Microbial Physiology & Metabolism

(CC - 6)

Nitrogen Metabolism (UNIT - 6)

• Within living cell, biomolecules are constantly being
synthesized & transform into some other biomolecules
• These synthesis & transformation constantly occur
through enzymatic reactions
• Together all the interconnected chemical reactions
occurring within a cell are called metabolism
• Metabolism serves two fundamentally different purposes
– a) generation of energy to drive vital functions & b) the
synthesis of biological molecules
• When an inorganic compound, such as, NO3- , SO42-, CO2
is reduced for use as a nutrient source, it is said to be
assimilated, and the reduction process is called
assimilative metabolism
• In assimilative metabolism, only enough of those
compounds are reduced to satisfy the needs for cell
growth
• The reduced atoms are eventually converted to cell
material in the form of macromolecules
• However, the use of these compounds as electron
acceptors in energy metabolism is called dissimilative
metabolism
• In dissimilative metabolism, a comparatively large amount
of the electron acceptor is reduced, and the reduced
product is excreted into the environment
• Many organisms, for example, bacteria, Achaea, fungi,
algae & higher plants carry out assimilative metabolism of
compounds, such as, NO3- , SO42-, CO2 whereas only a
restricted variety of organisms, primarily prokaryotes, carry
out dissimilative metabolism
Introduction to Nitrogen Fixation
• A continuous series of natural processes by which
nitrogen passes through successive stages in air, soil,
organisms involving nitrogen fixation, ammonification,
nitrification and denitrification is known as nitrogen cycle
 Nitrogen Fixation

• The metabolic assimilation of atmospheric nitrogen by

soil microorganisms especially Rhizobia and the
production of nitrogenous salts that are available for
plant use
• It is the reduction of atmospheric nitrogen gas (N2) to
ammonia (NH3)
• The complete equation for nitrogen fixation is –
N2 + 6e- + 6H+ + 12ATP + Mg2+ =
2NH3 + 12ADP + 12Pi + Mg2+
• Nitrate is the major form of N taken up by plants in soil
• Microbial oxidation of ammonium to nitrite (Nitrosification) is
carried out by Nitrosomonas, Nitrosococcus, Nitrosospira,
Nitrosolobus etc. (ammonium oxidizers/ Nitrosifyers)
NH4+ > NO2-
• Nitrite is then oxidized to Nitrate (Nitrification) by Nitrobacter,
Nitrococcus etc. (Nitrogen oxidizer/ Nitrifyers)
• Some nitrates are converted back into nitrogen gas through the
process of Denitrification. It is the conversion of nitrates to
nitrous oxide & nitrogen gas. It is brought about by
Pseudomonas, Thiobacillus denitrificans, Micrococcus
denitrificans etc.
• After being used by plants and animals, nitrogen is then
disposed off in decay and wastes
• Detritivores and decomposers decompose plants and animals,
thus converting the nitrogen into ammonia (ammonification)
• Non-symbiotic nitrogen fixation -
• Free living microbes involved in nitrogen fixation are –
• Aerobes (chemoorganotrophs) – Azotobacter, Klebsiella,
Beijerinckia, Bacillus polymyxa, Methylomonas,
Methylococcus
• Alcaligenes, some Thiobacillus (chemolithotrophs)
• Anaerobes (chemoorganotrophs) – Desulfovibrio,
Clostridium, Desulfotomaculum
• Chlorobium, Chromatium, Rhodospirillum, Rhodobacter,
Thiocapsa, Heliobacterium (phototrophs)
• Archaea, Methanococcus, Methanosarcina
(chemolithotrophs)
• Nitrogen fixation is highly sensitive to the presence of O 2,
so Azotobacter developed a special defensive mechanism
against O2 – a significant intensification of metabolism that
reduces the concentration of oxygen in cells
• A special nitrogenase -protective protein protects the
enzyme and is also involved in protecting the cells from
oxygen
• Mutants not producing this protein are killed by oxygen
during nitrogen fixation in the absence of a nitrogen source
in the medium
 Nitrogenase
• Biological process of nitrogen fixation is catalyzed by an
enzyme complex called nitrogenase complex
• There are 3 different forms of nitrogenase that differ in
their requirement for molybdenum, vanadium or iron as
metallic component
• Most of the nitrogenases that have been studied contain
a Mo cofactor
• Nitrogenase consists of 2 proteins – a dinitrogenase
reductase & dinitrogenase
• The dinitrogenase reductase (Fe protein) is a dimer of
identical 30 kDa subunits bridged by a 4Fe-4S cluster
• Dinitrogenase is a tetramer with 2 copies of two different
subunits
• It contains both Fe & Mo
• Because molybdenum is present in the cluster, the
dinitrogenase component is also called the molybdenum-
iron protein (MoFe protein)
• The MoFe cofactor is the site of nitrogen fixation
• The genes involved collectively in the synthesis of
nitrogenase & the catalytic process of nitrogen fixation
are called nif genes
• Accessory genes are called fix genes; they are also
necessary for the function & regulation of nitrogenase in
aerobic nitrogen-fixing bacteria
• The nitrogenase complex is very sensitive to oxygen & is
irreversibly inactivated by oxygen
• Hence, nitrogen fixation must occur under anaerobic
conditions
• For anaerobic prokaryotic organisms, there is no
problem
• Facultative prokaryotic organisms such as Purple
photosynthetic bacteria fix nitrogen only in anaerobic
conditions
• In aerobic organisms such as Cyanobacteria, anaerobic
conditions are created in specialized cells called
heterocysts
• Symbiotic nitrogen-fixing prokaryotes such as
Rhizobium, maintain a very low concentration of free
oxygen in root nodules of leguminous plants by
producing leghemoglobin, a homolog of hemoglobin,
which is present in the cytoplasm of infected nodule cells
at high concentrations
 Cross – Inoculation Groups of Rhizobium

• Scientists have studied and categorized the matching

system for many important legumes and Rhizobia
partners into cross inoculation groups.
• Each of these consists of the entire legume species that
will develop nodules when inoculated with Rhizobia
obtained from any other member of the same group
• This grouping is a convenient and satisfactory way to
classify Rhizobia into species.
 Leghaemoglobin

• It is an oxygen carrier and hemoprotein found in the

nitrogen-fixing root nodules of leguminous plants
• It is produced by legumes in response to the roots being
colonized by nitrogen-fixing bacteria (Rhizobia), as part of
the symbiotic interaction between plant and bacterium
• Roots not colonized by Rhizobium do not synthesize
leghaemoglobin
• It has close chemical and structural similarities to
hemoglobin
• It is red in colour
• It protects the oxygen-sensitive Nitrogenase, key enzyme
in nitrogen fixation
• It buffers free O2 concentration in the cytoplasm of
infected plant cells to ensure proper functioning of root
nodules
• It has a high affinity for oxygen, about 10 times higher
than the beta chain of human hemoglobin
• This maintains a low O2 concentration to allow
nitrogenase to function as well as provide the bacteria
with O2 for respiration
 Assimilation of Ammonium Ions

• Ammonia, the primary product of biological nitrogen

fixation, is toxic to cells in high concentrations
• Thus, it is converted to amides (eg. Asparagine,
glutamine) or ureides (eg. Allantoin, citrulline)
• These organic forms are transported to shoot via the
xylem
 Ammonia Assimilation
• Nitrogen is a key component of amino acids
• Nitrogen in the form of ammonium ion is the source of
nitrogen for all the amino acids
• Ammonia nitrogen can be incorporated into organic
material relatively easily & directly because it is more
reduced than other forms of inorganic nitrogen
• Some microorganisms from the amino acid alanine in a
reductive amination reaction catalyzed by Alanine
dehydrogenase
• Pyruvate + NH4+ + NADH (NADPH) + H+ > L-alanine +
NAD+ (NADP+) + H2O
• The two amino acids glutamate & glutamine provide the
critical entry point
• Glutamate is the source of amino group for most other
amino acids through transamination reactions
• There are two principal means by which ammonium ions
are incorporated into amino acids & eventually other
metabolites –
1. Reductive amination of α- keto acids:
• Glutamate dehydrogenase catalyzes the reductive
amination of α- ketoglutarate (a TCA cycle intermediate)
and forms glutamate
• This pathway is present in all organisms
• Different species vary in their ability to use NADPH &
NADH as the reducing agent in glutamate synthesis
2. Formation of the amides of glutamic acids:
• Ammonium ions are also incorporated into cell
metabolites by the formation of glutamine, the amide of
glutamate
• This amidation reaction is catalyzed by Glutamine
synthetase (present in all organisms) and is driven by
the hydrolysis of ATP
• Glutamate + NH4+ + ATP > Glutamine
• Then the amide nitrogen of glutamine is transferred to α-
ketoglutarate to generate a new glutamate molecule with
the help of Glutamate synthase
• Both ATP and a source of electrons, such as, NADPH or
reduced Ferredoxin, are required
• α- ketoglutaric acid + Glutamine + [NADPH + H+] (or
Fdreduced) > 2Glutamic acids + NADP+ (or Fdoxidized)
• Transamination:
• Many amino acid biosynthetic pathways involve
transamination reactions
• Once either alanine or glutamate has been synthesized,
the newly formed α-amino group is transferred to an α-
keto acid to produce a new amino acid
• This reaction is catalyzed by the enzyme
Aminotransferases (formerly called transaminases)
• The enzyme requires the participation of an aldehyde-
containing coenzyme, pyridoxal-5-phosphate
• Microorganisms have a number of transaminases, each
of which catalyzes the formation of several amino acids
using the same amino acid as an amino group donor
• When glutamate dehydrogenase works in cooperation
with transaminases, ammonia can be incorporated into a
variety of amino acids
 Assimilatory Nitrate Reduction
• The nitrogen in nitrate (NO3-) is much more oxidized than
that in ammonia
• Nitrate must be reduced to ammonia before the nitrogen
can be converted to an organic form
• This reduction of nitrate is called assimilatory nitrate
reduction
• In this process, nitrate is incorporated into organic
material & does not participate in energy generation
• The process is widespread among bacteria, fungi &
algae
• It takes place in the cytoplasm in bacteria
• It is an important step of nitrogen cycle
• The first step in nitrate assimilation is its reduction to
nitrite by Nitrate reductase, an enzyme that contains
both FAD & Molybdenum
• NADPH is the electron source
• NO3- + NADPH + H+ > NO2- + NADP+ + H2O
• Nitrite is next reduced to ammonia with a series of two
electron additions catalyzed by Nitrite reductase &
other enzymes
• Hydroxylamine may be an intermediate
• The ammonia is then incorporated into amino acids
 Dissimilatory Nitrate Reduction
• Inorganic nitrogen compounds are some of the most
common electron acceptors in anaerobic respiration
• One of the most common alternative electron acceptors
is nitrate (NO3-), which is reduced to N2O (nitrous oxide),
NO (nitric oxide) & N2
• As all these products of nitrate reduction are gaseous,
they can easily be lost from the environment, the
process is called Denitrification
• Gaseous nitrogen is formed biologically following this
process
• Numerous soil microbes use nitrate as an electron
acceptor in anoxic soils
• When this occurs in agricultural soils, it depletes the soil
nitrogen & decreases the yield from that field
• It also forces farmers to use nitrogen-containing
fertilizers that can have detrimental consequences (such
as contamination of nearby wells, streams & rivers) as
nitrate is toxic to humans and animals
• However, for sewage treatment, denitrification is
beneficial because it converts nitrate to nitrogen,
effectively decreasing the amount of available nitrogen in
the sewage treatment effluent that can stimulate algal
growth (eutrophication)
• The use of nitrate & other electron acceptors by
chemoorganotrophs links the carbon cycle to other
biogeochemical cycles such as the nitrogen cycle & the
sulfur cycle
• The anaerobic reduction of nitrate makes it unavailable
for assimilation into the cell
• Therefore, it cannot be used to construct N-containing
molecules such as amino acids & nucleotides
• Therefore this process is called dissimilatory nitrate
reduction
• It leads to the production of gaseous compounds such
as N2 that are released into the atmosphere
• The denitrifying bacteria are metabolically diverse in
terms of alternative energy-generating mechanisms
• Most denitrifying bacteria are facultative aerobes
• Many denitrifying bacteria also reduce other electron
acceptors anaerobically, such as Fe3+
• Some dinitrifiers can even ferment
• The enzyme that catalyzes the first step of dissimilative
nitrate reduction is Nitrate reductase, a molybdenum-
containing membrane-integrated enzyme whose
synthesis is repressed by O2
• The first product of nitrate reduction is NO2- & the
enzyme Nitrite reductase reduces this to NO
• Some organisms can reduce NO2- to ammonia in a
dissimilative process
• The biochemistry of dissimilative nitrate reduction has
been studied in detail in Escherichia coli, in which NO3- is
reduced only to NO2- & in Paracoccus denitrificans &
Pseudomonas stutzeri, in which true denitrification
occurs
• The E. coli nitrate reductase accepts electrons from a b-
type cytochrome
• In P. denitrificans & P. stutzeri, nitrogen oxides are
formed from NO2- by a series of enzymes including
Nitrite reductase, Nitric oxide reductase & Nitrous
oxide reductase
• During these electron transport reactions, a proton
motive force is established & ATPase couples this to the
synthesis of ATP
 References
1. Rhizobium ppt - https://
www.slideshare.net/sujatadandale/rhizobium-ppt
2. An Effective and Beneficial Plant Growth Promoting Soil
Bacterium “Rhizobium”: A Review - https://
www.researchgate.net/publication/271774673_An_Effec
tive_and_Beneficial_Plant_Growth_Promoting_Soil_Bac
terium_Rhizobium_A_Review
3. Brock Biology of Microorganisms by Madigan, Martinko,
Bender, Buckley, Stahl, 14th Edition, Pearson education,
Inc.
4. Prescott’s Microbiology by Willey, Sherwood,
Woolverton, 9th Edition, McGraw - Hill