ABR2: How do bacteria multiply? How do they defend themselves?

Recitation Worksheet
Fall 2024

This week’s learning objectives

By the end of this week, you should be able to:
Relate the development of resistance in bacterial populations to the mutations occurring in the targets of antibiotics in
the bacterial cell.
1. Describe how template polymerization is involved in DNA replication, RNA transcription, and protein synthesis.
2. Explain how all genetic information is encoded in DNA molecules.
3. Explain the processes leading to mutations, define the mutation rate, and estimate the number of mutations per
replicative cycle.
4. Discuss how a mutation in the unique sequence of a gene may affect the function of a protein.

After each week, evaluate your learning with respect to these learning objectives. This will prepare you for the exams!

1. DNA structure
The picture on the right shows the structure of a DNA sequence. Color coding for the bases is also shown. Answer the following
questions (You may use lecture notes).
a) How many DNA strands are present in the DNA sequence? Identify each strand in the picture shown here.
b) How many hydrogen bonds are found in this DNA molecule within the segment shown here? Identify them in the
picture.
c) Circle the phosphodiester linkage between two
nucleotides on the DNA sequence shown here.
d) Label the phosphate, base and the deoxyribose sugar on
a nucleotide.
e) A DNA strand has a direction specified as 5’ and 3’. Show
the 5’ and 3’ ends on a DNA strand shown here. Explain
how you recognize the ends of DNA strands. Write the
sequence of one of the strands.
f) Which chemical groups are found on 5’ and 3’ ends of a
DNA strand?
g) Why is there a need to label the ends of the strands?
2. DNA replication, transcription, point mutation
Rifampicin is an essential antibiotic drug for the efficient control of multidrug-resistant tuberculosis (MDR-TB) caused by
Mycobacterium tuberculosis bacteria. Rifampicin-resistant strains of M. tuberculosis evolve due to mutations in the sequence
of the gene encoding an RNA polymerase. This sequence is given below.

a) Write the complementary DNA strand for the region shown as (----------------) and label the directions.
b) When DNA is transcribed into RNA, there are key concepts (template strand, coding strand, direction of transcription,
promoter, terminator, kinds of bases) you have to pay attention to. Answer the following questions related to those key
concepts. Discuss these questions with your group members and explain answers in your own words.
i. What is the difference between template and coding strands? Which one of the above DNA strands is the
template strand?
ii. What is the direction of transcription? Why?
iii. What are the roles of the promoter and terminator sequences in transcription?
c) Write down the sequence of the mRNA strand that will be encoded by the DNA sequence.
d) Write down the corresponding amino acid sequence using the three-letter-code. Make sure you start translating at the
correct place. Use the codon table for the translation.
e) Assume that a number of point mutations occurred during replication of the DNA sequence given. Mutations are
underlined on the DNA sequence below.

5’- TCATGGTCGACCAGGCTGAGCAGTTCGTCCAGTGAGCGA-3’
Which mutations may cause changes in the amino acid sequence encoded by the DNA strand? Why? Discuss it with your
group and explain your answer in your own words.

3. Mutation rates
Mutation rates are used to determine how many mutations can accumulate over many generations in a single individual. This
is important to consider because as we have mentioned before, mutations can be linked to traits which are selected for if they
are beneficial to the organism and this can eventually lead to antibiotic resistance.
a) Define the term mutation rate.
b) Explain why a mutation rate might be different in one species from another one.
c) Look up the average mutation rate of a human and compare it to a bacteria. Which one has the higher mutation rate?
Which organism is more likely to pass on this mutation to the next generation and have it expressed, why?
d) A type of bacteria, Escherichia coli, has about 4400 genes, each coding for a protein. Given that the average length of a
protein in bacteria is 300 amino acids, what percent of the genome of this bacterium is made-up of non-coding bases?
The genome size of E. coli is 4.6 million bps.
4. DNA replication and mutation rates
Assume that scientists found a microbe on Europa (Jupiter’s icy moon), named Glacialis. Glacialis shows similar characteristics
to the soil bacteria-Eleftheria terrae which produces Teixobactin (an antibiotic, a chemical compound) but is sensitive to this
antibiotic.
After 10 years of research, scientists determined the following characteristics:

Genome size Doubling # of mutations /bp Growth rate

(base pairs) time (in one replication) constant (hr-1)

Glacialis 108 25 min 10−9 1.95

a) What is the rate of DNA synthesis (in bp/min) for the DNA of Glacialis in a single cell?
b) Similar to bacterial species on Earth, DNA polymerases of Glacialis also make mistakes during the replication process.
These mistakes are called replication errors, which, if not corrected, remain as mutations.
Based on the numbers given in the Table above, calculate how many nucleotides (DNA base pairs) are mutated in
genomes of Glacialis on average as a result of one replication?
c) Answer the following questions below.
i. Starting from a single cell, what would be the population size after 12 hours? Assume that resources are
unlimited and Glacialis grows exponentially.
ii. How many mutations will accumulate in this population of Glacialis at the end of 12 hours of replication?
iii. Are these mutations all of the same type? Do you think the results of these mutations will be the same?
Explain.
iv. Do you think this time period will be sufficient for some of the bacteria in the Glacialis population to develop
antibiotic resistance? Why or why not? Discuss it with your group members and explain your answer in your
own words.
Hint: One can assume that one out of 109 bacteria can develop resistance once an antibiotic is introduced.
(See ref: Antimicrobial resistance induced by genetic changes, J Med Life. 2009 Apr 15; 2(2): 114–123)

5. Connecting the Concepts [Work on this if you have time left]

Drawing a concept map can be a great way to organize your thoughts and help you connect various concepts that
you are learning. More about concept maps: “Concept Maps: What the heck are these?”

With your group members, create a “concept map” by using the following terms:
Antibiotic resistance (start with this), DNA, mutation, replication, DNA polymerase, RNA polymerase, ribosome, protein,
translation, transcription, mRNA, antibiotic inactivating enzymes, efflux pump, replication error rate, protein synthesis,
tRNA, amino acid, antibiotic targets.

End of the Worksheet