FIG 4.

The effect of osmolality on R67 and Quad4 DHFR function in restoring the E. coli ΔfolA strain to prototrophy. Panel A shows the effect of sorbitol on cell growth in BV media supplemented with thymidine, adenine, pantothenate, glycine, and methionine and containing kanamycin. Cells can grow up to osmolalities between 1.24 and 1.52. Panel B shows the effect of sorbitol on growth in minimal BV media containing kanamycin, ampicillin, and 200 ng/ml tetracycline. Cell growth is reliant on enzyme activity encoded by the DHFR genes cloned into the pKTS plasmid. Cells carrying the R67 DHFR-pKTS plasmid can grow only until osmolalities between 0.28 and 0.57 are reached, with isolated colonies appearing at higher osmolalities (0.88 to 1.15). In contrast, cells carrying the Quad4-pKTS plasmid can grow confluently until 1.81 osmol is reached. Table S1 lists the osmolalities at which cells stop growing. The drawings indicate the positioning of the various streaks on the plates. Two independent colonies were streaked per transformant. Figure S4 in the supplemental material shows the titrations for NM522 and NM522 thyA.