PMC

A. The gene pgi catalyzes the isomerization of glucose 6-phosphate to fructose 6-phosphate in upper glycolysis. Removal of this gene forces glycolytic flux through the pentose phosphate pathway, creating a redox imbalance due to excess NADPH production. B. The genes udhA and pntAB catalyze the interconversion of NAD/NADH and NADP/NADPH. UdhA is a soluble protein whereas PntAB is membrane-bound.

The strains were constructed by introducing five of the six rpoS mutations detected after adaptive evolution back into the starting unevolved Δpgi strain through site-directed mutagenesis. A similar knock-in strain containing the pgi_gluc3 rpoS mutation was not constructed. Growth rate data for the starting unevolved Δpgi strain is also shown for comparison. Error bars represent the standard deviation from three biological replicates. A full description for the strain abbreviations can be found in . Abbreviations – hr: hour.

A. Growth rates for the three single, three double and triple knock-in strains constructed based on the three mutations that appeared in pgi_gluc2. The growth rates for the starting unevolved Δpgi strain and the evolved pgi_gluc2 strain are also shown for comparison. B. Corresponding glucose uptake rates for the seven knock-in strains. Data for the unevolved and evolved strains are again shown for comparison. Error bars for both represent the standard deviation from three biological replicates. A full description for the strain abbreviations can be found in . Abbreviations – gDW: gram dry weight; hr: hour.

A. Growth rates for the starting unevolved Δpgi strain and all ten evolved strains after adaptive evolution. The growth rate of unevolved and 50-day evolved wild-type E. coli K12 MG1655 in the same medium was 0.69(0.0069 and 0.79(0.0092 hr-1. B. Glucose uptake rates for the unevolved (pgi strain and the ten evolved strains after adaptive evolution. Unevolved and 50-day evolved wild-type E. coli had a glucose uptake rate of 8.43(0.72 and 11.6(0.41 mmol/gDW/hour, respectively. C. Acetate secretion rates for the unevolved (pgi strain and the ten evolved strains after adaptive evolution. Unevolved and 50-day evolved wild-type E. coli had an acetate secretion rate of 12.8(8.87 and 5.61(0.24 mmol/gDW/hour, respectively. The symbol * indicates strains that also secrete formate (pgi_gluc1: 0.49(0.13 mmol/gDW/hour; pgi_gluc3: 0.22(0.02 mmol/gDW/hour). All error bars represent the standard deviation from three biological replicates. Abbreviations – gDW: gram dry weight; hr: hour.

A. Structure and location of the e14 prophage. It is integrated within the icd gene in the E. coli chromosome at position 1,195,432 to position 1,210,646. B. PCR analysis of the unevolved (pgi, pgi_gluc2 triple knock-in (KI2_3KI) and evolved pgi_gluc2 strains confirms loss of the e14 prophage in pgi_gluc2. Numbers 1 through 4 correspond to PCR amplification regions as indicated in the top panel. Regions 1 and 3 both span terminal segments of the integrated prophage and adjacent chromosomal DNA. Region 2 spans a segment wholly within the prophage. Region 4 spans the integration site and is amplified using the left primer from Region 1 and the right primer from Region 3.