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Adaptive Evolution of Escherichia coli: Fitness and Genetic Changes Beyond 100th Passage

by Aw, Zhen Qin; Loo, Bryan Zong Lin; Low, Samuel Xin Zher

Abstract (Summary)
Escherichia coli lives in the human intestine and any form of adaptation may affect the human body. The effects of food addictives on E. coli has been less studied compared to antibiotics. Since food addictives and preservatives are being consumed by humans so often nowadays, it is important to investigate this relationship. In this project, we continue to study the evolution of E. coli in different food additives (sodium chloride, benzoic acid, monosodium glutamate) in different concentration, singly or in combination, for over 83 passages. Adaptability of the cells was estimated with generation time and cell density at the stationary phase. Polymerase Chain Reaction (PCR) / Restriction Fragments Length Polymorphism (RFLP) were used with 3 primers and 3 different restriction endonucleases to analyze the adaptation at genomic level. The PCR product and the digestion profiles using the 3 different restriction enzymes were analyzed using Nei-Li Dissimilarity Index. Our results showed that adaptation started to slow down and the gradients of generation time against passage are less steep compared to the first 70 passages of the experiment, suggesting that most adaptive mutations occurred within the first 500 generations. In the genomic level, ecological specialization was observed as we found that the cells adapted through a different mechanism and diverge from each other although the resulting effect of the medium is the same. It could further suggest that different concentrations of food additives cause different types of chemical stress, instead of different levels of chemical stress.
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Advisor:Maurice HT Ling

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Source Type:Other

Keywords:escherichia coli, adaptation, experimental evolution, food additives

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Date of Publication:01/08/2011

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SINGAPORE POLYTECHNIC

SCHOOL OF CHEMICAL AND LIFE SCIENCES

Diploma in Biotechnology
General Biotechnology Option

Adaptive Evolution of Escherichia coli:
Fitness and Genetic Changes Beyond 100th Passage

Project Code: DBTBTech1005

Aw Zhen Qin (0819404)
Loo Zong Lin Bryan (0819996)
Low Xin Zher Samuel (0853633)

Year of Study: Year 3

Project Supervisor: Maurice Ling

AY2010/2011


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Abstract

Escherichia coli lives in the human intestine and any form of adaptation may affect the
human body. The effects of food additives on E. coli has been less studied compared to
antibiotics. Since food additives and preservatives are being consumed by humans so often
nowadays, it is important to investigate this relationship. In this project, we continue to study
the evolution of E. coli in different food additives (sodium chloride, benzoic acid,
monosodium glutamate) in different concentrations, singly or in combination, for over 83
passages. Adaptability of the cells was estimated with generation time and cell density at the
stationary phase. Polymerase Chain Reaction (PCR) / Restriction Fragments Length
Polymorphism (RFLP) were used with 3 primers and 3 different restriction endonucleases to
analyze the adaptation at genomic level. The PCR product and the digestion profiles using the
3 different restriction enzymes were analyzed using Nei-Li Dissimilarity Index. Our results
showed that adaptation started to slow down and the gradients of generation time against
passage are less steep compared to the first 70 passages of the experiment, suggesting that
most adaptive mutations occurred within the first 500 generations. In the genomic level,
ecological specialization was observed as we found that the cells adapted through a different
mechanism and diverge from each other although the resulting effect of the medium is the
same. It could further suggest that different concentrations of food additives cause different
types of chemical stress, instead of different levels of chemical stress.

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Acknowledgement

Firstly, we would like to thank Dr. Maurice Ling Han Tong of Singapore Polytechnic for his
precious guidance and attention, his time and effort in helping us throughout the entire
project.

We would also like to extend our appreciations to Mdm Sun Wei and Mdm Kanchini
Manivannan, Technical Officers of Microbiology Laboratory; Ms. Ye Song, Ms. Cheong
Yoke Fun and Mr Myo Min, Technical Officers of Life Science Laboratory; from Singapore
Polytechnic for their assistance in all of our laboratory work.

Special thanks to the group of friends around us who provided assistance and showered their
encouragement to us: Jian Ann, Joshua, Desmond, Chin How and their Final Year Project
members.

Lastly, we would like to thank Singapore Polytechnic for financially sponsoring our project,
Account Number: 11-27801-45-2550.

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Table of Contents

Abstract ..................................................................................................................................... 2
Acknowledgement..................................................................................................................... 3
List of Figures and Tables......................................................................................................... 5
Abbreviations ............................................................................................................................ 7
1 Introduction ................................................................................................................ 8
2 Literature Review ....................................................................................................... 9

2.1 Sources and Effects of Genetic Variation................................................................... 9
2.2 Experimental Advantages of Escherichia coli.......................................................... 14
2.3 Examples of Evolution Experiments with Bacteria.................................................. 15
2.4 Long-Term Experimental Evolution in Escherichia coli.......................................... 17
2.5 Effects of Chemicals on Bacteria ............................................................................. 22
2.6 Aims and Hypothesis of Project ............................................................................... 25
3 Materials and Methods ............................................................................................. 27

3.1 Main Culture Experiment ......................................................................................... 27
3.2 Swap Experiment...................................................................................................... 28
3.3 Polymerase Chain Reaction / Restriction Fragments Length Polymorphism .......... 29
3.4 Data Analysis............................................................................................................ 30
4 Results ...................................................................................................................... 36

4.1 Total Generations across 83 Passages ...................................................................... 36
4.2 Day 5 Day 7 Cell Density......................................................................................... 36
4.3 Generation Time ....................................................................................................... 42
4.4 Swap Experiment...................................................................................................... 45
4.5 Polymerase Chain Reaction / Restriction Fragment Length Polymorphism............ 54
5 Discussion ................................................................................................................ 56

5.1 Nutrient Broth Does Not Prime Cells for Adaptation in Other Treatments through
153 Passages............................................................................................................. 56

5.2 Decreased Adaptation Rates of Cells ....................................................................... 56
5.3 Ecological Specialisation in Adaptation of Cells to Their Individual Treatments ... 57
5.4 Different Concentrations of Food Additives Render Different Types, Instead of
Levels of Chemical Stress ........................................................................................ 59

5.5 Similar Cell Densities Regardless of Aerobic or Anaerobic Conditions.................. 59
5.6 Increased Growth Ability in Specialised Media....................................................... 60
5.7 Occurrence of Stepwise Adaptation ......................................................................... 60
5.8 Cells of different treatment shows genetic divergence............................................ 61
6 Recommendations .................................................................................................... 64
7 Conclusion................................................................................................................ 65
8 References ................................................................................................................ 66
Appendix A – Number of Generations ................................................................................... 72
Appendix B – Generation time Estimation ............................................................................. 81
Appendix C – Cell Density at Stationary Phase...................................................................... 82
Appendix D – Swap Treatments ............................................................................................. 94
Appendix E – Gram Staining Pictures .................................................................................... 96
Appendix F – Agarose Gel Electrophoresis of PCR-RFLP .................................................... 98
Appendix G – Dissimilarity Index ........................................................................................ 185

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List of Figures and Tables

Figure 3.1: Complete Experimental Design
Table 3.1: Effects of the 28 pair-wise comparisons among the 8 treatments
Figure 4.1: Total Generation Number across 8 Passages Intervals
Table 4.1: Tabulation of Coefficient of Variation of all Treatments for 83 Passages
Figure 4.2: Coefficent of Variation of all Treatments for 83 Passages
Figure 4.3: Ratio of Day 7 to Day 5 Cell Density of High and Low MSG Treatments over 83
Passages
Figure 4.4: Ratio of Day 7 to Day 5 Cell Density of High and Low BA Treatments over 83
passages
Figure 4.5: Ratio of Day 7 to Day 5 Cell Density of High and Low Salt Treatments over 83
Passages
Figure 4.6: Ratio of Day 7 to Day 5 Cell Density of High and Low Combination Treatments
over 83 passages
Figure 4.7: Generation Time of 8 Different Treatments Starting from H COMB on the Top,
followed by H SALT, L MSG, H MSG, H BA, L BA, L SALT and L COMB Across
a Total of 80 Passages Starting from 73 to 153
Table 4.2: Comparison of Generation Time gradient between Passage 0-70 and Passage 73-
153
Table 4.3: P-values of the eight different treatments of the Jackknife resampling technique
Table 4.4: Linear Regression Equations of Generation Time Trend of Low Salt Cells into
Non- Salt Treatment media (Passages 75 to 153)
Table 4.5: Linear Regression Equations of Generation Time Trend of Low Salt Cells into
Non- Salt Treatment media (Passages 10 to 74)(Lee et al., 2010)
Figure 4.9: Generation Time Trend of High Treatment Cells Inoculated into Low Treatment
Media over 14 Swaps (H MSG cells to L MSG Media, H BA cells to L BA Media, H
SALT cells to L SALT Media, H COMB cells to L COMB Media)
Table 4.6: Linear Regression Equations of Generation Time Trend of High Treatment Cells
Inoculated Into Low Treatment Cells
Table 4.7: Linear Regression Equations of Generation Time Trend of High Treatment Cells
Inoculated into Low- Salt Treatment Media (Passages 10 to 74)(Lee et al., 2010)
Figure 4.10: Generation Time Trend of Low Treatment Cells Inoculated into High Treatment
Media over 14 Swaps (L MSG cells to H MSG Media, L BA cells to H BA Media, L
SALT cells to H SALT Media, L COMB cells to H COMB Media)
Table 4.8: Linear Regression Equations of Generation Time Trend of Low Treatment Cells
Inoculated into High Treatment Media
Table 4.9: Linear Regression Equations of Generation Time Trend of Low Treatment Cells
into High Treatment media (Passages 10 to 74)(Lee et al., 2010)
Figure 4.11: Generation Time Trend of Low Single Treatment Cells Inoculated into Low
Combination Treatment Media over 14 Swaps (L MSG cells to L COMB Media, L
BA cells to L COMB Media, L SALT cells to L COMB Media)
Table 4.10: Linear Regression Equations of Generation Time Trend of Low Single Treatment
Cells Inoculated into Low Combination Treatment Media
Table 4.11: Linear Regression Equations of Generation Time Trend of Low Treatment Cells
into Low Combination Media (Passages 10 to 74)(Lee et al., 2010)
Figure 4.12: Generation Time Trend of High Single Treatment Cells Inoculated into High
Combination Treatment Media over 14 Swaps (H MSG cells to H COMB Media, H
BA cells to H COMB Media, H SALT cells to H COMB Media)

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Table 4.12: Linear Regression Equations of Generation Time Trend of High Single
Treatment Cells Inoculated into High Combination Treatment Media
Table 4.13: Linear Regression Equations of Generation Time Trend of High Treatment Cells
into High Combination Media (Passages 10 to 74)(Lee et al., 2010)
Figure 4.13: Generation Time Trend of H SALT and L SALT cells inoculated into Higher
Salt Treatment Media over 9 Swaps (H SALT cells to Higher Salt media, L SALT to
Higher Salt media
Table 4.14: Linear Regression Equations of Generation Time Trend of High/Low Salt
Treatment Cells inoculated into Higher Salt Treatment media

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Abbreviations

BSA
CC
DI
DNA
dNTPs
EDTA
H BA
H MSG
H SALT
H COMB
L BA
L MSG
L SALT
L COMB
NB
STE

Bovine Serum Albumin
Correlation Coefficient
Dissimilarity Index
Deoxyribonucleic Acid
Deoxyribonucleotide Triphosphates
Ethylenedinitrilotetraacetic Acid
High Concentration of Benzoic acid
High Concentration of Monosodium Glutamate Treatment
High Concentration of Salt Treatment
Low Concentration of Combination Treatment
Low Concentration of Benzoic acid Treatment
Low Concentration of Monosodium Glutamate Treatment
Low Concentration of Salt Treatment
Low Concentration of Combination Treatment
Nutrient Broth
Sodium Chloride–Tris–EDTA

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1 Introduction

Escherichia coli is a Gram negative bacterium commonly found in the lower intestines of
warm blooded organisms. Most E. coli strains are harmless, but others strains like O157:H7,
can cause food poisoning in humans. As part of the normal flora of the gut, they can benefit
the host by producing vitamin K and also preventing the establishment of pathogenic bacteria
within the intestine. However, these harmless bacteria could adapt and evolve which could
then cause harm to our human body.

E. coli evolutionary studies on antibiotics resistance have been widely studied but the
mechanism on non-antibiotic agents, like food preservatives and food additives has not been
well established yet. Food preservatives are used frequently to inhibit the growth of microbes
and food additives can delay the spoilage of food. Since E. coli in our gut are constantly
interacting with such chemicals, it is important to examine their relationship.

Experimental works had been done to study the effects of 4 different food preservatives of 2
different concentrations on bacterial cells for over 70 passages. In general, the cells started
adapting after 25 passage and the cells have continued to adapt to their individual treatment
until over 70 passages.

This project continues to observe the adaptation of E. coli cultured in different concentration
of food additives, namely sodium chloride, benzoic acid and monosodium glutamate. E. coli
cells are cultured in 8 different media over 83 passages and swapped at intervals among the
treatments, making up a total of an estimated 992 generations through 153 passages including
those of earlier studies. Adaptability over time is estimated by generation time and cell
density of stationary phase. Polymerase Chain Reaction (PCR) and Restriction Fragments
Length Polymorphism (RFLP) were used to characterize adaptation/evolution at genomic
level.

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2 Literature Review

Evolution is the result of genetic changes within a population from one generation to the
other. These changes refer to the modification to the DNA sequence resulting in genetic
variation. The genetic traits in the individual are inherited down from one generation to the
other. As these individual genetic changes accumulate over time, a population can be formed
through the process of genetic divergence (Lenski et al., 1991). These traits may vary within
population and show heritable difference of the organisms. Genetic changes originated in any
generation are usually small and the difference accumulated in each successive generations
can caused substantial changes in the population. Eventually, new species may be emerged
from the ancestor (a speciation event).

2.1 Sources and Effects of Genetic Variation

Genetic variation arises from different sources such as from mutation, gene flow and
independent assortment during sexual reproduction. A mutation is defined as a permanent
change in the DNA sequence of the genome, which ranges from a single nucleotide change
(point mutation) to one or more nucleotides being inserted or deleted (frame shift mutation).
The result of accumulation of many mutations may result in evolutionary changes in the
given population (Stanek et al., 2009) due to protein structures changing as an effect of
nucleotides sequences changing after the mutations (You et al., 2007).

There are two types of mutation. The first is through heredity mutation, which is the mutation
passed down from parents to the offspring. Successive generation will contain the inherited
mutations. The second is mutation acquired during the organism’s lifespan from exposure to
the environmental, physical or chemical stress. The acquired mutation may be beneficial to
improve survivability and adaptability (Travisano, 1997). An example will be in the case of
sickle cell disease found inhabitants of Sub-Saharan Africa (Hanchard et al., 2007), where
malaria rate are very high. The sickle cell mutation allows the survival rate of the individuals
carrying the sickle cell allele to improve as it halts the infestation of Plasmodium malariae
(Hanchard et al., 2007).

Gene flow, also known as gene migration, is defined as the transfer of alleles from one
population to another (Gayden et al., 2007). Gene flow may result in an addition of new
genetic variant to a pool of established gene population (Faure et al., 2009). For example,

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technological advances allow humans to travel across the globe. Therefore, allowing
individuals may search for mates in other geographical regions. If a child is successfully
conceived, and subsequently delivered, the allele is considered to have been transferred from
one population to the other. However, physical barriers exists in between the species, such as
the Himalaya Mountains, can hinder the transfer of alleles as haplotype differences have been
reported between populations on different side of the mountains (Gayden et al., 2007). Gene
flow within or across population can have different effects in evolution. When gene flow
occurs within the same population, it will increase the amount of genetic recombination and
increase the variety of genetic variants. Gene flow allows distant population to be genetically
similar to each other (Faure et al., 2009) which helps to reduce chances of speciation.
Speciation is the evolutionary process by which a new biological species arises by diverging
itself from the parental species (Schluter and Conte, 2009).

Sexual reproduction is the production of offspring by combining genetic materials from
parental genome which allows the chance for gene recombination into population, resulting
in genetic diversity. Sexual reproduction is important as it is a method to introduce new
combination of genes by homologous recombination to every successive generation which
increases the ability for the organism to adapt to new environments (Levin and Cornejo,
2009). An example will be one of the parents having brown eyes and curly hair, and the other
having blue eyes and straight hair and the conceived child having brown eyes and straight
hair through independent assortment during sexual reproduction, However, there is also a
possibility that good combination of genes may be removed (Dawkins, 2006).

The two main mechanisms responsible for evolution (Koonin, 2009) are natural selection and
genetic drift. The process whereby the heritable traits are passed on to successive generations
to improve the survivability of organism is known as natural selection (Hurst, 2009). Each
trait is linked to a gene; therefore, when traits are inherited, it will also mean that the gene
that is linked to the trait has been passed down. For natural selection to occur, heritable
variation for the particular traits must be present and able to exist within the population. In
addition, there must be differential survival and reproduction associated with the possession
of that trait. Through natural selection, the advantageous or traits are passed on to the next
generation and more offspring will be able to survive and adapt better. On the other hand, a
trait that does not confer an advantage is unlikely to be passed over to the next generation due
to them not being favourable for survival. In other words, if a gene is lethal, it will tend to be

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