1
Q
What are the 2 types of reduction pathways? What is the role of each?
A
- Assimilatory pathways
- Incorporation of reduced inorganic compounds into organic compounds
- NH3 –> Glu/Gln
- S/H2S –> cysteine - Dissimilatory pathways
- Reduced products are used as electron acceptors (anaerobic respiration)
- NO3 –> N2
- SO4 –> S/H2S
2
Q
What system incorporates the ammonia (product of reducing nitrate) into glutamine and glutamate?
A
GS/GOGAT system
3
Q
What are glutamine and glutamate used for?
A
Provide amino groups for the other nitrogen-containing organic compounds
4
Q
Give an overview of nitrate assimilation.
1. What is nitrate first reduced to ?
2. What are the oxidation states of the N in nitrate and ammonia?
3. How many electrons must be transferred?
A
- Nitrate is first reduced to ammonia
- Oxidation state of the N in nitrate is +5, N in ammonia is -3
- 8 electrons must be transferred to nitrate to reduce it to ammonia
5
Q
How many electrons must be transferred to nitrate to reduce it to ammonia?
A
8 electrons
6
Q
What are the enzymes in nitrogen assimilation?
A
- Cytoplasmic nitrate reductase (reduces nitrate to nitrite)
- Cytoplasmic nitrite reductase (reduces nitrite to ammonia)
7
Q
What are the electron donors in nitrate assimilation?
A
- NADH
- Ferredoxin
- Flavodoxin
8
Q
Give an overview of sulfate assimilation.
1. What is sulfate first reduced to?
2. What are the oxidation levels of S in sulfate and sulfide?
3. How many electrons must be transferred?
A
- Many bacteria can use sulfate as their principal source of sulfur
- Sulfate first reduced to sulfide and then incorporate into cysteine
- Oxidation level of S in sulfate is +6, S in sulfide is -2
- 8 electrons must be transferred to reduce sulfate to sulfide
9
Q
How many electrons is required to reduce sulfate to sulfide?
A
8 electrons
10
Q
What are the steps and enzymes of sulfate assimilation?
A
- Formation of APS
- ATP sulfurylase - Phosphorylation of APS to form PAPS
- APS kinase - PAPS is reduced to sulfite with the release of AMP-3’-phosphate
- PAPS reductase - Sulfite is reduced by NADPH to hydrogen sulfide
- Sulfite reductase - O-acetylserine sulfhydrylase
11
Q
Expalin what is happening in the assimilatory nitrate reduction pathway.
A
12
Q
Draw out assimilatory sulfate reduction pathway.
A
13
Q
Describe the dissimilatory pathways.
1. What molecules are used as electron acceptors during it?
2. What happens to the reduced products?
3. What types of bacteria use them?
A
- Nitrate and sulfate are used as electron acceptors during anaerobic respiration
- Reduced products are excreted rather than being incorporated into cell material
- Used by many facultative anaerobes
- Only obligate anaerobes use sulfate as an electron donor (sulfate reduceres)
14
Q
When is dissimilatory nitrate reduction used? Where does it occur? What are its products?
A
- Occurs as a substitute for aerobic respiration when the oxygen levels become very low
- Takes place place in membranes and a Δp is usually made
- Products: nitrite, ammonia, or nitrogen gas
15
Q
What is denitrification? Why is it important? When does it occur?
A
- When nitrate or nitrite is reduced to nitrogen gas
- Important drain of nitrogen from the soil
- Occurs when conditions become anaerobic and during composting and sludge digestion
16
Q
Why are denitrifiers important?
A
Important in anaerobic niches for the breakdown of undesirable biodegradable materials, plant materials, complex organic compounds, etc.
17
Q
What are the most commonly isolated denitrifiers?
A
- Alcaligenes
- Pseudomonas
- Paracoccus denitrificans (to a lesser extent)
18
Q
Explain the electron transport pathway in P. denitrificans.
1. What is it?
2. What is reduced to what?
3. What inhibits its enzymes?
4. How many electrons flow in and out of the cell membrane?
A
- Denitrification
- Reducing nitrate to nitrogen gas
- Cyt bb3
- See enzyme in figure
- Note: the synthesis and activity of denitrifying enzymes are inhibited by oxygen
- A total of 5 electrons flow in and out of the cell membrane through electron carriers from UQ to various reductases
19
Q
What are the steps of denitrification?
A
- Reduced coenzyme Q provides electrons for nitrate reductase (NaR)
- NaR reduced NO3 coupled to the oxidation of quinol
- Cyt c donates electrons to nitrite reductase (NiR) to reduce NO2 to nitric oxide (NO)
- Cyt c donates electrons to nitric oxide reductase (NOR) and nitrous oxide reductase (N2OR)
20
Q
Give a general description of the sulfate reducers.
1. What type of organism are they? (heterotrophs vs. autotroph, aerobe vs. anaerobe)
2. Where do they grow?
3. What do they do?
A
- Heterotrophic anaerobes
- Grow in anaerobic muds, mostly in anaerobic parts of fresh water, and in seawater
- Carry out anaerobic respiration during which sulfate is reduced to H2
- Gram-negative and Gram-positive bacteria and archaea
21
Q
What carbon sources can sulfate reducers use?
A
- Simple compounds (e.g. formate, lactate, pyruvate, malate, fumarate, ethanol)
- Straight-chain alkanes
- Variety of aromatic compounds
22
Q
What are some examples of sulfate reducers?
A
Archaeoglobus: hyperthermophilic archaea found in sediments near hydrothermal vents
23
Q
What genus do Gram-positive, spore-forming sulfate reducers belong to?
A
Desulfotomaculum
24
Q
Give an overview of the path of electrons to sulfate in Desulfovibrio.
A
- Electrons flow int he cell membrane to sulfate as the terminal electron acceptor
- Electron flow is coupled to the generation of a Δp
- Δp is used fro ATP synthesis (respiratory phosphorylation)
- Electrons may come from oxidation of organic compounds
25
How do assimilatory and dissimilatory sulfate reduction differ?
1. Assimilatory
- Cytoplasmic pathway
- Does not generate a Δp or ATP
2. Dissimilatory
- Membrane pathway
- Involves cytochromes
- generates a Δp
26
What are the steps and enzymes of the hydrogen cycling model in *Desulfovibrio*?
1. Lactate is oxidized to pyruvate in the cytoplasm, yielding 2 electrons
- Lactate dehydrogenase
2. Pyruvate is oxidized to acetyl-CoA and CO2
- Pyruvate-ferrodoxin oxidoreductase
3 and 4. Acetyl-CoA is used to generate ATP via SLP
- Phosphotransacetylase
- Acetate kinase
5. Electrons are transferred from lactate dehydrogenase and pyruvate–ferredoxin oxidoreductase to a cytoplasmic hydrogenase and then to H+, producing H2, and the H2 diffuses out of the cell into the periplasm
- Cytoplasmic hydrogenase
6. H2 is oxidized and the electrons are transferred to cytochrome c3
- Periplasmic hydrogenase
7, 9 and 10. Electrons travel through a series of membrane-bound electron carriers to APS reductase and sulfite reductase in the cytosol
- ATP sulfurylase
- APS reductase
- Sulfite reductase
8. Completion of the sulfurylation of ATP
- Pyrophosphatase
27
Why is a Δp generated in the hydrogen cycling model? Why is it important? Where to the 2 ATPs used up in reducing the sulfate come from?
- Inward flow of electrons across the membrane leaves the protons from the hydrogen on the outside
- Important because Δp is important for growth
- SLP
28
From the ecological perspective, why is nitrogen fixation so important?
Only prokaryotes can do it (eukaryotes cannot)
29
Why is the ability of prokaryotes to fix nitrogen necessary for maintaining the food chain?
Because fixed nitrogen is usually a limiting factor for plant growth
30
How is the ammonia produced via nitrogen fixation used?
It is incorporated into cell material via glutamine synthetase and glutamate synthetase
31
What types of organisms are capable of nitrogen fixation?
Many families of bacteria and archaea
32
What enzyme is responsible for nitrogen fixation?
Nitrogenase
33
What evidence suggest that the nitrogenase gene was transferred laterally between different groups of bacteria?
- Nitrogenase is very similar in different bacteria
- The nitrogen fixation genes have homologous regions
34
When does nitrogen fixation take place? Why?
- When N2 is the only or the major source of nitrogen
- Because the genes for nitrogen fixation are repressed by exogenously supplied sources of fixed nitrogen
35
Why is it remarkable that biological nitrogen fixation occurs?
Nitrogen molecule is so stable that very high pressures and temperatures in the presence of inorganic catalysts are necessary to make it reactive in nonbiological systems
- Prokaryotes can do it at atmospheric pressures and ordinary temperatures
36
What are the 3 types of nitrogen-fixing systems?
1. *Rhizobium, Sinorhizobium, Bradyrhizobium, and Azorhizobium* in symbiotic relationships with leguminous plants
2. Nonleguminous plants in symbiotic relationships with nitrogen-fixing bacteria
3. Many free-living soil and aquatic prokaryotes
37
Explain root nodule formation.
- Bacteria infect roots and stimulate the production of root nodules
- Bacteria fix nitrogen in the root nodules
- Plant responds by feeding the bacteria organic nutrients made during photosynthesis
38
List and describe the components of the nitrogenase enzyme complex What is the role of each?
1. Azoferredoxin (Fe protein)
- Homodimers
- 4Fe-4S cluster
- Role: Obtain electrons from ferredoxin or flavodoxin and transfer them to MoFe (electron transfer coupled to ATP hydrolysis)
2. Molybdoferredoxin (MoFe protein)
- Heterodimer (2 molecules of 2 proteins)
- Contain 2 molybdenum
- 28 iron and sulfur atoms
- Role: Reduces nitrogen (using electrons from azoferredoxin)
39
What is the formula for the nitrogenase reaction? Diagram it.
4e– + 0.5N2 + 4H+ + 8 ATP --> NH3 + 0.5H2 + 8 ADP + 8 Pi
40
In most known systems, nitrogenases are reduced by _____ or _____.
1. ferredoxins (FeS proteins)
2. flavodoxins (flavoproteins)
41
Do all nitrogen-fixing organisms have the same source of electrons for ferredoxin and flavodoxin?
No, the source of electrons varies with the metabolism of the organism
- Ex. pyruvate:ferredoxin oxidoruedtase or pyruvate:flavodoxin oxidoreductase
42
Some nitrogen-fixing bacteria are strict or facultative anaerobes. Some are microaerophillic (can grow only in low levels of oxygen). How do strict aerobes or cyanobacteria fix nitrogen if they are also exposed to oxygen?
They have methods for protecting nitrogenases from oxygen
43
How do the *Azotobacter* species protect its nitrogenase from oxygen?
- They have a very active respiratory system that is possibly uses oxygen fast enough to lower the intracellular concentrations in the vicinity of the nitrogenase
- They also associate their nitrogenase with protective proteins
44
How do the rhizobia in root nodules protect their nitrogenase from oxygen?
- They exist in plant vesicles in the inner cortex of the nodule as bacteriods (modified cells)
- Oxygen concentrations in the inner cortex are much lower than in the surrounding tissue
- Oxygen access to the inner cortex is controlled by regulating the intercellular spaces (either filled iwht air or water) within the boundary layer
45
How do filamentous cyanobactera (ex. *Anabaena* and *Nostoc*) protect their nitrogenase from oxygen?
They differentiate about 5-10% of their vegetative cells into heterocysts in the absence of combined nitrogen
46
What are heterocysts?
Vegetative cells that have been differentiated into nitrogen-fixing cells
47
In what ways do heterocysts differ from vegetative cells?
1.Heterocysts express the nitrogenase enzymes
2. They have only PS I --> do not produce oxygen
3. They do not fix CO2
4. They are surrounded by a thick cell wall made of glycolipid and polysaccharide (believed to serve as a permeability barrier to atmospheric oxygen)
5. They are not dividing
48
What are some of the inorganic compounds lithotrophs can use?
49
What is lithotrophy/llithotrophs?
Lithotrophs are organisms that metabolize inorganic compounds
50
What are chemolithotrophs?
Organisms that use chemicals as their energy source
- Inorganic electron source
- Variable carbon source
51
What are chemolithoautotrophs vs. chemolithoheterotrophs?
- Auto: use CO2 as major source of carbon
- Hetero: use organic molecules
52
What genera represent the hydrogen-oxidizing bacteria and the carboxydobacteria?
- *Pseudomonas*
- *Arthrobacter*
- *Bacillus*
- *Rhizobium*
53
Bacteria that oxidize ammonia as a source of energy are called _____.
nitrifiers
54
What are the 5 genera of nitrifiers? What do they have in common?
- *Nitrosomonas, Nitrosococcus, Nitrosospira, Nitrosolobus*, and *Nitrosvibrio*
- All are aerobic obligate chemolithoautotrophs that assimilate CO2 via the Calvin Cycle
55
What is the overall reaction of nitrification?
NH3 --> NO3-
56
What is nitrification?
The conversion of ammonia to nitrate
57
