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 in close 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 (N2) 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:

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. O2 has 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-

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-living heterotrophs 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.

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 responsible 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 protect 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.

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.