Research the answers to each of the questions below. Use whatever sources you finduseful – especially lecture notes and your textbook. You may work in groups. On thefinal exam these questions will make up the cumulative section of the exam. For thefinal exam, you will need to remember your answers and write them on the testpages: you may not write out your answers ahead of time and turn them in. (There 7questions, 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 nomemories of her numerous hospital stays, but over the years her parent’s stories about herearly struggles made a deep impression on Frederica, and she developed a keen interest inmedical 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 becomeone of the world’s leading experts on Streptococcus pneumoniae, and she has arranged animportant meeting with Melinda and Bill Gates and the Global Alliance for VaccineDevelopment (GAVI) to try to convince him to fund her research to develop a newvaccine that will protect people from all serotypes of S. pneumoniae. Like any goodscientist, Dr. Bimmel prepared extensively for this meeting, including putting together aPowerPoint presentation. On the cab ride from the airport to the Microsoft campus, shereceived a phone call from one of the Gates’s assistants with these words of advice: “Youknow, of course, that the Gates absolutely despises PowerPoint presentations, so I hopethat you plan on avoiding that…they really likes to see ideas scratched out in pen orpencil on a piece of paper. Is that going to be a problem for you?” You happen to besharing 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 basicdefinitions and ideas? I have some innovative ideas for how we could reduce S.pneumoniae infections by taking advantage of: (i) its metabolic properties; (ii) microbialecology; (iii) vaccine development. I’ll just tell you the ideas, and you can write them outon this paper!”The genome of the S. pneumoniae strain Dr. Bimmel is studying includes thefollowing 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 aphosphotransferase transport system that can transport glucose into the cytoplasmd. a gene encoding lactate dehydrogenase (ldh) and genes for each of theglycolytic enzymese. blaA, a secreted protein that can enzymatically inactivate β-lactam antibioticsf. A lac operon (lacZ lacY lacA) encoding proteins very similar to their counterpartsin Escherichia coli.II. A plasmid that carries:a. pilA, a gene encoding the major protein of the pilus that mediates attachment toepithelial cells in the lungsb. pilZ, A gene encoding the adhesin for the PilA pilusSeveral genes NOT PRESENT in the S. pneumoniae genome include:• those encoding TCA cycle enzymes• those encoding electron transport chain proteins1. Draw a single labeled diagram of S. pneumoniae that depicts this organism and thelocation of each of the features specified below:a. Functional anatomy of S. pneumoniaei. the shape of the organism;ii. the layers of the cell envelope including the capsule (Do label the layers; do NOTdraw individual lipid or carbohydrate molecules that are considered part ofthe layers.)iii. do clearly indicate whether this is a Gram-positive or Gram-negative bacteriumiv. the location of the attachment pilusb. 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 youwould expect to find: Teichoic acids, Pneumolysin, β-galactosidase (LacZ), PspA,β-lactamase (BlaA), PtsABCD (a multiprotein complex), PilZ, PilA, Autolysin(LytA), Phosphocholine, Hyaluronidase2. Dr. Bimmel realizes that the Gates may not understand the genetic nomenclaturecontained within her drawing of the cell, so she decides that it would be helpful toprepare an additional figure that would explain the concept of a gene. She decides thather drawing should highlight the ptsABCD operon. Prepare a simple figure thatshows how information is converted from a DNA molecule to a protein. Includein your figure the correct location of:a. 5’ and 3’ ends of each moleculeb. the location of transcriptional start and stop sitesc. any promotersd. ribosome binding sitese. start and stop codonsf. amino and carboxyl termini of the proteinsg. sites of operon regulation.3. Functional components of S. pneumoniae.!a. For each of the S. pneumoniae cell components listed below, briefly indicate how itcould contribute to the ability of this bacterium to cause disease in a human host. If itis an enzyme, what is the substrate, and what is the product? 1 or 2 sentences for each.i. Capsuleii. Attachment (PilA) Pilusiii. Pneumolysiniv. PspAv. Teichoic Acidvi. Autolysin (LytA)vii. Phosphocholineviii. Hyaluronidaseb. What term would a Pathogenic Microbiologist (or an Epidemiologist) use to refer tothe 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 3Glucose 20 g Sodium Citrate 50 g Lactose 20 gPeptone 5.0g Ammonium phosphate 1.0 g Ammonium phosphate 1.0 gBeef extract 3.0 g Sodium chloride 5.0 g Sodium chloride 5.0 gSheep blood 10 g Magnesium sulfate 0.2 g Magnesium sulfate 0.2 gSodium Chloride8.0 g Potassium phosphate 1.0g Potassium phosphate 1.0gAgar 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 literd. 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 ofS. pneumoniae to grow in medium 1?4. S. pneumoniae is highly dependentupon external sugars to meet its energyrequirements (recall that it has neither anelectron transport chain nor a TCAcycle). The transport system used byS. pneumoniae for glucose is depictedin Figure 1.a. What term best describes thistransport mechanism?b. !What is the immediate source ofenergy that drives this transport system and allows S. pneumoniae to scavenge lowlevels of glucose from its environment?!c. When S. pneumoniae is growing in the presence of oxygen using glucose as itsenergy source, which metabolic pathway (Aerobic respiration, fermentation oranaerobic respiration) would it use? Justify your answer.!d. What is the NET GAIN in ATP generated by S. pneumoniae catabolizing onemolecule of glucose during this type of metabolism? Remember to take intoaccount 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 makemetabolic decisions, opting for preferential use of one source of carbon over another inorder to maintain optimal growth. Simultaneous utilization of all available sugars wouldbe metabolically inefficient and would lead to slower growth. Like E. coli, the preferredsugar for S. pneumoniae is glucose. (The doubling time for S. pneumoniae growingminimal media + glucose is 40 minutes, and the doubling time for S. pneumoniaegrowing minimal media + lactose is 60 minutes)Figure 1. This is the phosphotransferase systemthat S. pneumoniae uses for the transport ofglucose. (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 presenceof lactose.iii. A third that would reflect the growth of S. pneumoniae growing in the presence ofboth sugars, where glucose is exhausted 80 minutes into log growth. Dr. Bimmelunderstands 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) youcan use it to practice your graphs before you present them, but do not feel compelled todo so. If you want, free hand your own graphs.6. Dr. Bimmel is concerned about eliminating the carrier state of S. pneumoniae. She isparticularly concerned about finding ways to prevent S. pneumoniae-caused otitis mediawithout the use of antibiotics. Antibiotic use against S. pneumoniae has becomeproblematic. Amoxicillin worked for her as a child, but today there are many antibioticresistant strains.a. Describe how amoxicillin affects bacterial cells. (What is the target site? What doesamoxicillin 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 tetracyclineand kanamycin)? Briefly explaine. Show (describe) how the blaA gene could be naturally transferred from aS. pneumoniae cell that is amoxicillin resistant to a S. pneumoniae strain that issusceptible to amoxicillin. Include in your response a description of the selectionconditions 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 forvaccines. Dr. Bimmel has always wanted to find a way to make a vaccine forS. pneumoniae that targets all of the serotypes! This may be her opportunity! Her goal isone 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 fromS. pneumoniae. Dr. Bimmel decides to try using yogurt as the delivery mechanism! Shewants to clone a gene from S. pneumoniae that encodes an antigen and to express thisgene in a microorganism that would grow in yogurt. Eating the yogurt would serve asthe 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 forDr. Bimmel’s vaccine preparation. What molecule have you chosen? What is thegene or genes needed to make that product?c. Explain how your choice would allow an immunized person protection from allstrains of S. pneumoniae.d. If your answer to (a) is not the same as (b) explain why that is. Describe anyadvantages your choice from (b) has over (a).For the vaccine she must incorporate the gene for the S. pneumoniae antigen into abacterium that will survive in the yogurt and will express the gene producing the antigenmolecule. One organism found in yogurt is Streptococcus thermophilus. Dr. Bimmel beginsher 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 agene encoding resistance to antibiotics is commonly used to select for recombinantorganisms (she does not want to include such a gene in her final construct for fear that itwould be transferred to normal flora in the gut of the immunized children). As such, herfinal 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 inanimals, it is ok.e. Prepare a drawing of the cloning vector that you would use. Be sure that your drawingcontains 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 wouldcontain the gene that encodes your antigen.!i. What is the role of restriction endonucleases in the process of constructingyour 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 needto add anything to the media to ensure expression? How will you determine if theprotein 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 antibioticresistance genes in the vector for the final product? What are the advantages (if any) ofleaving the antibiotic resistance genes in the plasmids?Graph Paper for Question 5