Questions for lecture 6 Be comfortable with the following terms

26 Jun Questions for lecture 6 Be comfortable with the following terms

Question
Questions for lecture 6

Be comfortable with the following terms:

Organotroph

Lithotroph

Chemotroph

Glycolysis

Entner-Doudoroff Pathway

Pentose Phosphate Pathway

b-oxidation

Krebs cycle

Methylotrophy

FAD/FADH2

Ferredoxin

Dehalorespiration (also known as reductive dehalogenation)

Nitrification

Denitrification

Anammox

Methanogenesis

Acetogenesis

You should use the redox table for many of these questions…

Does respiration require oxygen?

Is there any real advantage of using the Entner-Doudoroff pathway? Is it possible that evolution can produce organisms that are not as good as theoretically possible? How could this happen?

Compare the chemistry of the Entner-Doudoroff pathway and Glycolysis. What intermediates are common to both the Entner-Doudoroff pathway and glycolysis? What are unique to each?

How might you use a chemostat to tell the difference between a fermenting cell using glycolysis and one using the Entner-Doudoroff pathway? Describe the experiment you would use, and the results that you would expect. Now do it with batch culture.

Could a fermenting cell grow on fatty acids? What high-energy compound would it be likely to use for substrate-level phosphorylation? What waste product would it be likely to produce?

Why was it very surprising to see a fermenting bacterium that showed a reduced growth rate in the presence of an uncoupling agent? How does the use of both Fe- based e- carriers and organic e- carriers make this type of growth possible? How does this relate to the mechanism used in ETCs to make a proton gradient?

The E for the Ferredoxin(oxidized)/Ferredoxin(reduced) redox couple is -0.39V. Is the transfer of e- from reduced ferredoxin to NAD+ spontaneous or non-spontaneous?

Many organisms live in environments where many different types of sugars are available. Why might these organisms have both the pentose phosphate pathway and glycolysis? Why are enzymes in the pentose phosphate pathway useful for tasks such as making DNA? (hint—what is DNA made of?) What is the NADPH produced in this pathway useful for?

Many organisms do not use phospholipids for fuel, but still have enzymes that are used forb-oxidation. What cellular component(s) could be made using these enzymes (hint: most reactions are reversible, and most membrane fatty acids have an even number of carbon atoms).

Is glycolysis a necessary precursor to the Krebs cycle? Describe how carbon atoms from glucose, ribose (a 5-carbon sugar), oleic acid (a fatty acid), and benzoic acid can be completely oxidized to CO2. How many “turns” of the Krebs cycle can a C18 fatty acid power? How many turns of the Krebs cycle can a molecule of glucose power?

The Krebs cycle produces both NADH and FADH2; the reduction potential (E) for the FAD/FADH2 redox couple is -0.22V. Could a respiring organotroph use NADH oxidase to take electrons from FADH2 and feed them into an ETC?

If a respiring organotroph moved from an aerobic environment to one with only nitrate, what components of its ETC would you expect to change? Why?

What makes hydrogen such a good energy source? What biological process makes hydrogen? If an organism switches from using glucose to H2 as an e- donor, what aspect(s) of its electron transport chain need modification, if any?

Methanogens love H2 as an e- donor. Why are methanogens often found in the company of fermenters?

Why is it that methanogens can use H2 as an e- donor, but not hydrogen sulfide (H2S; use the redox table to answer). Why is the E of the CO2/methanol redox couple important for knowing whether or not methanogenesis is possible?

Is Methylotrophy simply Methanogenesis in reverse—the same enzymes and organism, just different conditions—or are they different? Why do methanogens fluoresce at a characteristic wavelength; do methylotrophs fluoresce?

How are methanogenesis and acetogenesis similar? How are they different?

Farmers spend lots of money injecting ammonia into their soil as a plant fertilizer. What bacterial activity is responsible for nitrification? What is the e- acceptor for nitrification? Is nitrification an example of anaerobic respiration or an example of lithotrophy? What bacterial activity is responsible for denitrification (the conversion of nitrate and nitrite to atmospheric N2)? What is the e- donor for this process? Is this an example of aerobic respiration or lithotrophy? Using the redox tabloe, which has the better energy yield?

Why is lithotrophy using Fe2+ as an e- donor challenging? At neutral pH, Fe2+ is soluble only under anaerobic conditions. Using the redox table, which could yield more energy: anaerobic iron oxidation at neutral pH, or aerobic oxidation at highly acidic pH?

Energetic lifestyles such as aerobic iron oxidation and methanogenesis have very poor energy yields. Why are they so common?

When mine tailings containing iron sulfides (Pyrites) are brought to the surface and exposed to the atmosphere, they are attacked by bacteria to create hyperacidic runoff such as the Rio Tinto. The sulfur in the pyrite changes from an oxidation state equivalent to –SH to an oxidation state of SO4. Is this an example of lithotrophy or anaerobic respiration? How does the utilization of sulfur make the utilization of Fe2+ possible under aerobic conditions? Can the resulting Fe3+ serve as an e- acceptor for the –SH under anaerobic conditions?

Thiomargarita namibiensis (the current record holder for largest Bacterium) has a vacuole that stores nitrate extracted from sea water and lives in an anaerobic environment rich in sulfides; “thio-margarita” translates to “sulfur pearl”, and refers to the blobs of elemental sulfur that accumulate in the periplasm of this organism. How does this organism make its ATP? What would you call its oxidase? Its reductase? Is the elemental sulfur just accumulating waste, or is it a snack that is saved for later? Would you expect the elemental sulfur to be used if H2S were still available? What difficulties might there be with using elemental sulfur, which is a solid? Why does the topology of the sulfur-using oxidoreductases matter?

Acetogenic organisms can use glucose as a growth substrate: essentially, they ferment glucose to 2 mol acetate, 2 CO2, and hydrogen. They then use the hydrogen and the CO2 to produce acetate. So, glucose goes in and acetate comes out; the same occurs in some fermentations. How might you use batch cultures to tell the difference between cells that are growing acetogenically, and cells that are fermenting glucose to acetate? How would uncoupling agents or ATPase inhibitors be useful in this determination?

Acetogens and aerobic hydrogen consumers both love to consume H2 as an e- donor. Acetogenesis produces Acetyl-CoA as an intermediate. How is the way acetogens produce ATP different from the way an aerobic hydrogen consumer produces ATP?

How is it possible that the anammox process can use N compounds both as e- acceptors and e- donors? Is it theoretically possible for an organism to use Sulfur in the same way? How about Carbon (remember to look at the redox table! The answers are there!)

Consider the diagram of the anammox process. How does the topology of the nitrate reductase (NIR) help with ATP synthesis? Hydrazine (N2H4) is a deadly poison (as well as a powerful rocket fuel). Why does having the anammoxosome compartment help the cell?

What kind of microscopy was used for the picture of Brocadiain the lecture slides, and why is it useful for showing the anammoxosome? Could it be visualized by freeze-fracture SEM? How could you use GFP technology to demonstrate that the hydrazine oxidase (HZO) was located in the anammoxosome membrane (AM)?

There are certain cases where the proper orientation of an oxidoreductase (e.g. the oxidoreductases in anammox bacteria or iron oxidizers) is very important. Compose a paragraph about the importance of proper orientation of oxidoreductases. Your paragraph should have a topic sentence briefly introducing what a respiratory oxidoreductase is and what is meant by its orientation. The meat of the paragraph should explain exactly why the proper orientation is critical, and what would happen if the oxidoreductase were “upside-down.”

If you observe an organism living in anaerobic conditions and producing CO2 and H2S, what would you conclude about its metabolic lifestyle?

If you observe an organism living in aerobic conditions and producing elemental sulfur, what would you conclude about its metabolic lifestyle?

If you observe an organism living in an aerobic environment and producing a lot of rust, what would you conclude about its metabolic lifestyle?

If you observe an organism living in an anaerobic environment, consuming H2S and Fe3+, what might it be producing?

If you observe an organism living in an anaerobic environment, consuming H2 and tetrachloroethylene, what might it be producing?

How do respiratory metabolic abilities allow microbes to clean up man-made disasters? How do respiratory metabolic abilities allow microbes to make man-made situations worse?

There seems to be a lot to memorize in looking at metabolism. It’s best to keep an eye on the big picture, and not worry too much about the details of whata-keto-glutarate is doing in the Krebs cycle. Focus on the essentials of these processes, and compare them with one another.

For instance, consider organotrophy:

Pathway

What is consumed?

What is produced?

Is O2 involved?

Is ATP made? How?

Distinctive to any group?

Glycolysis

Entner-Doudoroff

Pentose-Phosphate

Shunt

Krebs Cycle

b-oxidation

Aromatic catabolism (aerobic)

We can also consider different types of respiration. Of course, “lithotrophy” and “anaerobiosis” are very general terms; you can fill in the table with a specific example, or just be very general.

Type of respiration

e- donor

e- acceptor

Waste products

How is ATP made?

Is O2 involved?

Unique to any organism?

Aerobic organotrophy

Anaerobic organotrophy

Aerobic lithotrophy

Anaerobic lithotrophy

Methanogenesis

Acetogenesis

Reductive dehalogenation

(dehalorespiration)

What YOU do

Just for contrast, do the same for fermentation.