Isolation of Spontaneous Antibiotic-Resistant Mutants of E. coli by Direct Selection This exercise introduces basic techniques for manipulating E. coli, including aseptic transfers, serial dilutions, spread plates and streak plates. These methods are almost embarrassingly low-tech; but you must master them quickly to achieve reliable results from subsequent exercises in this course. The experimental procedure is designed to measure the frequency of several classes of spontaneous antibiotic-resistant mutants and to isolate a strain with a new antibiotic-resistant genotype. "Spontaneous" mutations are those that arise without direct intervention by the investigator, so their frequency in our experiment will shed some light on how frequently this type of mutation might arise in nature. The E. coli strains for this exercise are SC142 and SC116. You investigated the properties of SC142 earlier, and may want to review that information now. Strain SC116 is identical to SC142 except that it has a null mutation in gene dnaq. Both strains are sensitive to the antibiotics used in the exercise. The antibiotics will be rifampicin, ampicillin and kanamycin. These are briefly described in the following section. Basically we're going to spread a concentrated suspension of E. coli onto the surface of agar plates containing the antibiotics. Only the mutants that have become resistant to the antibiotic will form colonies on these plates. What you should observe is that the frequency of resistant mutants is variable, and depends on both the strain and on the antibiotic. Your challenge is to explain the molecular and genetic reasons for this variability based on the molecular mechanisms of the antibiotic and the strain genotypes. The relevance of these issues for understanding the evolution of antibioticresistant bacterial pathogens is almost too obvious to mention. Page 1 of 6
RIFAMPICIN Rifampicin is a semi-synthetic derivative of the natural antibiotic "rifamycin", which is produced by the soil bacterium Streptomyces mediterranei. Presumably, S. mediterranei uses rifamycin to kill other soil bacteria. The lethal effect is directed against the β' subunit of RNA polymerase, the enzyme responsible for transcription of all 3 classes of RNA in E. coli. The RNA polymerase rifampicin complex is permanently bound to the DNA template, thus blocking transcription even by RNA polymerase molecules that have not bound rifampicin. Rifampicin does not bind in the active site of RNA polymerase. It binds in the "exit channel" and blocks the progress of the nascent RNA transcript. Mutations leading to a rifampicin-resistant phenotype occur in rpob,the gene that codes for the target protein (β' subunit of RNA polymerase). These mutations apparently alter the structure of the exit channel, so that rifampicin is unable to bind to the enzyme, but since they do not alter the active site itself, the RNA polymerase retains its activity. Mutations that damage the active site of RNA polymerase would be lethal, and we would not observe them in our experiment. Rifampicin is used clinically, most frequently in treating tuberculosis. However, it is always administered simultaneously with some other antibiotic to mitigate the problem of spontaneous rifampicin-resistant mutants of Mycobacterium tuberculosis arising during prolonged therapy. Elizabeth A. Campbell (2001) Structural Mechanism for Rifampicin Inhibition of Bacterial RNA Polymerase Cell, 104, 901. Page 2 of 6
AMPICILLIN Ampicillin is a semi-synthetic derivative of the natural antibiotic Penicillin-G. It is in a general class called the "Beta-Lactam" antibiotics, after the curious 4-membered ring that is characteristic of this class. It is one of the most widely used antibiotics in clinical practice. Ampicillin interferes with the normal synthesis of the bacterial cell wall. Actively growing cells become sensitive to osmotic lysis when the tensile strength of the wall is compromised. The basis of ampicillin's effect on cell wall synthesis is not fully understood in all its details, but it involves inhibition of several similar enzymes in a closely related family that are essential for the peptide crosslinking reaction in peptidoglycan (cell wall) biosynthesis. Enzymes in this family are referred to as "transpeptidases" or sometimes as "penicillin-binding proteins". Ampicillin inhibits a half-dozen or so critical enzymes in this family by covalent binding to a serine residue of the active site of these enzymes. Page 3 of 6
PROCEDURE Day 1 Each student will get a small overnight culture of either strain SC142 or SC116 grown in LB medium. You need to set up and perform a serial dilution of the overnight culture using sterile 0.9% NaCl as the diluent. Follow the procedure outlined in the flowsheet. Make spread plates from 50 ul samples of the 10-6, and 10-7 dilutions of on LB Agar plates (without antibiotic). Counting colonies on these plates will allow you to estimate the total cell concentration in the overnight cultures. Also plate 50 ul samples of the undiluted (10 0 ) cell cultures on each of the following media: LB Agar LB + Ampicillin LB + Rifampicin LB + Triclosan Remember that these manipulations all must be performed using aseptic transfer technique. Also, be very aware that the most common source of error in this exercise is failure to thoroughly mix dilution tubes before withdrawing a sample. Incubate all plates at 37 C. Day 2 Make a detailed visual examination of all the plates and try to understand and interpret what you see. Record your observations using text and drawings. Form a team with a student who used the other E. coli strain and compare your results. Choose a triclosan-resistant mutant colony from strain 142 for isolation as a pure strain. Use the technique of streaking for single-colony isolation. Be sure to streak onto an LB + Triclosan plate. Then incubate your streak plate at 37 C. Next, count the number of colonies (if any) on the antibiotic plates, and record. Be sure that you re-streak before you count. The counting machine cross-contaminates all the colonies, so counting must be done after you have made your streaks. For the LB plates without antibiotic, count the number of colonies on whichever dilution plate has 30-300 colonies. Counting colonies on plates with < 30 colonies is imprecise because it introduces significant statistical sampling error. Counting colonies on plates with > 300 colonies is inaccurate because there would be significant coincidence counting (i.e. 2 cells close enough together that they grew into what appears to be a single colony). If both or neither dilution plate has between 30-300 colonies, then you probably screwed up your dilutions by not mixing the tubes thoroughly. Contribute your colony count results to the class spreadsheet. All teams in the lab did exactly the same experiment, so we are justified in combining the raw data for analysis. When you get the class data spreadsheet look it over to se if there are values you want to eliminate from consideration as "bogus". How can you objectively justify doing this? Page 4 of 6
Once you have filtered the class data for dubious values perform the following calculations: The total cell concentration (cells/ml; 2 sig. figs.) of each overnight culture. The concentration (cells/ml; 2 sig. figs.) of each type of antibiotic -resistant mutant. The frequency of each type of mutant (unitless; 2 sig. figs.). i.e. the value obtained by dividing the concentration of antibiotic-resistant mutant cells in the original culture by the total cell concentration of the overnight culture. IMPORTANT!!! Report cell concentration data with 2 significant figures only. The precision of the method allows no more. A calculated value of 2.06 X 10 8 cells/ml should be rounded to 2.1 X 10 8. If you have antibiotic plates with no colonies on them, then you report your result as a "<" value. It is not valid to enter "0". That is, if you examine 100 cells and none are resistant you cannot claim that the frequency is zero, only that it is less than 1/100. To calculate the appropriate concentration value in this case you must take into account the volume of cell suspension spread on the plate. The value would be is < 1 cell/ volume. (EXAMPLE: If you spread 0.1 ml and observe 0 colonies, your calculated value for cell concentration would be < 10 cells/ml.) Enter your data in an EXCEL spreadsheet. In EXCEL, scientific notation is formatted in an unusual way. 2.1 X 10 8 is entered as 2.1E+08, and so on. Page 5 of 6
DISCUSSION Once you calculate the frequencies of the antibiotic resistant mutants, what do they mean? Here we have a situation with 2 different experiments combined as one. The data allow you to make a comparison between the frequency of Rif R Tric R, and Amp R mutants. Why would the frequency of one type be different than the other? On the other hand, you also have data comparing the frequency of mutants in the 2 different E. coli strains. Why would the frequency in one strain be different from the other? Another fundamental issue you should try to address is "How does mutation rate affect fitness?" (i.e. Are mutations good, or bad?) Page 6 of 6