L35 + 36: N fixation applications Flashcards
Where does abiotic nitrogen fixation happen
electrical storms
combustion
fertiliser manufature
What is the haber bosch process
high temps and pressure
1.5kg of feul oil per kg fertiliser is manufactured and delivered, we depend on these fertilisers
*results in air and water pollution and 50% of applied N fertiliser is lose by leeching
explain properties of nirogenase
very sensitive to irreverible inactivation of O2 and needs a lot of energy and reduignn equivalents, essentially a continuous supply of ATP and electrons
Explain the genes involved in nitrogenase
encoded by nifH D and K
around 20 nif genses in a cluster of 24kb in 8 operon
expression is highly regulated due to energy requirements and senesiitvty
the expression is regulated many two component regulatory systems
What are the three main themes of nitrogen fixation and general understanding needed
- Induction by N-demand
- Inhibition by elevated ammonium concentration
- Regulation by [O2 ]
*cells need nitrogen but will stop fixing once there is enough
*O2 can irreversible damage nitrogenase
Explain Induction by N demand
induction of gene expression
so cells will only fix when needed to conserve energy
*the global nitrogen regulatory system encoded by ntr gene will sense when cells need N
then the expression of N fixation genes (nif and fix) is induced in response to N demand by NtrBC two comp system
to create a balance, cells balance N demand with NH4+ repression which is mediated by glutamine
What is the two-component regulatory system for signal transduction
it’s a senor and gene regulatory pair
for example, NtrB and NitrC
or FixL and FixJ
The (1st) sensor protein interacts with the regulatory (2nd) Protein
The detection by the sensor (to a stimulus) creates a conformational change => autophosphorylation.
The phosphorylation is transferred from the sensor to the regulator, which changes how it will react with the gene promoter to either increase or decrease gene expression.
What is inhibition by elevated ammonium concentration
end product inhibition of the enzyme activitites and metabolic pathways done by NH4+, this is an important control mechanism
it’s because N2 fixation needs a way to avoid ammonium repression until all the cells have N
to make sure this works we need rapid assimilation and displacement of ammonium to avoid inhibition of N2 fixation
What are ways that ammonium is rapidly displaced and assimilated
- enzyme activites by ammonium assimilation by GS/GOAT
- linked cells for example in cyanobacteria N fixed in heterocysts is displaced to vegetative cells
- symbiotic partners like plant cells of legume root nodules
Explain regulation of O2
O2 regulation isnt needed for aneorbic diazotrophs because aerobic/microaerophilic diaztropis do not produce nitrogenase when [O2] is high to inactivate it
so nif gene expression is induced by FixLJ two component system when O2 is low
other cells have ways to protect nitrogenase from irreversible O2 damage
Explain Regulation of O2 by genes
the FixLJ two-component regulatory system regulates nif genes
in response to [O2 ] in Rhizobia and some other aerobic N2 -fixers
- FixL is a heme-protein that binds/senses [O2 ]
- FixJ is a gene regulatory protein
FixLJ only induce nif gene expression when [O2 ] is low, if [O2 ] increases, nif gene expression will be repressed
High O2 and low O2 anf FixJ
High [O2] => FixJ dephosphorylated, nif genes not expressed
Low [O2] => FixJ phosphorylated, express nif genes
4 Strategies to protect nitrogenase from irreversible O2 damage
1.Anaerobic N2 -fixation
i. Strict anaerobes
ii. Facultative anaerobes
- Respiratory and conformational protection
- Microaerophilic N2 -fixation
- Compartmentation
i. Heterocysts
ii. Root nodules
Explain Anaerobic N2 fixation
- Strict anaerobes:
– obligate anaerobes that can fix N2 , such as Clostridium sp., only exist in anaerobic niches - Facultative anaerobes:
– species such as K. pneumoniae only fix N2 under anaerobic conditions, do not fix N2 under aerobic condition
Explain espiratory and conformational protection
– in aerobic N2 -fixing bacteria i.e., Azotobacter
– microaerophilic diazotrophs also use respiratory protection
two ways:
1. CONFORMATION PROTECTION
- FeSII (Shethna) protein
– protects nitrogenase from oxidative damage by binding to it
=> binds nitrogenase when oxidized (as below)
=> releases nitrogenase when reduced
- RESPIRATORY PROTECTION
– high rate of respiration uses up O2 at cell surface results in
intracellular [O2 ] low enough for nitrogenase function
– occurs at high [O2 ] and if energy source is not limiting
– Example: A. vinelandii respiratory chain adapts to [O2 ]
– NDH I is coupled to H+ pumping & membrane potential
– NDH II is uncoupled from H+ pumping, rapid reduction of [O2 ] confers respiratory protection
What is Microaerophilic N2 -fixation
microaerophiles Azospirillum has terminal oxidases with high affinity for O2 to allow aerobic respiration at low [O2]
==> Azospirillum is commonly used as a plant-growth promoting
bacterium (PGPR), inoculated onto cereal crops etc
– high-affinity terminal oxidase “burns up” the O2 at the cell surface, resulting in a very low intracellular [O2 ]
– respiratory protection of nitrogenase
– aerobic respiration allows cells to generate ATP very efficiently to support N2 -fixation
– microaerophilic diazotrophs perform a delicate balancing act between incompatible processes: N2 -fixation and aerobic respiration
– can use aerotaxis to move to zone with optimal low [O2 ]
Explain Compartmentation method 1 + caviat
- Heterocysts
when N-demand is sensed in cyanobacteria that grow in chains and that can fix N2 , some of the vegetative cells differentiate into heterocysts which can fix N2 day or nigh
caviat: some single-celled cyanobacteria can also fix N2 , but only at night/dark, when oxygenic photosynthesis is not active
– spatial separation of N2 -fixation and oxygenic photosynthesis
– no PS2 (oxygenic) in heterocyst
– thick cell wall = O2 barrier
– rapid NH4+ -assimilation and movement of fixed N out of
heterocyst
– heterocysts supplied by vegetative cells with disaccharides as
carbon and energy source
– exchange of C and N via microplasmadesmata
Regulation of nif gene expression in hetercysts
– in Anabaena vegetative cells nifHDK operon and fdxN gene are non-functional (and N2 fixation cannot occur)
– nifD (below) and fdxN genes are disrupted by DNA insertions
– during heterocyst development these insertions are excised
– via XisF recombinase and XisA recombinase
– nifHDK operon and fdxN gene are re-formed by recombination
– thus, heterocysts can fix N2
Explain compartmentation method 2
A. Root nodule O2 barrier: tightly linked, water-filled plant cells on periphery
B. O2 transport and binding within the root nodule
– leghemoglobin
– maintains low [dissolved O2 ], high flux from outside
C. High rate of respiration by bacteroids
– terminal oxidase has high-affinity for O2
– also functions as a form of respiratory protection
Explain 4 symbiotic relationships with N2 fixation
- Cyanobacteria with plants
– aquatic fern Azolla with cyanobacterium Anabaena azollae
– A. azollae grows in chains, forms heterocysts - Cyanobacteria with fungi in lichens, via heterocysts or dark
- Actinomycete bacteria with woody plants
– Frankia (filamentous bacteria) with plants such as Alnus (alder, common in bogs, etc.)
– N2 -fixing form (diazovesicles) have thick cell walls
– O2 barrier - Rhizobium-legume symbiosis
– interaction between plant and bacteria species is usually very specific
