NH3 + 16ADP + 16Pi +H2

This wastes a certain amount of ATP, but is unavoidable,as nitrogenase just isn't specific enough.


Complex Nitrogenase

This enzyme consists of twoproteins - an iron protein and amolybdenum-iron protein.The reactions occur while N2 isbound to the nitrogenaseenzyme complex.The Fe protein is first reducedby electrons donated byferredoxin.Then the reduced Fe proteinbinds ATP and reduces themolybdenum-iron protein, whichdonates electrons to N2,producing HN=NH. In twofurther cycles of this process(each requiring electronsdonated by ferredoxin) HN=NHis reduced to H2N-NH2, and thisin turn is reduced to 2NH3.

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Pathway forconverting (fixing)

atmospheric nitrogen,

N2, into organicnitrogen, NH3.

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

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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.

Nitrogenase, showingiron, molybdenum andADP cofactors.


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Microbial Physiology-

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

Myoglobin and leghaemoglobin are only 10% homologous, but have11

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extremely similar tertiary structures. Nisa RM

A point of special interest is that the nitrogenase enzyme complex is highlysensitive to oxygen. It is inactivated when exposed to oxygen, becausethis 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 aerobicbacteria of soils, such as Azotobacter and Beijerinckia. These organismshave various methods to overcome the problem.

Like other nitrogenases, Azotobacter nitrogenase is oxygen-sensitive, butit is believed that the extremely high respiration rate of Azotobacter(possibly the highest of any living organism) soaks up free oxygen withinthe cells and protects the nitrogenase by maintaining a very low level ofoxygen 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 bacteriacan limit the diffusion rate of oxygen to the cells.


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In the symbiotic nitrogen-fixing organisms suchas Rhizobium, the root nodules can containoxygen-scavenging molecules such asleghaemoglobin, which shows as a pink colourwhen the active nitrogen-fixing nodules oflegume roots are cut open. Leghaemoglobin mayregulate the supply of oxygen to the noduletissues in the same way as haemoglobinregulates the supply of oxygen to mammaliantissues.

Some of the cyanobacteria have yet anothermechanism for protecting nitrogenase: nitrogenfixation occurs in special cells (heterocysts)which possess only photosystem I (used togenerate ATP by light-mediated reactions)whereas the other cells have both photosystem Iand photosystem II (which generates oxygenwhen light energy is used to split water to supplyH2 for synthesis of organic compounds).

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Nitrification

The term nitrification refers to the conversion ofammonium to nitrate This is brought about by thenitrifying bacteria, which are specialised to gaintheir energy by oxidising ammonium, while usingCO2 as their source of carbon to synthesiseorganic compounds. Organisms of this sort aretermed chemoautotrophs - they gain theirenergy by chemical oxidations (chemo-) and theyare autotrophs (self-feeders) because they do notdepend on pre-formed organic matter.

The nitrifying bacteria are found in most soils andwaters of moderate pH, but are not active inhighly acidic soils. They almost always are foundas mixed-species communities (termedconsortia). The accumulation of nitrite inhibitsNitrosomonas, so it depends on Nitrobacter toconvert this to nitrate, whereas Nitrobacterdepends on Nitrosomonas to generate nitrite.


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Nitrosomonas europaea(phase contrast microscopy2,200X).

Nitrobacter

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Energy generation in Nitrosomonas. Only two enzymes, ammoniamonooxygenase (AMO) and hydroxylamine oxidoreductase (HAO) are involvedin the oxidation of ammonia to nitrite.

http://dwb.unl.edu/Teacher/NSF/C11/C11Links/www.bact.wisc.edu/microtextbook/metabolism/lithotrophs.html

NH3 + 1 1/2 O2 →HNO2 + H2O

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NitrificationThe reduction of ammonia by nitrifying bacteria (Nitrosomonas)



Nitrogen uptake andconversion by varioussoil bacteria

Images from Purves et al.,Life: The Science ofBiology , 4th Edition, bySinauer Associates(www.sinauer.com) andWH Freeman(www.whfreeman.com)

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Denitrification

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

The nitrogen cycle is completed by denitrifying bacteria, which convert nitratesback to nitrogen gas. They include bacteria like Pseudomonas, Alkaligenes andBacillus 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 organicmatter, and (2) absence of oxygen but availability of reducible nitrogensources. A mixture of gaseous nitrogen products is often produced because ofthe stepwise use of nitrate, nitrite, nitric oxide and nitrous oxide as electronacceptors in anaerobic respiration.

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


Ammonification

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

The amount of nitrogen released for plant uptake by this process is mostdirectly related to the organic matter content. The initial breakdown of a ureafertilizer 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)

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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 thatgenerate ammonia for their own use (e.g. bacteria living in soil but not associated witha root) (30 % of all N2 fixed in world)

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

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

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

Anabaena + Azolla (water fern).Microbial Physiology- Nisa RM


Azospirillum is a highly efficient species of

non-symbiotic bacteria capable of fixingatmospheric nitrogen and making it availableto the plant. Azospirillum is found in looseassociation with roots, and can fix Nitrogeneven under microaerophyllic conditions.

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

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Azotobacter species can fix nitrogen in the rhizosphere of several plants. Thereare around six species in the genus, some of which are motile by means ofperitrichous flagella, others are not. They are typically polymorphic, i.e. ofdifferent sizes and shapes. Their size of the cells ranges from 2-10 m long and1-2 m wide. Old cells tend to form thick-walled, optically refractile cysts, whichhave capsules consisting largely of alginates and other polysaccharides,enhancing their resistance to heat, desiccation and adverse environmentalconditions. However, these cysts cannot withstand the extreme temperatureswhich 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 photosyntheticorganisms. Most of them can assimilate atmospheric nitrogen under reducedoxygen pressure. Some can do nitrogen fixation under ordinary oxygenpressure. and most of them have large cells, called heterocyst, specializedfor nitrogen fixation. Cyanobacteria are either free-living or in symbiosis withplants.

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

Anabaena

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The photosynthetic cyanobacteria often live as free-living organisms inpioneer habitats such as desert soils or as symbionts with lichens in otherpioneer habitats. They also form symbiotic associations with otherorganisms such as the water fern Azolla and cycads.

Azolla is useful as a "soybean plant in rice field", because it can assimilateatmospheric 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. Inexchange Azolla provides the Anabaena with a protected environment and afixed 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 perha per day under the optimum condition.


A symbioticassociation ofcyanobacteriawith cycads

The first imageshows a pot-grownplant. The secondimage shows aclose-up of the soilsurface in this pot.Short, club-shaped,branching rootshave grown into theaerial environment.

These aerial rootscontain a nitrogen-fixingcyanobacterialsymbiont.

A symbiotic association

of cyanobacteria withAzolla

Anabaena azollae

Azolla pinnata

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Symbiotic N fixers

It has been estimated that nitrogen fixation in the nodules of clover roots orother leguminous plants may consume as much as 20% of the totalphotosynthate.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 adeterminate structure. It has a short, predestined life-span. Consequently,new nodules are being formed as the root grows in the soil and others arebeing 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 rootcortex and has a specialized anatomy.The second nodule type is illustrated by Clover. In this case the nodule has anapical meristem which functions for many months. It is called Indeterminate inthat meristematic activity is theoretically unlimited.These are elongate compared to the determinate nodules. They are tumescent,(swollen). The apical meristem continuously produces new cells which becomeinfected with bacteria from older cells.These nodules have a much more extensive vascular system which surroundsthe nitrogen-fixing parenchyma that occupy the center of the nodule. This

Microbial Physiology- Nisa RM 25central location is not a coincidence.

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

This helps to create a low oxygenenvironment.

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

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Sections through Soybean Determinate Nodules

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

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


The roots of an Austrian winter pea plant (Pisum sativum) with nodules harbouringnitrogen-fixing bacteria (Rhizobium). Root nodules develop as a result of asymbiotic 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 rootthrough an infection thread that allows bacteria to enter root cells, (C) which divideto form the nodule.

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

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

Koa root nodules (above) andadventitious stem nodule (right)

Cross Section of an Infected koaNodule: The dark areas are

parenchyma cells that contain N-fixingBacteria.

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


Long section through an clover

nodule: The Apical Meristem isat the upper right. The red cellsare the most recently infectedcells.

Apical region of an clovernodule. The Meristematic cellsare small and stain densely.The newly infected cells areenlarged and isodiametric.

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

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

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


Actinomycete root nodules fromAlder (Alnus): similar nodules may fixmore atmospheric nitrogen thanlegume/rhizobium nodules

Frankia fix nitrogen while living in rootnodules on “actinorhizalplants”. Frankia thus can supply mostor all of the host plants' nitrogen needs.Consequently, actinorhizal plantscolonize and often thrive in soils thatare low in combined nitrogen.

http://web.uconn.edu/mcbstaff/benson/Frankia/FrankiaHome.htm

Documents

1 9/30/2010 Nitrogen Metabolism Nisa Rachmania Mubarik Major Microbiology Department of Biology, IPB 1212 Microbial Physiology- Nisa RM Nitrogen Cycle