FIG. 3.

Alkali cation tolerance and efflux in strains of S. cerevisiae transformed with DhENA1 and DhENA2. (A) Tolerance to alkali cations was tested in solid YPD in the S. cerevisiae B31 null mutant (ena 1-4⁻ nha1⁻) transformed with DhENA1 (AD1) or with DhENA2 (AD2). The same strain containing the plasmid without any insert (AD0) or with ScENA1 and a D. hansenii strain were used as controls. Plates were inoculated with 10-μl drops of cultures at the middle of the exponential phase, and growth was scored after 60 h. The efflux of Na⁺ (B) or Li⁺ (C) was evaluated in AD1 (●), AD2 (▴), and AD0 (■). For determination of Na⁺ and Li⁺ efflux rates, cells were grown in YNB medium and loaded with the cations by incubation in the same medium plus 200 mM NaCl or 100 mM LiCl for 3 h. Cells were harvested and resuspended in the assay buffer (pH 5.5) supplemented with 50 mM KCl to trigger the efflux process. Samples were taken at regular intervals, filtered, and treated as previously described (24, 25). The experiments were repeated at least three times. The standard errors were 20% lower than the corresponding mean values.