Mutated E. Coli’s Resistance to Streptomycin
Rowaida Wardak
Abstract
Streptomycin is an antibiotic used against various bacteria that acts by inhibiting protein synthesis. E. coli is the bacteria of interest and was mutagenized under UV irradiation to create various mutant strains that were analyzed to determine their degree of resistance against Streptomycin. The gene rpsL was used to identify the location of the mutation in my 2 minutes irradiated + Streptomycin strain and was cloned into a plasmid and performed PCR and the bacteria was plated on various conditions to determine the antibiotic resistance. The mutation identified in the bacteria was a point mutation that led to a difference amino acid being expressed. Most colonies grew on the Ampicillin and Streptomycin plate. A simple mutation can lead to great impact on the expression of a gene. Cloning the rpsL gene into the plasmid allowed us to test the antibiotic resistance of the gene.
Introduction
Streptomycin is a common antibiotic that functions as a protein synthesis inhibitor, meaning it binds to the small 16S rRNA of the 30S subunit of the ribosome which interferes with the binding of a certain tRNA to the 30S subunit. This can lead to codon misreading, a frameshift mutation and eventual cell destruction. It functions by preventing the initiation and elongation processes during protein synthesis, which eventually destroys the microbial cells. Streptomycin only has this function against bacteria, not humans who have different ribosomes. It acts against gram-positive and gram-negative bacteria and is ultimately a very broad-spectrum antibiotic. Streptomycin can be utilized to attack various bacteria including Escherichia coli, a bacterium that resides in the intestines of warm-blooded organisms of which particular strains can cause food poisoning.
The genome of E. coli is comprised of a single 4.6 Mb circular chromosome, like many other bacteria organisms. Under favorable conditions, it can grow in under 20 minutes which is why it’s widely used for research purposes. It has a high genetic diversity and throughout all of the strains, only 20% of the genes are shared among them.
Mutations can be caused by several factors – like errors during the DNA replication, spontaneously, or by chemicals – but we are focusing on UV radiation. UV light destroys cells by damaging the DNA. The light begins a reaction between two thymine molecules that form a thymine dimer which is stable, but the repair necessary (removing the bases and replacing them) is too difficult to complete when the damage is excessive. The more exposure to the UV irradiation, the more dimers that are created. When there are more errors in the DNA, there’s a greater risk of the dimer error not being caught, and it will likely remain undetected.
The protein coding region of the gene rspL is the protein that’s most commonly mutated in Streptomycin-resistant strains. Therefore, we are focusing on that particular section to analyze and pin point the exact location of the mutation occurring with our bacteria. We did so by cloning the gene into a plasmid with an inducible promotor, allowing us to analyze the antibiotic resistance against various conditions.
Methods
1. Mutagenesis of E. coli
Irradiate the E. coli for 0, 2, and 15 minutes and transfer 150 micrograms of the culture into the appropriately labeled Eppendorf tube: 0min+str, 0min, 2min+str, 15min+str, and 15min.
In the end, we had 5 plates – one that had 0 minutes irradiated bacteria with the streptomycin, 0 minutes irradiated drug free, 2 minutes irradiated bacteria with the streptomycin, 15 minutes irradiated drug free, and 15 minutes irradiated with the streptomycin. Incubation is necessary for the cells to regenerate and synthesize their newly mutated proteins. After the cells recovered, we transferred 100 micrograms of the culture into their respective plates. It was imperative that we tilted the plate in order for the culture to properly spread and absorb into the agar. These plates were then incubated at 37 degrees Celsius for two days.
We were given three agar plates (0 streptomycin, 50 microgram/mL streptomycin, and 1000 microgram/mL streptomycin) to grid into 15 squares and pick colonies from our original five strains. We picked colonies and placed them in their respective grid, noting down the grid number. For example, we picked strain 0 min + str and put it on squares #1, 6 and 7. Then the plates were incubated to grow.
2. PCR rpsL gene
We analyzed our plates and examined where most colonies grew. My 2 min + str strain had the most colonies so we chose that for PCR. We completed the PCR of our 2 min + str strain through a mixture of polymerase mix, primer mix, and our colony.
We purified our rpsL-strR PCR product by using the Zymoclean PCR purification kit which utilized DNA binding buffer and DNA wash buffer and elution buffer. The purpose was to efficiently purify the DNA through spinning and elution. After purifying the DNA, our PCR product was complete. We then prepared the sequencing reaction by adding primer and PCR product to a pre-labeled sequencing tube that was #585.
3. rpsL DNA sequence analysis
We analyzed our rpsL DNA through ApE and used a good read (Z1 base calls). We found the start codon, converted them to lowercase, and translated the whole sequence into a one letter code. We then used BLAST to detect any changes by aligning the two sequences – normal wild type and our mutated sequence.
4. Plate & transform pET21-rpsL strR
The Gibson assembly was created by mixing our PCR product, vector, and master mix into an Eppendorf tube. With this, an exonuclease creates long overhangs, a polymerase will fill in the gaps of the annealed single strand regions, and a DNA ligase seals the ends. This process replicated and inserted our DNA into the vector to allow for the gene to be cloned.
We transformed and plated our pET21-strR onto 3 agar plates: one with Ampicillin, one with Ampicillin + Streptomycin, and one with Ampicillin + Streptomycin + IPTG. The purpose of this was to analyze the resistance of the bacteria on different antibiotics.
Results & Discussion
1. Mutagenesis of E. coli
After completing the plating, I expected the non-irradiated cells (0 min) to have maximum growth on the drug-free plate because there was no streptomycin antibiotic present to attack the bacteria. For the streptomycin plated with the non-irradiated (0 min) cells, I expect no bacteria growth because the bacteria is not mutated and the antibiotic is expected to function optimally, destroying all the E. coli. On the drug-free plates plated with 2 min irradiated cells, I expect stable growth because while the bacteria is mutated, there is no antibiotic to kill them. On the streptomycin plates plated with the 2 min irradiated cells, there should be less growth than that on the drug-free plates. The bacteria is mutated so the antibiotic should kill some, whereas others survive due to their mutation. On the drug-free plates plated with 15 min irradiated cells, there should be plenty of E. coli growth because there’s no antibiotic present to attack them. Instead, there should be mutated bacteria heavily resistant to the streptomycin. Plating 15min irradiated cells on the streptomycin plates should lead to plenty of bacteria growth because the E. coli is heavily mutated and most resistant out of all the strains.
The mutagenesis of bacteria gave interesting results. The most growth was observed in the 2 min + str, 15 min + str, and 0 min strains. The least amount of growth was in 15 min, which was interesting since I would have expected for the most growth to be there. I was correct in predicting a lot of growth on the 0 min plate. Something I noticed repeatedly was that in certain areas of high density of colonies, the color of the colonies was orange and not cloudy-white like the others.
Ultimately, we had most colonies grow on the 2 min + str plate. There was least amount of growth on the 0 min + str plate because the antibiotic killed virtually all of the bacteria since it wasn’t irradiated to mutate.
Then, I examined the streptomycin-resistant mutant strains later incubated. There was barely any growth in the plate with 1000 microgram/mL streptomycin. This makes sense because the more of the antibiotic drug to attack the bacteria, the less E. coli there will be. Most of the colonies were on plates 1 and 2 which had 0 and 50 micrograms of streptomycin. Plate 1, which had 0 microgram/mL of streptomycin, had the most growth and interestingly even had orange colored colonies, unique to squares 3 and 11 which were the 15 min strain. On plate #3, which had the most streptomycin, there was no growth of the 15 minutes, 2 minutes with streptomycin, and 0 minutes.
Before completing the lab, I expected that there would be most growth on plate 1, which was not irradiated, and that the amount of growth would decrease by the concentration of streptomycin and this was true – plate 3 had barely any growth. However, contrary to my initial expectation, plate 2, which had 50 microgram/mL of streptomycin, had almost the same degree of growth as plate 1. This led me to believe that there was a trend between low radiation time and mutation. It appeared as though the lower radiation time strain had more colonies than the 15 minutes irradiated.
2. PCR rpsL gene
PCR is completed in order to amplify a desired region of DNA and is essentially accelerated DNA replication of a specific section. It requires primer, which essential to begin the DNA elongation, and polymerase, which extends the primer, and dNTPS – a mixture of various nucleotides that create the actual strand. PCR uses denaturing, or heating to separate the DNA into two separate strands, and annealing, cooling for DNA primers to attach to the template DNA.
Polymerase mix contains water, polymerase buffer, dNTP mix and Taq polymerase. Taq polymerase is valued over other DNA polymerases because it can withstand high temperatures that would normally denature other types of polymerase. Interestingly, it comes from a bacterium that lives in hot springs!
3. rpsL DNA sequence analysis
The mutation identified in the sequence was a real single substitution point mutation. There was a one letter change in our mutated DNA at amino acid 55 that had E instead of K, like in the wild type.
From BLAST:
Query 2 VFTKQKLKPGAI*WQQLTSWYANHVLAKLRKATCLRWKHARKNVAYVLVYILPLLENRTP 61 VFTKQKLKPGAI*WQQLTSWYANHVLAKLRKATCLRWKHARKNVAYVLVYILPLL+NRTP Sbjct 1 VFTKQKLKPGAI*WQQLTSWYANHVLAKLRKATCLRWKHARKNVAYVLVYILPLLKNRTP 60
The codon mutated from GAA to AAA, which is a single nucleotide change that led to a completely different amino acid coded (leucine to phenylalanine). Because of the repetitive nature of codons that allow for multiple nucleotide sequences to code for a single amino acid, it could have been possible for the mutation to be silent and result in the same amino acid. However, our mutation coded for a completely different amino acid. They appear to be identical in their chemical structures, however while phenylalanine has a benzyl side chain, leucine has an isobutyl group. Both are hydrophobic, and phenylalanine can be substituted with other aromatic or hydrophobic amino acids which is why I determined that the mutation is semi-conservative.
4. Plate & transform pET21-rpsL strR
We PCR’ed the rpsL-strR DNA from the strR mutant and using the Gibson assembly, inserted this gene into the plasmid and transformed it into the E. coli. The ampicillin antibiotic resistance gene was only present in specific cells that had the plasmid and
Protein genes were placed after the lac promoter in a plasmid that also had the gene for lac repressor protein. The binding of the lac repressor will prevent the expression from the lac promoter.
Plate 1, which only contained Ampicillin, had the most growth. I expected for third plate to have the most growth, since it had the IPTG inducer that would allow for transcription and protein production. However, it still had a lot of growth, just a little less than plate 1. I didn’t expect to see many colonies on plate 2 since it was only Ampicillin and Streptomycin, lacking the inducer present in plate 3. However, I was shocked to see that not even one colony grew.
Analyzing how the bacteria becomes resistant and the antibiotics’ effect changes could be very beneficial to medicine. We could use this phenomenon to prevent antibiotic resistance so that the effectiveness will be at its maximum. Antibiotic resistance is one of the biggest health issues in America and finding out how to reverse this effect could change lives.
E. coli strains change because bacteria and viruses are always mutating themselves. With these experiments, when there’s a new strain of the bacteria, we can create a specific antibiotic that’s unique for that strain.
Additionally, a single point mutation caused such a great change in the expression of the protein and led to a great mutation that could be seen in the figure below – the mutation versus the control had vastly different results. And this was just due to one simple amino acid change!
Although a mutated amino acid results in resistance, it may still have ribosomal function. In missense mutations, one amino acid is changed to another. The amino acid it is changed to could have similar properties to the original or could be in a specific area that doesn’t affect the protein’s secondary structure or function.
References
Amino Acid Properties and Consequences of Substitutions from
https://pdfs.semanticscholar.org/3587/5cc233aefffaafdf1228c51d02500a28525a.pdf
Phenylalanine. (2018, November 02). Retrieved from https://en.wikipedia.org/wiki/Phenylalanine
Biolabs, N. E. (n.d.). Gibson Assembly®. Retrieved November 14, 2018, from
https://www.neb.com/applications/cloning-and-synthetic-biology/dna-assembly-and-cloning/gibson-assembly
Dr. Pokala Lab Sessions 5, 6, 7, 8, 9, 10 and Lecture 16
Escherichia coli. (2018, October 27). Retrieved from
https://en.wikipedia.org/wiki/Escherichia_coli
Leucine. (2018, November 05). Retrieved November 16, 2018, from
https://en.wikipedia.org/wiki/Leucine#Chemistry
Streptomycin. Retrieved November 13, 2018, from
https://pubchem.ncbi.nlm.nih.gov/compound/streptomycin#section=Top
Streptomycin. Retrieved November 14, 2018, from https://en.wikipedia.org/wiki/Streptomycin
How does ultraviolet light kill cells? (n.d.). Retrieved November 12, 2018, from https://www.scientificamerican.com/article/how-does-ultraviolet-light
Figures and Tables
Mutagenesis of E. coli
Picture of the growth of different irradiated strains. From left to right: plate of 0 microgram/mL streptomycin, 50 microgram/mL, 1000 microgram/mL.
As we can see, plate 3, which had the most Streptomycin, allowed the least amount of growth.
I constructed this table to outline the degree of growth of each strain in each plate. I described them in terms of percentages relative to the growth on the other plates. For example, the plate with the highest growth is 100% and then compared to that, I determined whether the other plates had 0%, 25%, or 50% of that particular strain.
Strain 0 microgram/mL 50 microgram/mL 1000 microgram/mL
1. 0 min + str 100% 0% 100%
2. 15 min +str 50% 100% 50%
3. 15 min 100% 0% 0%
4. 2 min + str 100% 50% 0%
5. 0 min 100% 0% 0%
6. 0 min +str 100% 100% 100%
7. 0 min + str 100% 100% 100%
8. 2 min + str 50% 100% 0%
9. 0 min 100% 100% 0%
10. 15 min + str 100% 100% 100%
11. 15 min 100% 50% 25%
12. 2 min+ str 100% 100% 25%
13. 0 min 100% 100% 0%
14. 15 min 100% 25% 0%
15. 15 min + str 100% 0% 25%
Transformation with pET21-strR plasmid
Left to right: Ampicillin, Ampicillin + Streptomycin, Ampicillin + Streptomycin + IPTG
Plate 1, with only Ampicillin, has the most growth while plate 3 is not far behind.