Midterm Exam #2 : MB 409 Microbial Diversity

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

Bacteria, Archaea, & Eukarya

2. How does the helical shape of vibrios and spirilla (not spirochaetes!) improve the efficiency of their motility? (5 points)

Cells driven by the rotation of helical flagella will counter-rotate due to the resistance of the media to the rotational force of the flagella. This counter-rotational energy is lost in rod shaped bacteria, but is recaptured in vibrios and spirilla because their the rotation of the curved cell provides additional forward force.

3. What do cyanobacteria get out of 2-photosystem, oxygenic photosynthesis that green and purple Bacteria do not get out of single-photosystem, cyclic photophosphorylation? (5 points)

The ability to produce reducing power (NADPH) for fixing CO2 from the energy of light. Purple and green Bacteria generally produce NADPH by reverse electron flow, using a strong reducant (usually H2S or H2) and ATP.

4. Why is it believed that Bacteria evolved from a thermophilic ancestor? (10 points)

Because the earliest branches of Bacteria (Aquifex, Thermotoga, and the green non-sulfurs) are all predominated by thermophiles, and the mesophilic Bacteria are almost all descended from the later main radiation of the bacterial tree. In addition, the Archaea are generally thermophilic.

5. On the diagram of the electron transport chain below, show how a sulfur-oxidizing (H2S + 1/202 --> S° + H2O) organism would generate an electron gradient for ATP production. (10 points)

6. Describe a specie, genus, or other phylogenetic group of Bacteria you find interesting. Be sure to include its development or life cycle. Bacillus and Thermocrinus ruber are off-limits. (10 points)

e.g. Myxococcus

Unicellular aerobic gliders with a complex life cycle - members of the delta-purple Bacteria. They grow freely in rich environments, especially rotten wood, but when starved, aggregate into 'slugs' than move as a single organism. The cells in the slug differentiate into fruiting bodies, with base, stalk, sporangium, and spores cells. This is a terminal differentiation - only the spore cells have a future, and so this really is a multicellular organism. Spores are released into the wind, & those that land in a better environment germinate to produce a new crop of free-living cells.

7. Describe another specie, genus, or other phylogenetic group of Bacteria you find interesting. Be sure to include its development or life cycle. Bacillus, Thermocrinus ruber, and the organism you described in the previous answer are off-limits. (10 points)

e.g. Arthrobacter

This is probably the single most common soil organism. They are pleomorphic - they are rods during log-phase growth, then develop into cocci at the transition to stationary phase. This is a clear example of how different log and stationary phase cells are. They are members of the high G+C Gram-positive Bacteria, and are aerobic heterotrophs.

8. Describe a specie, genus, or other phylogenetic group of unicellular eukaryotes you find interesting. Yeast are off-limits. (10 points)

Choanoflagellates are small, marine non-photosynthetic (phagotrophic) protists that feed by beating a single flagellum to produce a flow of water, from which they trap bacteria with tenticle-like cilia. They often have intricate external structures called 'lorica', and resemble the individual cells of sponges. These organims probably resemble the ancestral protists from which animals arose.

9. Describe the pink filamentous organism Thermocrinus ruber, it's habitat, and how it was isolated in pure culture. (10 points)

This organism grows as pink filaments in the direct outflow from Octopus Spring, Yellowstone National Park. The pink filaments were characterized without cultivation by molecular phylogenetic analysis, and this information was used in the successful enrichment, isolation, and pure cultivation of the organism:

1. Pink filament biomass was collected following growth for several weeks on cotton filter pads in situ (to make sure they actually grew in that environment rather than being toasted groundwater organisms)
2. DNA was isolated from a washed pink filament sample, and ssu-rRNA was amplified by PCR using universal ssu-rRNA primers, & analysed phylogenetically just like we've talked about in class.
3. Most of the sequences obtained were a novel sequence (EM17) related to Aquifex. There were two other sequences that showed up less often among the clones: EM19 is a less-close relative of Aquifex, and EM3 is a relative of Thermotoga (see below).
4. A fluorescently-labeled oligonucleotide probe complementary to a species-specific (highly variable) region of the EM17 sequence was used to probe environmental samples for the EM17 organism. It did fluorescently-label the pink filaments, and not other inhabitants of the sample, showing that the EM17 ssu-rRNA sequence really is that of the pink filament microbe.
5. Since this organism is apparently related to Aquifex, and all of the cultivated members of this phylogenetic group are physiologically similar, a series of enrichment media based on the optimal growth conditions for Aquifex was innoculated them with pink filaments. The enrichments obtained growth of a rod-shaped pink bacterium that also hybridized to the EM17-specific probe.
6. In order to isolate this organism in pure culture, a 1000nm (1 micron) wavelength focused infrared laser was used as 'optical tweezers' to capture a single cell, which was used to start a pure culture. The ssu-rRNA sequence of this organism, which was named Thermocrinus ruber, is very closely related to that of EM17, although not exactly the same.
7. Thermocrinus ruber grows as rods in suspension, but when cultivated in media prepared to match the composition of Octopus Spring water in an artificial 'creek', grows as nice pink filaments that are just like those seen in the wild.

10. Give an example genus or species from 20 of these 24 phylogenetic groups. Please cross out the four you choose not to answer. (1 point each)

  Chloroflexus Green non-sulfur Bacteria    
     Treponema Spirocheates     
    Thermotoga Thermotoga & relatives
       Aquifex Aquifex & relatives		
       Thermus Deinococcus & relatives		
 Agrobacterium Alpha purple Bacteria		
   Escherichia Gamma purple Bacteria		
  Helicobacter Epsilon purple Bacteria		
       Frankia High G+C Gram-positives	
   Trypanosoma Euglenozoans			
     Spirogyra Chlorophytes			
       Cyclops Choanoflagellates & animals	
    Chlorobium Green sulfur Bacteria
      Anabaena Cyanobacteria
Flavobacterium Cytophaga & relatives
 Parachlamydia Chlamydia & relatives
     Pirellula Planctomycetes
   Rhodocyclus Beta purple Bacteria
 Desulfovibrio Delta purple Bacteria
      Bacillus Low G+C Gram-positives
      Hexamita Diplomonads
    Paramecium Alveolates
   Penicillium Fungi
Dictyostellium Slime molds

11. Now name three bacterial groups that contain few or no cultivated species and are defined primarily by ssu rRNA sequences from environmental samples. (5 points)

e.g. OP11, Coprothermobacter, green non-sulfur Bacteria