Microbiology 11/13

What was the only mechanism of antibiotic resistance found in the C. diff strains tested?

target modification

What are two ways that antibiotics binding to the decoding site could alter translation?

Block tRNA from binding (tetracyclines) (1) Cause mismatches between tRNA and mRNA making junk proteins (aminoglycosides)

How did the scientists look for mutations that caused resistance?

By looking at the gene sequence of certain genes (1)

Penicillin is effective against Gram-positive, but not Gram-negative bacteria. Is penicillin a broad or narrow spectrum antibiotic?

narrow spectrum

What type of nucleic acids is in adenovirus? HPV?

Adenovirus and HPV both have double-stranded DNA

What is a superinfection and how does it relate to antibiotic use

A superinfection is a drugresistant bacterial infection (1). It can result from taking antibiotics because antibiotics kill all the suspectible bacteria (1), leaving only the resistant strains (1). The bacteria that did not get killed are resistant to the antibiotics.

Compare and contrast adenoviruses and HPV in terms of the five steps to a viral infection? Add any additional steps if necessary.

Adenovirus goes through the lytic cycle; the virus goes through all five stages of infection attachment, penetration, biosynthesis, assembly, and release (2.5). HPV goes through all five steps, but after penetration (1), the viral genome gets integrated into the host genome (1) and is propagated through vertical gene transfer (1) instigated by the viral proteins E6 and E7. At some later point, the HPV genome is excises and continues through the rest of the five steps of biosynthesis, assembly and release (1.5).

Examine the two growth curves shown below. Identify which drug is the bactericidal agent and which is the bacteriostatic agent. Briefly state how you arrived at your answer.

Agent A - bacteriostatic (1) because the bacteria does not grow while the antibiotic is present, but grows again when it is removed (1) Agent B - bactericidal (1) because the number of cells actually decreases to 0 (1)

Which antibiotics in the definitions above block protein translation? How is translation blocked?

Aminoglycosides (block decoding center causing missense mutations) (1) Tetracyclines (block A Site so tRNA cannot bind) (1) Oxzolidinones (block 50S from binding to 30S-mRNA-tRNA complex during intiation) (1) Chloramphenicol (block peptide bond formation) (1) Macrolides (block peptide exit channel) (1) Lincosamides (block peptide exit channel) (1) Streptogramins (block peptide exit channel) (1)

What are three mechanisms of antibiotic resistance? Give one example for each.

Antibiotic Modification (1) - Adding a phosphate group to erythromycin (1) so it cannot bind to the ribosome Antibiotic Hydrolysis (1) - Beta-lactamase enzymes breaking bonds in beta-lactam antibiotics (1) so they cannot inhibit transpeptidase Membrane Modification (1) - Efflux pumps removing wide range of antibiotics from the cell (1), conferring multidrug resistance Target Modification (1) - Addition of a methyl group to the ribosomes (1) so that erythromycin cannot bind

Explain how antibiotics represent a selective pressure for mutations in genes that encode proteins that antibiotics inhibit. Use gyrase and quinolones as an example for your explanation.

Antibiotics kill cells that are susceptible, where the target protein or process can get disrupted (1). If bacteria have mutations that allow them to escape the antibiotic, then they will survive while the susceptible bacteria die (1). Some mutations in gyrase allow the enzyme to function but do not allow quinolones to bind (1). Cells with wildtype (normal) gyrase will be killed because they cannot replicate, while cells with the mutations will not die when the antibiotics are present (2).

What is the difference between antigenic drift and antigenic shift?

Antigenic drift happens when small mutations cause changes in viral proteins that might lead to changes in virulence (2). Antigenic shift happens when one host is infected with two or more viruses and the viruses can recombine using genome segments from both strains

How does the MIC correlate with the number of mutations compared to the parent strain (FA-19)?

As the number of mutations increases, the more antibiotic is needed to kill the cells

Why do bacteria make antibiotics if they kill bacteria?

Bacteria need to compete with other microbes and bacteria for resources in the environment. (1) Antibiotics give certain bacteria an edge by killing the competition (1)

Describe how the LacI protein and CAP regulate the binding of the RNA polymerase to the lac operator.

CAP recruits the RNA polymerase to the promoter (1), so it if CAP is bound the RNA polymerase is more likely to bind (1). LacI does not inhibit RNA polymerase from bindint to the promoter (1), but it does inhibit the RNA polymerase from moving on the DNA and transcribing (1).

What is the shape of a bacterial chromosome? What unique challenge does this create at the end of replication?

Circular (1) At the end of replication, the chromosomes are linked

Why is competence key to the transformation process?

Competency means that a bacterium is able to import DNA from the environment (1). If a cell is not competent, then it cannot get DNA into itself and cannot participate in the transformation process

What is the complementary sequence to the template DNA sequence: 5' TAC 3'? What amino acid does the template DNA encode for?

Complementary sequence (non-template): 3'-ATG-5' (2) Amino acid: Template: 3'-CAT-5' mRNA 5'-GUA-3' Amino Acid---Asp---

In one of the first experiments that modeled HGT, Frederick Griffith was studying pathogenicity factors in Streptococcus pneumoniae, a naturally competent microbe. Griffith found that strains of S. pneumoniae that appeared rough (R) on agar plates were non-pathogenic in mice, while those that appeared smooth (S) were pathogenic. The smooth bacteria had capsules, while those that appeared rough did not encode a capsule. Heat-killed, smooth S. pneumoniae was unable to cause disease in mice. However, when Griffith infected mice with heat-killed smooth S. pneumoniae and live, rough S. pneumoniae, the mice got sick. When Griffith isolated bacteria from the mice, he found rough and smooth colonies. Explain how transformation led to the observed results of Griffith's experiment. (9.3)

DNA from the dead smooth cells was taken up by the live rough cells through transformation (2). The DNA that changed the virulence and the outer appearance of some of the cells (1) so that they were able to make the mice sick

How do DNA mutations occur? What can lead to their formation?

DNA mutations occur when DNA polymerase does not put the right nucleotide (1) into while replicating the chromosome. Sometimes the DNA polymerase can slip on the DNA (1) creating insertions (0.5) or deletions (0.5). Sometimes the base pairing does not happen correctly (1) and a wrong base gets added

Antibiotics are often described as being "selectively toxic". Explain and/or provide examples of this meaning. Theoretically, how is the therapeutic index of an antibiotic affected as selective toxicity decreases (i.e. it becomes less selective to bacteria)?

Due to the mechanisms of actions, antibiotics sometimes work better on certain types of bacteria (1). For example, beta-lactams block transpeptidase, but they can kill more Gram positive cells because there is not an outer membrane protecting the peptidoglycan layer. (2) On the other hand, monolactams inhibit only gram negative cells because the transpeptidase is different and binds to monolactams better than beta-lactams.

HPV strains are sub-divided into low-risk and high-risk strains based on the risk they pose to cancer development. Which viral protein(s) contribute to the risk level? Explain one way a protein could function in both types of HPV, but have different cancer-risks associated it.

E6 and E7 proteins are oncogenes (1) that force the host cell to continue dividing (1). Cells dividing out of the control is a hallmark of cancer (1). E7 might be able to kick-start the the G1-to-S transition for both types of HPV, but not strongly bind to the targets in the low-risk HPV.

Describe how an efflux pump removes antibiotics from a bacterial cell.

Efflux pumps use ATP (or energy; 1) to actively remove antibiotics from the cell. Mutations can occur to make the targets work on more general molecules

Explain how enveloped and non-enveloped viruses are different. How do they penetrate into eukaryotic cells?

Enveloped viruses have a membrane around the viral capsid (1), and they penetrate the host cell by the membranes merging (1). Non-enveloped viruses do not have a membrane around them (1); they get into the cell by endocyctosis

What is the difference between generalized transduction and specialized transduction?

Generalized transduction happens during a lytic infection (1) when host DNA gets packaged into the phage capsid instead of the viral genome (1). Specialized transduction happens during a lysogenic infection (1). When the viral DNA is excised, some chromosomal DNA is excised as well (1), so the phage particles have both phage and host DNA

What are the roles for the following proteins? helicase, ligase, primase, single-strand DNA binding protein.

Helicasse - unwinds the DNA (2) Ligase - binds together fragments of DNA (Okazaki fragments on the lagging strand during replication) (2) Primase - lays down an RNA primer that DNA polymerase can extend from (2) Single-stranded DNA binding protein - coats the lagging strand (or any single-stranded DNA) to protect it from attacks (2)

What two measurements are used to calculate a therapeutic index?

Highest concentration (in kilogram of body weight) of the drug tolerated by the host divided by the lowest concentration (per kilogram body weight of drug that will eliminate the infection.

Which type of gene transfer would affect a current population of bacteria?

Horizontal gene transfer (2) (current population means no time has elapsed

How does horizontal gene transfer differ from vertical gene transfer?

Horizontal gene transfer affects the current population of bacteria and does not happen through growth and binary fission (2). Vertical gene transfer relies on growth and binary fission (2).

The majority of hospital-acquired infections with Clostridium difficile occur after an individual has been taking a broad spectrum, oral antibiotic. How might a broad spectrum antibiotic lead to an infection in the gastrointestinal tract? Hint: Think about where an oral antibiotic enters, and where the excess of it exits the body, and where it might come into contact with microbes.

If antibiotics are not absorbed in the small intestine or blood, they exit through the large intestine (1). Broad spectrum antibiotics kill a wide range of bacteria (Gram negative and Gram positive) (2). So excess antibiotics leaving the body will kill large amounts of bacteria in the large intestine (1). C. diff is not killed by many antibiotics so without competitors it can grow faster and take over the gut; it is an opportunistic pathogen

The majority of hospital-acquired infections with Clostridium difficile occur after an individual has been taking a broad spectrum, oral antibiotic. How might a broad spectrum antibiotic lead to an infection in the gastrointestinal tract? Hint: Think about where an oral antibiotic enters, and where the excess of it exits the body, and where it might come into contact with microbes.

If antibiotics are not absorbed in the small intestine or blood, they exit through the large intestine (1). Broad spectrum antibiotics kill a wide range of bacteria (Gram negative and Gram positive) (2). So excess antibiotics leaving the body will kill large amounts of bacteria in the large intestine (1). C. diff is not killed by many antibiotics so without competitors it can grow faster and take over the gut; it is an opportunistic pathogen (1).

What might you expect the results to look like if a mutation occurred in the trp repressor's DNA-binding domain that inhibited its binding to the operator?

If the DNA binding domain could not bind DNA, then the trp repressor could not bind the operator (1) and repress the operon (2). This would mean that the operon would always be turned ON

Why would it be advantageous for a phage to carry out a lysogenic cycle rather than a lytic cycle?

If there are not many bacteria around, the phage would not want to kill all their hosts (2). So they choose the lysogenic cycle to bid their time until there are more bacteria around.

If deoxyribonucleotides that lack the 3'-OH groups are added during replication process, what do you expect will occur?

If there is no 3' -OH, then the next nucleotide in the sequence cannot be added to the DNA strand that is being synthesized (2). This would cause an incomplete chromosome (1).

Explain how the lytic cycle differs from the lysogenic cycle?

In the lytic cycle, the virus goes through all five stages of infection attachment, penetration, biosynthesis, assembly, and release (2.5). In the lysogenic cycle, the virus goes through all the steps, but after penetration (1), the viral genome gets integrated into the host genome (1) and is propagated through vertical gene transfer (1). At some later point, the viral genome is excises and continues through the rest of the five steps of biosynthesis, assembly and release

What can cause a thymine dimer and what are the consequences of one forming?

Ionizing radiation (1) causes thymine dimers. When DNA polymerase encounters a thymine dimer, it cannot read the bases and synthesis the new strand (2). The polymerase either stalls or actually fall off.

Imagine an E. coli strain containing a lacI gene with a mutation that encodes a variant of the LacI protein that is unable to bind lactose. Will this strain experience still preferentially use glucose before using lactose as an energy source?

LacI would always be bound to the operator (2) if it could not bind lactose. This strain could not use lactose as an energy source (2) because the operon would never be fully turned ON.

How could bacteria develop resistance to sulfa drugs (sulfonamide)? Be sure to state a possible resistance mechanism. (9.1)

Make more of the enzyme that makes folic acid (the target) - target modification (1) Mutate the binding site of sulfa drugs - target modification (1) Mutate a membrane transport to either stop the drugs from getting in or actively pump them out - membrane modification

AUG GCG AUA GUU AAA CCC GGA GGG UGA

Met-Ala-Ile-Ala-Lys-Pro-Gly-Gly-STOP

Given all these data, explain how antibiotic resistance develops.

Mutations in genes that arise naturally (2) are selected for (the bacteria with them will survive) and carry that mutation onto the next generation (2). Then additional mutations can develop in addition to the original

Based on the percentage of fluoroquinolone-resistant strains from 1991 and 1997, is this type of resistance increasing in France? Explain.

No (1), because the resistant strains are 7% in both 1991 and 1997 (2).

What is an operon? What is an operator?

Operon - set of genes controlled by one promoter (2); contains a promoter (0.5), operator (0.5) and structural genes (0.5) Operator - part of DNA (1) that repressor proteins bind to (1) between the promoter and the structural genes

Explain why penicillin would not be a useful antibiotic to use against Gram-negative bacterium or Mycobacterium.

Penicillin does not allow the cross-links between the polysaccharides in the peptidoglycan layer (1). It would not work well on Gram-negative bacteria because it cannot get through the outer membrane to get to the cell wall (2). It would not work well on Mycobacterium because it could not get passed the mycolic acid (2) to get to the cell wall.

How do polymyxins differ from other cell wall/membrane antibiotics?

Polymyxins disrupt the membrane by acting like a detergent (1), while other antibiotics act on the cell wall and disrupt peptidoglycan

What structural features of Pseudomonas aeruginosa make it a challenge to treat with antibiotics?

Pseudomonas is a Gram-negative bacteria, which means that it has an outer membrane (1) so that antibiotics cannot easily get to the cell wall (1). In addition, its membrane/wall are less permeable than other bacteria (1) so antibiotics cannot get to the cytoplasm

Compare and contrast the mechanisms of action for isoniazid and quinolones

Quinolones inhibit gyrase (1), stopping replication and have very broad bacterial targets (1). Isoniazids are very narrow-spectrum antibiotics that only work on Mycobacterium (1) that inhibit cell wall synthesis

Explain the differences between the three types of polymerases.

RNA-dependent RNA polymerase makes RNA using RNA as a template (1) RNA-dependent DNA polymerase makes DNA using RNA as a template (1) DNA-dependent DNA polymerase makes DNA using DNA as a template

In which process of the Central Dogma might you discuss the role of the oriC, gyrase, and DNA polymerase?

Replication

Explain how a population of Staphylococcus aureus may gain resistance to a low dose of methicillin

Some bacteria in the populations might have mutations that allow them to be resistant to methicillin (1) - mostly increasing the efflux from the bacterium so that the drugs cannot act (1). When the susceptible cells are killed by the antibiotics, only the resistant cells are left (2). Now the entire population is resistant to methicillin.

3) From the coding (nontemplate) sequence 5'- AAG -3': d) Write one nonsense mutation and the amino acid encoded by the new codon.

Template DNA: 3' - ATC - 5' mRNA: 5' - UAG - 3' (2) Amino Acid: ---Stop---

3) From the coding (nontemplate) sequence 5'- AAG -3': c) Write one missense mutation and the amino acid encoded by the new codon.

Template DNA: 3' - TTA - 5' mRNA: 5' - AAU - 3' (2) Amino Acid: ---Asn---

3) From the coding (nontemplate) sequence 5'- AAG -3': b) Write one silent mutation and the amino acid encoded by the new codon.

Template DNA: 3' - TTT - 5' mRNA: 5' - AAA - 3' (2) Amino Acid: ---Lys---

3) From the coding (nontemplate) sequence 5'- AAG -3': a) What amino acid does it encode?

Template: 3' - TTC - 5' mRNA: 5' - AAG - 3' Amino Acid:----Lys---

What is the F-plasmid? How is it transferred?

The F-plasmid is a plasmid that has gene for conjugation (1). It can either be a separate plasmid or integrated into the chromosome (1). It is transferred through conjugation

A patient with streptococcal sore throat takes penicillin for 2 days of a prescribed 10-day regimen. Because he feels better, he then saves the remaining penicillin for some other time. After 3 more days, he suffers a relapse of the sore throat. Discuss the probable cause of the relapse. (9.3)

The antibiotic susceptible cells were killed by the initial two days of antibiotics (1). However, not all cells were killed because there were mutations that allowed the cells to escape the antibiotic killing (2). The resistant cells were able to multiply and reestablish the infection (1). But he now has an infection resistant to penicillin (2).

How does vancomycin differ from the beta-lactam family of antibiotics?

The beta-lactam family of antibiotics competitively inhibit transpeptidase (1) by binding to the protein itself (1), while vancomycin binds to the pentapeptides of the peptidoglycan layer before they can get crosslinked (1), blocking transpeptidase activity.

What is the main conclusion of this paper?

The ciprofloxacin resistance of C. diff did not increase in France between 1991 and 1997 (1), and any resistance was through mutations in gyrase

The antibiotic resistance issue can be argued from two perspectives. Some people contend that because of the side effects and microbial resistance, antibiotics will eventually be abandoned in medicine. Others see the future development of a 'superantibiotic', a type of miracle drug. Given what we discussed about antibiotic resistance, what do you think about the likelihood of a superantibiotic?

The likelihood of a superantibiotic is very low (1) because bacteria constantly have different mutations within their populations that might confer resistance to antibiotics (2). The susceptible ones would then die, but the resistant cells would spread the resistant genes (2), eventually getting around the superantibiotic

Phage only attacks bacterial cells and are very numerous. If phage got into your gut, would they be in the lytic cycle or lysogenic cycle? Explain your reasoning

The phage would choose the lytic cycle (2) because there are many hosts (1) and the phage will be able to propagate through many of them.

When lactose binds the repressor protein, does the repressor bind to the lac operator or not?

The repressor does not bind the operator

There are over 50 serotypes of adenoviruses. Which protein(s) in the adenovirus is most likely the target of antibiodies?

The spikes (1) of adenoviruses most likely are targeted by antibodies because they are the main proteins on the outside of the virus

In the lab, you are trying to study the growth of a new bacterial species. Every time you grow the bacteria, the culture starts to grow and becomes cloudy. But then the cloudiness goes away. Explain what is happening.

There is a phage infecting the culture (1). The phage is in the lysogenic cycle when there are not very many cells (2). Once the bacteria grow to a high enough numbers, the phage excise from the chromosomes and move to a lytic cycle to produce an active infection (2).

What are advantages of cephalosporins over the penicillin family of antibiotics?

They have a broader range of bacterial targets than penicillin (1). They might be resistant to some beta-lactamases (1). They are used when the patient has an allergy to penicillin

Contrast vertical gene transfer and horizontal gene transfer.

Vertical gene transfer is when genetic mutations are inherited from the mother to daughter cells (1) through binary fission (1). Horizontal gene transfer happens in the same generation of bacteria (1) and does not rely on binary fission, but on either transformation conjugation or transduction (1).

Oxzolidinones

block 50S from binding to 30S-mRNA-tRNA complex during intiation

Tetracyclines

block A Site so tRNA cannot bind

Aminoglycosides

block decoding center causing missense mutations

Chloramphenicol

block peptide bond formation

Lincosamides

block peptide exit channel

Macrolides

block peptide exit channel

Streptogramins

block peptide exit channel

What is cAMP? What protein involved in regulating the lac operon does cAMP bind to?

cAMP is a signal molecule (1) and signifies that glucose concentrations are not high (1) cAMP binds to CAP (or CRP)

Of the 14 strains that showed fluoroquinolone resistance, in which genes did the scientists find mutations?

gyrA and gyrB (1)

What type of life style do the viruses likely to go through in order to be oncogenic?

lysogenic