1) The Aquificiaeand Thermotogaeare both primitive and (probably) deep-branching phyla. Is it possible to draw a tree with deep branches that are not primitive, and with primitive branches that are not deep? Do you know of any examples of either? Justify your answer.

2) Organisms such as Thermotogamaritimathat reduce protons to generate hydrogen can do so only if very low ambient hydrogen concentrations make it energetically favorable. How do you suppose this hydrogen is gotten rid of so efficiently in their environment?

3) What do you think the absolute limiting issue for high-temperature growth might be? What do you think is the highest temperature at which any familiar type of organism could grow?

4) Can you think of any opportunities that extreme thermophiles might have that are not available to mesophiles? In other words, what might be some advantages of life in high-temperature environments?

5) Thermocrinusruberis found in Yellowstone hot springs separated by many miles of inhospitably cold (for them) territory. How do you suppose they colonize new hot springs when they emerge? Would you predict that the same organisms exist in similar hot springs in other parts of the world? Why or why not?

6) How many continuously or intermittently high-temperature microbial environments can you identify close to where you live right now? List them. Do you think you could isolate thermophiles from these environments? How would you go about it?

7) For the following sequence:

5`- C T A A C G T T G C A A C G C T C A G T G -3`

A) Calculate the Tmof the sequence
B) Determine the reverse complement of the sequence

8) Determine the melting temperature of the 16S rDNA primers used in the “I, Microbiologist” recipes described in Chapter 3 if PCR is performed in a reaction mix containing KCl at 65 mM. (Hint: use one of the suggested web sites in Chapter 3 of “I, Microbiologist”)

9) Suggest three ways in which to increase the stringency of the reaction described in question #8.

10) What sorts of artifacts result from performing PCR under low stringency conditions at room temperature? Why are these artifacts especially problematic for metagenomic DNA samples?

11) If the DNA target you would like to amplify via PCR is expected to be a total length of 4.3 kb, how long must your extension time be during thermal cycling?

12) Spectrophotometric analysis confirmed that you successfully purified ample amounts of genomic DNA from a bacterial isolate. However, you have encountered problems at the PCR amplification step of the project. Despite repeated attempts using standard procedures, you have not been able to amplify the 16S rDNA product. You hypothesize that the GC content in the genome of your isolate is unusually high, and thus refractory to PCR by conventional methods. Devise a PCR experiment in which you must change either the components of the reaction mix and/or thermal cycling conditions to promote amplification of the 16S rRNA gene from this troublesome isolate. Briefly explain how the changes you make will affect the reaction.

13) DNA extraction using a direct lysis approach results in the co-purification of prokaryotic and eukaryotic nucleic acids from lysed cells as well as the extracellular environment. Whereas this type of ‘contamination’ can interfere with the construction of bacterial ‘shotgun libraries’, why is this method suitable for the libraries generated for your project?

14) On the DGGE gel diagram below, draw bands representing 16S rDNA as you would expect to find them after performing PCR on communities derived from the Great Salt Lake in Utah (lane 1) versus Lake Michigan in the Midwestern part of the United States (lane 2). Briefly explain your reasoning for the depicted patterns.

15) Microbes can be detected in the environment using microscopic, cultivation, and molecular approaches. Discuss one advantage and one disadvantage of each technique.
16) You isolate a facultative anaerobe from the soil that is capable of reducing nitrate (NO3) to nitrite (NO2), ascertained using a standard biochemical test in which a medium containing a large amount of nitrate (KNO3) is inoculated. Following incubation, alpha-napthylamine and sulfanilic acid are added, turning red upon interaction with nitrite produced by the organism. There are two distinct enzyme complexes responsible for anaerobic respiration of nitrate, NAP and NAR. The genes encoding the catalytic subunits for each complex are napA and narG, respectively. Devise a community sampling approach to visualize the cells expressing these genes in the soil sample.

17) What distinguishes a keystone species from other inhabitants of an ecosystem?

The following questions refer to the primary literature article assigned for this week (also discussed in Chapter 19 of Principles of Microbial Diversity:

18) Sulfide is very toxic to animals (largely because it displaces oxygen in the blood and in the mitochondria), and yet vent animals such as worms and snails swim in it. How do you think the vent animals can be so resistant?

19) Describe a way to determine whether it is the snail or the biofilm, or some combination of the both, that creates the iron sulfide plating on the scales.

20) The scaly snail endosymbionts are in the esophageal gland. Given that these are egg-laying animals, how do you think the snail’s offspring get their endosymbionts? How would you test your hypothesis?

21) Likewise, do you think young snails get their (presumably) protective biofilm or armor-plating organisms?

22) The authors of the paper go to great lengths to remind the reader that the relative number of sequences they obtained is not a good measure of the relative abundance of the different kinds of organisms. How, then, could you use the sequence data to either count the organisms in or on the snail, or identify them specifically among the complex biofilm community?

23) Is there some confirmation experiment the authors have failed to perform to show that the single bacterial SSU rRNA sequence they obtained really is from the organism they can see in the bacteriocytesof the esophageal gland? Explain.