Microbiology • BIO 340 • Winter 2014Key for Exam 1 1. Vancomycin is unable to pass through porins. This means that __________ bacteria have __________ 2. The blood agar that you used for your throat swab is what kind of medium? (Circle all that apply) 3. Methanogens could be cultured on a medium containing only (NH ) SO , Na HPO , KCl and trace minerals if the plates were incubated in an atmosphere containing: 4. Which of the following would likely be a good target for an antibiotic used therapeutically in humans? a. an enzyme that links cellulose molecules to form the cell wall b. an enzyme involved in synthesizing phospholipids for the cell membrane c. a structural region unique to the 80S ribosome 5. If the bacterial growth equation is expressed as B = b × 2n, the variable n represents: 6. Some bacteria can directly use nitrate (NO !) as a source of energy. In these bacteria, nitrate serves as the _______________ in the process of ___________________.
d. electron acceptor, anaerobic respiration 7. Which of the following antibiotics works by preventing cross-linking of peptidoglycan? 8. Bacteria use all of the following mechanisms to resist antibiotics except: 9. An antibiotic resistance gene on a mobilizable plasmid could be transferred from a resistant bacterium a. the resistance gene recombined with the chromosome of the resistant cell b. the susceptible cell contained a self-transmissible plasmid c. the susceptible cell were infected by a bacteriophage that degrades the host genome e. the mobilizable plasmid had both tra and mob genes 10. Bacteria that are not normally pathogenic but could cause disease under some conditions are: 11. Antibiotic resistance was predicted by Fleming in his 1945 Nobel lecture.
12. Consumer Reports found that organic ground turkey tested positive for potential pathogens much less frequently than turkey not raised organically.
13. Vancomycin is valuable as a “last resort” antibiotic because no vancomycin resistance has 14. According to a study by Courvalin, many antibiotic-resistance genes existed long before the antibiotics they work against began to be used therapeutically.
15. Lack of antibiotic-resistant bacteria in the gut of European honeybees suggests that the European Union’s restrictions on agricultural antibiotics are reversing the spread ofresistance.
16. Increased diversity of resident bacteria in the gut appears to be correlated with obesity and b. Add a drop of TMPD to a colonyc. Add a drop of iodine to a colonyd. Grow the bacteria in a Durham tube e. Grow the bacteria on MSAf. Grow the bacteria on EMBg. Grow the bacteria on blood agar h. Grow the bacteria on Simmons citrate agarj. Grow the bacteria on Hinton salt agar b. filtrationc. ethylene oxide gasd. gamma radiation e. QACsf. hydrogen peroxideg. sodium pentathol 30. Sterilizing disposable plastic pipets 31. You are supposed to have a culture of bacteria with an optical density (“absorbance”) of 2.0 when lab starts at 1:30 p.m. But, you forgot to start the culture the night before, yikes! When you remember,you rush to lab at 2 a.m. and start the culture. At noon, you measure its optical density and it’s onlyat 0.25. If g = 30 min. for this species in this culture medium, will the culture be ready on time? Remember that optical density is proportional to cell number. If the number of cells doubles, so doesthe optical density. Thus, if the OD is 0.25 at noon and the bacteria double every 30 min, then at12:30, the OD will be 0.5 (one generation), at 1:00 it will be 1.0 (two generations) and at 1:30 it willbe 2.0. Whew, just in time! (You could work this out with the growth equation, because again OD is proportional to cell numberand could be substituted for it. I think that’s a slower method, but it works: 2.0 = 0.25 × 2n, then 2n= 8 and n must be 3 generations or 1.5 hours.) 32. You have developed a new hand sanitizer based on an inorganic catalyst (Dr. Brant would be proud) that attacks the NAG-NAM linkage in peptidoglycan.
You want your sanitizer to have a broad spectrum of activity: that is, you’d like it to work againstboth Gram-positive and Gram-negative bacteria. What will likely be the key factor that willdetermine whether it’s broad-spectrum or not? Your sanitizer needs to get to the peptidoglycan layer; in Gram-positive cells, it can do thisdirectly, but in Gram-negative cells it will have to go through the outer membrane. So the key willbe whether it is able to pass through the outer-membrane porins of the Gram-negative cell.
b. Most current hand sanitizers are ethanol-based. Explain why there is not much concern about bacteria developing resistance to ethanol.
Ethanol denatures proteins and disrupts membranes. It has no specific target, so there’s no oneor few genes that can mutate to prevent it from working.
Do you think you will have to worry about bacteria developing resistance to your new sanitizer?Explain why or why not.
There is a specific target for your sanitizer, so it’s certainly possible for resistance to occur. Forexample, a mutation in the enzyme that joins NAG to NAM might alter this linkage in some way.
d. Suppose bacteria can develop resistance to your catalyst. You don’t think this is a concern, because this chemical is only used as an antiseptic, not internally: resistance to the catalyst willthus not increase antibiotic resistance. But, you’re not looking at the whole picture. Explain howyour product could potentially increase antibiotic resistance.
If there is selective pressure for bacteria to develop resistance to your catalyst (because it is beingused by lots of people and killing bacteria on hands), then resistant bacteria will grow and passon their genes. Some of these bacteria will have become resistant by mutation, but since that’spretty low-probability, over time it will be increasingly likely for resistance to be passed byhorizontal gene transfer of a resistance plasmid. Resistance plasmids can acquire new resistancesby interaction with transposons or recombination, so it’s entirely possible that selecting forbacteria resistant to your sanitizer will wind up selecting for the simultaneous transfer ofantibiotic resistance on the same plasmid.
33. You have isolated some tetracycline-resistant Bacteroides from the human intestine. When you mix a culture of these bacteria with a culture of E. coli, you find that some of the E. coli becometetracycline-resistant. You would like to know if this transfer of resistance is due to transformation,transduction or conjugation. Describe an experiment that would distinguish among these possibilities.
There are a number of ways you could go about this. Here’s one: (1) You know that conjugationrequires cell-cell contact. If culture medium from the resistant strain that has been centrifuged orfiltered to remove all cells can still cause the sensitive strain to become resistant, then you can ruleout conjugation. If this does not work, conjugation is the likely mechanism. (2) If the culture mediumworks, then you need to know whether the resistance results from naked DNA in the medium or froma phage. Treating the filtered medium with a protease or heating it to denature proteins wouldinactivate a phage; if it still works, then transformation is the likely mechanism; if not, it wasprobably transduction.
34. The most common mechanism of resistance to chloramphenicol is an enzyme, chloramphenicol acetyl transferase (CAT) that transfers an acetyl group to chloramphenicol, inactivating it.
Efflux pumps are commonly used by bacteria to resist antibiotics, but are not observed in the caseof chloramphenicol. Why wouldn’t an efflux pump be a very useful mechanism of resistance? Unlike tetracycline (where we talked about efflux pumps being the main mechanism ofresistance), chloramphenicol is a small, highly permeable molecule. If it can readily diffusethrough cell membranes, a pump won’t be very effective in keeping it out.
b. Mutations in ribosomal protein genes can result in resistance to chloramphenicol. But, resistance due to CAT is seen much more widely in nature. Why do you think this is true? If both of these mechanisms exist, why is the CAT mechanism better able to spread in thepopulation? Even if a ribosomal protein mutation ends up on a plasmid and can readily move byHGT, it would be transferred to cells that still had wild-type ribosomal genes as well. These cellswouldn’t be as resistant and thus not as strongly selected as cells that get the CAT enzyme andimmediately become fully resistant.
35. A major argument made by those opposed to limitations on agricultural use of antibiotics is that we do not have enough evidence to demonstrate that resistance of agricultural bacteria to agriculturalantibiotics results in resistance of human pathogens to human therapeutic antibiotics.
Give two specific lines of evidence for a connection between the resistant agricultural bacteriaand resistant human pathogens.
(1) Salyers’ experiments showing >95% conservation of resistance genes across very differentspecies of human and animal bacteria support the hypothesis that HGT occurring in the gut cantransfer resistance between ingested agricultural bacteria, resident microbiota and any pathogensthat may be passing through. (2) Decline in resistance has been observed in EU countries thathave banned or significantly restricted agricultural antibiotics. (3) Resistant bacteria are observedin the gut of completely unrelated organisms such as honeybees in the U.S. but not in antibiotic-restricting EU countries.
(Note that many people gave the reservoir hypothesis or a related hypothesis here—but the pointwas to give the evidence that supports the hypothesis.) b. The “precautionary principle” states that when science identifies an important risk and there is good evidence to support it, we should act on the evidence rather than waiting for the finalresolution of the matter. Do you think this applies in the case of limiting agricultural antibiotics,or should we in this case delay action while “the jury is still out?” Give details to support youranswer.
You could say either yes or no here, but you needed to support your answer carefully. It was notsufficient to just say that resistance is dangerous and you support a ban: what is your reasoningfor supporting a ban even before we have incontrovertible evidence that agricultural antibioticuse is the cause of this resistance? 36. The resident microbiota (or “normal flora”) of humans were at one time seen as commensals: harmless passengers that gain food and a stable environment from us; any benefit to us was seen as incidental.
Give one specific example to show that the resident microbiota are not always harmless.
Clostridium difficile is a good one: it can cause severe disease if allowed to grow out of controlin the gut even though most of the time it has no negative effects. Another example would beperitonitis caused by ordinary gut bacteria entering the peritoneal cavity via a ruptured appendix.
b. Give two lines of evidence to suggest that our relationship with our resident microbiota should A mutualism implies a significant degree of specificity in an association. Several people said thatthe fact we can gain nutrients from our gut bacteria is evidence of mutualism, but really that couldbe an incidental benefit of commensalism, because any bacteria that happened to be able tometabolize polysaccharides in the gut could provide some nutrients. Likewise, just crowding outpathogens is something that could be accomplished by any bacteria that happened to be in ourcolon.
Better evidence for mutualism would be something like the vaginal Lactobacillus, whereregulated synthesis of glycogen beginning at puberty specifically encourages the growth ofprotective acid-producing organisms. Another piece of evidence would be the use of specificreceptor-ligand interactions to allow specific organisms to colonize specific sites. Even somethinglike the maintenance of a smaller bacterial population in the small intestine where absorption isimportant would suggest some specific mechanisms.
37. Facultative anaerobes in the colon, including E. coli and its relatives, usually live by breaking down the plentiful undigested polysaccharides to release glucose and fermenting the glucose to someorganic waste product. Sketch one specific example of a fermentation reaction, then explain why thisreaction is essential to an organism that cannot carry out respiration.
Any one example would be fine; these pathways start with pyruvate, end with an organic wasteproduct (and in come cases CO and/or H ) and include oxidation of NADH to NAD+. For example, pyruvate to lactic acid or pyruvate to ethanol + CO would work. This reaction is essential in order to regenerate oxidized electron carriers (NAD+), which in turn are needed to keep glycolysis running in the absence of respiration.
38. Sketch the electron transport chain as it would function in the membrane of a methanogen. Show and label the electron donor, and the electron acceptor and show how ATP is made.
show the carbon and energysources for the four major 40. You are studying three bacterial species: two obligate anaerobes and an obligate aerobe. All three can carry out respiration. The aerobe (species A) of course uses oxygen (E0N = +0.82) as its electronacceptor. One of the anaerobes (species B) uses elemental sulfur (S0; E0N = –0.27), and the other(species C) uses fumarate (E0N = +0.03). If each of these species is given the same amount of the samecarbohydrate, order them from greatest to least in terms of the amount of ATP they will produce, andbriefly explain your ranking.
Energy generation in respiration depends on the amount of energy that can be extracted from high-energy electrons on a reduced electron donor. At each step where electrons reduce a more oxidizedcarrier, they give up some electricity along their “downhill” path. The number of such steps that canoccur depends on how much difference there is between the reduced donor and the oxidized finalacceptor. The reduction potential (E0N) is a measure of this difference (can also be thought of as theelectronegativity of the electron acceptor). Oxygen, fumarate and sulfur go in this order from higherto lower E0N, so this is also the order of energy capture: A, C, B.
BONUS (2 points): list one specific way that microbiology has been mentioned in the news since the beginning of this term. (For example, “smallpox” is not specific, but “there was a large outbreak ofsmallpox in Wheaton” would be fine.)


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