Lecture 22

Question 1

Chemolithotrophy

1. How is energy obtained from chemolithotrophy?
2. What chemolithotrophs are…?
3. Who discovered chemolithotrophs?
4. What else can chemolithotrophs be?
5. What is an example of chemolithotrophy?

Answer 1

1. Energy from the oxidation of inorganic compounds (examples: H₂S, NH₄, H₂).
2. Autotrophs (carbon from fixation of CO₂): chemolithotrophic autotrophy.
3. Sergei Winogradsky, Russian microbiologist, during the 1800s.
4. Can be aerobic, using O₂ or can be anaerobic, using alternative terminal electron acceptors, NO₃^-, SO₄^2-, etc.,
5. Hydrogen oxidation (Ralstonia eutropha)

Question 2

Example of chemolithotrophy:

1. What bacterial species carries out iron oxidation?
2. What is oxidized during this process?
3. What conditions does this bacterial species live in?
4. What does ferric iron form?
5. What does ferric iron act as?
6. What does the picture show?

Answer 2

1. Acidithiobacillus ferrooxidans
2. Aerobic oxidation of ferrous iron (Fe²⁺) to ferric iron (Fe³⁺).
3. Lives under acidic conditions (pH 2-3)
4. Ferric iron forms insoluble ferric hydroxide [Fe(OH)₃)] in water; red-brown color.
5. Ferrous iron acts as an electron donor - Fe²⁺ to Fe³⁺ is an oxidation.
6. Acid mine drainage, from a creek draining a coal mining area. The creek is acidic and contains a high level of ferrous iron (Fe²⁺). At low pH, ferrous iron does not oxidize spontaneously in air. The red-brown color is due to oxidation of ferrous iron to ferric iron (Fe³⁺) by A. ferrooxidans, the ferric hydroxide formed, together with complex ferric salts, make a colored precipitate.

Question 3

1. How does A. ferrooxidans make ATP?
2. What do the cells have to do to use that natural gradient of protons from ATP synthesis?
3. This bacterium is an autotroph, where does the reducing power come from for the fixation of CO₂ in the Calvin cycle?

Answer 3

1. Making of ATP:
   
   1. rusticyanin (periplasmic, copper-containing protein) oxidizes Fe²⁺ to Fe³⁺, a one electron transition.
   2. rusticyanin then transfers the electron to cytochrome c, which then transfers the electron to cytochrome aa₃
   3. cytochrome aa₃ then passes the electron to O₂ and at that step, protons are pumped out of the cytoplasm
2. Build up of protons can make the environment too acidic. Conversion to water and pumping them out of the cell neutralizes the pH.
3. There is a reverse flow of electrons that are passed to different cytochromes. Thus, reducing NAD⁺ to NADH.

Question 4

Anaerobic Respiration:

1. What is anaerobic respiration?
2. What is its electron transport chain like?
3. It which organisims does this form of respiration occur?

Answer 4

1. respiration using a terminal electron acceptor other than O₂
2. membrane-associated electron transport chain similar to that of aerobes (cytochromes, quinones, iron-sulfur proteins).
3. in facultative aerobes (which can carry out aerobic respiration if O₂ is available) and obligate anaerobes (which cannot use O₂).

Question 5

1. What is an example of anarobic respiration? Hint: E. Coli use this method
2. What kind of aerobe is E. Coli?
3. When is this mode of respiration useful?

Answer 5

1. nitrate (NO_3-) respiration, nitrate is the terminal electron acceptor. The use of nitrate as an electron acceptor in energy metabolism is dissimilative metabolim: Taking a massive amount of materials, converting it to energy and releasing it as waste product.
2. E. Coli is a facultative aerobe; present in the gut tract of mammals, in soil, on vegetable, in fresh-water streams.
3. When O₂ gets used up, it is beneficial to be able to shift to another terminal electron acceptor, if one is present.

Question 6

1. How is E. Coli different from Pseudomonas stutzeri?
2. What is nitrate reductase?
3. What does the dual control of nitrate reductase synthesis require?
4. How many proton-translocating steps occur during nitrate reduction?
5. What does this mean for the cell?

Answer 6

1. nitrate is just one of several inorganic nitrogen compounds used by bacteria in anaerobic respiration. E. Coli is only able to carry out the first step of the process called denitrification: the reduction of nitrate to nitrite.
2. nitrate reductase is the enzyme that catalyzes this first step. Membrane-integrated, molybdenum-containing enzyme, synthesis of which is repressed by oxygen.
3. the absence of oxygen and the presence of NO_3-
4. There are only two proton-translocating steps that occur during nitrate reduction, compared to three for oxygen respiration.
5. This means the cell grow a little slower. Less ATP is made under anaerobic respiration than under aerobic respiration.

Question 7

Nitrogen Fixation:

1. What is nitrogen fixation?
2. What is N₂ fixed to?
3. What organisms use this process?
4. What is the ecological advantage of nitrogen fixation?
5. What are some free-living aerobes that use this process?
6. What are some free-living anaerobes that use this process?
7. What are some symbiotic that use this process?

Answer 7

1. the use of N₂ as a source of cell nitrogen (for amino acids, nucleotides, etc.)
2. fixation of N₂ (gas, from air) into ammonia (NH₃).
3. this form of metabolism is strictly bacterial (Bacteria or Archaea); no member of Eukarya has been found to have this ability. This bacterial activity is the major source of fixed nitrogen for all organisms.
4. if other sources of nitrogen (ammonia, nitrate, amino acids) are not available or become limiting (as in nitrogen-depleted soils). Then bacteria with the ability to fix N₂ as a source of nitrogen for biosynthetic need would have a major competitive advantage.
5. Azotobacter (Chemoorganotrophs) and Anabaena (Phototrophs)
6. Clostridium (Chemoorganotrophs) and Methanococcus (Chemolithotrophs)
7. Rhizobium (With leguminous plants).

Question 8

Mechanism of Bacterial Nitrogen Fixation:

1. How many moles of ammonia are produced from one mole nitrogen gas?
2. How many moles of ATP does it cost?
3. N₂ is very inert (non-reactive), what does this mean for bond breaking?
4. What three steps does this process occur in?
5. What is this reaction catalyzed by, and what is this complex composed of?
6. What is this the site of?

Answer 8

1. Two moles of ammonia are made from one mole of nitrogen gas.
2. Cost 16 moles of ATP and lost of electrons and protons.
   
   1. N₂ + 8 H⁺ + 8 e^- + 16 ATP → 2 NH₃ + H₂ + 16 ADP + 16 Pi (carried out by a nitrogenase complex)
3. The three N-N bonds are very hard to break and produce NH₃
4. 3 N-N bonds to two, two N-N bonds to one, one N-N bond to none. A series of reductions.
5. Catalyzed by nitrogenase, an enzyme complex of an iron protein (dinitrogenase reductase) and a molybdenum-iron protein (dinitrogenase)
6. Site where reduction of N₂ to NH₃ occurs - FeMo-co. the iron-molybdenum cofactor of dinitrogenase.

Question 9

1. What happens when nitrogenase is exposed to oxygen?
2. What reaction occurs when it’s exposed to oxygen?
3. What type of bacteria is this not a problem for?
4. What type of bacteria is this a major problem for?

Answer 9

1. Dinitrogenase reductase is inactivated when exposed to oxygen. It is sensitive to oxygen.
2. Oxygen reacts with the iron component of the protein, inactivating the enzyme.
3. This is not a problem for anaerobic nitrogen fixing bacteria, because oxygen is not present in their habitats.
4. This a major problem for oxygenic phototrophic nitrogen fixers (cyanobacteria) and free-living aerobic soil bacteria (Azotobacter, Beijerinckia)

Question 10

1. In what ways do different aerobic nitrogen-fixing bacteria protect the enzyme? Hint: There are three ways
2. Why is nitrogen fixation a highly regulated activity?

Answer 10

1. 3 ways:
   
   1. Rapid removal of oxygen by respiration (a high rate of respiration)
   2. Formation of a slime layer that functions as a diffusion barrier for oxygen (Azotobacter). Cells grown at 2.5% oxygen, very little slime is produced. Cells grown at 21% oxygen, a lot of slime is produced.
   3. Compartmentalization of nitrogenase in specialized cells, heterocysts, in some cyanobacteria.
2. Highly regulated because:
   
   1. Energetically expensive, so make the enzyme only when needed (when nitrogen is not available).
   2. Sensitive to oxygen, so avoid making nitrogenase when oxygen is present (or protect it in some way).

Question 11

1. What is the nif regulon? What is a regulon?
2. In Klebsiella, what is the nif regulon composed of?
3. What does nifD and nifK code for?
4. What does nifH code for?
5. nifHDK genes are highly…?

Answer 11

1. Nitrogen-fixing regulon. A regulon is sets of genes (different operons) that are coordinately regulated, often having a common function.
2. The nif regulon is composed of 20 genes (24 kb of DNA). These are nitrogenase genes, genes for FeMo-co synthesis, genes involved in electron transport, and regulatory genes.
3. Code for dinitrogenase (two subunits)
4. Code for dinitrogenase reductase.
5. Conserved in many bacteria.