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8/2/2019 Review BappaShonaBaroi
1/5
A REVIEW ON FREE LIVING NITROGEN FIXING BACTERIA
By Bappa Shona Baroi (08MS016)
Free living nitrogen fixing bacteria are those which donot have intimate associations with any plant.
They represent a range of bacteria including saprophytes living on plant residues, bacteria living inclose association with the rhizosphere of plant roots and bacteria which live entirely within plants
(endophytes).They include members of family of Klebsiella, Azotobacter, Clostridium,
Rhodospirillum, Aspirillum and various cyanobacteria. Let us first look at what nitrogen fixation
means. The basic processes of Nitrogen fixation in both symbiotic and non-symbiotic bacteria are
same. The key difference lie in the regulation of these mechanisms as symbiotic ones live in the
microenvironment created by the symbiont while, the non-symbiotic ones are open to natural
conditions.
The Process:
Nitrogen fixation is a process, biological, abiotic, or synthetic by which nitrogen (N 2) in the
atmosphere is converted into ammonia (NH3). Atmospheric nitrogen (N2) is relatively inert: it does
not easily react with other chemicals to form new compounds. Fixation processes free up the
nitrogen atoms from their diatomic form (N2) to be used in other ways. Microorganisms that fix
nitrogen are bacteria called diazotrophs. This process requires the activity of a very unique enzyme.
Lets get to know it.
Key Enzyme:
Nitrogen fixation requires an enzyme called nitrogenase, which converts gaseous nitrogen into the
more available nitrogen form ammonia. Nitrogenase activity consumes large amounts of energy.
Symbiotic nitrogen fixing bacteria receive energy from the host legume but free-living bacteria must
find their own source of energy within the soil. Nitrogenase requires the products of about 20 genes
for its synthesis and activity. The fixation part involves various reactions which well see shortly.
The Chemistry:
Nitrogenase is composed of the heterotetrameric MoFe(Fe-S protein + Fe-Mo protein) protein that
gets transiently associated with the homodimeric Fe protein. Electrons required for the reduction of
nitrogen are received in this association. The heterocomplex undergoes cycles of association and
disassociation to transfer one electron, which is the rate-limiting step in nitrogen reduction. ATP
supplies the energy to drive the transfer of electrons from the Fe protein to the MoFe protein.
Nitrogenase ultimately bonds each atom of nitrogen to three hydrogen atoms to form ammonia
(NH3).This ammonia ,in turn gets bonded to glutamate to form glutamine. The nitrogenase reaction
additionally produces molecular hydrogen as a side product.The overall reaction:
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Regulation of Nitrogenase from Free-living perspective:
Although a diverse group, the physiology and functional biology of all free-living N2 fixers are
markedly affected by the properties and requirements of the nitrogenase enzyme. In particular, the
enzymes activity is characterized by:
1). O2 sensitivity: Free-living N2 fixers can be rigid anaerobes, facultative anaerobes, or
wholly aerobes and thus exist in a range of environments that span gradients of O2 availability. O2has the potential to inhibit nitrogenase and thereby suppress N2 fixation. N2 fixers avoid the
potentially toxic effects of O2 by(a) isolating N2 fixation in space using cellular components where
O2 concentrations are kept low (e.g., heterocysts),(b) by separating N2 fixation in time from O2
evolving processes such as photosynthesis, or by increasing respiration to draw down O2 levels .
Azotobacter (an aerobic nitrogen fixer) has one of the highest respiratory rates of any organism.
This enables it to remove oxygen rapidly from its surroundings through its own respiration. So in a
nutrient rich medium, this acts as a two way beneficial mechanism (i) keeping nitrogenase active
and (ii) produce large quantities of ATP needed for the high energy demanding fixation process!! For
obligate aerobes, optimal conditions are met when O2 concentrations are balanced by respiratory
demand: Under low O2 concentrations, nitrogenase is limited by energy, but at higher
concentrations nitrogenase can be inhibited directly by O2 .O2 inhibition can occur at two levels,
both by reducing nitrogenase activity and by reducing nitrogenase production.
2). ATP and Reducing Power: Nitrogen fixation is one of the most metabolically costly
processes on Earth. Photosynthetic N2 fixers use the suns energy, and heterotrophic fixers rely on
catabolic pathways to derive energy from organic matter. The efficiency of N2 fixation depends on
the particular energy source used and is also regulated by environmental conditions. The switching-
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off of the nitrogenase by ADP-ribosylation(enzyme DraT) in response to high NH3 and energy
depletion also serves as a regulatory mechanism of nitrogenase activity.
3).Metals: All known forms of the nitrogenase enzyme require Fe and most also contain Mo
or V. Alternative nitrogenases are widely distributed among N 2 fixers (Figure 2 ), but it is commonly
accepted that Mo-nitrogenase is most efficient at N2 fixation, and that N2 fixers use alternative
nitrogenases when Mo is in relatively low concentrations.
4).Nitrogen: Nitrogen fixers may meet their nitrogen demands by (a) fixing N2, (b)by
acquiring mineral N from the external environment, or(c) by enzymatic breakdown and reallocation
of internal cellular N. When mineral forms of N (i.e., NH4+or NO3) are readily available in the
environment, many N2 -fixing organisms will switch off N2 fixation. Free-living soil bacteria under
low nitrogen conditions exhibit maximum fixation rate. However the level of nitrogen should be
above a minima because very low nitrogen hampers the synthesis of nitrogenase.
5).Temperature & Carbon supply: Nitrogenase like any other enzymes, follow enzyme
kinetics with rates increasing with temperature to a certain level and then fall abruptly. Free-livingheterotrophs in litter and soil environments often use organic matter(Carbon) as a resource both to
fix N2 by increasing ATP production, and to maintain high respiration rates to avoid O2 deactivation
of nitrogenase.
All the above explained regulators are valid in case of symbiotic nitrogen fixers too, but ,as the
symbiotic ones live in the microenvironment created by the symbiont plant, the effects are not as
prominent. However as the free living ones are completely of their own, the fate of nitrogen fixation
is, to a large extent, depend on these.
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Examples of strategies implemented by different non-symbiotic nitrogen fixing bacteria:
The most important problem that free living bacteria has to tackle is that of Oxygen as it is the only
regulator that the microbe can control. Others, whether it be metals, ATP, Nitrogen, temperature or
carbon supply, are more or less out of its hands. Different strategies have been developed by
different bacteria.
i)Free living Azospirillum: The fundamental difference between symbiotic rhizobiaand
free-living diazotrophs is reflected by the differences in regulation of the nif genes (genes re-
sponsible for N2 fixation). Transcription of nif genes is induced by NifA. In rhizobia, it is tightly
oxygen regulated, whereas in free living ones, it is tightly nitrogen regulated. The less oxygen
dependence comes from the fact that a lot of the oxygen is used up for ATP synthesis while in
symbiotic ones most of the ATP comes from the symbiont.
ii)O2 protection in free-living N2-fixing Azotobacter: Nitrogenase of Azotobacter is
adequately protected to be able to cope with highly fluctuating O2 concentrations. A well-integrated
system of protection, comprising conformational protection, respiratory protection,
autoprotection(high rate of respiration) and other changes, allow Azotobacter species to grow under
fully aerated conditions . Activity of NifA in these bacteria is inhibited by the flavoprotein NifL, which
is rapidly oxidized in the presence of air. In its oxidized state, NifL forms a complex with NifA,
thereby inhibiting NifA activity. This produces the switched off state of the enzyme. In this state,
the enzyme is inactive, but protected from damage. By reduction of NifL, the NifA-NifL complex
dissociates and the inhibition is relieved. Hydrogenases like Superoxide Dismutase and catalase
perform this reduction High respiratory activity by the uncoupled respiratory chain makes reduction
of NifL compatible with potentially elevated O2 levels. Also, at high ambient O2 concentrations a
partially uncoupled cyt bd oxidase with low apparent in vivo O2 affinity is expressed. This
oxidase probably acts in concert with an uncoupled NADH-dehydrogenase. Electron flow through
this uncoupled chain allows high respiration rates and fast consumption of the intracellular O2
without exhausting the ATP and NADH pools.
iii) O2 protection in other free-living N2 fixing bacteria: In other free-living N2 fixing
bacteria, the protection system is not as elaborately studied as in Azotobacter and
Asperogillum. In Azospirilla carotenoids, produced at intermediate O2 levels are believed to pro-
tect cells against oxidative damage by quenching singlet O2. Carotenoids might also reduce O2
diffusion in the cytoplasm by reinforcing the membrane bilayer. Instead of excess respiration,
as observed in Azotobacter , the surplus energy generated during micro-aerobic respiration is
used for de novo nitrogenase synthesis of NifA. An O2 shift irreversibly damages the nitrogenase
and inactivates the NifA protein of Azospirillum. However, new tran-scription of nifA allows for
synthesis of nitrogenase immediately after the removal of oxygen stress.
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Uses of Free living Nitrogen fixers:
Whatever the mechanism might be for securing the nitrogenase activity, each of the different free
living nitrogen fixers have performed well in their respective niche. The non symbiotic nitrogen fixers
are of extreme ecological importance, especially in agricultural soils. Forest, desert, and prairie
ecosystems are dependent on nitrogen fixation by free-living species to replace the annual nitrogen
loss. Without it, growth of a number of plant species would suffer drastically, and food chains in
these ecosystems would soon be disrupted. A number of studies are investigating the advantages of
incorporating free-living nitrogen fixers into non-legume crop production, but clear benefits are
uncertain. Further experiments are most likely to provide positive results. Biofertilizers fortified with
free-living nitrogen fixers are already in use in paddy fields in China and other South-East Asian
countries with good results.
References:
Nitrogen fixation article on Wikipedia
Nitrogenase article on Wikipedia.
Functional Ecology of Free-Living Nitrogen Fixation:A Contemporary Perspective . Sasha C. Reed,Cory
C. Cleveland,and Alan R. Townsend.
The oxygen paradox of dinitrogen-fixing bacteria. Kathleen Marchal 7 Jos Vanderleyden.
Nitrogenase Activity and Regeneration of the Cellular ATP Pool inAzotobacter vinelandiiAdapted to
Different Oxygen Concentrations KERSTIN LINKERHA & GNERAND JURGEN OELZE.