Cloning and expression of two genes coding for sodium pumps in the salt-tolerant yeast Debaryomyces hansenii

Abstract

Two genes encoding Na(+)-ATPases from Debaryomyces hansenii were cloned and sequenced. The genes, designated ENA1 from D. hansenii (DhENA1) and DhENA2, exhibited high homology with the corresponding genes from Schwanniomyces occidentalis. DhENA1 was expressed in the presence of high Na(+) concentrations, while the expression of DhENA2 also required high pH. A mutant of Saccharomyces cerevisiae lacking the Na(+) efflux systems and sensitive to Na(+), when transformed with DhENA1 or DhENA2, recovered Na(+) tolerance and also the ability to extrude Na(+).

FIG. 1

Phylogenetic tree of Na⁺- and Ca²⁺-ATPases from fungi. Alignment of the sequences was performed with the CLUSTALX program (31) using the core sequences starting at the beginning of the f and ending at the end of the j conserved regions (28). Accession numbers are as follows: ScENA1, X67136; ZrENA, D78567; cta3Sp, P22189; NcENA1, AJ243520; DhENA2, AF263248; SoENA2, AF030861; DhENA1, AF247561; SoENA1, AF030860.

FIG. 2

Northern blot analyses of DhENA1 and DhENA2 transcripts in D. hansenii cells grown under the conditions indicated at the top of the figure. Total RNA was fractionated, transferred to a nylon membrane, and probed. Filters were stripped and stained with methylene blue as a loading control (bottom).

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.