Lecture 33

Question 1

What is the purpose of nitrogen fixation? What organisms can do it?

Answer 1

All cells require N, but the atmosphere is 79% N2. Only certain bacterial and archaeal species can fix it into a useable form, NH4+

Question 2

What is the formula for N2 fixation? What catalyzes it?

Answer 2

N2 + 8H+ + 8e- -> 2NH3 + H2
Catalyzed by nitrogenase

Question 3

Is nitrogen fixation a relatively energy-intensive reaction? Why or why not?

Answer 3

Yes - it is very energy intensive because the triple bonded N2 must be broken and converted to NH4+.

Question 4

What is abiotic N fixation used for?

Answer 4

Mostly industrial uses - combustion, fertilizer manufacture, etc.

Question 5

Abiotic N2 fixation occurs by what process?

Answer 5

The Haber-Bosch process.

Question 6

Explain how the Haber-Bosch process works, including the required conditions and the outputs.

Answer 6

It requires a high temperature (300-400ºC) and pressure (35-110 MPa), as well as a lot of fuel oil per kg of fertilizer manufactured. It results in artificial fertilizer as well as air and water pollution due to loss of fertilizer to leaching (up to 50%).

Question 7

What conditions does nitrogenase enzyme need to function?

Answer 7

It is extremely sensitive to irreversible inactivation by O2 - since it’s a strong reducing agent, it requires very anaerobic conditions. This is why nitrogen fixation is ALWAYS an ANAEROBIC process.

It also requires a continuous supply of ATP and electrons via NADH.

Question 8

What genes encode nitrogenase? Explain.

Answer 8

It is encoded by the nifH, nifD, and nifK genes. However, about 20 nif genes are needed to make nitrogenase function.

Question 9

To what extent is nitrogenase regulated? Why?

Answer 9

It is highly regulated because of its high energy requirements and extreme sensitivity to oxygen.

Question 10

What are the 3 themes in the regulation of nitrogenase expression?

Answer 10

1. Induction by N demand
2. Repression by ammonium
3. Regulation by [O2]

Question 11

Induction by N demand is induced in response to […] by the […].

Answer 11

N demand, NtrBC two-component regulatory system.

Question 12

Describe the proteins present in the nitrogenase two-component regulatory system and their function. Give 2 examples and explain how the system functions in general.

Answer 12

There is always a pair of proteins: a sensory protein and a gene regulatory protein (DNA binding). Two example: NtrB (sensor) and NtrC (regulator), FixL (sensor) and FixJ (regulator).

The sensor will sense a stimulus such as O2 or N2 demand. The detection of the stimulus will result in conformational change and autophosphorylation, which is then transferred to the regulator. This changes how it interacts with gene promoters to increase or decrease gene expression.

Question 13

Describe what happens in the NtrCB two-component regulatory system under high nitrogen conditions.

Answer 13

Under high intracellular nitrogen conditions, there is high [glutamine]. This means that NtrB will not phosphorylate NtrC, and nif genes will not be expression. Nitrogen fixation will therefore not take place.

Question 14

Describe what happens in the two-component regulatory system under low nitrogen conditions.

Answer 14

Under low intracellular nitrogen conditions, there will be low [glutamine]. This means that NtrB will phosphrylate NtrC, and nif genes will be expressed. Nitrogen fixation will therefore take place.

Question 15

Ammonium repression of nitrogenase expression is controlled by […]

Answer 15

End-product inhibition (NH4+)

Question 16

Explain the principle behind ammonium repression and the challenge it causes.

Answer 16

The idea is that cells conserve energy by only fixing N2 (expression of nitrogenase) when NH4+ is low. They will therefore be subject to end-product inhibitition when NH4+ is abundant, and nif genes will not be expressed.

Cells must therefore balance N-demand and NH4+ repression, as N2-fixing cells in multicellular systems must avoid ammonium repression until all cells have sufficient N. They must therefore rapidly assimilate or displace ammonium so that they can keep producing more N.

Question 17

What are the 3 ways in which ammonium can be displaced and/or assimilated? Explain each one.

Answer 17

1. Enzyme activities
   Ammonium can be assimilated into glutamate via GS-GOGAT
2. Linked cells
   N fixed in heterocysts can be displaced to vegetative cells
3. Symbiotic parters
   Examples: plant cells of legume root nodules, mycobiont of lichens, etc.

Question 18

Explain the principle behind regulation by O2 in nitrogenase expression. What system is involved in this?

Answer 18

Aerobic and microaerophilic cells will not produce nitrogenase when [O2] is high enough to inactivate it. The FixLJ two-component regulatory system is involed in this, and will induce nif gene expression when [O2] is low.

Question 19

Explain the function of FixL and FixJ in regulation by O2. Include what will happen in low and high [O2] scenarios.

Answer 19

FixL is a heme protein that senses [O2] and FixJ is a gene regulatory protein.

In high oxygen scenarios, FixL will not phosphorylate and will thus not phosphorylate FixJ.

In low oxygen scenarios, there’s no O2 bound to FixL’s heme, so FixL will phosphorylate and transfer the phosphorylation to FixJ, which will expression nif genes.

Question 20

Name the 3 strategies used by aerobes to protect nitrogenase from O2 damage.

Answer 20

1. Respiratory and conformational protection
2. Microaerophilic N2 fixation
3. Compartmentation

Question 21

Give an example of an aerobic N2-fixing bacteria that uses respiratory and conformational protection

Answer 21

Azobacter - can fix nitrogen under full aerobic conditions.

Question 22

Explain how conformational protection works, including the relevant proteins involved.

Answer 22

Bacteria that do this make a protein called FeSII (Shethna) protein. It protects nitrogenase from oxidative damage by binding to it.

It binds nitrogenase when oxidized (high oxygen concentrations) and will release nitrogen when reduced (low oxygen concentrations).

Question 23

Explain how respiratory protection works.

Answer 23

When [O2] is high and the energy source is not limiting, the respiratory chain will adapt. NDH II, the respiratory chain, will uncouple from H+ pumping. The rate of respiration will increase, using up O2 so that intracellular [O2] is low enough for nitrogenase function.

Question 24

Give am example of an organism that is capable of microaerophillic N2 fixation. Provide its function.

Answer 24

Azospirillum. It is commonly used as plant-growth promoting bacterium that is inoculated onto cereal crops.

Question 25

Explain how microaerophillic N2 fixation works.

Answer 25

A high-affinity terminal oxidase burns up the O2 at the cell surface, resulting in a very low intracellular [O2]. This is a form of respiratory protection of nitrogenase.

These cells still do aerobic respiration because it allows for efficient generation of ATP to support N2-fixation. They therefore have to balance N2 fixation, an anaerobic process, and aerobic respiration.

They therefore tend to use aerotaxis to move to zones with optimal low [O2].

Question 26

Give 2 examples of compartmentation.

Answer 26

Heterocysts and root nodules.

Question 27

Explain how heterocysts work.

Answer 27

In single-celled cyanobacteria, they can normally only fix N2 at night, when oxygenic photosynthesis is not active.

However, when N-demand is sensed in cyanobacteria that grow in chains, some of the vegetative cells will differentiate into heterocysts that can fix N2 day or night. This provides separation of N2 fixation and oxygenic photosynthesis, as there is a thick cell wall that blocks out O2.

Vegetative cells provide heterocysts with disaccharides as carbon and energy source, and vegetative cells receive N in exchange. This exchange happens via microplasmadesmata.

Question 28

Describe how photosystems differ between heterocysts and vegetative cells, and explain how this affects their functioning.

Answer 28

Vegetative cells have the standard PS1 and PS2, so they generate sugars from light and CO2, producing O2. These sugars can enter the heterocyst.

The heterocyst only has PS1 and NOT PS2. PS1 produces ATP from light, and this powers the N2 fixation reaction, generating NH4+. This is also fueled by carbon metabolism. The organic acids generated from carbon metabolism can also fuel the next step, assimilation of NH4+ to glutamine. The glutamine is then transferred to the vegetative cells.

Question 29

Describe the gene regulation of nif expression in heterocysts vs vegetative cells.

Answer 29

In vegetative cells, the nifHDK and fdxN gene are non-functional, so N2 fixation cannot occur.

During heterocyst development, nifD and fdxN genes are disrupted by DNA insertions via XisF recombinase and XisA recombinase. The nifHDK operon and fdxN gene are re-formed by recombination, and so heterocysts can fix N2.

Question 30

Give 2 examples of organisms that have root nodules.

Answer 30

Rhizobia (legumes) and Frankia (alder)

Question 31

Explain how root nodules work to fix N2.

Answer 31

N2 fixation occurs in fully developed bacteroids within root nodules. The root nodules have an O2 barrier. This barrier is compoosed of water-filled cells that can let in more or less oxygen - they have a protein called leghemoglobin that allows for oxygen fluxes that keeps [dissolved O2] low. Oxygen movement (flux) is therefore rapid without being at a high concentration. This allows the inside of the cell to stay anaerobic for N2 fixation while the parts of the cell that need the O2 (ETC) can receive O2. The TEA has a high affinity for O2 and will therefore burn it off quite quickly so that it doesn’t hurt the rest of the cell.