Research the answers to each of the questions below. Use whatever sources you find
useful – especially lecture notes and your textbook. You may work in groups. On the
final exam these questions will make up the cumulative section of the exam. For the
final exam, you will need to remember your answers and write them on the test
pages: you may not write out your answers ahead of time and turn them in. (There 7
questions, most with multiple parts, spread over 5 pages in this document).
Background:
Frederica Bimmel (the infant from Case 3: Wanda’s Woes) needs your help! Of course,
she’s all grown up now, having survived a rough first year back in 1975. She has no
memories of her numerous hospital stays, but over the years her parent’s stories about her
early struggles made a deep impression on Frederica, and she developed a keen interest in
medical microbiology. This interest motivated her as she completed her M.D. degree,
then her Ph.D. degree, followed by a Master’s degree in epidemiology. She has become
one of the world’s leading experts on Streptococcus pneumoniae, and she has arranged an
important meeting with Melinda and Bill Gates and the Global Alliance for Vaccine
Development (GAVI) to try to convince him to fund her research to develop a new
vaccine that will protect people from all serotypes of S. pneumoniae. Like any good
scientist, Dr. Bimmel prepared extensively for this meeting, including putting together a
PowerPoint presentation. On the cab ride from the airport to the Microsoft campus, she
received a phone call from one of the Gates’s assistants with these words of advice: “You
know, of course, that the Gates absolutely despises PowerPoint presentations, so I hope
that you plan on avoiding that…they really likes to see ideas scratched out in pen or
pencil on a piece of paper. Is that going to be a problem for you?” You happen to be
sharing the cab, and so Dr. Bimmel turns to you for help: “What do you know about S.
pneumoniae? Can you help me draw a few simple diagrams and write out some basic
definitions and ideas? I have some innovative ideas for how we could reduce S.
pneumoniae infections by taking advantage of: (i) its metabolic properties; (ii) microbial
ecology; (iii) vaccine development. I’ll just tell you the ideas, and you can write them out
on this paper!”
The genome of the S. pneumoniae strain Dr. Bimmel is studying includes the
following features that she needs to highlight in a simple diagram;
I. Several important chromosomal genes including:
a. pspA, a gene encoding pneumococcal surface protein A.
b. plyA, a gene encoding the S. pneumoniae pneumolysin.
c. a operon including ptsA ptsB ptsC ptsD encoding the components of a
phosphotransferase transport system that can transport glucose into the cytoplasm
d. a gene encoding lactate dehydrogenase (ldh) and genes for each of the
glycolytic enzymes
e. blaA, a secreted protein that can enzymatically inactivate β-lactam antibiotics
f. A lac operon (lacZ lacY lacA) encoding proteins very similar to their counterparts
in Escherichia coli.II. A plasmid that carries:
a. pilA, a gene encoding the major protein of the pilus that mediates attachment to
epithelial cells in the lungs
b. pilZ, A gene encoding the adhesin for the PilA pilus
Several genes NOT PRESENT in the S. pneumoniae genome include:
• those encoding TCA cycle enzymes
• those encoding electron transport chain proteins
1. Draw a single labeled diagram of S. pneumoniae that depicts this organism and the
location of each of the features specified below:
a. Functional anatomy of S. pneumoniae
i. the shape of the organism;
ii. the layers of the cell envelope including the capsule (Do label the layers; do NOT
draw individual lipid or carbohydrate molecules that are considered part of
the layers.)
iii. do clearly indicate whether this is a Gram-positive or Gram-negative bacterium
iv. the location of the attachment pilus
b. Genetic anatomy of S. pneumoniae: Depict the location of pspA, plyA, ptsABCD,
ldh, lacZYA, pilA, pilZ, and blaA in your cell diagram.
c. Biochemical anatomy of S. pneumoniae – show (on your diagram) where you
would expect to find: Teichoic acids, Pneumolysin, β-galactosidase (LacZ), PspA,
β-lactamase (BlaA), PtsABCD (a multiprotein complex), PilZ, PilA, Autolysin
(LytA), Phosphocholine, Hyaluronidase
2. Dr. Bimmel realizes that the Gates may not understand the genetic nomenclature
contained within her drawing of the cell, so she decides that it would be helpful to
prepare an additional figure that would explain the concept of a gene. She decides that
her drawing should highlight the ptsABCD operon. Prepare a simple figure that
shows how information is converted from a DNA molecule to a protein. Include
in your figure the correct location of:
a. 5’ and 3’ ends of each molecule
b. the location of transcriptional start and stop sites
c. any promoters
d. ribosome binding sites
e. start and stop codons
f. amino and carboxyl termini of the proteins
g. sites of operon regulation.
3. Functional components of S. pneumoniae.!
a. For each of the S. pneumoniae cell components listed below, briefly indicate how it
could contribute to the ability of this bacterium to cause disease in a human host. If it
is an enzyme, what is the substrate, and what is the product? 1 or 2 sentences for each.
i. Capsule
ii. Attachment (PilA) Pilus
iii. Pneumolysin
iv. PspA
v. Teichoic Acid
vi. Autolysin (LytA)
vii. Phosphocholine
viii. Hyaluronidase
b. What term would a Pathogenic Microbiologist (or an Epidemiologist) use to refer to
the collection of cell components listed above?c. Below are 3 media recipes. Which of these is (are) “complex”? Justify your answer.
Medium 1 Medium 2 Medium 3
Glucose 20 g Sodium Citrate 50 g Lactose 20 g
Peptone 5.0g Ammonium phosphate 1.0 g Ammonium phosphate 1.0 g
Beef extract 3.0 g Sodium chloride 5.0 g Sodium chloride 5.0 g
Sheep blood 10 g Magnesium sulfate 0.2 g Magnesium sulfate 0.2 g
Sodium Chloride
8.0 g Potassium phosphate 1.0g Potassium phosphate 1.0g
Agar 15.0 g All 20 amino acids
(200 µg each)
All 20 amino acids
(200 µg each)
Water 1 1iter Guanine, adenine, thymine,
cytosine, uracil (100 µg each)
Guanine, adenine, thymine,
cytosine, uracil (100 µg each)
All known vitamins
(25 µg each)
All known vitamins
(25 µg each)
Water 1 liter Water 1 liter
d. S. pneumoniae can grow in Medium 1 and Medium 3, but not in Medium 2. Why?
e. Would E. coli be able to grow in media 2? Justify your answer.
f. A mutation in what gene would prevent S. pneumoniae from growing in medium 3.
Justify your answer. Why would this mutation have no effect on the ability of
S. pneumoniae to grow in medium 1?
4. S. pneumoniae is highly dependent
upon external sugars to meet its energy
requirements (recall that it has neither an
electron transport chain nor a TCA
cycle). The transport system used by
S. pneumoniae for glucose is depicted
in Figure 1.
a. What term best describes this
transport mechanism?
b. !What is the immediate source of
energy that drives this transport system and allows S. pneumoniae to scavenge low
levels of glucose from its environment?!
c. When S. pneumoniae is growing in the presence of oxygen using glucose as its
energy source, which metabolic pathway (Aerobic respiration, fermentation or
anaerobic respiration) would it use? Justify your answer.!
d. What is the NET GAIN in ATP generated by S. pneumoniae catabolizing one
molecule of glucose during this type of metabolism? Remember to take into
account the cost of glucose transport. Show how you generated this answer.
5. When faced with a wide variety of carbon and energy sources, a bacterium has to make
metabolic decisions, opting for preferential use of one source of carbon over another in
order to maintain optimal growth. Simultaneous utilization of all available sugars would
be metabolically inefficient and would lead to slower growth. Like E. coli, the preferred
sugar for S. pneumoniae is glucose. (The doubling time for S. pneumoniae growing
minimal media + glucose is 40 minutes, and the doubling time for S. pneumoniae
growing minimal media + lactose is 60 minutes)
Figure 1. This is the phosphotransferase system
that S. pneumoniae uses for the transport of
glucose. (PEP is Phosphoenolpyruvate)Draw three growth curves:
i. One that would reflect the growth of S. pneumoniae growing in the presence of glucose.
ii. A second that would reflect the growth of S. pneumoniae growing in the presence
of lactose.
iii. A third that would reflect the growth of S. pneumoniae growing in the presence of
both sugars, where glucose is exhausted 80 minutes into log growth. Dr. Bimmel
understands this is a sketch of the results, but is insisting you label your x- and yaxis properly and plot it properly using the data she supplied you.
b. Explain what is happening in each phase of the growth curve for all three curves.
c. On the curve you drawn for 5.a.iii, describe β-galactosidase activity over the time course.
Dr. Bimmel found some graph paper in her bag, (See the end of this document) you
can use it to practice your graphs before you present them, but do not feel compelled to
do so. If you want, free hand your own graphs.
6. Dr. Bimmel is concerned about eliminating the carrier state of S. pneumoniae. She is
particularly concerned about finding ways to prevent S. pneumoniae-caused otitis media
without the use of antibiotics. Antibiotic use against S. pneumoniae has become
problematic. Amoxicillin worked for her as a child, but today there are many antibiotic
resistant strains.
a. Describe how amoxicillin affects bacterial cells. (What is the target site? What does
amoxicillin do?)!
b. What is the gene product of the blaA gene?!
c. How does this gene product allow bacteria to survive exposure to amoxicillin?!
d. Does blaA confer resistance to antibiotics other than amoxicillin (like tetracycline
and kanamycin)? Briefly explain
e. Show (describe) how the blaA gene could be naturally transferred from a
S. pneumoniae cell that is amoxicillin resistant to a S. pneumoniae strain that is
susceptible to amoxicillin. Include in your response a description of the selection
conditions required to select for amoxicillin-resistant S. pneumoniae.
f. Is this a genotypic change? Design an experiment to support your answer.
7. GAVI is very interested in funding projects that provide novel delivery mechanisms for
vaccines. Dr. Bimmel has always wanted to find a way to make a vaccine for
S. pneumoniae that targets all of the serotypes! This may be her opportunity! Her goal is
one vaccine that would be completely protective against all strains of S. pneumoniae.
She has an idea to make an edible vaccine that would contain the antigen derived from
S. pneumoniae. Dr. Bimmel decides to try using yogurt as the delivery mechanism! She
wants to clone a gene from S. pneumoniae that encodes an antigen and to express this
gene in a microorganism that would grow in yogurt. Eating the yogurt would serve as
the vaccine delivery. Consider the various molecules expressed by S. pneumoniae.
a. What is/are the immunogenic compounds of the current pneumococcal vaccines?
b. Choose one molecule that would be an appropriate choice to serve as the antigen for
Dr. Bimmel’s vaccine preparation. What molecule have you chosen? What is the
gene or genes needed to make that product?
c. Explain how your choice would allow an immunized person protection from all
strains of S. pneumoniae.
d. If your answer to (a) is not the same as (b) explain why that is. Describe any
advantages your choice from (b) has over (a).For the vaccine she must incorporate the gene for the S. pneumoniae antigen into a
bacterium that will survive in the yogurt and will express the gene producing the antigen
molecule. One organism found in yogurt is Streptococcus thermophilus. Dr. Bimmel begins
her plan to transform S. thermophilus with a plasmid vector encoding the antigen gene.
First Dr. Bimmel must choose a plasmid cloning vector. Dr. Bimmel understands that a
gene encoding resistance to antibiotics is commonly used to select for recombinant
organisms (she does not want to include such a gene in her final construct for fear that it
would be transferred to normal flora in the gut of the immunized children). As such, her
final recombinant S. thermophilus strain will need some further genetic manipulations.
However, for a phase one study, where the vaccine will be tested in the laboratory in
animals, it is ok.
e. Prepare a drawing of the cloning vector that you would use. Be sure that your drawing
contains all of the essential information needed to demonstrate:!
i. That the plasmid will replicate in S. thermophilus!
ii. Any other genes that you will need to identify your transformants.
f. Prepare a diagram that shows how to construct a recombinant plasmid that would
contain the gene that encodes your antigen.!
i. What is the role of restriction endonucleases in the process of constructing
your plasmid?!
ii. What is the role of DNA ligase in this process?
iii. Consider the plasmid that you have designed. How will you identify S.
thermophilus cells that have been transformed with this plasmid?
iv. For the vaccine to be functional, the antigen needs to be expressed. Do you need
to add anything to the media to ensure expression? How will you determine if the
protein is expressed?
g. Is Dr. Bimmel right to be concerned about the presence of antibiotic resistance genes,
even in the absence of selection? What are the risks (if any( of leaving the antibiotic
resistance genes in the vector for the final product? What are the advantages (if any) of
leaving the antibiotic resistance genes in the plasmids?Graph Paper for Question 5