this presentation is about what is the genetics involvement in nitrogen fixation i.e which gene is responsible etc....
the contents include Genetics of N2 fixing microorganisms, Bacterial Nodulation Genes and Regulation of nod Gene Expression, Nif Genes and their Regulation in K. Pneumoniae & Cyanobacteria, Nitrogen fixation mechanism
Nitrogenase Types, Structure and Function, Alternative nitrogenase, Substrate for Nitrogenase, Electron proteins and Hydrogen evolution
Presentation on genetics of nitrogen fixation by Tahura Mariyam
1. Genetics of Nitrogen
Fixation
Submitted by:
Tahura Mariyam
M.Sc. Microbiology
19MSCMB009
Submitted to:
Dr. S. Ahirwar
(Microbial Genetics)
JIBB, SHUATS
DEPARTMENT OF INDUSTRIAL MICROBIOLOGY
Jacob Institute of Biotechnology and Bio-Engineering
Sam Higginbottom University of Agriculture
Technology and Sciences, Prayagraj
2. Content
Introduction
Genetics of N2
fixing microorganisms
a) Bacterial Nodulation Genes and Regulation of nod Gene Expression
b) Nif Genes and their Regulation in K. Pneumoniae & Cyanobacteria
Nitrogen fixation mechanism
Nitrogenase Types, Structure and Function
Alternative nitrogenase
Substrate for Nitrogenase
Electron proteins
Hydrogen evolution
3. Introduction
At very first Beijerinck isolated the N2
fixing bacteria from the root nodules and
named it Bacillus radicicola.
The symbiotic association between legumes and rhizobia is by far the most
important contributor to the world’s supply of biologically fixed N2
to agriculture.
In 1893 , a German botanist Winogradsky, discovered nitrogen fixation in a free-
living heterotrophic bacterium, Clostridium pasteurianum.
The element nitrogen is an essential constituent of all - living things, the proteins
and nucleic acids are their major nitrogenous constituents but some other also
contain nitrogen atoms, and the gas which composes 78% of our atmospheric N2 is
correctly termed ‘dinitrogen’.
This dinitrogen is fixed (N2 → NH3) by bacteria; hence these are called
“diazotrophs”.
4. ► Nitrogenous fertilizer is made from atmospheric dinitrogen by following
reaction is called “Haber Process”.
N2
+ 3H2
→ 2NH3
► It is clear now to accept that about 60% of the input of world's soil and water
nitrogen is today supplied by N2
fixing microorganisms.
► And nitrogen fixation plays a major role in ‘global biological productivity’.
Nodule formation in
roots by Rhizobium
T.S of root
nodule
Nostoc
5. Genetics of N2
fixing microorganisms
a) Bacterial Nodulation Genes and Regulation of nod Gene Expression :
In rhizobium species nodulation genes together with other symbiotic genes,
are located on large - plasmids (Sym plasmids). Sym plasmids vary from 50 to
over 600 kb in R. leguminosarum bv. trifoli to 1200 to 1500 kb in R. meliloti.
Nodulation genes, nod and nol genes, are classified as regulatory, common
and host specific.
Regulation of nod genes is controlled by the nodD gene, of which all rhizobia
tested so far contain one or more copies. In conjunction with plant flavonoids
or other phenolic compounds, nodD proteins act as transcriptional activators
of inducible nod genes.
The common nodABC genes are structurally and functionally conserved among
Rhizobium, Bradyrhizobium and Azorhizobium strains.
6. Protein Homologies
NodC Chitin and cellulose synthases
NodD LysR family of DNA-binding proteins
NodE β- ketoacyl synthases
NodF Acyl carrier proteins
NodH Sulphotransferases
NodL Acetyltransferases
NodM D-Glucosamine synthase
NodP ATP sulphurylase
NodQ APS kinase
Table : Possible functions of rhizobial Nod proteins
(Based on Michiels and Vanderleyden, 1995)
7. Genes Function
Q Mo uptake or processing
B Synthesis or processing of FeMoCo
A Regulatory –nif A product activates the other operons
L Regulatory
F A flavoprotein involved in electron transfer in nitrogenase
M Processing of Fe-protein
V Influences specificity of FeMoCo-Protein
S Not known
U Not Known
X Not defined genetically; presence deduced from physical map and cloning of nif DNA
N Like B
E Like B
Y Discovered in the same way as X
K Codes for β-subunit of MoFe-protein
D Codes for α-subunit of MoFe-protein
H Codes for subunit of Fe-Protein
J May be involved in electron transfer to nitrogenase
Table : Nif gene products & their function in
Klebsiella pneumoniae
8. b) Nif Genes and their Regulation :
K. Pneumoniae : The N2
fixation (nif) genes are organized into a regular of 17 genes,
consisting of seven or eight operons each of which is transcribed into a single, usually
polycistronic mRNA. Although only 5 of the gene products have been purified and properly
characterized, functions have been assigned to all of the gene except for nifX and nifY.
Regulation of nif gene expression has 2 elements, an external system designated ntr and an
internal system mediated by nifA and nifL. 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, in the last case
by switching on the nif genes. The inter-relationships between external and internal
regulation of the nif genes in K. pneumoniae and the conditions under which nitrogenase
syntheses occurs.
Actually, the ntrA gene product (NtrA) is a-factor of RNA
polymerase which recognizes the nif ans, 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 there. The ntrB gene product
(NtrB) is an enzyme that function both as a protein kinase and as a phosphatase, the
substrate of which is NtrC (the ntrC gene product).
9. Whether kinase or phosphatase activity predominates depends upon the nitrogen status of the
bacterium, and the consequence of this is that, under conditions of starvation, NtrC-P acts as an
activator of, among other operons, nifL and nifA. 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 O2, inactivate the nifA product, thereby preventing
transcription of other nif genes.
10. In cyanobacteria - In heterocystous cyanobacteria, the acquisition of
nitrogenase activity in response to nitrogen deficiency is accompanied by the
differentiation of vegetative cells into a specialized structure called ‘heterocysts'. This
process has been studied by Haselkorn (1986). All the non-heterocystous
cyanobacteria possess the gene nifH, nifD, nifK as a cluster. In the DNA of vegetative
cells of heterocystous cyanobacteria the gene nifK is separated from the gene nifD
and nifH as observed by Haselkorn et al. (1986). During the differentiation the
intervening DNA of about 11000 base pairs is excised as a circle resulting in a
clustered nifHDK operon as studied in Anabaena PCC 7120. This excision is catalyzed
by the product of a gene, xisA located with in excised 11 kb region.
A second rearrangement has been observed during
heterocyst differentiation by Haselkorn et al. (1986). This occurs, the region of nifS, a
gene involved in K. pneumoniae. In this rearrangement a operon with structure of
nifB: ORF -1: nifS; ORF-2 is formed. The excision occurs between ORF-1 and nifS,
which is not catalyzed by the xisA product . It is interesting to note that no
expression of nifH gene from Anabaena variabilis was found when interested with its
promoter into K. pneumoniae which gave the evidence that nif gene promoters in
Anabaena PCC 7120 differ in structure from both E. coli promoters and the nif
promoters of K. pneumoniae.
11.
12. Nitrogen fixation mechanism
a) Nitrogenase Types, Structure and Function :
The biological conversion of atmospheric N2
to ammonia taken place with
the help of an enzyme called Nitrogenase.
This enzyme is anaerobic in nature and when it comes in contact with O2
or air, it becomes inert.
The proteinaceous enzyme is made up of 2 subunits mainly called large
MoFe protein and another small Fe protein.
The nitrogenase purified from three species of bacteria :
i. The Cp type.
ii. The Kp type (Klebsiella pneumoniae)
iii. The Ac type (Azotobacter chroococcum).
13. b) Alternative nitrogenase :
► The genetic evidences revealed that their normal genes for nitrogenase
(nifYKDH) deleted.
► This nitrogenase was later isolated from A. chroococcum. It consists of 2
proteins, one large and heteromeric, one smaller very like the regular Fe -
protein; both are sensitive to O2
.
c) Substrate for Nitrogenase :
► For enzyme activity a suitable substrate is required so as to bind all the active
sites of enzyme to get a product. The overall reaction in the enzymic reduction
of atmospheric nitrogen to ammonia could be postulated as follow:
N2
(N≡N) → HN═NH → H2
N−NH2
→ H3
N + NH3
► This enzyme system catalyzes the transfer of electrons from pyruvate or
hydrogen to ferredoxin or flavodoxin.
14. a) Ferredoxins or dinitrogenase : Ferredoxins are electron carrier,
discovered by Mortenson and Caruahan in the year 1962 from C.
pasteurianum. It is naturally occurring electron carrier iron – Sulphur (Fe-S)
protein (reversible). It has now been isolated from number of cyanobacteria,
photosynthetic bacteria and even from higher plants. The ferredoxins are
involved in various physiological process such as photosynthesis in plants and
pyruvate metabolism in anaerobic bacteria.
b) Flavodoxins or dinitrogen reductase : The bacteria grow under limited
iron supply i.e. nutritional stress condition and produce flavodoxin. It was also
isolated in the beginning from C. pasteurianum. It is interesting that it was
found to replace ferredoxin as an electron carrier in a large number of
reactions. An electron carrier named ‘azotoflavin' has been isolated from
Azotobacter vinelandii possessing biological activity similar to ferredoxins.
15. Electron Proteins
MoFe (kp1
) proteins play a key role in nitrogen fixation (substrate binding and
reduction) with Fe proteins (kp2
) that assists in transfer of electron from flavodoxin
to the bigger subunits and ATP consumption. Neither of the two proteins subunits
can function independently. Fe proteins has four Fe centers and equal number of
Sulphur, whereas the number of Fe centers in MoFe protein varies. An equivalent
number of inorganic Sulphur is also present. In contrast to Fe protein, MoFe
protein possesses two additional Mo atoms. Mo is suggested to play a vital role in
H2
evolution which accompanies N2
fixation.
N2
reduction process starts with the transfer of single electron (2 ATP molecules)
from Flavodoxin to smaller Fe protein subunit Kp2
. At this stage two ATP
molecules combine with two Mg2+
ions to form complex, Mg ATP which attaches
to Fe protein and energizes it to transfer electron from the iron atoms in Fe
protein to Mofe protein, prior reaching the bound substrate.
16. The MoFe protein becomes unstable and to counter balance this electron a H
+
ion formed
by dissociation of H2
O comes to attach at Mo atom of MoFe protein . The electron flow from
flavodoxin to MoFe protein via Fe protein is a repeated process . Each time an electron is
transferred, there is a consumption of two molecules of ATP . The second electron is again
balanced by another H+
ion. When the third electron is transferred to MoFe protein, the H+
ions are displaced by nitrogen leading to the evolution of one molecule of hydrogen. This
third electron again balances by attachment of one H
+
ion to nitrogen i.e. HN = NH .
17. Hydrogen Evolution
For every N2
molecule fixed, one molecule of H2
is evolved. This process is
expensive to nitrogen fixation due to involvement of 16 ATP molecules,
whereas similar fixation can be held by using 12ATP molecules. It means
four molecules are just wasting. To overcome this process, the enzyme
responsible for normal hydrogen evolution is called hydrogenase, and it
can catalyse both the uptake and evolution of hydrogen.
The hydrogen is trapped by hydrogenase in Azotobacter and recycled.
