Article Figures & Data
Figures
- 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
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
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
- TABLE 2
Lysine decarboxylase activity of clones from acid-evolved populationsa
No. of generations No. of isolates in the following population testing negative for lysine decarboxylase activity: A7 B7 C7 D7 E7 F7 G7 H7 A9 B9 C9 D9 E9 F9 G9 H9 A11 B11 C11 D11 E11 F11 G11 H11 760 1 1,400 1 2 3 2 1 1 2 2 3 1 2 3 1 1 1 2,000 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 3 3 3 3 3 3 3 ↵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
Gene Function Mutation Identification of a mutation in clone: H9-1 H9-2 B11-1 B11-2 F11-1 F11-2 F9-2 F9-3 adiY Activator of adiA Missense (R119P) X X flu AG43 transport (biofilm) Missense (A162E) X X ftsX Cell division (IM transporter) Missense (A101V) X X fucI l-Fucose isomerase Missense (R363S) X X gcvP Glycine decarboxylase Missense (T854A) X hepA RNAP recycling factor Insertion (1139, +G) X X icd Isocitrate dehydrogenase Missense (D398E) X X X X X X menH Menaquinone biosynthesis Missense (D79Y) X X metE Homocysteine transmethylase Missense (R337S) X mhpB Phenylpropionate degradation Missense (D226Y) X mppA Murein tripeptide transporter Insertion (970, +A) X X msbA Lipid A transport IM to OM Missense (G150C) X X rlmE 23S rRNA methyltransferase Missense (P157L) X rpoB RNAP beta subunit Missense (A679V) X X rpoC RNAP beta prime subunit Missense (I774S) X X rpoC RNAP beta prime subunit Missense (V507L) X X rpoD RNAP sigma 70 Missense (M273I) X X spr Murein endopeptidase Missense (Q73K) X wzzE ECA polysaccharide chain length Missense (P314Q) X X yeiH IM protein Missense (A245S) X yejM IM hydrolase Insertion (position 531) of 9 bp (TTTATCGCC) X X X X yfdI IM protein Missense (V7L) X yfjK Ionizing radiation resistance Missense (T400I) X yjiK IM protein Silent (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: H9-1 H9-2 B11-1 B11-2 F11-1 F11-2 F9-2 F9-3 matP →/← ompA Intergenic (+40/+36, C to A) X racR ←/→ ydaS Intergenic (−61/−63, C to A) X Y75_p4289 ←/→ eaeH Intergenic (−545/−1035, C to A) X proL → Noncoding (57/77 nt, C to A) X X nuoA ←/← lrhA Intergenic (−87/+544, C to A) X X yfjL ←/← yfjM Intergenic (−258/+102) +8 bp (GCACTATG) X rhaA →/→ rhaD Intergenic (+308/−143, C to A) X X rrsC ← Noncoding (1417/1542 nt, +A) X X pabA →/→ argD Intergenic (+34/−52, A to G) X X yjbI →/→ ubiC Intergenic (+84/−139, G to T) X X dcuA ←/← aspA Intergenic (−202/+104, G to T) X X acs ←/→ nrfA Intergenic (−228/−165, G to A) X X ↵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.
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
- Supplemental file 1 -
