Lecture 35 part 2

Question 1

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

Answer 1

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

Question 2

Describe activation of Nitrogenase by N demand

Answer 2

• Cells only fix N2 when needed
  - To conserve energy
• This theme is inextricably linked to repression by ammonium
• The global nitrogen regulatory system (encoded by ntr genes) senses when cell needs N
• The expression of nitrogen fixation (nif and fix genes) genes is induced in response to N demand by NtrBC

2 COMPONENT REGULATORY SYSTEM

Question 3

Describe the 2-component regulatory systems of Nitrogenase?

Answer 3

• Sensor and gene regulatory (DNA-binding) protein pair
• 2 examples specific to nitrogen fixation
  - NtrB and NtrC
  - FixL and FixJ
• Detection of the specific stimulus by the sensor causes a conformational change in the protein
• The different conformations of the sensor protein interact differently with the regulator protein
• Interaction with the sensor stimulates phosphorylation of the regulator which changes how it interacts with gene promoters to increase or decrease gene expression

Question 4

Explain ther relation of NtrB and C under low and high N.

Answer 4

Under Low N

• NtrB (sensor) will phosphorylate NtrC
• Ntr C will turn on nif genes to fix N

Under High N

• NtrB will dephosphorylate NtrC
• Ntr C will NOT turn on nif genes to fix N

Question 5

How is activation of N controlled? (repression…)

Answer 5

End-product inhibition is an important control

• Conserve energy by only fixing N2 when N is needed
• Recall the end-product inhibition of assimilatory nitrate reduction by NH4+
• Cells balnce N-demand and NH4+ repression
  - NH4+ repression can be mediated via [glutamine]
• N2-fixing cells in multicellular systems need strategies to avoid ammonium repression until all cells have sufficient N
  - RAPID ASSIMILATION and/or DISPLACEMENT of ammonium is needed to avoid ammonium repression of N2-fixation

Question 6

How is ammonium rapidly displaced and/or assimilated?

Answer 6

1. enzyme activities
   
   • ammonium assimilation : GS-GOGAT
2. Linked cells
   
   • In cyanobacteria, N fixed in heterocysts is displaced to vegetative cells
3. Symbiotic partners
   
   • Plant cells of legume root nodules
   • Mycobiont cells of lichens

Question 7

Explain regulation of N2 fixation by O2?

Answer 7

Aerobic and microaerophilic cells do not produce nitrogenase when [O2] is high enough to inactivate it
- nif gene expression in some species is induced by the FixLJ 2 component regulatory systen when [O2] is low

Some cells have strategies to protect nitrogenase from ireeversible O2 damage and/or other [O2]-sensing systems

Question 8

How does the FixLJ regulation system by O2 work?

Answer 8

FixLJ is a 2-component regulatory system that regulates nif genes in response to O2

       - FixL is a heme-protein that 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