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Microbiology Exam Questions

Question 1

A) (2 points) What does the term “Hfr” stand for? 
High frequency recombination 
B) (4 points) What feature of Hfr strains makes it possible for them to readily transfer genomic sequences of the donor cell to recipient cells? 
In Hfr strains, a conjugative plasmid, e.g. F-plasmid, has integrated into the genome of the donor cell. Thus, when transfer to a recipient cell occurs, transfer starts at the origin of transfer and continues through to genomic DNA. 
C) (2 points) Of the three types of horizontal gene transfer that we discussed in class, which type is used by Hfr strains? 
2. You are a scientist studying horizontal gene transfer among bacteria. A colleague of yours claims to have isolated a bacterium that uses a new mechanism of horizontal gene transfer, in which a donor bacterium generates modified RNA molecules that are released when the cells are lysed, then taken up by recipient cells, and then converted into DNA in the recipient cell. Your colleague has an established assay in which a donor can transfer genetic material that confers resistance to ampicillin. (4 points) Describe a simple experiment that you could propose to your colleague to test whether the transferred material was truly RNA. Be sure to describe the result you expect if it is or is not RNA.  Set up a transfer experiment including one set of donor + recipient cells in which transfer is achieved as per your colleague’s assay (control sample), and a second set of donor + recipient cells (test sample) in which RNAse is added after lysing the donor cells. Test for transfer to the recipient cells by asking if they becomes resistant to Ampicillin. If RNA is the transferred material, adding RNAse will block transfer and the recipient will remain sensitive to ampicillin. If RNA is not the material, then RNAse is expected to not affect transfer. 
3. (3 points) Genes carried by transposons are often found to be genes that confer drug resistance, but not genes for general functions of a cell, e.g. DNA replication or mRNA translation. Why is this the case? 
Genes conferring drug resistance provide a selective advantage to cells that receive them, therefore such genes are more likely to be preserved in transposons than are genes that do not confer a special trait. 


4. Electron micrographs below are taken from a wild type cell and a mutant cell.


A). (2 points) What structure or structures are missing in the mutant? 
Radial Spokes 
B). (2 points) What defect (phenotype) do you expect to observe in this mutant? 
The mutant would be unable to swim/move like WT; …would be defective in motility/movement. 
C). (3 points) The image below shows ameboid movement through pseudopod extension and retraction. What protein polymer is used to drive this motility and what happens to this protein polymer at the leading edge and trailing edge of the cell to enable this motility? 

Question 2


Actin – polymerizes at the leading edge to push the pseudopod outward, depolymerizes at the trailing edge to allow retraction. 


6. Eukaryotic motility depends on motor proteins. 
A) (3 points) What motor proteins are used to power motility in eukaryotes? 
Myosins; Kinesins; Dyneins 
B) (3 points) Describe in your own words how these motor proteins power motility. 
Motor proteins move along tracks that are composed of protein polymers/filaments. The motor binds the track, then uses power from ATP hydrolysis to drive movement along the track. 
7. You are studying chemotaxis in E. coli. You have an E. coli mutant that you hypothesize it is defective at chemotaxis. To test this, you conduct a capillary assay for chemotaxis as described in lecture, using a known and confirmed attractant in the test capillary versus the control capillary. 
(A) (2 points) If the cells have a defect in chemotaxis, what numbers of E. coli do you expect to find in the test capillary compared to the control capillary?

(B) (5 points) Results from your experiment in part A support your hypothesis that the E. coli cells are defective in chemotaxis. A colleague tells you that she thinks the defect is not chemotaxis specifically, but that the cells simply have a defect in motility. How might you distinguish between these two possibilities, i.e. defective chemotaxis specifically versus general defect in motility. Be sure to describe what you expect as results if the defect is chemotaxis and what you expect if the defect is motility. 
There are a few potential good/correct answers. Basically, you must have a way to examine the cells to see if they are motile, e.g. visualize live E coli and look for movement from point A to point B – expect…; one could use a tethered cell assay or other method to view flagellum movement (technically, that doesn’t necessarily equate to cell motility, but OK answer when provided with expected outcomes.) 
 8. (8 points) You are trying to identify compounds that act as repellents for E. coli, i.e. compounds that E. coli will avoid. To test each compound, you use a tethered cell assay, as described in lecture. Describe how you would set up your tethered cell assay and you using this determine if a compound is a repellent, i.e. what do you expect to see in your tethered cell assay if a compound used is indeed a repellent.  
Describe the assay set up (4 points) 
Describe the results expected for a repellent (4 points) – frequency of CW rotation expected to increase when adding repellent 

Question 3


9. (2 points) What provides power for flagellar rotation in E. coli? 
A proton motive force; a gradient of H+ concentration across a membrane; movement of protons down their concentration gradient;, etc… 
10. (3 pts) Consider the phylogenetic tree below, with each letter corresponding to an individual organism. 
A) What organism(s) is(are) the closest relative(s) to F?  (all are equidistant) 
B) Which of D, E and F is more closely related to C:  is it D, E, F, or all the same distance?  (all the same) 
C) Are E and F monophyletic?  (No) 


11. (3 pts) In 2015 a group from Sweden reported results from metagenomics analysis from samples obtained in ocean sediments near the Loki’s Castle hydrothermal vent near Norway. They discovered a novel organism and named it “Lokiarchaeum”. Based on phylogenetic analysis, they proposed that Lokiarchaeum is monophyletic with eukaryotes but not with other archaea.  Which of the phylogenetic trees below depicts such a relationship? (B = bacteria, A = Archaea, E = Eukarya, L = Lokiarchaeum. 



12. (6 points) You have identified a brand new cellular organism and you name it Myown organism (“M. organism”). Describe three experimental approaches, other than DNA sequence, that can be used to assess whether a given organism is within the domain of Bacteria, Archea, or Eukarya. You do not have to give experimental details, but do indicate what is expected for placing the organism in the correct domain. 

Approach 1: Compare RNA Polymerase subunits of M. organism with that from known members of each domain. M. organism is placed within the domain that shows most similar subunits to M. organism. 
Approach 2:  Examine the membranes of M. organism – if ether linkage instead of ester, or if monolayer instead of bilayer, then placed within Archae.  (If Ester or bilayer, then need more experiments to distinguish.) 
Approach 3:  Compare sensitivities of M. organism to a range of translation inhibitors. Place in the domain that shows sensitivity and resistance to the same inhibitors as M. organism.

13. Fish
A) (2 points) In your own words, briefly describe how fluorescence in situ hybridization fish is performed. 
Probes corresponding to short fragments of DNA are fluorescently-labeled and then hybridized to DNA within a cell, i.e. and unbound probe is washed away. If DNA sequences complementary to the probe sequence are present in the target cell, then a fluorescent signal will be revealed.

B) (3 points) How can fish be used to distinguish between two organisms from different domains? 
Probes can be selected that are specific to one domain, e.g. bacteria (i.e. not found in the other domains). Now, if one sees a fluorescent signal in the target cell, the cell belongs to the domain that harbors the sequence used in the probe. If no signal, then the target cell must be in a different domain (assuming control experiments show the experiment worked properly). 
14.  (4 points) List names of 4 of major eukaryotic clades and include one distinguishing feature of each clade (or one specific organism within that group). 

Question 4


Opisthokonta (includes humans and other metazoan) Amoebazoa (includes slime molds) Rhizaria (= amoebas with filamentous pseudopods) Viridiplantae (includes algae) Alveolata (includes important human pathogens, e.g. malaria; includes examples of organisms arising through 2ry endosymbiosis) Heterokonta (includes brown alage; includes examples of organisms arising through 2ry endosymbiosis) Euglenozoa (includes human pathogens) Metamonada (includes human pathogens, organisms that lack mitochondria 


15. (2 points) Name one type of protozoan pathogen that infects humans and one protozoan pathogen that infects non-human animals. 
Human:  Plasmodium
Non-human:  Dinoflagellates

16. (4 points) During fruiting body development in Dictyostelium, cells detect a pulse of camp from pacemaker cells, begin migrating toward the pacemaker, secrete a secondary pulse of camp, and then become resistant to camp pulses for a brief period. What is an advantage of this short period of resistance? 
This allows chemotaxing cells to continue moving toward the pacemaker (the original source of camp and not get misdirected to other cells that have produced a pulse of camp.

17. You are working in a cell biology lab and growing mammalian cells in tissue culture. You are examining your cultured cells by electron microscopy and notice an extra structure with the cells that appears to be a virus. You have several additional cultures, and you use these to conduct a series of experiments that demonstrate this structure is indeed a virus.   
(3 points) What is an approach you might take to assess if this structure is a virus, and if a virus, whether it is a virus with an RNA genome? State how your results will answer this question. 
Use FISH and as your probe, use RNA-dependent RNA-polymerase, and use Reverse-transcriptase. If it’s an RNA virus, it will have either RdRp, or RT, and the RNA for these would be visible in the host cytoplasm, or in the packaged virus genome.

18. (3 points) Studies of poliovirus indicate it cannot infect mice. What is a potential explanation for why poliovirus infects humans, but not mice? 
Mouse cells do not express the receptor for poliovirus, so it cannot bind or enter into mouse cells. 
19. (2 points) True or False:  Lysogeny is a step in the events associated with specialized transduction? 
Antimicrobial Therapy, Mechanism, & Resistance  
20. (13 points) Liam suffers from Inflammatory Bowel Syndrome (IBS) and has noticed that when he eats yogurt (supplemented with probiotics), it alleviates his symptoms and discomfort. However, starting from a few days after his visit to a urologist specialist for his urinary tract infection, Liam has noticed that eating yogurt no longer works. Nothing in Liam’s life has changed beside being put on a course of combinatorial antibiotic therapy using broad spectrum antibiotics that can affect both Gram-positive and Gram-negative bacteria.  
A. (3 points) Explain why Liam’s IBS symptoms no longer disappear despite Liam eating the same yogurt as before, which is supplemented with probiotics.
Liam is taking antibiotics that are broad-spectrum and are potentially affecting the probiotics in the yogurt, so that these bacterial population are killed (3 pts). 


B. (3 points) Which of the following two drug combinations is unlikely to be prescribed together? (1 pt) Explain why those two drugs would be unlikely to work well together (2 pts). 
Drug combination A: Tetracycline (a bacteriostatic drug) and Ampicillin (a bactericidal drug)  Drug combination B: Sulfonamides (a bacteriostatic drug) and Trimethoprim (a bacteriostatic drug) 
Drug combination A (1 pt); Students should know from lecture that Sulfonamides and Trimethoprim are in fact often prescribed together (synergistic effect).  Problem: Ampicillin targets cell wall synthesis and since tetracycline is a bacteriostatic drug, the two drugs have an antagonistic relationship (2 pts) 
C. (3 points) What antibiotic resistance mechanism is used by bacteria to resist tetracycline? (1 pt) Briefly describe how this resistance mechanism is employed (2 pts) 
Efflux pump (1 pt); carries a transposon that codes for a membrane protein that transports tetracycline out of the cell (2 pts) 
D. (4 points) Propose a test you would use to determine whether an antibiotic is: 
a. (2 points) bacteriostatic  
Kirby-Bauer Test Or E Test

Or Broth Dilution Test      

(2 points) bactericidal 
Broth Dilution Test 


Microbial Immunity: DNA restriction & modification and crispr interference  
21. (3 points) Bacteria defend against phage infection through use of crispr and restriction endonucleases. Which of these defense systems is more conceptually similar to human innate immunity and which is more similar to human adaptive immunity, and why? 
DNA restriction endonucleases are analogous to the innate immune system in humans in that it is a general defense mechanism against phages, while CRISPR interference is analogous to adaptive immune system and the response is based on memory of past infection.  
CRISPR interference in antibiofilm phenotype  

22. (4 points) What is the significance of CRISPR interference and the antibiofilm phenotype employed by bacteria in a biofilm?  
To banish a lysogenic cell from a biofilm, so that phage produced by the lysogen cannot kill the rest of the biofilm community.




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