FIG. 2.

Functional expression of L. casei FolT and ThiT in L. lactis. (A) SDS-PAGE (12% gel) of membrane fractions from L. lactis harboring pNZ8048 alone (lane 1; 50 μg protein), or containing FolT (lane 2; 25 μg protein) or ThiT (lane 3; 25 μg protein). Staining was with Coomassie brillant blue. The arrows indicate FolT and ThiT bands. Positions of molecular mass markers (kDa) are shown. (B to E) Binding of ³H-labeled folates or thiamine to L. lactis cells harboring pNZ8048 alone (open squares) or expressing FolT or ThiT (filled squares). Assays (total volume, 1 ml) were performed in phosphate-buffered saline (PBS), pH 7.4, at 30°C with stirring. Cells were washed and resuspended (optical density at 600 nm, 20), and 0.5-ml aliquots were pretreated for 5 min with 2-deoxyglucose (25 mM final concentration). Assays were started by adding 0.5 ml of PBS containing ³H-labeled vitamin (final concentration, 12.6 to 14.5 nM). At various times, cells (100 μl) were harvested by vacuum filtration on a cellulose nitrate membrane (0.45 μm). Filters were washed twice with 2 ml of ice-cold PBS, and their ³H content was determined by scintillation counting. The arrows show when unlabeled vitamin was added to give a final concentration of 50 μM. Cells expressing FolT were incubated with (6S)-[3′,5′,7,9-³H(N)]folinic acid diammonium salt (Moravek; 10 Ci/mmol) (B), [3′,5′,7,9,-³H]folic acid diammonium salt (Moravek; 25.9 Ci/mmol) (C), or [³H]folic acid polyglutamates (45 Ci/mmol) comprising 40% tri-, 56% tetra-, and 4% pentaglutamates (D). Cells expressing ThiT were incubated with [³H(G)]thiamine hydrochloride (ARC; 10 Ci/mmol) (E). ³H-Labeled substrates were chromatographically purified before use (4, 13).