Midterm exam #2
MB 409 - Microbial Diversity
March 27, 2000

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

Bacteria, Archaea, Eukarya

2. What is the fundamental distinguishing feature of the "Gram-positive" cell envelop? (5 points)

The absence of an outer membrane

3. Why is it believed that Bacteria have a thermophilic ancestry? (5 points)

Because the deepest branches of the Bacterial Kingdom (Aquifex, Thermotoga and green non-sulfurs) and the Archaea are predominantly thermophilic.

4. Describe one mechanism used by Bacteria for motility. (5 points)

Possible answers: gas vacuoles, flagella, gliding, or spirochaete

Many aquatic & marine organisms can move themselves up & down in the water column by fine-tuning their bouyancy using gas vacuoles. These can be bound by a lipid membrane similar to the cytoplasmic membrane, or by a protein layer. Gas vacuoles usually contain CO2 generated by metabolism; how the cell controls the inflation and deflation of these vacuoles is unknown, but it must be a dynamic process. Maintaining a particular position in the water column requires continuous adjustment of bouyancy, because as the cell rises, the water pressure decreases and the vacuole will expand, making the cell more bouyant and therefore causing it to rise faster and
faster. Likewise, if a cell sinks, the vacuole is compressed by the increase in pressure, making it less bouyant and causing it to sink further.

5. How is photosynthesis different in cyanobacteria and other photosynthetic Bacteria? (10 points)

2 photosystems are used to generate both ATP and NADPH. PS I corresponds to the single photosystem in other Bacteria, and can be run either cyclicly or non-cyclicly. The ratio of cyclic vs noncyclic PS I electron flow regulates the ratio of ATP:NADPH generated. For every round of non-cyclic flow, the PS I chlorophyll has to be re-reduced by PS II. PS II uses H2O is the electron donor (transfering it to PS I) and therefore generates O2.

6. Briefly describe a specie, genus, or other phylogenetic group of Bacteria you find interesting. (10 points)

e.g. magnetotactic gamma-purple bacteria

These organisms have an internal string of magnetite beads that orient them along the magnetic lines of the earth. In the Northern hemisphere are North-going species, and in the Southern hemisphere are South-going species. Following the magnetic lines takes them quickly downward to the sediment, where they want to be (they are anaerobes).

7. Briefly describe a specie, genus, or other phylogenetic group of microbial eukaryotes you find interesting. (10 points)

Dinoflagellates

Usually photosynthetic flagellates - the 2 flagella are oriented at right angles, and they often have cellulose cell walls. Although they are photosynthetic and have chloroplasts, like the euglenoids they are not related to plants and aquired their photosynthetic symbiontic cyanobacteria independently of plants.

They are quite versitile, and often have complex life cycles. They are common symbionts of animals, such as reef-building corals and marine clams. On the other hand, they are also the causitive agent of red tide and ciguatera poisoning. Pfisteria is notorious in North Carolina at this time. Interestingly, these organisms generally lack histones and do not condense their chromosomes during mitosis or meiosis.

8. Describe any bacterial developmental process or life cycle (do not use the same example you described in question 6). (10 points)

e.g. log to stationary phase transition

The shift to stationary phase (& later back to log phase) is a complex developmental process, controlled by a sigma-factor cascade (like sporulation in Bacillus). Log-phase cells are very different from stationary-phase cells: log cells are adapted for maximal growth rates in nutrient-saturated conditions, where growth rate is often limited only by the organisms physical ability to import nutrients, process them into cell material, and replicate. They are usually large cells with ability to replicate DNA & make RNA/proteins rapidly. Stationary cells are usually small, and are adapted for maximum competitiveness. In many species, the morphology of the cells types are different - such as Arthrobacter described above. Keep in mind, as well, that microbes generally spend most of their time in stationary phase in the environment.

9. How can the electron transport chain be the foundation for such a wide range of phenotypes (e.g. photosynthesis, sulfur oxidation, iron oxidation, heterotrophy, to name a few)? (10 points)

Bacteria seem to switch lifestyle readily between sulfur oxidation or reduction, photosynthesis, heterotrophy, nitrogen oxidation or reduction, etc. All of these lifestyles are based on the same electron transport chain - all that's changed are the inputs & outputs (electron donors & acceptors). Most heterotrophs oxidize organic compounds into CO2 to generate NADPH, which serves as the electron donor for ATP synthesis, using O2 as the 'terminal' electron acceptor. Sulfur oxidizing autotrophs use H2S as an electron donor (--> sulfate) and oxygen as the electron acceptor. Other electron donors are thiosulfate, elemental sulfur, activated photosystem chlorophylls, hydrogen, methane, and ammonia. Other electron acceptors are sulfate, sulfite, sulfur, nitrite, nitrate, ferric ion (and many other oxidized metal ions), and reduced photosystem chlorophylls. Most of the electron flow is in the 'forward' direction for making ATP. Reverse electron flow is generally reserved for the synthesis of NADPH by autotrophs (who don't use organic carbon to make NADPH) for fixing carbon.

10. Name 15 of the major evolutionary branches of Bacteria. Some of these will, of course, need to be groups that are mostly or entirely known only from rRNA sequences rather than cultivated species. (1 point each - 15 points total)

Cytophaga and relatives Spirochaetes
Gram-positive Bacteria Green sulfur Bacteria
Green non-sulfur Bacteria Deinococci & Thermus
Thermotoga & relatives Chlamydia
Planctomycetes Proteobacteria
Cyanobacteria Aquifex & relatives
Fusobacterium & relatives Verrucomicrobium & relatives
Acidobacterium & relatives OP11 & relatives

11. How did Dr. Reysenbach obtain the sequences of the rRNA from the pink filaments biosystem? How did she then determine which of the sequences obtained was that of the pink filamentous organism? (10 points)

First, she isolated DNA from a sample of the pink filaments, and amplified ssu-rRNA sequences ny PCR directly from this DNA. The sequences were separated by cloning, and sequenced. The correct sequence was identified by "in situ hybridization". Dr. Reysenbach made a fluorescently-labeled oligonucleotide probe complementary to a unique sequence in EM17, and probed a sample from the pink filements environment. She showed at only the pink filaments could be labeled with the sequence-specific fluoresecent probe.

12. How did Dr. Huber use the rRNA sequence information to cultivate the pink filaments organism? (5 points)

The phylogenetic placement of EM17 in the tree near Aquifex lead Dr. Huber to use media based on the growth requirements for Aquifex, as well with the chemical composition of the outflow stream. A specific fluorescent probe based on the EM17 sequence (the same one used by Dr. Reysenbach) was used to screen and monitor enrichment cultures. This same fluorescent probe was used to label cells in an enrichment during the isolation of a single cell using optical tweezers. The identity of the pure culture was confirmed by comparing the sequence of its ssu-rRNA, which is almost exactly the same as EM17.