Article Figures & Data
Figures
- FIG. 1.
Phylogenetic relationships among E. coli isolates that evolved in a single population. The sympatric divergence of phenotypic, genotypic, and metabolic characteristics of evolved clones was analyzed by the neighbor-joining method rooted with the ancestral strain, BW2952, as described by Pupo et al. (37). Cluster A contains rpoS mutants, and cluster B strains are low-ppGpp strains (27). The dendrogram (a) was based on the 11 characteristics of 41 isolates described in reference 27. The metabolome tree (b) was based on spot matching and quantitation as described in reference 29. The number of informative spots compared for all isolates was 177. The Biolog tree (c) was based on the 23 informative substrates differentially utilized by the isolates and ancestor shown in Table 2, including all members of cluster B. The bootstrap values at nodes are percentages based on 1,000 replications.
- FIG. 2.
Heterogeneity of metabolic processes in coevolved bacteria. The thickness of the arrows indicates the magnitude of the measured fluxes in the ancestral strain (light box) and isolates (shaded boxes). The rates of glucose and oxygen uptake and the rates of production of biomass, CO2, and acetate are based on the data in Table 1. The numbers in the ellipses are the last two digits of the strain numbers in Table 1. Dashed lines indicate undetectable rates.
- FIG. 3.
Metabolome profiles for chemostat-evolved and ancestral clones. The metabolites were extracted (28) from E. coli K-12 strain BW2952 (a and d), the ancestral strain in chemostat experiments (33), and BW3767, a cluster A isolate obtained after 90 generations (b and e). The images are PhosphorImager outputs for 14C-labeled metabolites separated by two-dimensional HPTLC using pairs of solvent systems. In panels a and b the compounds were separated using solvent system A, and panels d and e were obtained using solvent system B (28, 44). Resolved spots in metabolome fingerprints were matched using the Phoretix software (30). (c and f) Results of an analysis of matched spots in the evolved and ancestral metabolomes with solvent systems A and B, respectively. ▿, metabolite spots that were down-regulated; ▵, metabolite spots that were up-regulated; −, metabolite spots that were not present; +, metabolite spots that were unique in the evolved clone. Spots with no symbol were the metabolites whose proportions were the same in the two metabolomes. (g) Variation in the metabolome proportions for four metabolite spots in the isolates. The spots identified as glutamate, UDP-hexose, and fumarate using methods described previously (28, 44) were quantitated in four replicate metabolome estimations. The error bars indicate standard deviations.
- FIG. 4.
Metabolome profiles of ancestral and evolved clones. The images are the phosphorimages of 14C-labeled metabolites of ancestral strain BW2952 and isolates BW3767, BW4001 to BW4006, BW4029, and BW4036 that coevolved in the 90-generation chemostat population. Extracts were prepared and separated by two-dimensional HPTLC using solvent systems A (bottom 10 images) and B (top 10 images) as described in the legend to Fig. 3.
Tables
- TABLE 1.
Properties of the ancestor (BW2952) and isolates coadapted to glucose limitationa
Strain Maximal growth rate on glucose (h−1)b Glucose uptake rate (pmol/min/108 cells)c Contribution of Mgl to glucose transportd Contribution of PtsG to glucose transporte Oxygen uptake rate (nmol O2/min/108 cells)f Biomass yield (g g−1)g Acetate production (g g−1)h Rate of glucose conversion to CO2 (pmol CO2/min/108 cells)i Fitness relative to ancestorj Ecostrategyk BW2952 0.69 ± 0.01 120 ± 19 49 ± 13 64 ± 1 5.9 ± 0.22 0.41 ± 0.01 <0.01 180 ± 11 = Ancestor BW3767 0.77 ± 0.02 890 ± 74 1600 ± 349 100 ± 11 8.8 ± 0.33 0.52 ± 0.02 <0.01 250 ± 28 +++ Mixed K and r BW4001 0.53 ± 0.01 180 ± 81 230 ± 10 150 ± 26 10.8 ± 0.11 0.29 ± 0.01 <0.01 560 ± 44 +++ R BW4002 0.77 ± 0.03 400 ± 190 240 ± 34 270 ± 100 8.3 ± 0.22 0.48 ± 0.03 <0.01 210 ± 23 +++ Mixed K and r BW4003 0.69 ± 0.02 130 ± 41 37 ± 17 86 ± 56 7.5 ± 0.11 0.40 ± 0.01 <0.01 270 ± 25 ++ ? BW4004 0.69 ± 0.05 960 ± 76 1700 ± 189 200 9.2 ± 0.90 0.46 ± 0.01 <0.01 230 ± 21 +++ Mixed K and r BW4005 0.66 ± 0.01 97 ± 6 27 ± 32 63 ± 23 7.2 ± 0.11 0.34 ± 0.02 0.08 ± 0.01 240 ± 22 − New ecotype BW4006 0.50 ± 0.02 370 ± 250 149 210 ± 145 10.1 ± 0.67 0.29 ± 0.01 <0.01 530 ± 30 +++ r BW4029 0.69 ± 0.01 140 ± 17 67 ± 26 85 ± 29 4.8 ± 0.22 0.52 ± 0.03 <0.01 96 ± 13 +++ K BW4036 0.69 ± 0.01 92 ± 4 35 ± 4 49 ± 4 7.2 ± 0.00 0.40 ± 0.02 <0.01 81 ± 8 = ? ↵ a The values are means ± standard deviations for replicates (n ≥ 2).
↵ b Growth rates were determined from the exponential growth phase for isolates and the wild type in minimal medium with 0.05% (wt/vol) glucose.
↵ c Measured using the rate of uptake of [14C]glucose by bacteria growing in a chemostat at a rate of 0.1 h−1. See Materials and Methods for details.
↵ d Measured using the Mgl-specific substrate [14C]galactose. The galactose transport rate shown is expressed in pmol/min/108 cells.
↵ e Measured using the PtsG-specific analog methyl-α-[14C]glucoside. The methyl-α-glucoside transport rate is expressed in pmol/min/108 cells. The large errors in some of the values reported were due to instability between replicate cultures, probably due to unstable genomic rearrangements affecting transporter genes.
↵ f The glucose-dependent respiration rate was measured for cultures transferred to a Clark oxygen electrode. See Materials and Methods for details.
↵ g Biomass yields (expressed in grams of cell dry mass produced per gram of glucose) for isolates and the wild type in the chemostat culture were determined from individual dry weight determinations.
↵ h Acetate production (expressed in grams of acetate per gram of glucose) by isolates and the wild type was determined by using the culture medium of 48-h-old glucose-limited chemostats grown at a rate of 0.1 h−1.
↵ i Rates of [14C]glucose oxidation to 14CO2 were determined as described in Materials and Methods.
↵ j Levels of fitness in glucose-limited chemostats were compared by using 50/50 starting population mixtures of the ancestor and an isolate. For ancestor-ancestor competition, a neutral marker was introduced into one competitor as previously described (27). +++, isolate eliminated ancestor within 72 h; =, no change; −, ancestor dominated but did not eliminate isolate; ++, isolate dominated but did not eliminate ancestor within 72 h (27).
↵ k The groups are discussed in the text.
- TABLE 2.
Informative substrates from Biolog analysis of evolved isolatesa
Substrate Response BW2952 BW3767 BW4001 BW4002 BW4003 BW4004 BW4005 BW4006 BW4009 BW4007 BW4008 BW4010 BW4011 BW4012 BW4013 BW4029 BW4036 Dextrin − + − + − + − − − − − − − − − − − Glycogen − − − − − − − + + + + − − − − − − Tween 80 − − − − + − − − − − − − − − − − − Lactulose − + + + − + − + + + + + + + + − − Maltose − + − + − + − − − − − − − − − − − β-Methyl-d-Glucoside − + − + − + + + − + − − + + + − − Turanose + − − − − − − − − − − − − − − − − Pyruvic acid methyl ester − + + + + + + + + + + + + + + + + Succinic acid mono methyl ester − + − − − − − − + + + + + − + − − Acetic acid + − + − − − + + + + + + + + + + + Formic acid − − + − − − − + + + + + + + + − − α-Ketoglutaric acid − + + + − + − + + + + + + + + − − α-Ketovaleric acid − − − − − + − − − − − − − − − − − Sacchiric acid − + + + − + − − + + + + + + + − − Succinic acid − + + + − + − + + + + + + + + − − Bromosuccinic acid − − − − − − − − − + + − − − + − − d-Alanine − + − + + + − − − − − − − − − − − l-Alanyl-glycine + − + + + + + + + + + + + + + + + l-Asparagine − + + − − + − + + + + + + + + − − l-Aspartic acid − − − − + − − − − − − − − − − − − Glycyl-l-aspartic acid + − + − − − + + + + + + + + + + + Glycyl-l-glutamic acid − − − − − − − − − − + − + + + − − l-Threonine − − − + − − − − + − − − − − − − − Thymidine + − + + + + + + + + + + + + + + + l-Serine + + + + + + + + + + + + + + + + − ↵ a The cutoff point between negative responses and positive responses in the Biolog assay was an optical density at 600 nm of 0.2. The responses of substrates giving values close to the cutoff point were considered positive or negative if there was at least a fourfold difference in replicate readings between the ancestor and the isolate (see Materials and Methods for details).
