Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance

1. Construction of the Experimental System Encoding Antibiotic Resistance Gene

Note: Here E. coli MG1655 was used as the recipient strain of the plasmid- or chromosome-encoded antibiotic resistance gene. The antibiotic resistance gene is encoded in the chromosome or a multicopy plasmid in an otherwise isogenic strain (Figure 1).

1. Insertion of the antibiotic resistance gene in the λ phage integration site (attB)²⁰ of the chromosome of MG1655.
   1. Amplify 500 bp-long regions at both sides of the chromosomal attB site by PCR. Use oligonucleotides YbhC-F (5'- CCTGTACCGTACAGAGTAAT-3') and attB-R (5'- GCCCGCCACCCTCCGGGCCGGTATAAAAAAGCAGGCTTCA-3') for the left homology region and attB-F (5'- AGCGCCCTAGCGCCCGCTCCTTATACTAACTTGAGCGAAA-3') and YbhB/R (5'- TGGCGATAATATTTCACCGC-3') for the right one. Amplify the bla_TEM-1 resistance gene using primers Tem1-pBAD-F (5'- TGAAGCCTGCTTTTTTATACCGGCCCGGAGGGTGGCGGGC-3') and Tem1-pBAD-R (5'- TTTCGCTCAAGTTAGTATAAGGAGCGGGCGCTAGGGCGCT-3'). Design primers with approximately 20 bp (in the 5' end) of sequence showing complementarity to the fragment that is going to be fused to.
   2. Fuse homology regions to the antibiotic resistance gene PCR product (including its promoter region) using isothermal assembly²¹, at 50 °C for 30 min.
   3. Electroporate MG1655 cells containing plasmid pKOBEG.
      Note: pKOBEG is a thermosensitive vector that contains the λ Red machinery, promoting homology-based allelic exchanges between the chromosome and PCR products²².
      1. Use 1 µL of the product from step 1.1.2²³ into 40 µL of electrocompetent cells in 2 mm cuvettes at 4 °C and 2.5 kV. Resuspend cells in 1 mL of LB broth + 0.2% arabinose to maintain the expression of the λ Red machinery.
      2. Transfer the total volume to a microfuge tube and incubate 2 h at 30 °C (permissive temperature for pKOBEG) with intense shaking (200 rpm in an orbital shaker) to allow for the phenotypic expression of the resistance markers inserted in the chromosome.
      3. Plate the cells on LB agar containing the appropriate antibiotic to select for the antibiotic resistance gene (ampicillin or carbenicillin at 100 mg/l for bla_TEM-1).
   4. Plate 100 µL on one Petri dish, and spin down the rest of the volume, resuspend it in 100 µL of fresh LB broth and plate it on another Petri dish. Incubate overnight at 42 °C.
      Note: This temperature is non-permissive for the replication of pKOBEG. The colonies able to grow in these conditions will have lost pKOBEG and will present the resistance gene integrated into the attB site (for simplicity this strain will be called MG1655::resA from here on).
   5. Test for the loss of pKOBEG by replicating the colonies of interest in plates with chloramphenicol (the antibiotic against which pKOBEG contains a resistance marker).
      Note: Lack of growth in chloramphenicol plates at the permissive temperature of 30 ºC is indicative of the absence of the plasmid.
   6. In order to verify clones, PCR amplify the construction using external primers YbhC-Extern (5'-TTTGTGACCAGAAGACCGCA-3') and YbhB-Extern (5'-CTCATCAGTAACGATCTGCG-3'), verify through gel electrophoresis the correct size of the amplicon and sequence the purified product to ensure that the sequence of the inserted gene is correct.
2. Insert the antibiotic resistance gene in a multicopy plasmid.
   Note: Because plasmids with very-high copy number tend to impose a large reduction in bacterial host fitness, we recommend the use of multicopy plasmids with a natural origin of replication, such as p3655 (pSU18T-pBADgfp2, ColE1-type origin of replication²⁴). These plasmids usually present a moderate copy number of about 15-20 copies per cell.
   1. PCR amplify the antibiotic resistance gene of choice (including promoter region), phosphorylate the PCR product and ligate it to the PCR-amplified backbone of the plasmid:
      1. PCR amplify the antibiotic resistance gene; for bla_TEM-1 use primers Tem1-pBAD-F (5'- TGAAGCCTGCTTTTTTATACCGGCCCGGAGGGTGGCGGGC-3') and Tem1-pBAD-R (5'- TTTCGCTCAAGTTAGTATAAGGAGCGGGCGCTAGGGCGCT-3').
      2. Phosphorylate the purified product using T4 polynucleotide kinase, following manufacturer's instructions.
      3. PCR amplify the plasmid backbone using a high-fidelity polymerase and oligonucleotides pBAD-F (5'-CGTTGATCGGCACGTAAGAG-3') and pBAD-R (5'-AAACGACGGCCAGTGCCAAG-3').
      4. Ligate both PCR fragments using T4-ligase following manufacturer's guidelines.
   2. Electroporate, as described previously, 40 µL of electrocompetent E. coli DH5-α cells with a maximum of 1 µL of the ligation product and select on the appropriate antibiotics for the antibiotic resistance gene (ampicillin or carbenicillin at 100 mg/L for bla_TEM-1) and the plasmid backbone.
      Note: Supplementing LB with arabinose here is unnecessary.
   3. Extract the plasmid using the commercial mini-prep kit of the reader's choice:
      1. Harvest 5 mL of an overnight culture by centrifuging at 12,000 x g at room temperature. Resuspend the cells in 250 µL of resuspension solution and mix well. Add 250 µL of lysis solution and mix well. Add 350 µL of neutralization and mix well.
      2. Transfer the supernatant to the DNA binding column provided with the kit, centrifuge for 1 min and discard the flow through. Wash twice the column with 500 µL of washing solution, centrifuging for one minute and discarding the flowthrough at each wash. Centrifuge the empty column for 1 additional min to remove residual washing buffer.
      3. Place the column in a clean tube and add 50 µL of ultra-pure water to the membrane to elute the purified DNA. Centrifuge for 1-2 min and collect the flow through that contains the purified plasmid. Sanger sequence the gene of interest in the plasmid using primers from outside the inserted sequence to confirm that the sequence is correct and that no mutations are present in the resistance gene prior to the evolution experiments.
   4. Once the sequence is verified, electroporate the plasmid in the E. coli MG1655 strain, as described above.
      Note: This yields strain MG1655/pRESA.

2. Evolutionary Rescue Approach to Experimentally Evolve Antibiotic Resistance (Figure 1)

1. Streak out the strains under study on LB plates: MG1655::resA and MG1655/pRESA plus the susceptible parental strain, MG1655.
   Note: Plasmid pRESA should be stable in MG1655, so there is no need to add antibiotics to the LB plates. Mutation rates in E. coli are low enough so once the construction is verified it is not necessary to verify plasmid sequence at each step.
2. Prepare 96-well plates with 200 µL of LB in each well and inoculate 48 isolated colonies of each strain in independent wells (one plate per strain). Incubate the plates overnight at 37 °C and 200 rpm. Keep a frozen stock of these founder populations.
   Note: To prevent and control for culture cross-contamination in the 96-well plates, use a checkerboard plate design by intercalating inoculated wells with bacteria-free medium throughout the 96-well plate. Use this plate design during the entire experimental evolution.
3. Start the evolutionary rescue experiment by inoculating 2 µL from each well of the plates with the founder populations in new 96-well plates with 198 µL of LB in each well with a sub-inhibitory concentration of the antibiotic under study. For the evolutionary rescue approach start with ¼ or 1/8 of the minimal inhibitory concentration (MIC, determined previously²⁵) of the antibiotic in LB for each of the three strains and double the concentration of the antibiotic daily. Use this approach to maximize the chances of populations to acquire resistance mutations²⁶. Incubate the plates at 37 °C and 200 rpm for 20 h.
   Note: Measure the overnight OD of the founder populations. If there are significant differences in OD among strains correct the initial inoculum to start the experiment with the same number of cells per well.
4. Every day, measure the OD of each population after overnight culture and perform a transfer of the cultures, as described in 2.3, to new 96-well plates with double the concentration of the antibiotic than the day before. Incubate the plates 20 h at 37 °C and 200 rpm.
   Note: The 1:100 dilution factor produces approximately 6-7 generations per day.
5. In parallel, propagate control populations of each strain in the same conditions as described in 2.3 and 2.4, but in the absence of antibiotics.
   Note: Control populations will help discriminate between mutations arising due to the presence of the antibiotics and general mutations helping bacteria to adapt to the experimental conditions. Parallel mutations arising in the absence of antibiotics are likely to be helping bacteria to adapt to the experimental conditions and not related to antibiotic resistance.
6. Track the number of surviving populations every day by measuring the absorbance at a wavelength of 600 nm (OD) of the cultures using a plate reader.
   Note: Optical density values lower than 0.1 indicate the extinction of the population. See Figure 2 for an example of survival curves.
7. Keep a frozen stock of all of the populations periodically (every 3-5 days).
8. Use log-rank tests [package "survival" in RStudio (Version 0.99.486)] to determine statistical differences in the survival of populations of the different strains over time under increasing concentration of antibiotics¹².
   Note: This experiment will determine if multicopy plasmids potentiate the evolution of antibiotic resistance for the particular antibiotic and gene under study.

3. Molecular Basis of the Evolution of Antibiotic Resistance (Figure 1)

1. Perform total DNA extractions from a representative number of populations and clones across strains and treatments. Include the parental strains (MG1655, MG1655::resA and MG1655/pRESA) to be able to detect the mutations accumulating during the experiment.
   Note: For examples of DNA extraction kits see the table of materials. Each kit has different protocols; follow manufacturers instructions.
2. Quantify DNA quality and concentration. There are different methods to determine DNA quality and concentration. Determine the quality measuring the ratios of absorbance 260 nm/280 nm and 260 nm/230 nm. Use a fluorescent, DNA-binding dye to measure DNA concentration following manufacturer's instructions, and agarose gel electrophoresis to confirm that there is no DNA degradation or RNA contamination.
3. Use deep DNA sequencing from samples of whole populations and individual clones to investigate the genetic basis of antibiotic resistance.
   Note: We conducted all sequencing at the Wellcome Trust Centre for Human Genetics, Oxford, UK. For more details see San Millan et al. 2016¹².
4. Use the breseq 0.26.1 pipeline^27,28 to detect the mutations, using polymorphism mode to estimate the frequency of mutations in populations. Compare the different evolved genomes to the parental genome to detect mutations that have accumulated during the experiment.
5. Compare the mutations accumulated in parallel in the control populations with those of the populations evolved under increasing concentration of antibiotics to differentiate between mutations helping bacteria to adapt to the general experimental conditions (those found in the control populations) and those involved in antibiotic resistance (those found exclusively in populations subjected to antibiotic pressure).
   Note: The number of parallel mutations is usually low, so different parallel mutations between treatments can be easily assessed. Apart from the mutations in the antibiotic resistance gene under study it is quite likely to find other mutations associated with antibiotic resistance in the chromosome, such mutations in porins or efflux systems¹².
6. Reconstruct the mutations in the antibiotic resistance gene in the parental strain using the same approach as in section 1 of this protocol. Follow points 1.1 and 1.2 of the protocol to introduce the new mutations in the parental background (using the evolved genes as a PCR template). Analyze the antibiotic resistance phenotypes of these new constructions to confirm the role of the mutations.