Final Exam - MB409 - Spring 2001

1. What are the 3 primary evolutionary branches of life? (5 points)

Bacteria, Archaea, and Eukarya

2. What are the two major phylogenetic groups of Archaea? (5 points)

Crenarchaea and Euryarchaea

3. What are the three major phenotypic groups of Archaea? (5 points)

Suflur-metabolizing thermophiles, methanogens, and extreme halophiles

4. Give one biochemical/molecular trait in which Archaea resemble Bacteria but not Eukarya. (5 points)

Genes are organized in operons rather than being transcribed one-at-a-time

5. Give one biochemical/molecular trait in which Archaea resemble Eukarya but not Bacteria. (5 points)

Promoters are recognized by transcription factors rather than directly by RNA polymerase

6. Give one biochemical/molecular trait in which Archaea resemble neither Eukarya nor Bacteria. (5 points)

Membrane lipids are isosoprenyl alcohols linked to glycerol-phosphate via ether bonds, rather than fatty acids linked via esther bonds

7. Acidophiles will always have a proton gradient across their cellular membrane, since the outside is much lower in pH than the cytoplasm. Why, then, can't they make ATP for free? Why do they still have to run electron transport, pumping protons out? (5 points)

Because production of this 'free' ATP would acidify the cytoplasm (not good), and would anyway quickly run to a stop because of the formation of a counteracting electrical charge gradient.

8. The three major types of sulfur metabolism used by Archaea (and Bacteria, for that matter) are sulfur oxidation, sulfur reduction, and sulfur respiration. What are the electron donors, acceptors, and products for each of these? How is fixed carbon obtained by the organism (i.e. are they autotrophic or heterotrophic?)? Are the organisms that carry out each of them anaerobes or aerobes? For each type of sulfur metabolism, write the name of a genus that carries out that type of metabolism. (20 points)

Sulfur oxidation

sulfur (e- donor) + O2 (e- acceptor) + H2O ---> H2SO4 (product)

These organisms are aerobic, and either autotrophs or heterotrophs, e.g. Sulfolobus

Sulfur reduction

sulfur (e- acceptor) + H2 (e- donor) ---> H2S (product)

These organisms are anaerobic autotrophs, e.g. Thermoproteus

Sulfur respiration

sulfur (e- acceptor) + organics (e- donor) ---> H2S + CO2 (products)

These organisms are anaerobic heterotrophs, e.g. Thermococcus

9. The aphid symbiont Buchnera aphidicola gets a place to live (in the bacteriome), food, and assured transmission to the offspring of the animal. How does the animal benefit from the symbiosis? (5 points)

Essential amino-acids and vitamins that are not present in their sap diet.

10. Esther Angert used PCR to amplify ssu-rRNA sequences from samples of Epulopiscium picked one-at-a-time from fish guts. How did she show that the sequences she got, which were related to Gram-positive Bacteria, were actually from Epulopiscium and not contaminating residue from the fish gut contents? (5 points)

She designed an oligonucleotide primer specific to the ssu-rRNA sequences she obtained, fluorescently-labeled them, and used them in in situ hybridizations of fish gut contents. Sure enough, the Epulopiscium, and nothing else, lite-up!

11. In the paper by Birger Rasmussen (Rasmussen, B. 2000 Filamentous microfossils in a 3,235-million-year-old volcanogenic massive sulphide deposit. Nature 405:676-679), the fossil remains of filamentous prokaryotes in a massive sulfide deposit in western Australia were described. Describe the local environment these organisms were living in 3.25 billion years ago? (5 points)

The environment was a deep-sea (at least 1000 meters deep) hydrothermal vent field. The organisms were living in fissures and perhaps 'black smoker' chimneys in the volcanogenic rock from which hydrothermal fluid was flowing.

12. You are a graduate student in the lab of Ed DeLong, and you and he have decided that for the next 4-6 years, working toward your Ph.D., your task is to obtain a pure culture in vitro of a free-living, marine, mesophilic, open water Archaeon. How would you go about this? (10 points)

I would set up enrichment cultures based sea water basal media (30C) and selected to test a wide range of phenotypic possibilities - certainly heterotrophic (with a wide range of possible growth substrates) and perhaps phototrophic. I would then test each enrichment by fluorescent in situ hybridization using Archaea-specific probes to find cultures that contained archaeal growth. These I would try either to grow ass colonies on plates (screening colonis by hybridization using the same probe), or if not possible, fish out using optical tweezers (using the same probe to identify the correct cells) and cultivate from a single cell.

13. Describe an example of an interesting microbiological question you could answer using organisms or DNA isolated from museum samples? How would you go about getting this answer? (10 points)

What is the host and geographic range of the sponge symbiont Cenarchaeum symbiosum? This could be answered by isolating DNA from preserved samples of all kinds of sponges from all over the world, and using ssu-rRNA primers specific to C.symbiosum to test for the presence of it's DNA in each sample by PCR amplification, similar to the tests used to confirm the presence of Y.pestis in the teeth of plague children.

14. Below is a well-established ssu-rRNA tree of a small sampling of Gram-positive Bacteria. Next to it is another tree, based on the gene fauX from the same species. After carefully checking your alignments, tree statistics (including bootstrap values), etc, you come to the conclusion that the fauX gene has been transferred horizontally somewhere amongst this tree. Show, on either the ssu-rRNA tree or the fauX tree, or in a new drawing, where this transfer must have taken place. How would you go about testing this hypothesis? (10 points)

This could be easily tested by comparing the genes for other organisms in this phylogenetic group - especially relative of Streptomyces and groups that branch off between the Bacillus/Lactobacillus split and the Mycoplasma to determine the exact source and recipieent of the horizontal transfer.