He, Amanda

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Digital Kenyon: Research, Scholarship, and Creative ExchangeKenyon Summer Science Scholars Program Summer Student Research ScholarshipSummer 2013Laboratory Evolution of Acid-Adapted Escherichiacoli StrainsAmanda HeFollow this and additional works at: https://digital.kenyon.edu/summerscienceprogramPart of the Biology CommonsThis Poster is brought to you for free and open access by the Summer Student Research Scholarship at Digital Kenyon: Research, Scholarship, andCreative Exchange. It has been accepted for inclusion in Kenyon Summer Science Scholars Program by an authorized administrator of Digital Kenyon:Research, Scholarship, and Creative Exchange. For more information, please contact noltj@kenyon.edu.Recommended CitationHe, Amanda, "Laboratory Evolution of Acid-Adapted Escherichia coli Strains" (2013). Kenyon Summer Science Scholars Program. Paper231.https://digital.kenyon.edu/summerscienceprogram/231Laboratory Evolution of Acid-Adapted Escherichia coli Strains Amanda He ’16 and Joan L. Slonczewski Department of Biology, Kenyon College, Summer Science 2013 Abstract Results I would like to thank Dr. Joan Slonczewski for her endless support and guidance throughout the project. I would especially like to thank Keith Martinez, M. Michael Harden, Jr., and R. Luke Kresslein for starting and maintaining the ALE experiment during the 2012-2013 school year. Without their contribution, this project would not have been possible. In addition, I would like to thank Meredith Dunn and fellow Slonczewski lab members for media preparation and entertainment throughout the duration of this experiment. The experience would not have been the same without this group. This work was funded by the NSF grant MCB-0644167 and the Kenyon College Summer Science Program.  1. Blount, ZD, Barrick, JE, Davidson, CJ, Lenski, RE. 2012. Genomic analysis of a key innovation in an experimental Escherichia coli population. Nature 489: 513-518. 2. Foster, J.W., Escherichia coli acid resistance: tales of an amateur acidophile. Nat. Rev. Microbiol., 2004. 2: p. 898-907. 3. Haneburger, I., Fritz, G., Jurkschat, N., Tetsch, L., Eichinger, A., Skerra, A., Gerland, U., Jung, K. 2012. Deactivation of the E. coli pH Stress Sensor CadC by Cadaverine. Elsevier, 424(1-2): 15-27. 4. Hughes, BS., Cullum, AJ, Bennett, AF. 2007. Evolutionary Adaptation to Environmental pH in Experimental Lineages of Escherichia Coli. Evolution 61(7): 1725-1734. 5. Sleight, SC., Orlic, C., Schneider, D., Lenski, RE. 2008. Genetic Basis of Evolutionary Adaptation by Escherichia coli to Stressful Cycles of Freezing, Thawing, and Growth. Genetics 180: 431-443. 6. Woods, RJ., Barrick, JE., Cooper, TF., Shrestha, U., Kauth, MR., Lenski, RE. 2011. Second-Order Selection for Evolvability in a Large Escherichia coli Population. Science 331: 1433-1435. Figure 2: Evolved strains show higher growth rates than W3110 in moderate acid with malate. A) Doubling rate of evolved isolates and parental strain in presence of malate B) Doubling rate without malate present. Error bars = SEM. GLM, PpH and PpH*malate < 0.05 for all three groups. Pmalate < 0.05 for W3110, but Pmalate > 0.05 for evolved isolates. Introduction Methods Conclusions Acknowledgements References The gram negative bacterium, Escherichia coli, is able to grow in an external pH range of 5 to 9 [1]. E. coli have an intricate regulatory network, which allows for survival in acidic environments [2]. One of these regulatory networks is the utilization of lysine decarboxylase (CadA). CadA is an enzyme which converts the amino acid, lysine, into cadavarine [3]. In this process, CO2 is removed from lysine and replaced with a proton.   By evolving a standard strain of E. coli to maintain growth in low pH, it would be easier to identify what genes were utilized to maintain acid resistance. Preliminary experiments have shown that E. coli are capable of evolving traits in response to their environment [1].   In these series of experiments, we anticipated the E. coli populations grown in moderate acid for 1500 generations would show increased growth in low pH in comparison to the parental E. coli strain. Laboratory Evolution Since September 17, 2012, an overnight culture of W3110 was diluted 1:4000 into LBK, 100mM HOMOPIPES 6g/L D/L malate, pH 4.8. E. coli growth was monitored with a spectrophotometer, set at 37°C to measure the absorbance for twenty-two hours at OD450 in intervals of fifteen minutes daily [5]. Within two hours of reading completion, cultures from the plate were diluted 1:4000 into fresh medium in a new sterile well plate. Over the summer, the medium was changed to LBK, 100mM HOMOPIPES 10g/l D/L malate, pH 4.6, and dilutions increased to 1:100. m Growth Curves Overnight cultures were grown in LBK, pH 7.0 at 37°C, rotating. Overnights were diluted 1:400 into 20ml of buffered LBK with and without malate. The dilutions were shaken in aerobic conditions in the water shaker, set at 37°C. Cultures were read at OD600. Each strain had three replicates. Growth rates and mean doubling rate was determined from linear growth. m Lysine Decarboxylase Test Isolated colonies were transferred into a 96-well plate, each well containing 200µl of lysine decarboxylase broth, pH 6.5. The well plate incubated anaerobically at 37°C overnight. Color change from purple to yellow indicates that the pH is <5.2; lysine decarboxylase is not present.       1-30-2013              2-15-2013              3-15-2013              4-19-2013               6-19-2013   1-30-2013        2-15-2013         3-15-2013        4-19-2013         5-31-2013       6-17-2013 H9 A7 A11 Figure 3: Evolved strains lost function of lysine decarboxylase (CadA). A) Enterotube tests showed that isolates in H9 population had lost CadA by mid-April. Purple indicates that lysine decarboxylase is present. Yellow represents lack of a functional lysine decarboxylase. B/C) Using the lysine decarboxyase broth test, we estimated CadA loss appeared in H9 population during late-May, in A7 population around mid-April, and in A11, in late-January. A) B) C) Figure 1: The growth curves of evolved strain populations. Each well contains a population of strains grown in moderate pH for over 1500 generations. Populations from column 7, 8, and 9 were bifurcated from columns 1, 3, and 5 respectively on January 7, 2013 in pH 4.6 media, while column 1, 3, and 5 were continued on pH 4.8 media until May 17, 2013.  Evolved strains are able to utilize the presence of malate to grow in moderate acid.  • The evolved strains consistently had a higher growth rate in pH 4.5 and 4.6 than the parental strain (W3110) only in the presence of malate (Figure 2)  • Without malate, the evolved strains do not consistently show faster growth rates than the parental strain  Acid-adapted strains showed slightly slower growth than parent at high pH.  • As evolved strains have been grown in a moderate acid, it is possible that the ability to grow in moderately basic environments was reduced or lost in order to upregulate the acid growth  Evolved strains lost lysine decarboxylase.  • Although lysine decarboxylase has been a known enzyme used in bacteria to regulate acid stress, the evolved strains have lost this expression (Figure 3)  • Populations such as A11 show loss of lysine decarboxylase prior to a change in environment from pH 4.8 to 4.6  •The loss of lysine decarboxylase could be an adaptation to accommodate moderate acid growth, which could be detrimental to its survival in extreme acids  As we continue growing our acid-adapted strains in moderate acid, we anticipate the populations to further develop its acid resistance. We will track the progress of this development through fitness competition assays with parental strains in moderately acidic environments. In addition, we plan to determine at what time period each population began to lose lysine decarboxylase and how it has affected their growth and survival in various pH. In the near future, we foresee submitting isolates from the populations as well as the parental strain to be sequenced for genomic analysis for comparison.  Escherichia coli are normally able to grow at an external pH range of 5 to 9 [2]. In this experiment, we investigated whether E. coli were able to grow below pH 5. We grew twenty-four replicates of E. coli in moderately acidic media with malate, a carbon source which consumes acid. These populations grew and adapted in pH 4.8 media for 730 generations in pH 4.8 since September 17, 2012 before being lowered on January 7, 2013 to pH 4.6 for 770 generations. We predicted that the evolved strains would have a higher growth rate than the parental strain in moderate acid. The growth curves showed that the evolved strains had adapted specifically to the media they were grown in. Evolved strains showed faster growth in acid with malate than parental strains. Without the presence of malate, the evolved strains showed varied growth in comparison to the parental. An Enterotube test battery revealed the loss of lysine decarboxylase in wells A7, H9, and A11, which is known to be beneficial in combating acid stress in bacteria. As we continue to grow the evolved strains in moderate acid, we anticipate more acid resistant development. Glu Lys Orn  Ind Ado Lac Ara Sor  VP  Dul  Ure  Cit Jan 30   Feb 15   Mar 15   Apr 19  May 31    Jun 17 A) B)  1-30-13   2-15-13  3-15-13 4-19-13  5-31-13 6-17-13    1-30-13  2-15-13  3-15-13  4-19-13             6-17-13    1-30-13  2-15-13  3-15-13  4-19-13             6-17-13   H9       A7       A11     W3110 

Laboratory Evolution of Acid-Adapted Escherichia coli Strains

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Laboratory Evolution of Acid-Adapted Escherichia coli Strains

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