Microbial Diversity 2004 Final Exam KEY

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

Archaea, Bacteria and Eukarya

Multiple Choice - 2 points each

2. ___D__ In a molecular phylogenetic analysis of a microbial community using PCR-amplified ssu-rDNA sequences, what is chimera?

A. a holocephalic elasmobranch, e.g. the Spotted Ratfish (Hydrolagus colliei)
B. a part lion, part goat, part serpent creature from the Iliad
C. an out-of-context rDNA sequence aquired by the organism by horizontal transfer
D. a hybrid rDNA sequence from two different organisms created in the PCR reaction
E. a non-rDNA sequence that somehow appears in a sequence collection

3. ___B__ Robert Huber & collegues isolated the organism Thermocrinus ruber (that had been shown to be the pink filamentous growth in Octopus Spring) by...

A. plating out water samples & searching manually for pink fibrous colonies
B. capturing individual cells with the right morphology using a laser built into the microscope
C. recreating an exact-scale copy of Octopus Spring in their lab for enrichment cultures
D. tweezing out filaments one at a time and carefully washing away any adhering organisms
E. T. ruber has not yet been isolated, but has been studied only in situ

4. ___A__ Which of the following was not used in the class papers to sort rDNA clones into closely-related groups?

A. fluorescent in situ hybridization
B. partial, preliminary sequences
C. T-tracts
D. RFLP (restriction fragment length polymorphism)
E. full-length sequences

5. __E__ Which of the following pathogens might not be a good candidate for PCR-based detection from dental remains?

A. Bartonella quintana, which infects red blood cells and causes cat-scratch disease
B. Salmonella typhi, which enters the bloodstream via the Pyer's patchs in the gut
C. Marburg virus, which spreads throughout the body after contact with bodily fluids or aerosols
D. Bacillus anthracis, which grows systemically after entering via the lungs, gut, or skin break
E. Corynebacterium diphtheriae, which grows in the throat and secretes a toxin into the circulation.

6. ___B__ In the Juretschko paper on the analysis of the phenol-degrading microbial community in the industrial wastewater treatment facility, they designed and used a probe against Nitrosococcus mobilis even though they did not get it's rRNA sequence in the survey part of the analysis. They did this because...

A. they needed a negative control in the FISH experiments
B. they knew from previous work that N. mobilis was abundant in this ecosystem
C. they could see cells that looked like N. mobilis that didn't hybridize to any of their other probes
D. they'd spiked the samples with N. mobilis so they could quantitate hybridization efficiency
E. this probe should cross-hybridize to most nitrate or nitrite oxidizers.

7. ___C__ A monogram is...

A. an X-ray-based medical screening procedure for infectious mononucleosis
B. a chapter or subsection of Bergey's Manual
C. a published, formal description of a new species
D. a method to visualize hybrization when probes are radio-labeled rather than fluorescently-labeled
E. 1 x 10^-18 grams

8. ___E__ In the paper by Hugenholtz, et al., describing the phylogenetic survey of Bacteria in

Obsidian Pool, the authors assigned "inferred metabolisms" of sulfate reduction (47 clones), organotrophy (28 clones), or hydrogen oxidation (84 clones) to some of the sequences. On what did they base these assignments?
A. they tested representative sequences directly for their metabolic capability in vitro
B. they were identical to sequences from organisms with known metabolisms
C. from the microenvironmental conditions each sequence came from
D. from the sequences of metabolic genes flanking the rDNA sequence
E. the sequences fell into groups of organisms that have that type of metabolism

9. ___A__ The microbial population of Obsidian Pool seems to be...

A. predominantly bacterial
B. predominantly archaeal
C. predominantly eukaryotic
D. fairly evenly balanced between Bacteria and Archaea, few if any eukaryotes
E. a blend of all 3 phylogenetic groups

10. ___E__ The microbial population of surface ocean water seems to be (from the DeLong, et al. paper) ...

A. predominantly bacterial
B. predominantly archaeal
C. predominantly eukaryotic
D. fairly evenly balanced between Bacteria and Archaea, few if any eukaryotes
E. a blend of all 3 phylogenetic groups

11. ___B__ Proteorhodopsin seems to be a....

A. light-driven chloride pump
B. light-driven proton pump
C. type I light-specific sensor
D. type II light-specific sensor
E. cone-specific opsin

12. ___D__ In a stable-isotope probing (SIP) experiment on a microbial population, labeled rRNAs are separated from unlabeled rRNAs by...

A. denaturing gradient gel electrophoresis
B. tangental-flow filtration
C. atomic absorbtion spectroscopy
D. cesium-TFA density gradient centrifugation
E. cloning and probing with fluorescently-labeled oligonucleotides

13. ___A__ Medieval european "Black Death" seems to have been caused, at least in some cases, by...

A. Yersinia pestis
B. Bacillus anthracis
C. uncultivated members of the SAR86 group
D. Mycobacterium leprae
E. an Ebola-like virus

14. ___E__ What is the relationship between Bacillus and bees?

A. Bacillus causes a systemic infection in bees, killing them so they can become fossilized
B. the insect provides the proper environment for the sporulation phase of the Bacillus life cycle
C. the insect is the arthropod intermediate vector for Bacillus species that infect mammals
D. Bacillus is just in the gut of these insects because they eat soil, where Bacillus lives
E. Bacillus is a commesalistic symbiont of bees, involved in pollen digestion and disease prevention.

15. ___A__ The largest part of the paper by Vreeland, et al., on the isolation of a halotolerant Bacillus from a 250 million-year-old salt crystal was spent on...

A. controls for sterility and contamination-control
B. analyzing the phylogenetic placement of the Bacillus isolate 2-9-3
C. phenotypic characterization of the Bacillus isolate 2-9-3
D. determining the age of the inclusions
E. describing the containment procedures used to prevent the escape of this ancient organism

16. ___D__ The filamentous fossil microbes from the massive sulfide deposit described in the paper by Rasmussen look like the sulfur-oxidizing organisms that live in hydrothermal vent environments today, but they probably had some other form of metabolism because...

A. sulfur oxidizers hadn't evolved yet
B. the temperatures in the vent environment were too high for sulfur oxidation
C. the sulfur isotope ratios aren't consisteent with the fractionation seen in sulfur oxidizers
D. there weren't significant concentrations of oxygen at that time in Earth's history
E. there is no evidence for sulfur in this hydrothermal vent environment

Short answer (5 points each)

17. Give an example of an organism other than Thermocrinus ruber that was identified by molecular phylogenetic analysis before it could be cultivated (if it even has been cultivated).

Some acceptable answers:

18. What is the "full-cycle rRNA approach"?

An rRNA survey followed by FISH, using the data from the survey to design probes for the FISH.

19. What are the 2 fundamental reasons why rRNA such a good target for FISH?

20. What were the three things found in the martian asteroid ALH84001 that were repesented as evidence for the presence of life on Mars?

21. What are the features of the 3.2 billion-year-old filamentous fossils from the massive sulfide deposit that distinguish them from non-biological/mineralogical in origin?

22. What are the features of unerupted teeth that make them good for looking for the causative agent of Black Death in skeletal remains from Medieval times?

23. Dr. Jean Ristaino at NC State University and Dr. Carol Groves at the New England Plant, Soil and Water Laboratory have identified the fungus that caused the Irish potato blight, and resulting famine, in the mid-1800's. How do you suppose they did it?

By rRNA-based phylogenetic analysis of potato lesions from museum collections

24. Why do the rDNA PCR products from different organisms in a population show up as distinct bands on a denaturing gradient gel electrophoesis (DGGE) gel, even though they are the same length?

Because they differ in sequence, the denature at different places in the gradient of denaturant, and this denaturation essentially stops their migration in the gel.

25. In the paper by Beja (Ed DeLong's lab) on the proteorhodopsin, they study the photocycle of the proteorhodopsin to try to distinguish whether it is a pump or a sensor opsin. What was it about the light cycle that they were looking for to make this distinction?

It's fast, like an ion pump opsin, rather than slow like a sensory opsin.

Essay (10 points each)

26. In the paper on the phylogenetic identification of the pink filaments organism, the authors (Reysenbach, Wickham & Pace) seem to have been surprized by their lack of detection of Archaea in the pink filaments sample. Why were they surprized? What could they have done to investigate this further?

Because of the belief that life at these temperatures is dominated by Archaea - nearly all of the cultivated organisms that grow above 80C (extreme thermophiles) are Archaea.

They could, for example, have done exactly what Hugenholtx, et al, did with the Obsidian pool samples; use bacterial- and archaeal-specific probes to test for the relative proportion of Bacterial and Archaea rDNA in DNA extracted from the hot-spring community. In this case there is no reason they couldn't do the probing with the bulk DNA itself rather than PCR-amplified rDNA. They could also use group-specific rDNA PCR primers to get the rRNA sequences of those Archaea that do exist there.

27. Of the three lines of evidence for life on Mars from ALH84001, describe how one of these can be explained without the need for biological processes on Mars.

Here of course you could discusss any of the three major lines of evidence for life in this asteroid - PAHs, the carbonate "fossils", or the magnetite beads.

For example:

It has been shown since this paper came out that PAHs are abundant (as these things go) in interplanetary and interstellar space, and the amount and size distribution of PAHs in ALH84001 are entirely consistent with them having been absorbed by the asteroid during its 16 million years drift in space from Mars to Earth.

NCSU Honor Pledge:

I have neither given nor received unauthorized aid on this test.

Sign __________________________________________ Date _______________