Abstract

Saccharomyces cerevisiae strains belonging to the CEN.PK family are widely used in fundamental and applied yeast research. These strains have been reported to be hypersensitive to sodium ions and a previous microarray-based genotyping study indicated an atypical organization of the PMR2 locus. In other S. cerevisiae strains, this locus harbours one to five ENA genes that encode plasma membrane sodium-pumping ATPases. Sequence analysis of the PMR2 locus in S. cerevisiae CEN.PK113-7D revealed the presence of a new ENA gene that showed substantial sequence differences, both at the nucleotide level and at the predicted amino acid sequence level, with previously described ENA genes. The presence of this single and atypical ENA gene correlated with hypersensitivity to sodium and, in particular, to lithium ions. The native ENA6 gene was transcriptionally induced by sodium and lithium ions, but, apparently, the capacity for sodium export upon full induction was insufficient to achieve the levels of sodium and lithium ion tolerance observed in other S. cerevisiae strains. The sodium and lithium hypersensitivity of CEN.PK strains, which is potentially detrimental during cultivation in sodium-rich media, could, however, be suppressed by overexpression of ENA6.

Na+ and its analogue Li+ are generally toxic for living cells, including those of Saccharomyces cerevisiae, as a result of their competition with magnesium for specific binding sites on enzymes. The high charge density of Li+ and Na+ may displace magnesium from weak cationic binding sites and form reversible (Murguía, 1995) or dead-end complexes (Atack, 1995). The mechanisms for Na+ and Li+ entry into yeast cells are incompletely understood and probably involve several yeast transporters that contribute to ‘cation leak’ (Serrano, 1999). In order to maintain nontoxic cytosolic concentrations of these cations, S. cerevisiae harbours two transporter systems in its plasma membrane that function cooperatively. While the Na+/H+ antiporter encoded by NHA1 has been shown to play a role at low pH, the major Na+- and Li+-exporting system in S. cerevisiae is the Na+-ATPase encoded by the ENA/PMR2 locus (Kinclová, 2002). The composition of the PMR2 locus is strain-dependent and contains a cluster of one to five (ENA1-5) tandemly arranged and highly similar genes. The commonly used laboratory S. cerevisiae strains S288C, A364A and D273-10B harbour five copies of ENA genes, DBY746 and W303.1A harbour four copies and Σ1278b only contains a single ENA gene (Haro, 1991; Wieland, 1995; Bañuelos, 1998).

Saccharomyces cerevisiae strains of the CEN.PK family (Entian & Kötter, 2007) are widely applied in systems biology research (Bro, 2003; Kresnowati, 2006) as well as for application-oriented metabolic engineering research (Kuyper, 2005; Wattanachaisaereekul, 2008). While comparative genotyping with oligonucleotide microarrays showed that the PMR2 locus in CEN.PK113-7D was too small to contain more than a single ENA gene copy (Daran-Lapujade, 2003), comparative phenotypic analyses of laboratory and industrial S. cerevisiae strains revealed an extreme sensitivity of CEN.PK strains to Na+ (Warringer & Blomberg, 2003; Garay-Arroyo, 2004). As sodium salts are common ingredients of laboratory and industrial media, an insight into the sodium hypersensitivity of these strains is highly relevant for their application in fundamental and applied research. The aim of this work was therefore to characterize the PMR2 locus in S. cerevisiae CEN.PK113-7D and to investigate the presence of an ENA type of gene. Furthermore, the involvement of the PMR2 locus in this laboratory strain's hypersensitivity to Na+ and its responses to Li+ were explored.

Plating assays confirmed earlier observations that CEN.PK strains are very sensitive to Na+ and further revealed an extreme sensitivity to Li+ (Fig. 1a). While most other S. cerevisiae strains grow in the presence of 20 mM Li+, the growth of CEN.PK was completely inhibited at a concentration of 5 mM LiCl and higher [Fig. 1a and Garciadeblas (1993); Wieland (1995)]. This hypersensitivity was very specific to Na+ and Li+ as CEN.PK showed no exacerbated sensitivity to other salts (i.e. KCl, Supporting Information, Table S1) or to osmotic pressure (tested with sorbitol, data not shown). To quantify the sensitivity of CEN.PK to Na+ and Li+, this strain and the sequenced strain S288C were grown in a shake flask in the presence of 20 mM LiCl or 500 mM NaCl (Table S1). The tolerance of the CEN.PK strain to salt was significantly better in liquid media than on solid media. However, it was still markedly lower than that of S288C as shown by a 45% and 65% decrease in the specific growth rate in the presence of Na+ and Li+, respectively.

1

(a) Na+ and Li+ sensitivity of Saccharomyces cerevisiae S288C and CEN.PK113-7D, (b) Na+ and Li+ tolerance of S. cerevisiae parental strain and ena6Δ deletion mutant CEN.PK708-1A (c) Na+ and Li+ tolerance of S. cerevisiae S288C, CEN.PK113-7D, CEN.PK721-7A (control strain carrying an empty vector) and CEN.PK720-7A overexpressing ENA6 on a multicopy plasmid behind its own promoter. Saccharomyces cerevisiae strains were grown overnight at 30°C in shake-flask cultures containing YPD medium (Sherman, 1991), and were then washed once with water and resuspended in an equal amount of water. After counting, cell suspensions were diluted and spotted on agar plates containing defined minimal medium (Verduyn, 1992) supplemented with the specified concentration of salt and adjusted to pH 6 with potassium hydroxide. Two percent glucose was used as the carbon source. The strains and primer sets used in this work are described in Tables S2 and S3.

1

(a) Na+ and Li+ sensitivity of Saccharomyces cerevisiae S288C and CEN.PK113-7D, (b) Na+ and Li+ tolerance of S. cerevisiae parental strain and ena6Δ deletion mutant CEN.PK708-1A (c) Na+ and Li+ tolerance of S. cerevisiae S288C, CEN.PK113-7D, CEN.PK721-7A (control strain carrying an empty vector) and CEN.PK720-7A overexpressing ENA6 on a multicopy plasmid behind its own promoter. Saccharomyces cerevisiae strains were grown overnight at 30°C in shake-flask cultures containing YPD medium (Sherman, 1991), and were then washed once with water and resuspended in an equal amount of water. After counting, cell suspensions were diluted and spotted on agar plates containing defined minimal medium (Verduyn, 1992) supplemented with the specified concentration of salt and adjusted to pH 6 with potassium hydroxide. Two percent glucose was used as the carbon source. The strains and primer sets used in this work are described in Tables S2 and S3.

The hypersensitivity of CEN.PK strains to Na+ and Li+ might be the result of a previously suggested low copy number of ENA genes. Sequencing an amplicon from the CEN.PK PMR2 locus (sequencing information in Fig. S1) confirmed the presence of a single ENA allele (called ENA6) that was highly homologous to ENA1, ENA2 and ENA5. However, while the ENA2 and ENA5 coding sequences shared 98% and 97% identity with ENA1, respectively, ENA6 only shared 91% identity with ENA1. These differences in the nucleotide sequence affected the protein sequence, as 51 amino acids out of the 1091 forming the Ena proteins were unique to Ena6 as compared with Ena1, Ena2 and Ena5 (Fig. S1). Specific regions of Ena6, such as the sequence located between the amino acids 497 and 730, shared as little as 70% identity with the other Ena proteins. The Ena6 protein harbours amino-acid modifications in both transmembrane and catalytic domains, which could result in peculiar physical and kinetic properties. The transcript level of ENA6 was undetectable by quantitative reverse transcriptase-PCR in the absence of salt and increased strongly upon exposure to 20 mM Li+ or 500 mM Na+ (data not shown). This salt-dependent induction resembles that of ENA genes in other S. cerevisiae backgrounds (Wieland, 1995; Alepuz, 1997).

Deletion of ENA6 in CEN.PK resulted in the complete absence of growth at Na+ and Li+ concentrations that the parental strain could tolerate (Fig. 1b, strain construction described in Tables S2 and S3), thereby confirming the role of ENA6 product in its (low) Na+ and Li+ tolerance. Overexpression of ENA6 on a multicopy plasmid or by integration in the genome restored the hypersensitivity of CEN.PK strains to both Na+ and Li+ to the tolerance levels of S228C (Fig. 1c). Conversely, tolerance to potassium and sorbitol was not affected by deletion or overexpression of ENA6, thereby confirming the specific affinity of Ena6 for Na+ and Li+ (data not shown).

The present study demonstrates that CEN.PK's PMR2 locus harbours a single gene (ENA6) encoding a Na+ and Li+ pump similar to ENA1, 2 and 5. Ena6 is clearly the major exporter responsible for the low Na+ and Li+ tolerance in CEN.PK113-7D. Its overproduction restored the tolerance of the CEN.PK strain to these cations to the level of S. cerevisiae strains carrying up to five ENA genes. It could be concluded that the acute sensitivity of the CEN.PK strains to these salts resulted from an insufficient capacity of Ena6-mediated cation transport across the plasma membrane. Additional experiments will be required to define whether CEN.PK's hypersensitivity results from low ENA6 transcript levels, impaired transport kinetic properties (low Vmax or high Km) due to sequence differences or a combination of both. A similar situation was shown with Σ1278b, an S. cerevisiae strain carrying a single ENA gene. In this strain, which exhibits poor Na+ tolerance, but a high ENA gene induction, the authors concluded that low tolerance levels were due to a poorly active Na+ pump (Wieland, 1995).

Based on the results presented in this study, we recommend that for cultivation of strains belonging to or derived from the CEN.PK family, Na+- and Li+-rich media should be avoided. Especially for high cell density cultivation, NaOH as a pH titrant should preferably be replaced by KOH or ammonia. Alternatively, the sodium hypersensitivity of CEN.PK-derived strains can be complemented by overexpression of its native ENA6 gene (as shown in this study) or, probably, by the functional expression of other ENA genes. The large size of the ENA6 gene (3.3 kb) precludes convenient use in PCR-amplified deletion cassettes. However, it may be applicable as a selection marker for plasmid transformation into CEN.PK strains as transformation of ENA6-carrying plasmids into such strains results in a clear phenotype.

Growth inhibition of CEN.PK strains on Na+- and Li+-rich media may be used to check for potential contamination of cultures by non-Na+- and Li+-sensitive yeasts. In our laboratories, we routinely use plate assays with 20 mM LiCl to check and confirm culture purity during fermentations with CEN.PK strains.

The rapidly decreasing costs of whole-genome sequencing are bound to lead to the discovery of the genetic basis for many strain-specific phenotypes in S. cerevisiae and other commonly used microorganisms. The ENA6 case illustrates how such knowledge can enable rapid and targeted complementation of potentially undesirable phenotypes, without a requirement for backcrossing or other nontargeted approaches.

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Supporting Information

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Author notes

Editor: Monique Bolotin-Fukuhara