L35 + 36: N fixation applications

Question 1

Where does abiotic nitrogen fixation happen

Answer 1

electrical storms
combustion
fertiliser manufature

Question 2

What is the haber bosch process

Answer 2

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

Question 3

explain properties of nirogenase

Answer 3

very sensitive to irreverible inactivation of O2 and needs a lot of energy and reduignn equivalents, essentially a continuous supply of ATP and electrons

Question 4

Explain the genes involved in nitrogenase

Answer 4

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

Question 5

What are the three main themes of nitrogen fixation and general understanding needed

Answer 5

1. Induction by N-demand
2. Inhibition by elevated ammonium concentration
3. Regulation by [O2 ]

*cells need nitrogen but will stop fixing once there is enough
*O2 can irreversible damage nitrogenase

Question 6

Explain Induction by N demand

Answer 6

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

Question 7

What is the two-component regulatory system for signal transduction

Answer 7

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.

Question 8

What is inhibition by elevated ammonium concentration

Answer 8

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

Question 9

What are ways that ammonium is rapidly displaced and assimilated

Answer 9

1. enzyme activites by ammonium assimilation by GS/GOAT
2. linked cells for example in cyanobacteria N fixed in heterocysts is displaced to vegetative cells
3. symbiotic partners like plant cells of legume root nodules

Question 10

Explain regulation of O2

Answer 10

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

Question 11

Explain Regulation of O2 by genes

Answer 11

the FixLJ two-component regulatory system regulates nif genes
in response to [O2 ] in Rhizobia and some other aerobic N2 -fixers

1. FixL is a heme-protein that binds/senses [O2 ]
2. 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

Question 12

High O2 and low O2 anf FixJ

Answer 12

High [O2] => FixJ dephosphorylated, nif genes not expressed

Low [O2] => FixJ phosphorylated, express nif genes

Question 13

4 Strategies to protect nitrogenase from irreversible O2 damage

Answer 13

1.Anaerobic N2 -fixation
i. Strict anaerobes
ii. Facultative anaerobes

2. Respiratory and conformational protection
3. Microaerophilic N2 -fixation
4. Compartmentation
   i. Heterocysts
   ii. Root nodules

Question 14

Explain Anaerobic N2 fixation

Answer 14

1. Strict anaerobes:
   – obligate anaerobes that can fix N2 , such as Clostridium sp., only exist in anaerobic niches
2. Facultative anaerobes:
   – species such as K. pneumoniae only fix N2 under anaerobic conditions, do not fix N2 under aerobic condition

Question 15

Explain espiratory and conformational protection

Answer 15

– 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

2. 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

Question 16

What is Microaerophilic N2 -fixation

Answer 16

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 ]

Question 17

Explain Compartmentation method 1 + caviat

Answer 17

1. 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

Question 18

Regulation of nif gene expression in hetercysts

Answer 18

– 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

Question 19

Explain compartmentation method 2

Answer 19

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

Question 20

Explain 4 symbiotic relationships with N2 fixation

Answer 20

1. Cyanobacteria with plants
   – aquatic fern Azolla with cyanobacterium Anabaena azollae
   – A. azollae grows in chains, forms heterocysts
2. Cyanobacteria with fungi in lichens, via heterocysts or dark
3. 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
4. Rhizobium-legume symbiosis
   – interaction between plant and bacteria species is usually very specific