FIGURE 3.

First round of site-directed mutagenesis of the Slr0642 protein. A, in vivo folate uptake assays. The E. coli pabA abgT strain was transformed with pLOI707HE with no insert (V) or containing native slr0642 (WT) or 19 mutants thereof. Two independent clones of each mutant construct were streaked on minimal medium plus 0.5 mm IPTG alone (control) or containing 3.6 μm pABA or 11 μm 5-FTHF. Clones were streaked once on pABA-supplemented plates and three times in succession on the others. The pABA plates were photographed 1 day after streaking, and others were photographed after 2 days. Red, transport-critical residues; blue, non-critical residues. B, Western blot analysis of Slr0642 protein levels in membrane preparations from the above clones. Membrane proteins (25 μg/track) were separated by SDS-PAGE. The position of the 52-kDa molecular size marker is shown.