FIGURE 6.

Bacterial expression of PfFT1 and PfFT2. A, shown is growth in response to folic acid. The effect of folic acid (0–40 μm) on the growth of E. coli ΔpabA/ΔabgT-carrying recombinant constructs of the tac promoter plasmid pLOI707HE (32) with either PfFT1 or PfFT2 or the empty plasmid (pLOI was used as the control) was determined. If we accept a maximum growth effect at 40 μm, the folic acid-effective concentrations (EC₅₀) for PfFT1 and PfFT2 were calculated as 10.95 ± 2.4 and 10 ± 2.0 μm, respectively. In the absence of the transporter, pLOI, folic acid-induced growth stimulation was significantly lower and linear across the concentration range compared with the E. coli-expressing PfFT1 and PfFT2. A₆₀₀ on the y axis represents growth of the cultures based on the absorbance of the broth measured at 600 nm as under “Experimental Procedures.” Data taken from three different assays (n = 3) performed in triplicate. The differences in growth response to folic acid were significant (p < 0.001, two-way analysis of variance with Bonferroni post test) between bacteria carrying PfFT1 or PfFT2 and bacteria carrying the control plasmid pLOI at all FA concentrations. B, shown is growth in response to pABA. pABA (0–0.1 μm) stimulated the growth of the E. coli mutants with saturation occurring at around 0.1 μm. The maximum growth effect seen with pABA was achieved at 0.18 μm, and the EC₅₀ values were 3.7 ± 0.4 and 3.1 ± 0.6 nm for PfFT1 and PfFT2, respectively. In the case of the empty plasmid (pLOI), the EC₅₀was18 ± 2.4 nm. Differences between bacteria carrying PfFT1 or PfFT2 and the control plasmid pLOI were significant (p < 0.001, n = 3, data analysis as in A). C, growth response to folate precursors and derivatives is shown. pABA glutamated with one or two residues (pABAG₁; pABAG₂) was present at 20 nm final concentration. Pteroic acid-equivalent to folic acid without a glutamate (Pte), dihydropteroic acid (DiPte), and diglutamated folic acid (FA₂) were present at 20 μm. Data are from triplicate observations with at least two experimental replicate assays (n = 2). Statistical analysis was performed with two-way analysis of variance and Bonferroni post-tests. The differences in growth between bacteria carrying PfFT1 or PfFT2 and bacteria carrying the control plasmid pLOI were significant for pABA, pABAG₁, pteroic acid, dihydropteroic acid, and folic acid (p < 0.001) are denoted with an asterisk (*).