Hamdallah, Issam

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Digital Kenyon: Research, Scholarship, and Creative ExchangeKenyon Summer Science Scholars Program Summer Student Research ScholarshipSummer 2014The Effects of high pH on acid adapted EscherichiacoliIssam HamdallahKenyon CollegeFollow 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 CitationHamdallah, Issam, "The Effects of high pH on acid adapted Escherichia coli" (2014). Kenyon Summer Science Scholars Program. Paper255.https://digital.kenyon.edu/summerscienceprogram/255Conclusions Methods Introduction The Effects of high pH on acid adapted Escherichia coli   Issam Hamdallah ‘16 and Joan L. Slonczewski Department of Biology, Kenyon College, Summer Science 2014 Abstract Escherichia coli possesses the ability to grow under a wide range of pH stresses.  E. coli can grow in moderate acid (pH 4.5 – 5.0) and can grow in moderate base (pH 9.0).  After 2,000 generations of growth in moderately acidic buffered complex media (pH 4.6), twenty-four E. coli populations independently evolved to acquire a fitness advantage when co-cultured with the ancestral D8 W3110 strain in buffered pH 4.6 medium.  Each of the sequenced evolved strains possessed at a least one mutation to RNApolymerase. Of the evolved strains, four (JLSE0079, JLSE0083, JLSE0091, and JLSE0137) were co-cultured with the ancestral D8 W3110 strain in moderately basic buffered pH 9.0 medium to assess the effects of newly acquired mutations beneficial in moderate acid. The mutations in evolved strains JLSE0091 and JLSE0137 were only slightly antagonistic, whereas mutations in evolved strains JLSE0079 and JLSE0083 caused a more significant reduction of fitness in moderate base.  Furthermore, an adaptive laboratory evolution (ALE) experiment was established by growing and re-diluted twenty-four independent D-8 W3110 E. coli populations in buffered moderately basic medium (pH 9.0).  After ~600 generations of growth, we have seen a significant improvement in growth and have adapted our E. coli strains to survive in pH 9.2 media. • Escherichia coli must survive exposure to acidic stresses as low as pH 1.5 in the stomach and exposure to basic stresses exceeding pH 10 at the pancreatic duct.   • During growth, E. coli’s cytoplasmic pH is normally maintained within the range of pH 7.4 to 7.9, yet it has the ability to grow within the range of pH 4.5 to 9.0.  • After 2,000 generations of growth in buffered complex medium (pH 4.6), the evolved strains have developed a fitness advantage over the ancestral wild-type strain when co-cultured in moderate acid.  • Each evolved strain has at least one missense mutation in RNApolymerase which affects numerous genes.  • This study aims to compete the ancestral strain with four evolved strains (JLSE0079, JLSE0083, JLSE0091, and JLSE0137) in buffered high pH media (pH 9.0) in order to assess any fitness effects, under alkaline stress, of newly acquired mutations beneficial in moderate acid.  • We began an adaptive laboratory evolution (ALE) experiment to assess how E. coli grows under alkaline conditions (pH 9.0).  Competition Assays  Both the ancestral E. coli wild-type D8 W3110 strain and an acid adapted strain (JLSE 0079, JLSE 0083, JLSE 0091, and JLSE 0137) were co-cultured in buffered pH 9.0 LBK media to determine the fitness of the evolved strains under alkaline stresses.  The lac- ancestral wildtype and lac+ evolved strains were cultured for 24 hours and rediluted (1:100) in new media and allowed to overnight. The competition cultures were prepared by adding equal volumes of ancestral and evolved culture into buffered pH 9.0 media.  The competition medium was then diluted (1:400,000) in M63 minimal glucose media and plated on X-Gal (4 μL/mL) plates. The remainder of the competition media was shaken at 37°C with aeration in water bath for 24 hours.  The cultures were then diluted (1:400,000) in M63 minimal glucose media and plated on X-Gal (4 μL/mL) plates.  The colony counts of the plates before and after the dilutions were used to determine the fitness of both the ancestral strain and the evolved strain.  The strain with the greatest growth rate was determined to outcompete the other strain.  Each competition assay was replicated on 6 plates.   High pH (pH 9.0) Adaptive Laboratory Evolution (ALE)  Each day, twenty-four E. coli D8 wild-type strains are independently cultured in 200 µL of LBK pH 9.2 buffered with 150 mM AMPSO on a 96-well plate.  After the cultures on the 96-well plate are allowed to grow in a spectramax reader at 37°C for 22 hours, a 1:100 dilution is taken from the previous culture day’s cultures and placed into fresh media.  The old plated are stored in a -80°C freezer after glycerol buffered to pH 7.5 is added to each individual culture.  The spectramax measures the OD 450 at 15 minute intervals.   Results References Acknowledgements • Weiner, L., Model, P. 1994. Role of an Escherichia coli stress-response operon in stationary-phase survival. Microbiology. 91:2191-2195 •C. Bori, L. Theraulaz, V. Mejean and C. Jourlin-Castelli. 2003. Anticipating an alkaline stress through the Tor phosphorelay system in Escherichia coli. Molecular Microbiology. 48: 211-233 •P. Small, D. Blankenhorn, D. Welty, E. Zinser and J. L. Slonczewski. 1994. Acid and base resistance in Escherichia coli and Shigella flexneri: role of rpoS and growth pH. Journal of Bacteriology. 176: 1729. • H. Carola, E. Screpanti, M. Venturi, A. Rimon, E. Padd, H. Michel 2005. Nature. Structure of the Na+/H+ antiporter and insights into the mechanism of action and regulation by pH. Vol. 435: 1197-1202 •E. Padan, E. Bibi, M. Ito, T.A. Krulwich. 2005. Alkaline pH homeostasis in bacteria: new insights. Biochimica et Biophysica Acta. 1717:67-88. • K.A. Glass, Loeffelholz J.M., Ford J.P., Doyle M.P. 1992. Fate of Escherichia coli O157:H7 as affected by pH or sodium chloride and in fermented, dry sausage. Applied and Environmental Microbiology. 58(8): 2513. •Cabo, J.J.S., and J.L Slonczewski. 2012. Diagraming the Alkaline Stress Response in Escherichia coli: RpoS Controlled Loci are Vital to High pH Resistance. Unpublished data.   Figure 1:  Fitness of four of the 2000th generation isolates relative to the ancestral wild-type strain when co-cultured for 24 hours in buffered pH 9.0 LBK medium. Figure 2: lac+ JLSE0079 v. lac- W3110 D8. LBK X-gal plate co-cultured with  a lac- wild-type strain and a lac+ JLSE0079 strain after exposure a 24 hour exposure to basic medium (pH9.0).  lac- wild-type  colonies are white and a lac+ JLSE 0079 colonies are  blue. Figure 3: Procedure used to conduct  the competition assays. pH 9.0 (Gen 33) pH 9.2 (Gen 620) Figure 4:  Grow curves of ALE cultures on a 96-well plate after 22 hours of growth in buffered LBK media. I would like to thank Dr. Slonczewski for her guidance and support throughout my undergraduate research.  I would also like to thank Michelle Clark and everyone else in the Kenyon bacteria lab for their help and support throughout my summer research.  Lastly, I would like to thank Kenyon College for the funding and opportunity to conduct scientific research this summer. This work was in part funded by grant MCB-1050080 from the National Science Foundation. • Moderate to heavy reduction in fitness of the 2000th generation isolates relative to the ancestral wild-type strain when co-cultured for 24 hours in buffered pH 9.0 LBK media.  • Strains JLSE0079 and JLSE0083 demonstrated a significant reduction of fitness when co-cultured with the ancestral wild-type strain in buffered pH 9.0 media for 24 hours.  • JLSE0091 and JLSE0137 demonstrated a moderate reduction of fitness when co-cultured with the ancestral wild-type strain in buffered pH 9.0 media for 24 hours.  • High variability in colony size and colony counts were exhibited under alkaline stress conditions.  • Colony sizes, colony counts, and ultimately fitness were not as consistent as they were when co-cultured under moderate acidic stress (the evolved condition).  • This high variability has also been demonstrated in other experiments that observe effects of observed in beneficial mutation in Escherichia coli.  • E. coli strains evolved increased growth rate after ~600 generation in buffered pH 9.0 medium  • E. coli strains have evolved to grow well at pH 9.2 pH 9.0 (Gen 580) 

The Effects of high pH on acid adapted Escherichia coli

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The Effects of high pH on acid adapted Escherichia coli

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