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Adaptive Evolution of Escherichia coli: Growth Kinetics and Genetic Changes from 2 to 10% Halophilization

by Lim, Joshua Zhi Rui; How, Jian Ann; Goh, Desmond Jian Wei

Abstract (Summary)
Escherichia coli (E. coli) is a Gram negative, rod-shaped bacterium, commonly found in the lower human intestine. Salt (NaCl, sodium chloride) is present in the human diet and constantly interacts with E. coli in the intestine. High consumption of salt may pose adverse effects on the growth of E. coli. In order to adapt to the changes to its environment, E. coli may undergo halophilization. In this study, we observed the growth kinetics and genetic changes of E. coli under increasing concentrations of 3% - 10% NaCl over 80 passages. Adaptability of E. coli was estimated by generation time and cell density at stationary phase. Minimum Inhibitory Concentration Experiment (MIC) was used to confirm the resistance of E. coli to the range of salt stress. Colony MIC was conducted to observe the deviation in mutations of the E. coli cells more accurately. Our results demonstrated that E. coli adapted from 1% NaCl to 8% NaCl at the rate of about 1% increment per month. Our colony MIC results demonstrated that the area under the MIC curve where NaCl is above 7.5% increased from 5% at passage 44 (cultured in 5% NaCl) to 13% at passage 72 (cultured at 7% NaCl). Polymerase Chain Reaction and Restriction Fragment Length Polymorphism were used to analyse the adaptation and mutation of E. coli at a genomic level. The amplification and digestion profiles were analysed using Nei-Li Dissimilarity and demonstrated an increasing trend of genomic distance across passages suggesting that the genomes of the E. coli has changed over the course of the 80 passages.
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Advisor:Maurice HT Ling

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Source Type:Other

Keywords:escherichia coli, adaptation, experimental evolution, halophilization

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Date of Publication:01/08/2011

Document Text (Pages 1-10)

SINGAPORE POLYTECHNIC

SCHOOL OF CHEMICAL AND LIFE SCIENCES

Diploma in Biotechnology
General Biotechnology Option

Adaptive Evolution of Escherichia coli:
Growth Kinetics and Genetic Changes

from 2 to 10% Halophilization

Project Code: DBTBTech1006

Lim Zhi Rui Joshua (0819194)

How Jian Ann (0819842)
Desmond Goh Jian Wei (0819433)
Year of Study: Year 3

Project Supervisor: Maurice Ling

AY2010/2011


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Abstract

Escherichia coli (E. coli) is a Gram negative, rod-shaped bacterium, commonly found in the
lower human intestine. Salt (NaCl, sodium chloride) is present in the human diet and
constantly interacts with E. coli in the intestine. High consumption of salt may pose adverse
effects on the growth of E. coli. In order to adapt to the changes to its environment, E. coli
may undergo halophilization. In this study, we observed the growth kinetics and genetic
changes of E. coli under increasing concentrations of 3% - 10% NaCl over 80 passages.
Adaptability of E. coli was estimated by generation time and cell density at stationary phase.
Minimum Inhibitory Concentration Experiment (MIC) was used to confirm the resistance of
E. coli to the range of salt stress. Colony MIC was conducted to observe the deviation in
mutations of the E. coli cells more accurately. Our results demonstrated that E. coli adapted
from 1% NaCl to 8% NaCl at the rate of about 1% increment per month. Our colony MIC
results demonstrated that the area under the MIC curve where NaCl is above 7.5% increased
from 5% at passage 44 (cultured in 5% NaCl) to 13% at passage 72 (cultured at 7% NaCl).
Polymerase Chain Reaction and Restriction Fragment Length Polymorphism were used to
analyse the adaptation and mutation of E. coli at a genomic level. The amplification and
digestion profiles were analysed using Nei-Li Dissimilarity and demonstrated an increasing
trend of genomic distance across passages suggesting that the genomes of the E. coli has
changed over the course of the 80 passages.

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Acknowledgement

First and foremost, we would like to express our heartfelt gratitude to our project supervisor,
Dr. Maurice Ling Han Tong of Singapore Polytechnic for his valuable guidance, advice,
support and concern throughout the entire project.

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

Special thanks to the group of friends around us who provided assistance and given us
encouragement during the course of the project: Samuel, Zhen Qin, Bryan, Chin How, Jack,
Kun Cheng and Wei Chuan.

Lastly, we would like to thank Singapore Polytechnic (Account number: 11-27801-45-2551)
for financially sponsoring our project.

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Table of Contents

Abstract ..................................................................................................................................... 2
Acknowledgement..................................................................................................................... 3
List of Figures and Tables......................................................................................................... 6
Abbreviations ............................................................................................................................ 7
1. Introduction........................................................................................................................ 8
2. Literature Review............................................................................................................... 9

2.1 Evolutionary Studies Done on Enteric Bacteria ......................................................... 9
2.2 Halophilization of Bacteria....................................................................................... 10
2.3 Factors Affecting the Survival of Bacteria under Salt Stress ................................... 10
2.4 Past Research on Escherichia coli ............................................................................ 12
2.5 Biofilm Formation in Escherichia coli ..................................................................... 14
2.6 Adaptation Study on Escherichia coli ATCC 8739.................................................. 15
2.7 Minimum Inhibitory Concentration on Escherichia coli.......................................... 16
2.8 Objectives and Hypothesis of Project....................................................................... 18
3. Methods and Materials..................................................................................................... 19

3.1 Subculture................................................................................................................. 19
3.2 Glycerol Stock .......................................................................................................... 19
3.3 Generation Time and Cell Density ........................................................................... 19
3.4 Minimum Inhibitory Concentration ......................................................................... 19
3.5 Colony Minimum Inhibitory Concentration............................................................. 20
3.6 Extraction of Total Genomic DNA from E. coli Cell Cultures ................................ 20
3.7 Polymerase Chain Reaction...................................................................................... 20
3.8 Restriction Fragment Length Polymorphism ........................................................... 21
3.9 Data Analysis............................................................................................................ 21
4. Results.............................................................................................................................. 23

4.1 Number of Generations ............................................................................................ 23
4.2 Generation Time ....................................................................................................... 24
4.3 Day 7/Day 5 Cell Density Ratio ............................................................................... 27
4.4 Minimum Inhibitory Concentration (MIC) – R2 Values .......................................... 30
4.5 Minimum Inhibitory Concentration ......................................................................... 32
4.6 Colony MIC .............................................................................................................. 35
4.7 Polymerase Chain Reaction / Restriction Fragment Length Polymorphism............ 35
5. Discussion ........................................................................................................................ 37

5.1 Increase NaCl Concentration Increases Generation Time........................................ 37
5.2 Decrease in Generation Time in Same NaCl Concentration until Next Concentration
Increase................................................................................................................................ 37
5.3 Comparing Generation Time with Lee et al. (2010) ................................................ 38
5.4 Stationary Phase of Escherichia coli ........................................................................ 38
5.5 Generations of Escherichia coli................................................................................ 40
5.6 Fourth Power Polynomial Fitting of the Data Taken at 21 to 23 Hours Postinoculation
is Accurate for Analysis of MIC Data.............................................................. 40
5.7 E. coli Cells Adapt to 1% Increase in NaCl in about One Month ............................ 41
5.8 Cells from Later Passages are More Genetically Different from the Original Cells 45
6. Recommendations............................................................................................................ 47
7. Conclusion ....................................................................................................................... 48
8. References........................................................................................................................ 49
Appendix A – Generation Time .............................................................................................. 53
Appendix B – 2 Day Generations and Cell Density................................................................ 57

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Appendix C – Cell Density at Stationary Phase...................................................................... 67
Appendix D – Minimum Inhibitory Concentration (MIC) ..................................................... 72
Appendix E – Colony MIC ..................................................................................................... 78
Appendix F – Gram Staining Photos....................................................................................... 87
Appendix G – PCR/RFLP Agarose Gel Photos ...................................................................... 91
Appendix H – Dissimilarity Matrix ...................................................................................... 133
Appendix I – MIC Analysis Script........................................................................................ 135

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List of Figures and Tables

Table 2.1: Salt tolerance of different bacteria (Garrity et al, 2003).
Figure 4.1: Comparison of 2-Day Generations Across Tubes A, B, C and D.
Figure 4.2: Comparison of 2-Day Generations Across Tube A, B, C and D Trend Line.
Figure 4.3: Comparing the generation time of four tubes: Tube A, Tube B, Tube C and Tube
D across 80 passages.
Figure 4.4: Generation time of the four tubes: Tube D, Tube B, Tube A and Tube C
respectively in order across 80 passages.
Table 4.1: Tests of between – subjects effects in relation to generation time.
Table 4.2: Table of coefficient of variation for all four treatments, from 3% NaCl to 8% NaCl
Figure 4.5: Coefficient of variation for all four tubes over 80 passages.
Figure 4.6: Ratio of day 7 to day 5 cell density of Tubes A, B, C and D over 80 Passages.
Tube A (A), Tube B (B), Tube C (C), Tube (D).
Table 4.3: Fitting of the MIC graphs using different polynomial equations.
Figure 4.7: Fitting of Polynomial Equations to MIC (Tube A, 19 hours).
Figure 4.8: x4 fitted MIC for Tube A taken at 17, 19, 21 and 23 hours post-inoculation.
Figure 4.9: Concentration of NaCl (%) whereby the OD is at maximum.
Figure 4.10: Concentration of NaCl (%) whereby the OD is at half of maximum.
Figure 4.11: Area under the curve whereby the concentration of NaCl is more than 7.5%.
Figure 4.12: Growth Curve at 11% NaCl, 21 to 23 hours post-inoculation.
Table 4.4: Table of Means and Standard Deviations (SD) of Colony MIC for Passage 44, 53
and 72.
Figure 4.13: Dissimilarity Indices across 80 passages, error bars are +/-2σ.
Figure 5.1: Linear fittings of Day 5 Cell Density; A (Tube A), B (Tube B), C (Tube C) and D
(Tube D).

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Abbreviations

AUC
BSA
DI
DNA
dNTPs
EDTA
MIC
NB
STE

Area under Curve
Bovine Serum Albumin
Dissimilarity Index
Deoxyribonucleic Acid
Deoxyribonucleotide Triphosphates
Ethylenediaminetetraacetic Acid
Minimum Inhibitory Concentration
Nutrient Broth
Sodium Chloride–Tris–EDTA

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1. Introduction

Escherichia coli, a Gram negative bacterium, is commonly found in the lower intestine of
warm blooded organisms. They are part of the normal flora of the gut and can produce
vitamin K which is needed for clotting of blood and prevent establishment of pathogenic
bacteria in the intestine.

The adaption and evolution of E. coli to develop a resistance to antibiotics and drugs are
widely studied but the mechanisms to non-antibiotic agents, such as salt are less understood.
Salt, which is used as a common food addictive, is added to preserve food and inhibits growth
of microorganisms by drawing water out of the cells of both microbe and food through
osmosis. As E. coli is constantly exposed to the salt present in the faecal matter, it is
important to investigate their relationship on how E. coli copes with the change in
environment.

Our project aims to observe the adaptation of E. coli cultured in NaCl supplemented medium
over 80 passages. Adaptability over time is estimated by generation time and cell density of
the stationary phase. Polymerase Chain Reaction (PCR) and Restriction Fragments Length
Polymorphism (RFLP) are also used to characterize adaptation/evolution at genomic level.

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2. Literature Review

2.1 Evolutionary Studies Done on Enteric Bacteria
The enteric bacteria belong to a group of bacteria that live in the gastrointestinal tract of
humans and mammals. They are of special concern because they share a symbiotic
relationship to mammals and as humans are a mammalian species, it is also important for us.
For example, bacteria of the Lactobacilli strain are able to curb the growth of other
pathogenic bacteria such as Streptococcus pyogenes (Westbroek et al., 2010). Lactobacilli are
able to inhibit the growth of pathogenic bacteria because they produce lactic acid,
bacteriocins and hydrogen peroxide (Westbroek et al., 2010). Members of the enteric bacteria
family are also able to inhibit the growth of Escherichia coli O157:H7 (Toshima et al., 2007).
E. coli O157:H7 is one of the pathogenic strains of E. coli and is able to cause haemorrhagic
colitis. E. coli O157:H7 infects the large intestine and produces Shiga toxin that causes
bloody diarrhoea. If untreated, haemorrhagic colitis can cause death; hence, the enteric
bacteria are important as a defence and guarding mechanism which is able to keep the
pathogenic bacteria in check. Many of which, help protect us from diseases that arise from
the gastrointestinal tract. The non-pathogenic strain of E. coli which is found naturally in the
gastrointestinal tract is able to produce Vitamin K2, a vitamin that is important in the
coagulation of blood (Bently and Meganathan, 1982). Evolutionary studies (Cai et al., 2009;
Lozupone et al., 2008) had shown how these enteric bacteria have adapted to the conditions
within the host and how these bacteria share a symbiotic relationship with us. Comparison
between the genomes of enteric bacterial species shows that various genetic convergences
may arise due to the fact that the different species of bacteria are exposed to the same
environment (Lozupone et al., 2008). The balance of the symbiotic relationship between
these organisms and humans, which is the result of the many interactions between the
bacteria and the host, can be better understood.

Evolutionary studies were also done on various strains of enteric bacteria such as on
Lactobacillus casei ATCC 334 (Cai et al., 2009) or whole population of enteric bacteria
(Lozupone et al., 2008). These studies revealed that genetic interactions such as horizontal
gene transfer had occurred between certain bacterial species. These genetic interactions are
crucial in the evolution of the enteric bacteria as they can allow different bacterial strains to
acquire genes which originate from other strains. A study (Cai et al., 2009) compared the

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genomes between Lactobacillus casei and other members of the Lactobacilli family revealed
that L. casei contains many Insertion Sequence (IS) elements that result in the expression of
proteins that are involved in the metabolism of various carbohydrate substrates. The ability to
metabolize a wide variety of carbohydrate compounds is essential for the survival of L. casei.
The IS elements were also found to be similar in a genus of enteric bacteria, Enterococcus.
The IS elements could have been transferred over to L. casei from Enterococcus by
horizontal gene transfer between the two bacterial strains. Another study done on the
microbes in the gastrointestinal tract revealed that the metabolic genes of the microbes have
converged (Lozupone et al., 2008). The convergence is likely to be caused by horizontal gene
transfer and parallel gene loss between different species of bacteria. This is because all of the
microbes in the gastrointestinal tract have been exposed to the same environment. The
environmental stress experienced by all of the microbes should be the same; therefore, the
convergence in the metabolic genes could be because of the same stress that is experienced
by the microbes.

2.2 Halophilization of Bacteria
Halophilization is the gradual adaptation of the organism to the inhibitory effects of salt
(sodium chloride; NaCl) by introducing increasing concentrations of salt into the growth
environment of the organism. The organism normally thrives best in environmental
conditions where salt is present in low concentrations resembling the natural environment of
which the organism lives. The addition of salt presents a number of stresses, including
osmotic stress and ionic stress (Burg et al., 2007). These stresses can cause cell death if the
cells were not adapted to growing in higher salt concentrations.

2.3 Factors Affecting the Survival of Bacteria under Salt Stress
Halophiles are a category of extremophile organisms which thrive in environments of high
salt concentrations. They are generally classified as mildly, moderately and extremely
halophilic (Nester et al., 2004), with a range of 0-5% NaCl tolerance for mildly halophilic, 0-
6.5% NaCl tolerance for moderately halophilic, and 3-15% NaCl tolerance for extremely
halophilic organisms (Garrity et al., 2003). On another hand, halotolerant organisms are not
naturally salt adapted organisms, but had adapted to high salinity over the course of time.

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