That's all about role of microbes in nitrogen fixation Symbiotic Relationship of Microbes and plants

1. COURSE : GLYCOBIOLOGY
TOPIC: ROLE OF SYMBIOTIC ASSOCIATION
BETWEEN NITROGEN FIXING BACTERIA And PLANTS

2. LESSON OUTCOMES
WHAT IS NITROGEN FIXATION
NEED OF NITROGEN FIXATION
CLASSIFICATION OF NITROGEN FIXATION
MECHANISM OF NITROGEN FIXATION
GENES INVOLVED IN NITROGEN FIXATION
FACTORS AFFECTING NITROGEN FIXATION

3. What is nitrogen fixation
The conversion of this atmospheric nitrogen to the usable form of ammonia is
known as nitrogen fixation.
Nitrogen fixation can be defined as the process of creating ammonia from
atmospheric nitrogen.
Without this process, most plants and animals would not have the nitrogen
needed to live.
Nitrogen fixation is carried out by the enzyme nitrogenase, which are found in
microbes

4. Need of nitrogen fixation:
Nitrogen fixation is essential to life because fixed inorganic nitrogen compounds are required for the
Biosynthesis of all nitrogen-containing organic compounds, such as amino acids and proteins nucleoside
triphosphates and nucleic acids.
Nitrogen fixation is carried out naturally in soil by microorganisms termed diazotrophs
All biological reactions involving the process of nitrogen fixation are catalysed by enzymes
called nitrogenases.
Without bacteria to play a role in the nitrogen fixation process, most global photosynthesis would come
to a grinding halt within a year. At the same time, there would be no microbes to break down massive
amounts of accumulating waste.

5. HISTORY OF NITROGEN FIXATION:
Hermann Hellriegel German agricultural chemist, discovered that leguminous plants took atmospheric
nitrogen and replenished the ammonium in the soil through the process now known as nitrogen fixation.
He found that the nodules on the roots of legumes are the location where nitrogen fixation takes place.
Hellriegel did not determine what factors in the root nodules carried out nitrogen fixation.
Martinus Willem Beijerinck a Dutch microbiologist and botanist, explored the mechanism responsible,
discovering that the root nodules contained microbes.
He further demonstrated that these microbes were bacteria, which he named rhizobia. These rhizobia
perform the chemical processes of nitrogen fixation.

6. Importance of nitrogen
NITROGEN essential constituents of all biomolecules
PLANTS from soil in form of nitrates or ammonium ion
ATMOSPHERE 78% of molecular nitrogen but plant
unable to convert this molecular nitrogen into a useable
form

7. Nitrogen cycle
There
are five
stages in
the
nitrogen
cycle
Nitrogen
fixation
Mineraliza
tion
Nitrificati
on
Immobiliz
ation
denitrifica
tion

8. Nitrogen cycle

9. Model indicating plant-microbe interaction
microbe-plant
interaction
Rhizobia
nodule
Pest control
disease
phyllosphere
Symbiotic
N2 fixation
rhizosphere
Plant
tolerance
mycorrhizae
Stem
module

10. Classification of nitrogen fixation
Nitrogen fixation
Non-biological Biological
symbiotic
Non-symbiotic
Nitrogen fixation

11. Biological nitrogen Fixation

12. Symbioitic and non-symbiotic bacteria
Symbiotic bacteria
cyanobacteria (or blue-green
algae)
Anabaena
Nostoc
Genera such
as Azotobacter, Beijerinckia,
and Clostridium
Non-symbiotic bacteria
Rhizobium associated with leguminous
plants e.g., various members of
the pea family
Frankia, associated with certain
dicotyledonous species (actinorhizal
plants
Azospirillum species,
associated with cereal grasses

13. Symbiotic nitrogen-fixing bacteria
The symbiotic nitrogen-fixing bacteria invade the root hairs of host plants
They multiply and stimulate formation of root nodules, enlargements of plant cells
and bacteria in Intimate association.
Within the nodules the bacteria convert free nitrogen to ammonia which the host
plant utilizes for its development.
To ensure nodule formation and optimum growth of legumes (e.g., alfalfa,
beans, soybeans)seeds are inoculated with commercial
cultures of Rhizobium species, especially in soils poor or lacking in the required
bacterium.

14. Non-symbiotic nitrogen fixing bacteria
• It includes Aerobic bacteria, anaerobic bacteria
and blue green algae
• Bacteria types
• Free living anaerobic : Clostridium
• Free living aerobic : Azotobacter
• Free living photosynthetic : Rhodopseudomonas
• Free living Fungi : Yeast
• Blue green algae: Nostoc, Anabaena
Other nitrogen-
fixing bacteria are
free-living and do
not require a host.
They are
commonly found in
soil or in aquatic
environments

15. NODULE:
In legumes and a few other plants, the bacteria live in small growths on the roots
called nodules. Within these nodules, nitrogen fixation is done by the bacteria,
and the NH3 they produce is absorbed by the plant

16. Structure and function of nodule
Outermost layer of nodule-bacteriod zone-enclosed by cortical cells.
Rate of N- fixation of nodule directly proportional to the volume of the
nodule.Nodules are small-contain leghaemoglobin
Effective nodules are larger pink in colour due to the presence of red coloured
leghaemoglobin.This pigment is similar to haemoglobin of blood.
Found in nodules between bacteriods & membrane envelops, enclosing them.

17. Nitrogen fixing organisms found in nodules
Rhizobium
• Rod shaped,motile.etc
• Non sporing, non-acid fast
Rhizobia are susceptible to antibiotics, bacteriophages,
fungicides, herbicides etc.
• Named after the host plant
– Pea – Rhizobium leguminosarum
– Beans – R. phaseoli
– Soyabeans – R. japonicum
– Lupins – R. lupini
• Two types of Rhizobium-
– Bradyrhizobium – slow growing species
– Rhizobium - fast growing species

18. Site and mechanism of nitrogen fixation in
nodules
Bacteroids-site
of N-fixation.
Mechanism
1) Theory of Virtanen
• N fixation in roots appear immediately
after nodule formation.young plants fix
N than the old plants.A great part of N-
converted to-L-aspartic acid and
Lglutamic acid.
• Apart from this alpha-alanine present in
nodule-produced from L- aspartic acid
by decarboxylation.small amount of
Oxime-N and nitrite-N are also present.
2) Theory of
Burris and
Wilson
hydroxylamine is the
central compound of
N fixation from which
ammonia is formed
through reduction.

19. BASIC REQUIREMENTS FOR NITROGEN FIXATION
Nitrogenase
enzyme
complex
Protective
mechanism
against Oxygen
leghaemoglobin
Electron donor
carbohydrare via
Ferrodoxin
Constant supply
of ATP
Coenzymes
and cofactors
like CoA,
inorganic
phosphate and
Mg+2
Cobalt and
Molybdenum

20. • LHb combines with O2 to form oxyleghaemoglobin (OLHb) and
makes it available at the surface of bacterial membrane
• Fe-protein interacts with ATP and Mg++, and Mo-Fe-protein catalyses
the reduction of N2 to NH3, H+ to H2
• Ferredoxin act as electron transfer during N2 fixation
• This enzyme complex gets energy from Mg ATP produce in bacterial
respiration.
N2 + 16 ATP + 8e- + 10H+ 2NH+4 + H+2 + 16ADP + 16Pi
N2 FIXATION IN ROOT NODULES

21. Fig: N2 fixation in root nodules of legumes
N2 fixation in bacteria

22. Genes involved in nitrogen fixation:
Genes involved in root
nodule formation called
nodulin genes ( nod
genes)Nodulin genes
essential for infection of
plant root and nodule
formation by symbiotic
N₂ fixing bacteria
It is divided into 2
classes
1:include genes that
specify biochemical
composition of bacterial
cell surface such as the
synthesis of
exopolysaccharides ( exo
genes),Lipopolysaccharides
(lps gene),Capsular
polysaccharides or K
antigen & β 1,2 glucans
(ndu genes)

23. Genes involved in nitrogen fixation
2) consist of nodulation genes (nod or nol)
nod genes are involved in nodulation of particular host -called
host specific nod(hsn) genes.
• Fast growing rhizobium sps - nod genes are located on large sym plasmids
Slow growing bradyrhizobium sps carry late nod gene on the
bacterial chromosome.

24. Nod D genes
Genes directing specific steps in nodulation of a legume by a strain of rhizobium are called Nod
genes
Nod genes are born on large plasmids called sym plasmids
Nod genes are located between two clusters of genes for N2 Fixation called Nif genes
Nod gene consists of 8 genes
Nod A,B,C,D,E,F,L,M
NOD D controls the function of all nod genes

25. Nif genes
nif and fix genes - structural genes for nitrogenase enzyme
Sym plasmid carry hup genes coding for hydrogenase activity
Nif genes - 22 genes are involved , arranged in 7/8clusters
Nif Q,B,A,L,F,M,Z,W,V,S,U,X,N,E,Y,T,K,D,H,J

26. Non-nodulation
Lichens - cyanobacteria
Anthoceros - Nostoc
Azolla – Anabaena azollae
Cycas – Nostoc and anabaene
Gunnera macrophylla - Nostoc
Digitaria, Maize and Sorghum – Spirillum notatum
Paspalum notatum – Azotobacter paspali

27. Nitrogenase complex
Biological nitrogen fixation
is carried out by a highly
by a highly conserved
complex of proteins called
as nitrogenase complex
which is mainly consists of
2 important proteins
Nitrogenase complex

28. Action of nitrogenase enzyme
For reducing nitrogen into ammonia nitrogenase requires 8 electrons
At first dinitrogense is reduced by transferring of electrons to dintrogenase reductase
Dinitrogenase has a 2 binding sites for reductase
The 8 electrons are transferred from reductase to dinitrigenase one at a time
Reduced reductase binds to dinitrogenase and transfers single electron , oxidized form
dissociates in a repeating cycle.
Each cycle requires the hydrolysis of ATP molecule by dinitrogenase reductase
Immediate source of electrons to reduce reductase is reduced ferredoxin
Ultimate source of electrons to reduce ferredoxin is pyruvate

29. Action of nitrogenase enzyme

30. Factors affecting N2 FIXATION
Presence of
nitrate or
ammonium
Presence of
certain inorganic
substances Ca,
Co, Mo influence
N2 fixation along
with P
Availability of
energy source :
addn. of C source
increase N2
fixation
pH :
Neutral
favours
Azotobacter
Acidic-
Beijerinkia
Soil moisture :
Adequate is
good for
fixation
Temperature:
Mesophilic –
30°C.