FINAL EXAMINATION BIOLOGY 422 FALL, In the spirit of the honor code, I pledge that I have neither given nor received help on this exam.

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1 Name First Last (Please Print) PID Number - FINAL EXAMINATION BIOLOGY 422 FALL, 2012 In the spirit of the honor code, I pledge that I have neither given nor received help on this exam. Signature

2 1. Pathogenesis (6 points) You are working with Doctors Without Borders in Bangladesh. You see an adult patient with profuse watery diarrhea. The diarrhea is so massive that it amounts to more than 1 liter in an hour. There is no blood in the stool. What disease is this likely to be? What is the principle virulence factor for the organism which causes the disease? What is the mechanism of action of this virulence factor? Be sure to include biochemical detail in your answer. B. (2 points) You are treating a patient who has a genetic defect which prevents her from making plasma cells. She has developed a lung infection. Which of the following are you most concerned about? Circle one Streptococcal pneumonia TB chlamydia pneumonia fungal pneumonia (histoplasma) viral pneumonia She has also fallen and has a puncture wound. Do you give her a tetanus booster shoot? Yes or no If no, what do you do about the wound? 2

3 2. Regulation (8 points) In the course of studying adenine synthesis in E. coli you isolate a mutant which overproduces adenine. Suggest 3 possible different sites at which this mutation could be located You have available the following: The base sequences of the adenine biosynthesis genes, nucleotide primers, a pcr machine and all necessary reagents, a cloning vector and all necessary reagents and enzymes, and competent cells of your mutant, competent cells of the parent strain, regular cells of your mutant, and regular cells of the parent strain. You do not have the capacity to sequence your mutant. Design a protocol to test one of your hypotheses listed above. My protocol is designed to test my hypothesis #. Experiments performed by you suggest that your mutation is dominant. Does this fit with any of your hypothetical explanations? Yes or no If yes which one? #. If no, suggest a possible explanation which does fit with a dominant phenotype. 3

4 3. Viruses A. (4 points) What type of enzyme inhibitor would be useful against a picorna virus such as polio or yellow fever without being likely to harm the host? (i.e. what enzyme would you like to inhibit?) Would this type of inhibitor be useful against influenza? Yes or no Would this type of inhibitor be useful against Herpes? Yes or no Would this type of inhibitor be useful against a retrovirus such as HIV? Yes or no B. Above is shown a map of the phage λ. (7 points) When λ is introduced introduced into E. coli K12(-) what type of plaques are produced? circle one None pinpoint(very small) cloudy clear If I replace pr with the promoter of an operon containing some of the genes encoding the enzymes of glycolysis and introduce this phage into E. coli K12(-) what type of plaques are produced? circle one None pinpoint(very small) cloudy clear If I replace pr with the promoter of an operon containing some of the genes encoding the enzymes of glycolysis and introduce this phage into E. coli K12(λ) what type of plaques are produced? circle one None pinpoint(very small) cloudy clear If I cut out the λ DNA between the start codons of CI and of Cro and invert it and then replace it and introduce this phage into E. coli K12(-) what type of plaques are produced? circle one None pinpoint(very small) cloudy clear 4

5 If I cut out the λ DNA between the start codons of CI and of Cro and invert it and then replace it and introduce this phage into E. coli K12(λ) what type of plaques are produced? circle one None pinpoint(very small) cloudy clear If I take some bacteria from the cloudy part of a cloudy plaque and place them in Luria broth will they grow? Yes or no If I infect them with wild type λ what type of plaques will I get? circle one None pinpoint(very small) cloudy clear 4. Energy metabolism (10 points) When using glucose as an energy source to grow aerobically where do the electrons to generate reducing power come from? What is the final electron acceptor? When using glucose as an energy source to grow anaerobically where do the electrons to generate reducing power come from? What is the final electron acceptor? When a cyanobacterium uses light as an energy source where do the electrons to generate reducing power come from? What is the final electron acceptor? When a purple photosynthetic bacterium uses light as an energy source where do the electrons to generate reducing power come from? What is the final electron acceptor? When Halobacterium rubrum uses light as an energy source where do the electrons to generate reducing power come from? What is the final electron acceptor? 5

6 5. Genetics (5 points) You are performing a conjugation between an E. coli strain that you obtained from a wastewater treatment plant which appears to be an Hfr strain (that is it will conjugate its chromosome to another E. coli strain K12 (-)). Your donor strain requires no additions to minimal medium beyond glucose to grow and is antibiotic sensitive. It can also grow on maltose or arginine as a carbon source. Your recipient strain E. coli K12(-) is trp mal met rif R. It cannot grow on maltose or arginine as a carbon source. What do you put into the medium to isolate transconjugants which are trp +? What do you put into the medium to isolate transconjugants which are mal +? What do you put into the medium to isolate transconjugants which are able to use arginine as a carbon source? After about 30 min. incubating the strains together under conditions suitable for conjugation you do the platings described above and recover large numbers of all three types of transconjugants. However when you plate again on the same medium after 50 min. the number of transconjugants shows a marked decrease when compared to 30 min. Suggest a possible explanation for this observation. 6. Evolution and Ecology (6 points) Recently a scientific expedition drilled through the Antarctic ice sheet to a lake several thousand feet below the surface. They retrieved a water sample from this lake aseptically. On observation it was found to contain living prokaryotes. What is the likely energy source in this ecosystem? What type of organisms are likely the primary energy producers in this ecosystem? (Be more specific than just bacteria or fungi or whatever) How did you know that the prokaryotes were alive? 6

7 You suspect that some of these organisms may be Eubacteria and some may be Achaea. How do you find out if this is true? 7. Wastewater treatment and treatment of xenobiotic compound contamination (8 points) In a wastewater treatment plant where complex organic compounds are being metabolized what are the end products for C and N? If oxygen were pumped through to keep the system aerobic what would be the end products? What is the end product if ammonia is removed aerobically? What is the end product if nitrate is removed anaerobically? I am trying to get rid of (metabolize) a xenobiotic compound which my bacteria cannot use as an energy source but which they can metabolize. What are some of the problems I need to address so that the bacteria will be effective? Would it be preferable to use a pure culture of one type of bacteria or to allow a microbial community to develop? Why? 7

8 8. Gene cloning (5 points) You are working as an undergraduate intern in a lab at UNC. A graduate student in the lab knows that you recently learned about cloning in your microbiology class, and asks you for help troubleshooting their cloning problems. The student is trying to clone the gene conferring ampicillin resistance from DNA isolated from ampicillin-resistant Salmonella enterica. The bacterial isolate he has is virulent in mice and causes diarrhea. He has prepared DNA from the bacterium. After performing the cloning, the student got colonies to grow, but none of them contained the gene of interest. What is one possible reason he did not obtain any positive clones? He has gotten so frustrated with the experiment that he trashed the previous protocols, so you don t have any notes to go on. You want to go on winter break instead of work in the lab for the next month, so you offer to write a detailed protocol for the graduate student. The lab keeps every restriction enzyme in stock, materials to make solid and liquid media, antibiotics, E. coli competent cells, Agrobacterium competent cells, the enzymes DNA polymerase, DNA ligase, and topoisomerase, healthy mice, and DNA of the cloning vector p3853 on the last page. Preliminary sequencing shows that there are recognition sites for the enzymes SacII, HindIII, SnaBI adjacent to the suspected gene and recognition sites for BamHI, XhoI, and EcoRI adjacent to and within the gene. Please be specific about what steps you should take to clone the desired gene, starting with genomic DNA and ending with a positive clone. 8

9 9. Transposon mutagenesis (7 points) You come back from your break and find that the grad student finally managed to clone his gene using your protocol. Now he is making a muddle of trying to use transposon mutagenesis to identify virulence genes in his S. enterica strain (described above). He grew S. enterica in complex medium and added plasmid DNA of pdum (shown on the last page) to introduce a transposon into S. enterica by transformation. He also had available DNA of the plasmids pf2 and pg3 shown on the last page. None of these plasmids can replicate in S. enterica. After transformation he plated the cells on Luria agar with ampicillin. He obtained a lot of colonies and so thought everything was OK. However, none of the colonies showed any alteration in virulence. What did he do wrong? How do you fix these mistakes? He follows your suggestions and now obtains a reasonable number of colonies. He then proceeds to test them for virulence by making a suspension of each the colonies he isolated and injecting them into mice. All of the mice die but none of them developed diarrhea. What has gone wrong this time and how do you fix it? He wonders why the mice that he injected died. What is a likely reason? He has now obtained a transposon mutant which does not cause diarrhea. He needs to confirm that the strain is a mutation in his original S. enterica strain and that the transposon insertion is responsible for the phenotype. What should he do (briefly)? 9

10 10. Ecology (6 points) You are trying to help a friend who is a county agent for the Department of Agriculture. One of the farmers in his county has two large fields which appears very similar. However, he routinely obtains a much higher crop yield from the first field than the second. On closer examination it appears that the low yield in the second field is due to fungal disease of the crop. He wonders if the bacteria in the soil could be causing this difference in fungal disease. You take soil samples from both fields. How do you determine which bacteria are present in each field and whether the same bacteria are present in both fields? You suspect that the antifungal activity may result from bacterial production of phenazine, an antifungal antibiotic made by some pseudomonads. The enzymes involved in the synthesis of this compound and the genes which encode them are known. How do you determine if there are any organisms which would be capable of synthesizing phenazine present in your soil samples? Phenazine fluoresces under UV light with a characteristic color. How could you determine if any of the bacteria you identified are actually making phenazine? 10

11 11. Metabolism A.(5 points; you lose one point for each incorrect answer down to zero; note that failing to circle an appropriate answer counts as an incorrect answer). Which of the following processes can be used to generate energy for growth? Circle the appropriate processes. glycolysis aerobic respiration anaerobic respiration N2 fixation fermentation CO2 fixation methanogenesis chemotaxsis light absorption B. (6 points; you lose one point for each incorrect answer down to zero; note that failing to circle an appropriate answer counts as an incorrect answer).which of the following processes could be carried out by a mutant bacterium which lacked all cytochromes but had an adequate supply of ATP? Circle the appropriate processes. glycolysis aerobic respiration anaerobic respiration N2 fixation fermentation CO2 fixation methanogenesis photosystem I photosystem II bacteriorhodopsin energy generation C. (6 points; you lose one point for each incorrect answer down to zero; note that failing to circle an appropriate answer counts as an incorrect answer). Which of the following could be used as an electron acceptor in anaerobic respiration? Circle the appropriate compounds. CO2 CH4 N2 NO3 - NH3 Fe +2 Fe +3 Mo +2 Mo +4 Cu +1 Cu +2 H2SO3 D. (6 points; you lose one point for each incorrect answer down to zero; note that failing to circle an appropriate answer counts as an incorrect answer). Which of the following could be used as an energy source? Circle the appropriate compounds. CO2 CH4 N2 NO3 - NH3 Fe +2 Fe +3 Mo +2 Mo +4 Cu +1 Cu +2 H2SO3 11

12 12. Symbiosis (6 points) Fill in the following table with respect to symbiotic interactions Tube worms and prokaryotes Cows and cellulolytic bacteria Rhizobia and legumes 12

13 13. General properties of 3 major groups of living things (eubacteria, archaea, and eukaryotes) Fill in the table (17 points). Note that in order to receive credit for a row you must fill each space in that row. Property Eubacteria Archaea Eukaryotes(excluding chloroplasts and mitochondria) Has a nucleus (yes/no) Has nucleosomes and histones (yes/no) Contains many operons (yes/no) Contains many introns (yes/no) Has coupled transcription and translation (yes/no) Encodes DNA-dependent RNA polymerase (yes/no) Encodes RNA-dependent RNA polymerase (yes/no) Some species can obtain energy from light (yes/no) Some species can fix nitrogen (yes/no) Some species can grow at temperatures over 100 o C (yes/no) Some species can obtain energy from N2 NO2 (yes/no) Can carry out anaerobic respiration (yes/no) Contains 16S RNA in the cytoplasm (yes/no) Protein synthesis is initiated by (name amino acid) Some species generate CH4 (yes/no) Type of plasma membrane lipid linkages (ester or ether) Cell wall composition (state major chemical types) We have enjoyed teaching you this semester. We hope you have a good holiday. 13

14 General information and plasmids for specific questions Amino acids vitamins sugars antibiotics threonine (thr) biotin (bio) glucose (glu) streptomycin (sm) leucine (leu) thiamine (thi) lactose (lac) rifampicin (rif) histidine (his) maltose (mal) ampicillin (amp) tryptophan (trp) nucleic acid bases arabinose (ara) tetracycline (tet) arginine (arg) adenine (ade) neomycin (neo) cytosine (cyt) kanamycin (kan) Plasmids ampr IR IR pdum tpna kanr IR SmR IR IR pg3 pf2 IR tpna IR= inverted repeat tpna= transposase 14