Indian Journal of Biotechnology

Vol 3, October 2004, pp 502-510

Transgenic diazotrophs: Indian perspective

M N Jha* and S K Misra
Department of Microbiology, Faculty of Basic Sciences and Humanities, Rajendra Agricultural University, Pusa 848 125, India
Received 7 August 2002; revised 6 July 2003; accepted 6 November 2003
One of the major objectives of direct agronomic importance is either to engineer a microbe having better nitrogen
fixation rate and efficiency or to extend the symbiotic nitrogen fixation process to non-nodulated plants, especially cereals.
This could be achieved either by increasing the number of nif genes or by making nitrogenase specific to only N2 molecule.
Introduction of hup gene in symbiont and increasing the number of hetR gene in an asymbiont cyanobacteria may improve
the nitrogen fixing potential. Search for a better nitrogen fixer would be another ideal approach to achieve the goal. A
number of researchers have described the induction of nodule like structure in cereals, systemic distribution of some
diazotrophs in grasses and incorporation of cyanobacteria into higher plant protoplast or midrib air space. Such
developments suggest the possibility of creating artificial symbiosis. However, at present we can say In theory any plant
and at the moment no plant whatever! The authors briefly review this research and discuss the strategies associated with
better bionitrogen availability to cereals.
Keywords : transgenic, diazotroph, cyanobacteria, nif gene, artificial symbiosis
IPC Code: Int. Cl.7 C 12 N 15/00

Introduction
A number of viable strategies have been developed
to increase the copy number of a particular gene or a
set of genes usually constructed through an operon
like system. Perhaps the easiest and also the effective
strategy is to place the necessary gene(s) on a plasmid
vector and introduce it in such a manner inside the
cytoplasm/nucleus so that the relevant gene(s) is as
well expressed along with the constitutive gene(s).
The gene(s) placed on the plasmid vector should be
under proper regulating regime so that no excessive
expression of the gene(s) occurs, which could disturb
(in a sort of tumorous manner) the overall gene
expression of the cell. The construction of selftransmissible plasmid (Ti plasmid) bearing nif genes
made it possible to shift the nitrogen fixing capacity
to non-fixing species like Escherichia coli (E. coli CM 74), Salmonella typhimurium, Serratia marcescens
and Pseudomonas fluorescence or to non-fixing
mutant of diazotrophic species like Azotobacter
vinelandii. It is possible to transfer nif gene from one
species to another or even from one genus to another
by conjugation, transformation and transduction.
______________
*Author for correspondence :
Tel: 91-6274-240412; Fax:91-6274-240266
E-mail: mani_raksha@yahoo com

Since in many diazotrophy nif genes occur in cluster,

it is easy to cut out the relevant stretch of DNA and
insert the whole batch into another organism.
The regulation of nif gene and expression is highly
complex.
Recent
researches
have
clearly
demonstrated that 'nif' system is a highly complex
entity and there is surprising diversity in organization,
function and regulation of nif gene. For example, (i)
spatial arrangement of individual nif gene is not
always conserved at the interspecies level (nifW and
nifZ), (ii) a number of potential genes interspersed
among nif gene cluster in some diazotrophs, (iii) the
tight clustering of all nif gene(s) in Klebsiella
penumoniae is not conserved in all other organisms,
(iv) certain nif genes are duplicated in some
organisms (nifA, nifB, nifH), and (v) in some
diazotrophs alternative nitrogenase (other two or three
types) was recorded. Such diversity will be helpful in
constructing transgenic diazotrophs having better
nitrogen fixing ability. Transfer of alternative
nitrogenase (Vnf, anf) from Azotobacter chrococcum
or A. vinelandii or Anabaena variabilis or Calothrix
antarctica to Rhizobium sp./Bradyrhizobium sp. may
lead to better nitrogen fixation by constructed
Rhizobium species in acidic soils and cold
environments. Thus, with molecular techniques now
available, the goal should be within reach, especially

JHA & MISRA: TRANSGENIC DIAZOTROPHS

if we consider some of the simpler ways in which

nature herself has evolved successful system.
For achieving the goal (better nutrition of crop
through bionitrogen source) it is essential to decide
approaches which would be better in the context of
Indian agriculture and economy. There are two
options: (i) indirect feeding of plant through
transgenic diazotrophs and (ii) direct feeding of plant
through artificial symbiosis by using existing
symbionts, search for new symbionts or through
transgenic diazotrophs. In an era, where things are
moving very fast, as evident from Monsanto
researchers claim, to decipher genetic code of rice
(while International Rice Genomic Project, IRGP still
working on that aspect), both options should be
explored without delay.
Development of Transgenic Diazotrophs
Recent developments in understanding the
molecular basis of nif genes, gene cloning systems,
cloning, expression, transformation and conjugal
transfers of vectors, genes cloned in diazotrophs led to
a belief that construction of transgenic diazotrophs are
within our reach. Some success has already been
achieved in recent years. Expression of Rhizobium
leguminosarum bv viciae genes encoding for amino
acid transporters found essential for symbiosis1.
Complete genome of Bradyrhizobium japonicum
USDA
110,
Sinorhizobium
melliloti
and
Mesorhizobium loti has ben sequenced2-4. Complete
nucleotide sequence of symbiotic plasmid of
Rhizobium species NGR 2345, characterization of
nolO and noll of NGR 2346, identification of plant
signal interacting with nodD gene product7, oxygen
regulated nifA gene of S. meliloti8 and products of
nodDABC gene of rhizobia9 was reported. Nodulation
of non-legume Paraspornia by cowpea group
Rhizobium species strain NGR 234 was also
reported10. The molecular organization and regulation
of expression of nif genes has been also studied in
cyanobacteria. All non-heterocystous cyanobacteria
have a continuous nifHDK cluster, whereas the
heterocystous forms show a similar arrangement in
the heterocysts11, but the vegetable cell nifK and nifD
genes are separated by 11 kb intervening DNA
sequence. The rearrangement of these genes occurs by
a unique temporally and spatially regulated
phenomenon involving site-specific recombination
and an enzyme excises. This enzyme is encoded by a
XixA gene present in the 11 kb intervening sequence,

503

which restores continuity between nifK and nifD

genes. Hybridization with XisA gene in nonheterocystous cyanobacteria Plectonema sp., Lyngbya
sp. and Synechocystis sp.12, and heterocystous
cyanobacteria reveal the presence of nifHDK cluster
in Fischerella sp., Mastigocladus sp. and
Scytonematopsis sp. whereas in Tolypothrix sp. and
Scytonema sp. nifD & nifK was separated by 11 kb
intervening DNA sequence13 .
Genes
controlling
circardian
rhythm
in
cyanobacteria are widely distributed, and a clock
system is universal among cyanobacteria. The kai
ABC gene cluster is important for maintaining
circardian rhythm in Synechococcus PCC 7942. Forty
cyanobacterial strains possessed kaicC related
sequences14.
Improve Efficiency of Asymbiotic or Symbiotic
Diazotrophs
Increasing Efficiency of Existing Diazotrophs

Diazotrophy is an energetically costly process. In

symbionts like Rhizobium sp., 250-300 mg N fixed
per g carbon consumed but the efficiency is very low
in Azotobacter where 10-20 mg N is fixed per g
carbon consumed; thus, merely increasing the rate
may result in certain other discrepancy in the
metabolism of diazotrophs and it is essential that
increased nif gene activity fits the general
metabolism. Therefore, while developing transgenic
diazotrophs, attention must be given to efficiency.
There are various molecular techniques by which
efficiency could be increased.
One of the side reactions of nitrogenase is the
reduction of proton (H+) to dihydrogen resulting in the
evolution of hydrogen gas by some strains, which do
not have an uptake hydrogenase enzyme. The uptake
hydrogenase enzyme is responsible for the oxidation
of dihydrogen molecules back to proton and thus
could recoup lost reducing power for nitrogenase
activity. The gene responsible for the hydrogen
uptake is designated hup; it has been cloned and used
to improve strains which do not naturally contain the
enzyme. In R. leguminosarum, hup gene is located on
symplasmid. The cosmid PH 462, which contains the
hydrogenase genome was used to transform HUP
strain of B. japonicum, R. trifolii and R. meliloti. The
strains could then be induced to express hydrogenase
activity in the free living state. However, the best
approach would be making nitrogenase for absolute
specificity for molecular nitrogen which seems to be a
very difficult task at present.

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INDIAN J BIOTECHNOL, OCTOBER 2004

The hetA gene is required for envelope

polysaccharide synthesis. A wild type Anabaena gene
(hetR) introduced on a multi copy plasmid into either
wild type Anabaena or the original mutant, on
nitrogen free medium, the filaments produced cluster
of heterocysts, two to five cells in a row. Thus, in the
strains with multiple copies of hetR gene, the
heterocyst frequency could be increased in free living
diazotrophic
cyanobacteria
like
symbiont
cyanobacteria, which is considered to be efficient one.
Recombinant strains, which contain an introduced
nodD gene resulted in enhanced selectivity and
nodule occupancy. An alternate approach to have a
recombinant strain containing genes for rhizopine
biosynthesis and carabolism and such strains may
have a competitive edge through its ability to
synthesize and selectively utilize rhizopine and
related substrate as a source of nutrient. Further, by
mutation or recombination, the structure of nodD
could be altered such that either the expression of nod
genes is flavonoid independent or the nod genes are
induced by a broader spectrum of inducer molecule. A
hybrid nodD gene that does not require a flavonoid
for nod gene expression has been constructed15.
Unfortunately, the inhibitors of nod gene induction
are unstable in soil. Addition of multiple copies of
nifA from K. pneumoniae in R. meliloti increased
nodulation competitiveness and hence, efficiency of
introduced strain16.
Another approach would be incorporation of
alternative nitrogenase in a diazotroph in which it is
absent. Four types of nitrogenase (nitrogenase 1,
nitrogenase 2-Vnf, nitrogenase 3-anf and nitrogenase
4-Conf ?) have been observed in diazotrophs like A.
chrococcum, A. vinelandii, Anabaena variabilis,
Nostoc commune, Calothrix antarctica, Clostridium
pasteurianum and R. capsulatus. It was observed that
low temperature favoured N2 reduction by nitrogenase
2 and thus could be ideally suited for low temperature
diazotrophy. Further, at low pH, where Mo is known
to be biologically inactive, Vnf or anf may play a
major role. Thus, by incorporating alternative nitrogenase in rhizobia, it is possible to increase its
efficiency. Such incorporation is quite possible as
techniques for incorporation and idea of the genetics
of the alternative nitrogen-fixation systems are
available.
However, for increasing efficiency in relation to
plant particularly for asymbiotic diazotrophs, the level

of glnA also should be considered during construction

of transgenic diazotrophs.
Increasing Rate of Nitrogen Fixation

Transformation by DNA uptake, electroporation,

conjugation and biolistic transfer of particulate DNA
are some of the techniques that have been
standardized in cyanobacteria17,18. For recombinant
DNA work, the hosts should lack restriction
endonucleases, which, otherwise, would cleave
unmodified foreign DNA. Anacystis nidulans lacks
restriction enzymes, whereas, in Agmenellum
quadruplicatum, the enzyme Aqu I is thought to be
responsible for the lower efficiency of transformation,
when the unmodified hybrid plasmid is transferred
into the cyanobacterial host. The elimination of the
Aqu I restriction in the vector site resulted in a 10-fold
increase in transformation efficiency. Anabaena
strains also contain a number of restriction enzymes19.
In addition to removal of restriction enzyme sites
during the construction of cloning vectors, isolation of
mutants lacking restriction enzymes20, use of heat
treatment to selectively inactivate the restriction but
not the modification enzymes21 or the isolation of the
appropriate methylases to modify the foreign DNA
before introduction into the host22, could be the
possible solutions to this problem.
Cloning vectors could be constructed from
plasmids or viruses, and in cyanobacteria the use of
plasmids as vectors is much promising. Many
cyanobacterial strains have indigenous plasmids23,
ranging in size from 2.3 to over 100 kb and the
number of plasmids in a strain varies from 1-7 and the
average being 1-3. A. nidulans contains two plasmids,
pUH24 and pUH25. The smaller plasmid pUH24 has
been used for the construction of most hybrid vectors.
Vectors pCY 100, pCY 101 and pCY 102 for cloning
DNA fragments into Synechocystis sp. ARM 340 and
Anabaena cylindrica ATCC 29414 have also been
developed24.
Expression vectors are cloning vectors which carry
regulatory sequences, such as promoter and ribosomebinding site, which allows a high rate of transcription
of the cloned genes. Antibiotic resistance genes
originating from other bacteria could be expressed in
the cyanobacterium, Synechococcus25, indicating that
other prokaryotic promoters are recognized by
cyanobacteria.
The
coding
sequence
for
Saccharomyces cerevisiae copper metallothionein
(CUP1), the protein responsible for enhanced

JHA & MISRA: TRANSGENIC DIAZOTROPHS

sequestration of Cu2+ in yeast, was placed under the

control of an inducible synthetic E. coli promoter in
the cyanobacterial vector plasmid pTrc1K. Strain R2PIM8 (smt A) of Synechococcus sp. PCC 7942 was
transformed with the resulting construct (plasmid
pMcK2), and the yeast CUP1 gene was integrated into
its genome. In the transgenic cyanobacteria, the
integrated CUP1 gene transcribed and produced a
protein product with the expected metallothionein
characteristics26. For maximum expression, however,
it may be necessary to use a strong cyanobacterial
promoter.
Two strategies have been employed for gene
cloning in cyanobacteria:
1.

2.

One uses shuttle plasmid vectors that carry both

a cyanobacterial and an E. coli replicon, each of
which functions in its indigenous host.
Depending on the sequence of the cloned gene,
the selection applied, and the cyanobacterial
hosts, the cloned gene could be maintained on
the autonomous plasmid or integrated into the
cyanobacterial chromosome.
The other strategy exploits the efficient
recombination system of A. nidulans R2 and
Aphanocapsa PCC 6803 for a direct integration
of the cloned gene into the chromosome. By this
strategy, a DNA vector that lacks a
cyanobacterial replicon, but carries similar
chromosomal
sequences,
is
applicable.
According to the imposed selective conditions,
the cloned gene, either alone or together with the
vector
sequences,
integrates
into
the
chromosome.

In unicellular and non-heterocystous cyanobacteria,

the genes are dispersed in single contiguous nif HDK
while in heterocystous cyanobacteria contiguous nif
HDK is present only in the heterocyst. The vegetative
cell DNA of heterocystous forms like Anabaena
7120, contains two elements interrupting the nif gene
region. One of 11 kb interrupts the nifD gene; the
other of 55 kb interrupts the fds gene (Fischerella is
an exception having contiguous nifHDK). The 11 kb
element has been detected in many strains of
Anabaena and Nostoc, while the 55 kb element
appears to have a more restricted distribution in
nature. Such variation can even occur up to species
level for e.g. A. cylindrica contains both the elements
while Anabaena 29413 lacks 55 kb element. Excision
of the 11 kb element required the product of a gene

505

within the element called xisA. Another gene

equivalent to xisA, responsible for excision of 55 kb
element from the fdxN gene. Expression of xisA from
a plasmid introduced into Anabaena 7120 resulted in
a cured strain lacking the 11 kb element. xisA is
absolutely needed for nitrogen fixation. It is possible
to make contiguous nifHDK in vegetative cell
followed by creating laminated layer around
vegetative cell and making it to fix nitrogen.
Among the cyanobacteria itself lot of variation in
nitrogen fixation occurs. Unicellular forms show
temporal separation between photosynthesis and N2fixation; in Lyngbya, the nitrogenase is localized
mainly at the two ends of filament in those cells
where less thylakoids are present, in heterocystous, it
is the heterocyst which acts as a site of nitrogen
fixation. The unicellular bacteria accumulates enough
carbohydrate during the day to fuel N2-fixation most
of the night. However, ceasation of nitrogenase is due
to inactivation of nitrogenase by photosynthetically
generated O2. In Gloeothece. O2 is required for N2
fixation both in the dark and in the light27. In
Synechococcus, however, the diurnal appearance of
nifHDK messenger RNA, so it is likely that
nitrogenase is destroyed during the (oxygenic) day
time and must be synthesized a new each night28. In
the background of this knowledge, it would be useful
to promote a spatial separation of these processes in
other filamentous cyanobacteria, which may result in
increased nitrogen fixation.
Cyanobacterial nitrogen fixation rate could also be
increased by increasing the cytochrome oxidase
activity as observed in Trichodesmium29, by
destructing phycobiliprotein (which collects and
transfers energy to PSII reaction centre) and by
increasing the concentration of ferrodoxin (nifB, fdxN
and fdxH).
It is also observed that the presence of multiple
copies of nifA, nifH resulted in increased nitrogen
fixation in Rhizobium phaseoli and A. vinelandii16,30.
However, increasing nitrogen fixing rate means more
demand of photosynthate. It was proposed that
increased expression of dctA (structural gene for
dicarboxylate transport) could increase the rate of
nitrogen fixation in alfalfa and soybean16.
Transferring nif Genes to Other Prokaryotic or Eukaryotic
Microbes

If N2 fixation is so widespread, why do we persist

in thinking of it as a specialization? The authors think

506

INDIAN J BIOTECHNOL, OCTOBER 2004

this stems out from colicentric view of bacteria: E.

coli K-12 does not fix N2, therefore, anything that
does must be a specialist. N2 fixation is distributed in
green sulphur bacteria, Firmibacteria, Thallobacteria,
Heliobacteria, Cyanobacteria, Proteobacteria (alpha,
beta and gama subdivision), Archaeiobacteria and
even in Actinomycetes. The latest addition is
Streptomyces thermoautotrophicus31. Many of the
diazotrophs have still to be identified in nature and so
there is no need to transfer nif gene to non-fixer
microbes and it would be better to increase rate and
efficiency of existing microbes.
Potential of Creating Artificial Symbiosis or
Extending this Beneficial Process to Nonnodulated Plants, Especially Cereals
Search for New Symbiont

India is gifted with vast microbial diversity. Our

biological feature depends on the ability to search,
conserve and utilize rich genetic diversity occurring in
microbes. The extent of ignorance on the number of
species on earth needs no emphasis. The changed
international trade scenario after the WTO accord has
now posed greater challenge to be competitive. The
first question that should be considered is the
ownership of germplasm into which the trait of
interest is to be introduced. Further, the authors
believe that it is difficult to compete with the west so
far genetic tinkering is concerned but at the same time
it is very difficult for west to compete with us so far
microbial diversity is concerned. Unfortunately, all
the reports for new diazotrophs like Rhizobiumnonlegume Parasponia symbiosis32; Azospirillum33;
Acetobacter
diazotrophicus34;
Herbaspirillum
seropedicae and Streptomyces thermoautotrophicus31
has come from the west. Indian researchers have so
far really grown 1% of the existing microbial species.
Thus, attempts to isolate new symbiont i.e. Typhamycorrhiza35; or free living diazotrophs by examining
maximum plant types and environment should
continue and this could be achieved by motivating
microbiologists to continue to work as microbe
hunters. This is an area where sincere efforts might
place India at number one in the whole world and we
will be able to supply germplasm of interest. In
authors opinion, search for new diazotrophs or
symbiont is best suited in the context of our country.
Artificial Symbiosis in Maize and Rice

The most direct method to enhance bionitrogen

source for crop would be to produce a new symbiotic

association. Is this approach plausible? If yes, then

difficult decision arises in deciding which plants to
target, and which microbes would be the best
candidates and finally the techniques to be used.
Among the crops, rice and maize may be good
options. The bundle sheath cells of maize plant, in
which oxygen evolution does not occur, could provide
a relatively low oxygen environment comparable to
that found in the cyanobacterial heterocyst. The
transcripts of large subunits of ribulose biphosphate
carboxylase (rbcL) gene have also been found to be
expressed in bundle sheath cells and are not present in
photosynthetic mesophyll cells36. By linking the nif
regulatory genes to the rbcL promoter, the tissue
expression of nif gene might be achieved in the
bundle sheath cells of maize. Finally, it may be
possible to regulate the genes temporarily so that they
are expressed only in the dark, a situation analogous
to that found in non-heterocystous unicellular
cyanobacteria. Further, placing the specific diazotroph
in bundle sheath cells and manipulating the
environment for its colonization will also lead to
better nutrition of maize crop.
In rice, root respiration takes place through
arenchymatous tissue present in leaves and stem.
These air spaces could be used for the colonization of
microbes, especially cyanobacteria. It is an
established fact that many cyanobacteria produce
mucilaginous sheath and the production of mucilage
can be increased by inducing stress. Thus, if the air
space could be filled with mucilage of cyanobacterial
origin or the plant could be engineered so that the
intercellular space or air space becomes filled with
polysaccharide or other O2 excluding substances upon
infection, may lead to develop artificial symbiosis37.
Recently, formation of pseudonodules and
paranodules have been reported from wheat, rice, oil
seed, barley38,39,40. The infection of paranodules
induced by chemicals such as 2,4-D on wheat roots, by
rhizobia and free living diazotrophs has been
considered to be a potential vehicle to achieve
nitrogen-fixation in cereals41,42. In fact, nitrogen
fixation by Azospirillum barasilense and Azorhizobium
caulinodans in paranodules induced on wheat roots has
been reported43-45. Moreover, ammonium excreting
Azospirillum can become established intracellularly in
2,4-D induced paranodules in maize in some cases46.
The induction of nodule like structures on and
invasion of rice, maize and wheat seedling roots by
Parasponia and Aeschynomene rhizobia, either

JHA & MISRA: TRANSGENIC DIAZOTROPHS

spontaneously or after the treatment of roots with cellwall degrading enzymes, has also been described47.
It has been proposed that these approaches, involving
nodulation in non-legume crops by rhizobia may
lead to advances in obtaining nitrogen from the air
for non-legume crops48. A Rhizobium strain has been
constructed containing a plasmid carrying a nodD
allele able to respond to signals produced by rice
roots. This strain induced infected nodule like
structures on rice seedlings at a low frequency49. A
high frequency induction of infected nodule like
structures on rice root by Sesbania rhizobia and a
positive effect of such infection on paddy growth and
yield has been suggested50,51. Thus, there is also a
possibility for modeling Rhizobium to nodulate nonlegume genera, since it happens naturally in the
genera, Parasponia and the genetics of ParasponiaRhizobium nif gene is known. However, one actually
needs nodules or nodule like structure on cereal roots
to achieve symbiotic/endophytic nitrogen fixation. It
may, indeed, be sufficient to identify a stable
endophyte of rice roots and, if necessary, to engineer
this microbe to efficiently fix nitrogen and excrete the
fixed nitrogen for use by the plant. This endophyte
may not need to be stably maintained intracellularly,
like a rhizobial endosymbiont; it may be sufficient if
it colonizes the plant root intracellularly, as long as it
is able to evade the plant defense responses.
In this context, it is interesting to note that stable
endophytic diazotroph (Acetobacter diazotrophicus)
that contributes substantially to sugarcane growth has
been described52,53. Moreover, a strain of nitrogen
fixing bacterium, Azoarcus has been described that
colonizes and spreads systemically in grasses such as
rice54. This bacterium was found to invade roots inter
and intracellularly, spread through the xylem, and
increased rice yield.
Cyanobacteria, being endosymbiotic with all the
groups like Fungi, Bryophyta, Pteridophyta,
Gymmonsperm and Angiosperm, are obvious
candidates for trying to synthesize new symbiosis.
Heterocystous forms have their own oxygen
protection system and, therefore, would need fewer
control systems in the plant. Now, it is established
that chloroplast of plants have cyanobacterial origin.
Recently, a gene whose potential product is similar to
the nifH like gene from R. capsulatus is also encoded
within and expressed from the chloroplast genome of
Marchantra polymorpha. Several research groups
have succeeded in incorporating cyanobacteria into

507

higher plant protoplast or callus, though apparently

not yet in a long term stable relationship38,55,56.
Some success has also been achieved with Frankia.
Protoplast fusion between Frankia and the fast
growing actinomycete, Streptomyces has been
reported and nodulation of Alnus rubra by these
hybrids met with limited success.
Numerous successful attempts have been made to
grow rhizobia in association with callus tissue from
both legumes and non-legume plants. How stable
these are, is not clear and there has been no
reproducible evidence on intracellular location of
bacteria.
To achieve sufficient fixation in a plant controllable
fashion, intracellular location of endophytes in a
special structure such as nodules makes a great deal of
sense. To construct new nodules may be relatively
very simple, as evident from inducing paranodule in
many crops, however, getting the endosymbionts into
the nodules is likely to be more difficult40,57.
In genetically engineered host plants, it may also be
necessary to take into account the regulation of
defense genes. In this context, the recent use of
Agrobacterium rhizogenes to effect transgenic roots
and root nodules holds great promise.
The gene technologists disregarded the possibility
to transfer nitrogenase to green plants, because the
enzymes involved in nitrogen fixation are highly
oxygen sensitive. Discovery of nitrogen fixation in S.
thermoautotrophicus is a revolution in this regard
since in this case oxygen is involved in nitrogen
fixation31.
If successful invasion or placement of microbes in
host cells is achieved, the problem of maintaining the
symbiosis arises. Natural symbiosis varies greatly in
longevity, although individual, N2-fixing nodule cells
probably have a maximum active life span of a few
weeks. Thus, with the molecular techniques available
now (delivering DNA by a particle gun, using
microlaser beam to create holes in cell wall,
electroporation, biological vectors, microinjection,
microprojectible bombardment, etc.) the goal should
be within reach, especially if we consider some of the
simpler ways in which nature herself has evolved
successful system.
Conclusion
Due to unexpected complexity and diversity of nif
genes, molecular approaches have not improved the
performance of symbiotic or asymbiotic microbes, as

508

INDIAN J BIOTECHNOL, OCTOBER 2004

once expected. Even if, improvement is achieved in

laboratory (engineered Anabaena and Rhizobium), the
inability to ensure nodulation by engineered strain or
survival of strain in competition with naturally
occurring strain remains a major obstacle. However,
the kind of basic knowledge gained during the last
decade regarding nif genes, oxygen dependent nif
genes, genes involved in enhancing competitiveness,
the barrier may be overcome in coming decades.
Now the question is, is it proper and necessary to
enhance the copy number of such a complex genetic
system in diazotrophs, which is already fixing the
dinitrogen molecule ? The environmental problem
most likely would be increased, perhaps, dramatically
and this problem remains a potential danger that
under increased nitrogen fixation, the cell would not
be able to cope with the additional production of NH3
and other resulting biomolecule as suggested by the
researchers, who opposed genetic tinkering of nif
genes.
The addition of extra gene(s) or increasing the copy
number of gene(s) may reduce the competitive ability
of targetted organism due to the budgeting of more
energy towards the functioning of extra gene. The
situation will be more complicated in high energy
demanding nitrogen fixation process where only 1020 mg N fixed per g carbon consumed in case of
Azotobacter. Even in symbionts like Rhizobium only
250 mg N fixed per g carbon consumed, supplied by
host cell. Thus, the engineered organism (thereby)
probably be less fit than the present organism under
natural
agricultural
system.
Under
such
circumstances, the goal of augmenting bionitrogen for
plants will not be achieved because in agricultural
systems survival and proliferation of the introduced
organisms are key factors for extracting benefit.
Then, the question is What will be the best
approach in Indian context ? Is it transgenic ? Is it
search and selection of natural new diazotrophs ? At
present, it seems that the best way to solve the
nitrogen crisis is to introduce the natural efficient
strains and get them to do the real job needed. To
achieve this goal greater emphasis should be given to
the agromicrobial diversity research. Besides this, we
should also go for creating artificial symbiosis by
using indirect (releasing DNA into mature pollen,
stigma, style has also resulted in desirable
incorporation but frequency was low) or direct
method of DNA transfer. Introducing cyanobacteria
into the rice airspace and inducing heterocyst

formation or mucilage synthesis even by crude

manner may result in the development of artificial
symbiosis in rice because who knows that the story
of Alexander Flemming will not be repeated again.
References
1

10
11

12

13

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