Lecture 1 - Exam 5

Question 1

What organisms depend on nitrogen?
It is required in large amounts as an essential component of?

Answer 1

All organisms.
Required in large amounts as an essential component of proteins, nucleic acids, and other cell constituents. It is critical for agricultural production.

Question 2

Is N2 available for use by most organisms?
Why?

Answer 2

It is unavailable for use by most organisms because there is a triple bond between the two nitrogen atoms, making the molecule almost inert.

Question 3

What is often the limiting factor for growth and biomass production in all environments where there is suitable climate and availability of water to support life?

Answer 3

Nitrogen.

Question 4

In order for nitrogen to be used for growth, what must happen?

Answer 4

It must be fixed in the form of ammonium (NH4)*

Question 5

What does the overuse of ammonia as plant fertilizer result in?

Answer 5

Contaminated ground water, leading to eutrophication.
Increases in nutrient availability results in blooms of algae and cyanobacteria.

Question 6

N is cycled between:

Answer 6

NH4+ (ammonia, -3 oxidation state) and NO3- (nitrate, +5 oxidation state)

Question 7

What kind of organisms are critical for nitrogen cycling?
What are these specific processes?

Answer 7

Prokaryotes.
Nitrogen fixation, nitrification, denitrification, ammonification (through biomass decomposition).

Question 8

Fixing nitrogen is difficult because?

Answer 8

Of the strength of the N2 bond (triple bond).
-Reduction of N2 to NH3 requires a lot of energy to break the triple N2 bond (16 ATP to fix 1 N2 to make 2 NH3).

Question 9

Nitrogen fixation is critical for agriculture. What’s the deal with plants?

Answer 9

Plants cannot use N2 directly but need N in a usable form like ammonia (NH3).

Question 10

What is the method for nitrogen fixation?

Answer 10

Haber-Bosch method: ammonia production using catalyst with H2 and N2 at 200 atm and 500 C have negative environmental effect.
-More than 100 million tons of NH3 produced annually via Haber-Bosch method.

Question 11

More NH3 produced by ________ than Haber-Bosch method (~400 million tons/yr).

Answer 11

Bacteria

Question 12

Nitrogen fixation is confined to? What kind of organisms cannot fix nitrogen?

Answer 12

Confined to prokaryotes. Eukaryotic species cannot fix nitrogen.

Question 13

What kind of prokaryotes can fix nitrogen?

Answer 13

Prokaryotes and Archaea
-Symbiotic and free-living
-Aerobic and anaerobic

Question 14

Where were nitrogen-fixing bacteria first found?

Answer 14

In the nodules of clover roots.

Question 15

Formation of a root nodule in a legume infected by Rhizobium requires what?
In order to initiate a productive symbiosis, rhizobia must..?

Answer 15

Molecular communication to establish infection.
Rhizobia must recognize and then respond to the presence of host plant roots.

Question 16

What are the steps for the formation of the nodule?

Answer 16

1. Plant roots produce flavonoid compounds.
2. Rhizobium cells respond by producing nodulation (nod) factors.
3. In response to nod factors, plant roots grow root hairs. Rhizobium infects and grows within the root hair.
4. Bacteria in infection thread form a ‘bacteroid’ elongated cell type as they penetrate the root.
5. The infected root grows and forms a nodule, in which the Rhizobium fixes N2 and the plant provides sugars, amino acids, etc.

Question 17

During growth in the rhizosphere of a host plant, rhizobia sense?

Answer 17

Compounds such as flavonoids and betaines secreted by the host root and respond by inducing nod genes.

Question 18

What are nod genes?
What are nod factors?

Answer 18

Nod genes encode ~25 proteins required for the bacterial synthesis and export of Nod factor.
Nod factor is a host-specific lipooligosaccharide signal. They initiate developmental changes seen in the host plant early in the nodulation process, including root deformation and curling.

Question 19

In order to fix nitrogen, diazotrophic bacteria produce what?

Answer 19

A vital enzyme called nitrogenase.

Question 20

What is the nitrogenase enzyme is responsible for what?

Answer 20

It is solely responsible for replenishing the nitrogen cycle from atmospheric dinitrogen.
-Nitrogenase catalyzes the ATP-dependent reduction of N2 to ammonia.
-Can reduce 1 N2 per second (10 to 100 times slower than ribosomes and polymerases)
-It is very sensitive to oxygen*, and its activity is inhibited by even small amounts of O2.

Question 21

What is nitrogenase comprised of?

Answer 21

Nitrogenase is an enzyme complex comprised of two proteins:
A molybdenum-iron protein (MoFe) called dinitrogenase, and a smaller iron protein (Fe) called dinitrogenase reductase.

Question 22

Dinitrogenase (MoFe) does what?
Dinitrogenase reductase (Fe) does what?

Answer 22

Binds N2.
Acts as an electron donor to MoFe.

Question 23

Within each MoFe subunit is what?

Answer 23

An electron carrier complex called the P complex that accepts electrons from Fe. The Fe proteins delivers electrons one at a time to the catalytic MoFe protein.
This process occurs through a series of synchronized events collectively called the “Fe protein cycle.”

Question 24

Where does Fe get the electrons to transfer to MoFe?

Answer 24

Reduced Ferredoxin.
-Ferredoxin is an electron carrier that accepts e- from acetyl-CoA or pyruvate.

Question 25

The nitrogenase enzyme complex requires?

Answer 25

Reduced ferredoxin and ATP.

Question 26

What is the reaction for biological N fixation?

Answer 26

N2 + 8 H+ + 8 e− → 2 NH3 + H2
With a delta G > 900 kJ/mol
So, in order for these reactions to proceed, a lot of ATP is required.
16 ATP + 8e- to fix 1 molecule of N2

Question 27

N-N (triple bond) bond energy = ?

Answer 27

941 kJ/mol

Question 28

Describe the process of N2 fixation.

Answer 28

1. 8 ferredoxin molecules accept 8e- from 4 CoA and 4 pyruvate.
2. Reduced ferredoxin transfer an electron to Dinitrogenase reductase (Fe protein).
3. Reduced Fe protein then binds 2 ATP.
4. Reduced Fe + ATP transfers electrons ONE AT A TIME to Dinitrogenase (MoFe protein).
5. Dinitrogenase then transfers the electrons to N2, which catalyzes the conversion of N2 to 2NH4+/2NH3.
   *This process happens 8 times for each N2, with 2 ATP used each time.

Question 29

Because oxygen inhibits nitrogenase, plant nodule adaptations have developed to protect nitrogenase. What are they?

Answer 29

1) Leghemoglobin
2) An oxygen permeability barrier at the periphery of the nodule that reduces oxygen diffusion.
3) Rhizobium cells produce large amounts of exopolysaccharide that reduces oxygen diffusion.

Question 30

Describe leghemoglobin.

Answer 30

An oxygen scavenging molecule produce by root nodule cells which gives nodules their pink color.
It has a heme group that has a high affinity for O2, similar to hemoglobin in RBC.
Leghemoglobin binds O2 so it cannot associate with the nitrogenase enzyme complex.

Question 31

Nitrogenase enzyme complex is highly sensitive to oxygen. Who is this a problem for?

Answer 31

Aerobic bacteria.

Question 32

Cyanobacteria have evolved a mechanism to protect the nitrogenase from oxygen inhibition. What is this mechanism?

Answer 32

Nitrogen fixation occurs in special cells called heterocysts that possess only photosystem I, which can generate ATP but will not produce O2.

Question 33

What is an example of a symbiotic relationship?

Answer 33

Between the water-fern Azolla and the cyanobacteria Anabaena.
The water fern has 2 different cyanobacteria endosymbionts, the chloroplast that provides energy from photosynthesis and fixed carbon, and the Anabaena that provides fixed nitrogen.

Question 34

What is the first step of the nitrogen cycle?

Answer 34

Nitrogen fixation.
-Converts atmospheric N2 into Nh3 (ammonia)
-Nitrogen fixation requires energy.

Question 35

What is the second step of the nitrogen cycle?

Answer 35

Nitrification.
-NH3 is converted to NO3- in a two step process by nitrifying bacteria.
-Nitrification reactions provide a source of energy for different microorganisms by converting ammonia into nitrate (NO3-).
-Nitrate can be taken up and assimilated (another possible step after nitrification) into plant tissues.

Question 36

What happens after nitrification?

Answer 36

Denitrification.
-Under some conditions, nitrite or nitrate can be reduced back into N2 gas or N2O (nitrous oxide) by denitrifying bacteria. This returns nitrogen back to the atmosphere.

Question 37

What is ammonification?

Answer 37

A vital process of returning organic nitrogen to mineralized NH4+ (ammonium). It is the first step in decomposition of organic N.

Question 38

Describe nitrification a bit further (the two steps and who carries nitrification out).

Answer 38

It is the conversion of ammonia (or ammonium) to nitrite, and nitrite to nitrate. The oxidation of ammonia to nitrite is a two step process carried out by nitrosifying, or ammonia-oxidizing, bacteria (Nitrosamonas).
1) NH3 + O2 + 2e- –> NH2OH + H2O
2) NH2OH + H2O –> NO2- + 5H+ + 4e-
The oxidation of nitrite to nitrate is carried out by nitrifying, or nitrite-oxidizing, bacteria (Nitrobacter).
NO2- + 1/2O2 –> NO3-

Question 39

Overview of Nitrification

Answer 39

Ammonia (NH4) –> Nitrite (NO2) carried about by Nitrosomonas species.
Nitrite (NO2) –> Nitrate (NO3) carried out by Nitrobacter species.

Question 40

What about nitrite and nitrate causes their leaching from soils?

Answer 40

The negative charge of nitrite and nitrate results in their results in their leaching from soils, since clay and other soil particles have a net negative charge.

Question 41

Nitrite and nitrate leaching from soils ends up where? What does it cause?

Answer 41

The leached nitrite and nitrate end up in groundwater and can cause severe eutrophic conditions, and the resulting algal blooms can devastate freshwater and marine ecosystems.
These blooms in municipal water reservoirs can result in off-flavor and ingestion of cyanotoxins can result in illness.

Question 42

Nitrate, Nitrite, and Ammonium Assimilation
-What is it assimilated by?
-What is reduced and by what enzymes?
-What is it incorporated into, and by what?

Answer 42

Ammonium, nitrite, and nitrate can be assimilated by microorganisms.
-The nitrite or nitrate must be reduced to ammonium by the enzymes nitrate reductase (NR) and nitrite reductase (NiR).
-The resulting ammonium is incorporated into organic molecules by GS (glutamine synthetase) and GOGAT (glutamine amide 2-oxoglutarate amino transferase)

Question 43

Denitrification
-What is it?
-Who converts what to what? What is the result of this?
-What are some examples of denitrifiers?

Answer 43

-Denitrification is when NO3- (nitrite) is fully reduced to N2.
NO3 –> NO2 –> NO –> N2O –> N2
-Denitrifying bacteria convert nitrates in soil to free atmospheric nitrogen, thus depleting soil fertility and reducing agricultural productivity.
-Thiobacillus denitrificans and Micrococcus dentrificans are examples.

Question 44

Denitrification
-What kind of conditions does it occur under?
-Denitrification by ________ can reduce the amount of fixed nitrogen (as fertilizer) by up to 50 percent.

Answer 44

-Under anaerobic conditions, some bacteria use nitrate as a terminal e- acceptor, eventually reducing nitrate to N2.
-Pseudomonas aeruginosa