Study Questions Lectures 10, 12-19Bio119

Lecture 10

1) Both NO3- and NH3 are in the growth medium for a prokaryote; which will get used up first? Why?

2) What is a reaction used to incorporate NH3 into biomolecules?

3) What needs to be done to NO3- in order to incorporate it into the cell? What enzyme does this?

4) What does fixing nitrogen mean? Why doesn't every prokaryote do this?

5) What is the basic cycle of reactions that nitrogenase carries out in order to reduce N2 to NH3? Why do you think this uses so much ATP?

6) What does nitrogenase use as a source of electrons? Why doesn't it just use NADH? What are at least two possible sources for these electrons?

7) Why is the study of microorganisms like Rhizobium so important to agriculturists?

8) Describe the "dialog" between Rhizobium sp. and its host pea plant. i.e. what does the plant produce, what does Rhizobium sp. produce? What is the result of this dialog?

9) How does the Plant-Rhizobium symbiosis benefit each partner?

10) What is a bacteroid?

11) Why is it surprising that Trichodesmium sp. fixes N2 in the presence of O2? How might it do this?

(Lecture 11 was on first midterm and is in other study questions)

Lecture 12

1) How were cell division mutants identified? Why are these lethal? How can one get around that?

2) How do cell division mutants tell us about cell division?

3) Filamentous division mutants fell into two classes. Be able to describe these and how they differ.

4) How does stopping chromosomal replication stop cell division? Why?

5) What is the role of FtsZ in cell division? Does it act early or late?

6) What is the phenotype of ftsZ-? What about too much ftsZ? (Too much = overexpression)

7) Where does FtsZ localize in the cell? How did scientists figure that out?

8) What is the phenotype of minC, minD and minE-? How about too much of each? Know why.

9) Where do MinC/D/E localize? Why does the cell do this?

10) Why are errors in DNA replication rare? What enzymatic activity reduces errors?

11) Why must DNA replication start with a primer? Describe the steps in replicating the leading strand and the lagging strand. How do they differ.

12) Where does DNA replication begin in bacteria?

Lecture 13

1) If you discovered that a particular bacterium had two DNA molecules in it, one that was 450 kilobases and one that was 550 kilobases, how would you decide if it had one chromosome and one plasmid, or two chromosomes?

2) What are some things normally found on plasmids?

3) What polypeptides is core RNA polymerase composed of? What does the core do?

4) What is the sigma subunit and what does it do?

5) Why does sigma 70 recognize different promoters than the heat shock sigma factor does? What is the purpose of having these different promoters? Why are some promoters a good “match” and some a less good match to the consensus, and what does this match mean for gene expression?

6) What regions of the promoter are important for RNA polymerase binding and release? How are they defined?

7) Where do ribosomes bind mRNA? In the following sequence of mRNA, where would translation begin, if the ribosome binds at the 5' end? Can a ribosome bind at more than one site on an mRNA?

5'- AGGGGGCUUCCAUGCGAUUGUUCAAGUGACA-3'

8) What would be the result (in terms of the produced protein) if RNA polymerase initiated transcription one base upstream of its normal starting point and why? What would be the result (in terms of the produced protein) if translation began one base downstream of its normal starting point and why?

9) In a polycistronic mRNA, would there be multiple promoters, or multiple ribosome binding sites or both? Why?

10) How does a ribosome tell that it is at the end of a gene? What happens then?

11) Why do organisms have so many genes if they don't even express them all at once?

12) What are three levels at which an organism can regulate the activity of an enzyme? (include ways to regulate its synthesis)

13) How does arginine repress the genes for its own biosynthesis? Why does it do that? When would a bacterium that was mutant for the Arg Repressor (lacking it) express the arg operon?

14) How does this differ from the regulation of the maltose operon? What would you change about the maltose operon promoter so that it was repressed by the Mal Activator?

15) What is different about Fnr when there is oxygen, and when there is not? Be sure to relate the chemical difference to the activity difference.

16) What types of genes are activated by Fnr and what types are repressed? How can one protein do both?

Lecture 14

1) Why might bacteria prefer to do some activities-such as light production-only in a group?

2) How does the autoinducer enter and leave the cells?

3) Why does binding of autoinducer allow LuxR to bind DNA? To what DNA does it bind?

4) How does the autoinducer lead to activation of gene expression?

5) What would be the phenotype of luxR- V. fischeri? How about luxI-?

6) In the squid-V. fischeri symbiosis, what does the bacterium receive? What does the squid receive?

7) How is the frequency of "dark" mutants (V. fischeri mutants unable to make light) in the lab different from in the squid? Why is this so? What are four genes that, if eliminated, would yield a dark mutant?

8) Describe the typical two component system.

9) Where does the Sensor Kinase obtain phosphate from? For EnvZ, what determines if it is phosphorylated or not?

10) Where does the Response Regulator obtain phosphate from? For OmpR, what determines if it is phosphorylated or not?

11) What genes does OmpR regulate? How? Why?

12) How would the OmpR/EnvZ system change if the high affinity site in the ompF promoter was deleted?

Lecture 15

1. What are the parts of your average phage? What do each of them do for the phage life?

2. How would restriction enzymes protect a bacterial host?

3. What would be the fate of the T4 virus if the host cell were killed at 18 min. post infection?

4. Why is transcription of the T4 phage DNA under such tight temporal control?

5. What would happen to lambda phage if the host were a mal porin- mutant (missing maltose porin)?

6. Why are lysogenized E.coli strains resistant to re-infection by lambda?

7. Scientists detect phage using the plaque assay. What is a plaque? How is one produced?

8. What would a plaque assay done with WT lambda phage look like? How would it change in a cl repressor minus mutant?

9. Compare and contrast the lifecycle of T4 and phage lambda. What proteins do they each use for attachment? How do they escape?

10. How does lambda "decide" whether to become a lysogen or to become lytic? Be sure and know the molecular details (e.g. which promoters are transcribed to produce which proteins and how these act) of how this works.

11. What are some lambda genes that when mutated to be non-functional make lambda always go the lytic route? Always be lysogenic?

Lecture 16

1) From past lectures, homework and exams, identify the mutations that arose from the acquisition of DNA and those that arose from mutation of an existing gene.

2) What are point mutations? Given the genetic code, be able to draw a point mutation that would lead to a silent mutation, a nonsense mutation and a missense mutation, and know what each does at the protein level.

3) What effect does an insertion or deletion within a gene coding sequence have on the resultant protein? What’s the effect of these mutations if they occur within a promoter, terminator or ribosome binding site?

4) If E. coli was growing without any extra mutagens, would you expect its genome to have any mutations? How do mutagens change this?

5) E. coli can grow with a variety of sugars as its only carbon source. One of these is maltose. Mutant E. coli can be isolated that no longer grow on maltose (they can grow on other carbons, e.g. glucose). How would you find such a mutant? Include whether you would mutagenize, what you would plate the bacteria on etc—the steps. Would your plan be a screen or selection?

6) What if you had a bacterium that was sensitive to a certain antibiotic, and you wanted to find a mutant that was resistant. How would you do this, and would it be a screen or selection?

7) How does the Ames test work? What do you think the original his- mutation is? (point, insertion, deletion). How would the test change if the starting mutation was one of the other types?

8) Describe how transformation, conjugation and transduction occur, including the similarities and differences between them.

Lecture 17

What is the definition of normal microflora?
What organs of the human body are normally colonized by microorganisms? What do these organs have in common? What about the opposite—devoid of microorganisms?
What are some of the benefits we derive from our normal microflora?
What are the bacterial events that happen on your teeth every day? How does bacterial metabolism contribute to cavities?
How do germ-free mice (germ-) differ from germ+ mice?
How does intestinal epithelial cell surface sugar expression differ in germ- and germ+ mice? What was added to the germ- mice to make them more like germ+ mice?
What kind of mutant of Bacteroides thetaiotaomicron would not change the germ- mice? Why?
Different genes are expressed by the intestinal cells in response to B. thetaiotaomicron. What are a couple of these, and what does this tell you about the role of the intestinal microflora?

9. What differentiates pathogens from our normal microflora? When and how do normal microflora become pathogenic?

10. Know the steps that a pathogen must accomplish to cause disease. What would happen to a mutant that was defective in one of the steps? (answer this for all steps).

Lecture 18

1. This lecture included lots of vocabulary. Please be sure you know what the following are: Phagocyte, White Blood Cell, Macrophage, Leukocidin, Lysosome, B cell, T cell (and types), MHCII, TNF, IL4, T cell receptor, antibody, antigen, plasma cell, memory cell, septic shock.

2. Many different parts of the human body are colonized by lots of microbes. Some of these have the potential to be pathogenic. What keeps them in check? Describe a set of circumstances that would allow these microbes to cause disease.

3. Why are stressed individuals more susceptible to infectious disease? What do you think cortisone could act on?

4. What mammalian cells are first on the scene of an infection? Explain how these cells engulf and kill microbes? What microbes evade this response and how?

5. Describe the basic structure and function of MHCII molecule.

6. What are the two types of T helper cells and how do they differ?

7. Immunity can be generated by a process called passive immunity, in which a person receives an injection of antibodies to a specific pathogen (instead of antigen). Would you expect this form of immunization to lead to memory? Why or why not?

8. Trace the path of a pathogen that comes into the body via a wound in the calf. What cells will recognize the pathogen first? Where will the pathogen most likely cause an infection? What cells are involved in the destruction of the pathogen? What cells are involved in specific immunity to the pathogen?

9. TNF causes its cellular responses by acting through the TNF receptor. There are mutant mice that lack the TNF receptor. Would you expect them to get septic shock? Would you expect them to be able to fight infections? Explain your answers.

10. What are two ways that antibodies help fight off infectious agents?

11. We have not been able to make vaccines for all pathogens—e.g. HIV and malaria. Propose at least one reason that this is the case.

Lecture 19

1. For Mycobacterium tuberculosis, Helicobacter pylori, Treponema pallidum, and Chlamydia trachomatis know what (i) disease(s) they cause, (ii) how they are transmitted and the steps in causing disease, (iii) treatment, (iv) any virulence factors we discussed in class, (v) general microbiological features of the organisms.

2. Is tuberculosis a current problem? How is Mtb infection dealt with by public health officials? Is this different than how we deal with sexually transmitted diseases and why?

3. After a person inhales Mtb, there are a couple of possible outcomes--what are these and how do they differ? Why does one happen versus the others?

4. What is the PPD test? What does it tell you

5. What is LD50? How is this measured? How is this different from ID50?

6. How are virulence factors (proteins required to cause disease) identified?

7. If a gene for a virulence factor is eliminated, is the LD50 increased or decreased? Why?

8. How are virulence factors of syphilis identified? What makes this process difficult?