THE OXFORD COLLEGE OF SCIENCE

PG DEPARTMENT OF MICROBIOLOGY

SEMINAR REPORT

SYMBIOTIC NITROGEN FIXATION BY FRANKIA

Presented by

ANISHA P
MSc Microbiology IV semester

UNDER THE GUIDANCE OF

Deepika ma`am

Assistant professor

Department of Microbiology
INTRODUCTION
Nitrogen is the element that most often limits plant growth. Biosphere nitrogen is
continuously lost to the atmosphere by denitrification and can only be depleted by
nitrogen fixation. Only some prokaryotes can form the enzyme complex
nitrogenase that catalyzes the reduction of air dinitrogen to ammonia and thus
moves nitrogen from the atmosphere to the biosphere. Several plant species can
enter symbiosis with nitrogen fixing soil-bacteria. The most efficient of those are
root nodule symbioses.
Rhizobia symbioses are entered between a polyphyletic group of gram negative
soil proteobacteria, rhizobia and species from the legume family as well as form
one legume genus, Parasporia. Actinorhizal symbioses are between gram positive
soil actinobacteria of the genus Frankia and 25 genera of dicotyledonous plants,
from eight families belonging to three different orders, collectively called
actinorhizal plants.
In these symbioses, the host plants form special organs, the root nodules, upon
signal exchange with the microsymbionts. In root nodules, the microsymbionts fix
nitrogen while stably internally accommodated within nodule cells and export the
products of nitrogen fixation to the host plant, thereby rendering it independent of
soil nitrogen sources.

NITROGEN FIXATION BY FREE-LIVING HETEROTROPHS

Many heterotrophic bacteria live in the soil and fix significant levels of nitrogen
without the direct interaction with other organisms. Examples of this type of
nitrogen fixing bacteria include species of Azotobacter, Bacillus, Clostridium and
Klebsiella.
These organisms must find their own source of energy, typically by oxidizing
organic molecules released by other organisms or from decomposition. There are
some free living organisms that have lithotrophic capabilities and can thereby
utilize inorganic compounds as a source pf energy.
Because nitorgenase can be inhibited by oxygen, free living organisms behave as
anaerobes or microaerophiles while fixing nitrogen. Because of this scarcity of
suitable carbon and energy source for these organisms, their contribution to the
global nitrogen fixation ratio is generally considered minor.

ASSOCIATIVE NITROGEN FIXATION

Species of Azospirillum are able to form close associations with several members
of the poaceae, including agronomically important cereal crops, such as rice,
wheat, corn, oats and barley. These bacteria fix appreciable amounts of nitrogen
within the rhizosphere of the host plants. The level of nitrogen fixation is
determined by several factors, including soil temperature, the ability of the host
plant to provide a rhizosphere environment loss in oxygen pressure, the availability
of host photosynthates for the bacteria, the competitiveness of the bacteria, and the
efficiency of nitrogenase.

SYMBIOTIC NITROGEN FIXATION

Many microorganisms fix nitrogen symbiotically by partnering with the host plant.
The plant provides sugars from photosynthesis that are utilized by the nitrogen
fixing microorganisms for the energy it needs for nitrogen fixation. In exchange for
these carbon sources, the microbe provides fixed nitrogen to the host plant for its
growth.
One example of this type of nitrogen fixation is the water fern Azolla symbiosis
with a cyanobacterium Anabaena azollae. Anabaena colonizes cavities formed at
the base off azolla ferns.
There are cyanobacteria for significant amounts of nitrogen in specialized cells
called heterocysts. Another example is the symbiosis between actinorhizal trees
and shrubs, such as Alder with the actinomycete Frankia. These plants are native to
North America and tend to thrive in nitrogen poor environments. Actinorhizal
plants are found in many ecosystems including alpine forest, glacial hill, coastal
dune and arctic tundra environments.

LEGUME NODULE FORMATION

The rhizobium or Brady rhizobium bacteria colonize thee host plant root system
and cause the roots to form nodules to house the bacteria. The bacteria then begins
to fix the nitrogen required by the plant. Access to the fixed nitrogen allows the
plants to produce leaves fortified with nitrogen that can be recycled throughout the
plant. This allows the plant to increase photosynthetic capacity which in turn yields
nitrogen rich soil. The consequences of legumes not being nodulated can be quite
dramatic, especially when the plants are grown in nitrogen poor soil.

Frankia
Frankia are nitrogen fixing actinomycete symbionts that cause the formation of
perennial nodules on the roots of a botanically diverse group of bushes and small
trees belonging to eight families, 25 genera and will over 200 species. The
association is referred to as actinorhizal.
Frankia has the ability to convert atmospheric nitrogen gas (N2) into ammonia
(NH4), which can be used by plants as a source of nitrogen. This process is
essential for plant growth as nitrogen is a vital nutrient required for the synthesis of
proteins, nucleic acids, and other important compounds.
The symbiotic relationship between Frankia and the host plant involves the
formation of specialized structures called root nodules. These nodules are formed
through a series of chemical signaling exchanges between the plant and the
bacterium. Once the nodules are established, Frankia infects the root tissue of the
plant, forming colonies within the nodules.
Within the nodules, the bacteria receive carbohydrates and other nutrients from the
plant, while the plant benefits from the nitrogen fixation carried out by the
bacterium. The plant provides the bacteria with an environment rich in oxygen and
carbon compounds, which are necessary for nitrogen fixation to occur.
The bacteria use a unique enzyme called nitrogenase to convert atmospheric
nitrogen into ammonia. The enzyme is highly sensitive to oxygen and requires a
low-oxygen levels within the nodules to ensure optimal conditions for nitrogen
fixation. The ammonia produced by Frankia is then transported to the plants cells,
where it is used for various metabolic processes. In return, the plant provides the
bacteria with organic compounds such as sugars and amino acids, which serve as a
source of energy and carbon for the bacterium.
Overall, the symbiotic nitrogen fixation by Frankia is a mutually beneficial
relationship that allows certain plants to thrive in nitrogen poor environments. This
unique ability of Frankia to fix atmospheric nitrogen contributes to the ecological
success of these plants and their ability to play a crucial role in nitrogen cycling
with ecosystems.

ACTINORHIZAL PLANTS AND SYMBIOSIS

Actinorhizal plants have the ability to develop endosymbiosis with the nitrogen
fixing soil actinomycetes Frankia. The establishment of the symbiotic process
results in the formation of root nodules in which Frankia provides fixed nitrogen to
the host plant in exchange for reduced carbon. Actinorhizal plants represents a
diverse plant families distributed in the three orders, Fagales, Rosales, and
cucurbitales. All actinorhizal species belong to the Rosid I clade, thus sharing a
common ancestor with legumes, but differing from them in their wide distribution
in numerous botanical families.
Actinorhizal plants are woody shrubs and trees, except for the genes Dastica,
which is herbaceous. They are distributed worldwide from cold regions to warm
latitudes.

NITROGEN FIXING ACTINOBACTERIA – FRANKIA

Frankia is a genus of soil actinomycetes in the family Frankiaceae that fix nitrogen,
both under symbiotic and free living aerobic conditions, while most rhizobia do
not.
Frankia is a gram positive bacteria that is found on the roots of plants. The fact that
Frankia is gram positive means that the bacteria is made up of thick cell walls
made out of protein called peptidoglycan. This helps with the resistance of the
heavy metals that may be in the degraded soil. Frankia does not tolerate
temperature will, it grows best at around 30oC with an environment pH between
6.5 and 7. These facts shows that Frankia is very sensitive to its environment.

Actinorhizal plants are group of plants which have the ability to develop an
endosymbiosis with the nitrogen fixing soil actinomycete Frankia.
Frankia is a gram positive aerobic filamentous bacteria.
Frankiaceae that fix nitrogen, both under symbiotic and free living aerobic
conditions, while most rhizobia do not.
Frankia from nitrogen fixing root nodules with numerous genera of non-
leguminous angiosperms, such as alder, sea buckthorn and Casuarina.
Most Frankia strains are specific to different plant species. The bacteria are
filamentous and convert atmospheric nitrogen into ammonia via the enzyme
nitrogenase, a process known as nitrogen fixation.
They do this while living in root nodules on actinorhizal plants. The bacteria can
supply most or all of the nitrogen requirements of the host plant. As a result,
actinorhizal plants colonize and often thrive in soils are low in plant nutrients.
Frankia can resist low concentration of heavy metals such as Cu, Co, and Zn.
Frankia may be an advantage for degraded soil. Degraded soil is known as soil that
is heavy metal rich or nutrient depleted due to a drought. Frankia is a nitrogen
fixed organisms, explaining why it is able to resist heavy metals.
This genus was originally named by Jorgan Breenchorst in 1886 to honor the
German biologist Albert Bernhord Frank, Breenchorst considered the organism he
had identified to be a filamentous fungus.
During growth, the Frankia spp produce their cell types. Spores which occur in
sporangia and contribute to natural dissemination of the actinobacteria.
Hyphae- Diazo vesicles which are spherical, thick walled, lipid enveloped cellular
structures)
The diazo vesicles are responsible for the supplying of sufficient nitrogen to the
host plant during symbiosis, which are the sites of nitrogen fixation and
differentiate at the tips of hyphae under nitrogen limitation. The vesicles are
surrounded by a laminated envelope composed of hapanoid lipids that provide the
necessary O2 protection to prevent nitrogenase inactivation.
NODUALTION PROCESS
The infected zone of legume nodules is control and is normally contained within an
endorphins and an inner cortical layer of lightly packed cells, with the vascular
tissue lying outside these tissues. In contrast, Actinorhizal nodules normally have a
central state that has infected tissue adjacent to it or around it.
Frankia cells enter symbiosis by real hair infection (intracellular) or by a process of
intercellular penetration of root epidermis and cortex. Root hair infection is
characterized by root hair branching and curling. Only one root hair infection is
required for nodulation and where multiple root hairs are infected cortical infection
is related to only one root hair infection.
Cells of the hypodermis and cortex divide in response to the invasion to form a
prenodule. Prenodule formation resulting from mitotic activity in the root cortical
cells is observed only during the intracellular infection process.
Frankia hyphae penetrate through the prenodule tissue into the inner cortex of the
root. The nodule itself develops in the same manner as a lateral root, with primary
nodule primordia arising in the pericycle endodermis, or cortex at the same time as
prenodule development. The Frankia hyphae penetrate cells of the developing
nodule lobe primordia to form the infected nodule.
nif genes- Nitrogen fixation genes
The complex of genes encoding enzymes involved in the fixation of atmospheric
nitrogen. They are found in nitrogen fixing bacteria. They occur as an operon in
free living anaerobic nitrogen fixing bacteria such as Klebsiella pneumoniae,
Rhodospirillum ruburm, and Rhodobacter capsulants.
These genes may also be found on plasmids in symbiotic bacteria such as in
Rhizobium inhabiting the roots of leguminous plants.
The nif genes are genes that encode for enzymes involved in the fixation of
atmospheric nitrogen into a form of nitrogen available to living organisms.
The primary enzyme encoded by the nif genes is the nitrogenase complex which is
in charge of converting atmospheric nitrogen to other nitrogen forms such as
ammonia which the organism can use for various purposes.
Nitrogen fixation is important because many living organisms are unable to
metabolize directly the atmospheric nitrogen and would require the nitrogen
fixation capability of certain bacteria in order to produce a form of nitrogen that
can be readily utilized. The nif genes are found in both free living nitrogen fixing
bacteria and in symbiotic bacteria associated with various plants.

REGULATION
In most bacteria, regulation of nif genes transcription is done by the nitrogen
sensitive nifA protein.
When there isn’t enough fixed nitrogen available for the organisms use, ntrC
triggers nifA expression, nif A activates the rest of the nif genes.
If there is a sufficient amount of reduces nitrogen or oxygen is present, another
protein is activated. Nif L inhibits Nif A activity resulting in the inhibition of
nitrogenase formation.

The nif genes are organized into a regulon of 17 genes consisting of 7 or 8 operons
each of which is transcribed into a single, usually polycistronic mRNA.
Regulation of nif gene expression has two elements
An external system designated ntr (nitrogen regulatory)
An internal system mediated by nif A and nif L.
The ntr system responds to conditions of nitrogen starvation by activating genes
that enable the organism to utilize unusual nitrogen sources such as arginine,
proline and histidine as well as N2 itself.
The ntr system responds to conditions of nitrogen starvation by activating genes
that enable the organism to utilize unusual nitrogen sources such as arginine,
proline and histidine as well as N2 itself.
The ntr A gene product is a factor of RNA polymerase which recognizes the nif
and other ntr regulated genes.
These promoters have a structure different from that of typical bacterial promoters.
NtrA allows RNA polymerase to bind at the nif promoters and to initiate
transcription.
ntrB gene product NtrB is an enzyme that functions both as protein kinase and as
phosphatase the substrate of which is NtrC.
The nifA product is an activator of transcription of other nif genes, whilst the nifL
product, in the presence of either intermediate concentrations of fixed nitrogen or
inactivate the nifA product, thereby preventing transcription of other nif genes.

CONCLUSION:
Symbiotic nitrogen fixation by Frankia plays a crucial role in enriching the soil
with nitrogen and promoting the growth of various plants, especially woody
species. Through a mutually beneficial relationship Frankia convert atmospheric
nitrogen into a form that can be readily absorbed by plants, thus reducing the
reliance on synthetic fertilizers. This process not only improves agricultural
productivity but also enhances soil fertility and contributes to system sustainability.
Understanding and harnessing the potential of symbiotic nitrogen fixation by
Frankia can have significant implications for sustainable agriculture and
environmental restoration. Further research and technological advancements are
need to optimize the efficiency of this process and maximize its benefits in various
ecosystems.
BIBLIOGRAPHY
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Chapion A. (2016). Tolerance to environmental stress by the nitrogen fixation
actinobacterium Frankia and its role in actinorhizal plants adaptation. Symbiosis,
70, 17-29
2. Santi.C.BIgus.D, & Franchi.C (2013). Biological nitrogen fixation in non-
legume plants. Annals of Botany, 111 (5), 743-767.
3. Thang.W, Chen.Y. Hang.K, Wang.F, &Mei.Z, (2023). Molecular Mechanism
and Agricultural Application of the NIfA-NIfL. System for nitrogen fixation.
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