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Genetics and Molecular Biology

Acid-Adapted Strains of Escherichia coli K-12 Obtained by Experimental Evolution

Mark M. Harden, Amanda He, Kaitlin Creamer, Michelle W. Clark, Issam Hamdallah, Keith A. Martinez II, Robert L. Kresslein, Sean P. Bush, Joan L. Slonczewski
J. L. Schottel, Editor
Mark M. Harden
Department of Biology, Kenyon College, Gambier, Ohio, USA
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Amanda He
Department of Biology, Kenyon College, Gambier, Ohio, USA
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Kaitlin Creamer
Department of Biology, Kenyon College, Gambier, Ohio, USA
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Michelle W. Clark
Department of Biology, Kenyon College, Gambier, Ohio, USA
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Issam Hamdallah
Department of Biology, Kenyon College, Gambier, Ohio, USA
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Keith A. Martinez II
Department of Biology, Kenyon College, Gambier, Ohio, USA
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Robert L. Kresslein
Department of Biology, Kenyon College, Gambier, Ohio, USA
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Sean P. Bush
Department of Biology, Kenyon College, Gambier, Ohio, USA
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Joan L. Slonczewski
Department of Biology, Kenyon College, Gambier, Ohio, USA
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J. L. Schottel
Roles: Editor
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DOI: 10.1128/AEM.03494-14
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  • FIG 1
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    FIG 1

    Growth of evolved populations at pH 4.6. The endpoint growth of the 24 populations from generations 25, 730, and 2,000 was compared. Frozen populations were thawed and diluted 1:50 in LBKmal, pH 4.8, for initial recovery and then incubated in a microplate reader for 22 h. Culture was conducted for three subsequent days by daily dilution 100-fold at pH 4.6. On each day, cultures were incubated at 37°C in a microplate reader with shaking every 15 min. The final OD450 was measured after 22 h. Error bars represent SEMs (n = 24).

  • FIG 2
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    FIG 2

    Fitness changes in acid-evolved clones. For each pair of strains, the relative fitness (W) was calculated as described in Materials and Methods. To control for any fitness effect of the transduced lac-negative allele, the original lac+ ancestral wild type was cocultured with the lac-negative wild type (gray bars). (A) pH 4.6. Clones were serially diluted (1:200) in LBKmal buffered to pH 4.8 and incubated overnight twice prior to each experiment. Each isolate was then cocultured with a lac-negative variant of the ancestral wild type (or else the lac-negative isolate was cocultured with a lac+ ancestor) for 24 h in LBKmal buffered to pH 4.6. Error bars represent SEMs (n = 6 for evolved strains, n = 18 for ancestral strain W3110). (B) Benzoate. Strains were serially diluted 1:200 and incubated overnight twice in LBK medium buffered to pH 6.5. For the competition, each isolate and ancestor were cocultured for 24 h in LBK medium with 15 mM benzoic acid buffered with 100 mM PIPES at pH 6.5 (n = 4 for evolved strains, n = 8 for W3110) (C) pH 9.0. Strains were serially diluted and incubated overnight twice in LBK medium buffered to pH 8.5. For the competition, each isolate and ancestor were cocultured for 24 h in LBK medium buffered with 150 mM TAPS at pH 9.0 (n = 4 for evolved strains, n = 8 for W3110). *, P ≤ 0.05 (t test) in comparison with the results for W3110.

  • FIG 3
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    FIG 3

    Early-log-phase growth rates of acid-evolved clones in LBKmal buffered to pH 4.5. Clones were twice serially diluted (100-fold) and cultured overnight in LBKmal buffered to pH 4.8. Cultures were then diluted 1:200 into LBK medium buffered to pH 4.5 and then grown until mid-log phase at 37°C under aerated conditions. Growth rates were calculated from OD600 measurements taken every 20 min throughout early-log-phase growth. Error bars indicate SEMs (n = 3). *, significant differences (P < 0.05) from the results for ancestor strain W3110, based on t test.

Tables

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  • TABLE 1

    Strains isolated after 2,000 generationsa

    StrainPopulationIsolate no.
    JLSE0079H91
    JLSE0080H92
    JLES0083B111
    JLSE0084B112
    JLSE0091F111
    JLSE0092F112
    JLSE0137F92
    JLSE0138F93
    • ↵a The sequence data are available in the NCBI SRA under accession number SRP041420.

  • TABLE 2

    Lysine decarboxylase activity of clones from acid-evolved populationsa

    No. of generationsNo. of isolates in the following population testing negative for lysine decarboxylase activity:
    A7B7C7D7E7F7G7H7A9B9C9D9E9F9G9H9A11B11C11D11E11F11G11H11
    7601
    1,400123211223123111
    2,000333333333333333323333333
    • ↵a For each generation, three isolates from each population were assayed. The number of isolates of three isolates tested that lost lysine decarboxylase activity (indicated by a yellow color in Moeller broth) after 24 h of culture in a microplate is indicated.

  • TABLE 3

    Mutations identified in coding regions in the 2,000th generation clonesa

    GeneFunctionMutationIdentification of a mutation in clone:
    H9-1H9-2B11-1B11-2F11-1F11-2F9-2F9-3
    adiYActivator of adiAMissense (R119P)XX
    fluAG43 transport (biofilm)Missense (A162E)XX
    ftsXCell division (IM transporter)Missense (A101V)XX
    fucIl-Fucose isomeraseMissense (R363S)XX
    gcvPGlycine decarboxylaseMissense (T854A)X
    hepARNAP recycling factorInsertion (1139, +G)XX
    icdIsocitrate dehydrogenaseMissense (D398E)XXXXXX
    menHMenaquinone biosynthesisMissense (D79Y)XX
    metEHomocysteine transmethylaseMissense (R337S)X
    mhpBPhenylpropionate degradationMissense (D226Y)X
    mppAMurein tripeptide transporterInsertion (970, +A)XX
    msbALipid A transport IM to OMMissense (G150C)XX
    rlmE23S rRNA methyltransferaseMissense (P157L)X
    rpoBRNAP beta subunitMissense (A679V)XX
    rpoCRNAP beta prime subunitMissense (I774S)XX
    rpoCRNAP beta prime subunitMissense (V507L)XX
    rpoDRNAP sigma 70Missense (M273I)XX
    sprMurein endopeptidaseMissense (Q73K)X
    wzzEECA polysaccharide chain lengthMissense (P314Q)XX
    yeiHIM proteinMissense (A245S)X
    yejMIM hydrolaseInsertion (position 531) of 9 bp (TTTATCGCC)XXXX
    yfdIIM proteinMissense (V7L)X
    yfjKIonizing radiation resistanceMissense (T400I)X
    yjiKIM proteinSilent (TCG → TCA)X
    • ↵a The genomes of the experimental clones and the ancestral strain were sequenced and aligned with the sequence of the E. coli K-12 W3110 reference genome (35) using the breseq computational pipeline (http://barricklab.org/breseq). The mutations listed represent differences between the genome of the ancestor and the adapted clones, excluding intergenic mutations. An X signifies that the mutation is present in that clone. IM, inner membrane; OM, outer membrane; ECA, enterobacterial common antigen.

  • TABLE 4

    Intergenic mutations identified in the 2,000th generation acid-adapted clonesa

    Proximal gene(s)Mutation (positions, nucleotide change)Identification of a mutation in clone:
    H9-1H9-2B11-1B11-2F11-1F11-2F9-2F9-3
    matP →/← ompAIntergenic (+40/+36, C to A)X
    racR ←/→ ydaSIntergenic (−61/−63, C to A)X
    Y75_p4289 ←/→ eaeHIntergenic (−545/−1035, C to A)X
    proL →Noncoding (57/77 nt, C to A)XX
    nuoA ←/← lrhAIntergenic (−87/+544, C to A)XX
    yfjL ←/← yfjMIntergenic (−258/+102) +8 bp (GCACTATG)X
    rhaA →/→ rhaDIntergenic (+308/−143, C to A)XX
    rrsC ←Noncoding (1417/1542 nt, +A)XX
    pabA →/→ argDIntergenic (+34/−52, A to G)XX
    yjbI →/→ ubiCIntergenic (+84/−139, G to T)XX
    dcuA ←/← aspAIntergenic (−202/+104, G to T)XX
    acs ←/→ nrfAIntergenic (−228/−165, G to A)XX
    • ↵a The mutations listed indicate intergenic differences between the adapted clones and the ancestral wild type. A positive distance from a proximal gene represents the downstream location of the mutation relative to the gene's translation stop site, whereas a negative distance represents the mutation's upstream location relative to the gene's translation start site. nt, nucleotide position number.

Additional Files

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  • Supplemental material

    Files in this Data Supplement:

    • Supplemental file 1 -

      Sequence differences between E. coli K-12 strains W3110-D8 and W3110-D13 compared to the E.coli K-12 reference genome, NC_007779.1 (Table S1).

      PDF, 329K

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Acid-Adapted Strains of Escherichia coli K-12 Obtained by Experimental Evolution
Mark M. Harden, Amanda He, Kaitlin Creamer, Michelle W. Clark, Issam Hamdallah, Keith A. Martinez II, Robert L. Kresslein, Sean P. Bush, Joan L. Slonczewski
Applied and Environmental Microbiology Feb 2015, 81 (6) 1932-1941; DOI: 10.1128/AEM.03494-14

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Acid-Adapted Strains of Escherichia coli K-12 Obtained by Experimental Evolution
Mark M. Harden, Amanda He, Kaitlin Creamer, Michelle W. Clark, Issam Hamdallah, Keith A. Martinez II, Robert L. Kresslein, Sean P. Bush, Joan L. Slonczewski
Applied and Environmental Microbiology Feb 2015, 81 (6) 1932-1941; DOI: 10.1128/AEM.03494-14
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