Fig. 2. Galactose transport and metabolism are inhibited in the presence of the preferred carbohydrate glucose.
A. Galactose incorporation was measured for WT in the presence and absence of glucose using a radiolabeled carbohydrate incorporation assay. Cells preconditioned to galactose were washed and exposed to a mixture of unlabeled and C14-labeled galactose or a mixture of C14-labeled galactose, H3-labeled glucose and 100 μM of each unlabeled carbohydrate at 37°C. Accumulation of each radioisotope over time was measured by standard liquid scintillation methods. Black solid line represents galactose incorporation in the absence of glucose. Gray solid line represents glucose incorporation in the presence of galactose. Black dashed line represents galactose incorporation in the presence of glucose. Values are reported as the total nanomoles of carbohydrate accumulated / 109 cells by accounting for the concentration of unlabeled carbohydrate and the colony-forming units in each reaction. Each point is the average of three biological replicates, and error bars are the standard error of the mean.
B. The extent of galactose metabolism by WT in the presence and absence of glucose was determined by measuring the concentration of galactose remaining in culture supernatants. WT cells pre-grown in galactose chemically defined medium (CDM) were washed and inoculated into 5.6 mM galactose or 2.8 mM glucose plus 2.8 mM galactose CDM. OD600 was measured every 30 min (smooth lines). Supernatant aliquots were collected every 15 min and assayed for total galactose using the Amplex Red Galactose/Galactose Oxidase Kit (Molecular Probes) (dotted lines). A galactose standard curve was used to convert absorbance readings to moles of galactose present. Data are shown as the percentage of the original concentration of galactose remaining at the point of collection. A representative pair of experiments is shown.