Article Figures & Data
Figures
- FIG. 1.
Growth of wild-type S. cerevisiae strains A2 and W303-1B and terbinafine-resistant mutants in the presence and absence of terbinafine. The yeast strains were inoculated into 2 ml of YPD medium to an OD600 of 0.005 and grown at 30°C for 45 h. The medium was supplemented with 20, 50, and 100μ g of terbinafine per ml dissolved in ethanol. The control culture without terbinafine was supplemented with ethanol. Growth was monitored by measuring the OD600.
- FIG. 2.
Schematic presentation of the Erg1 protein of S. cerevisiae. The putative FAD, monooxygenase (MOX), and substrate binding (SQ) domains of squalene epoxidase are indicated together with the positions of the mutations identified in the terbinafine-resistant variants (*) and the respective amino acid exchanges.
- FIG. 3.
Northern blot analysis of the ERG1 mRNA of terbinafine-resistant mutants. Cultures of terbinafine-resistant mutants, wild-type strains A2 and W303-1B, and strain KLN1 with an erg1 disruption with the recombinant plasmids pNS1 and pNS2 were grown in YPD medium to early log phase. Total RNA was extracted from either lysed spheroplasts (strains H1B, H2, H6, and W303-1B in panel A) or mechanically disrupted cells [strains A2, A2M8, KLN1(pNS1), KLN1(pNS2) in panel A and all strains in panel B], and 10 μg of the RNA was separated by agarose gel electrophoresis. Hybridization was performed as described in Materials and Methods by using an ERG1-specific DNA probe to detect the transcript from the chromosomal ERG1 locus and low-copy-number recombinant plasmids pNS1 and pNS2. The amounts of ACT1 mRNA and 25S rRNA were used to normalize the amount of RNA loaded on the gel.
- FIG. 4.
Resistance to terbinafine of mutants overexpressing Pdr5p. ONCs of wild-type strain S. cerevisiae A2 and two mutants containing allelic forms of PDR1 (PDR1-12 in KPHJ1) and PDR3 (PDR3-33 in KPHJ2) causing overexpression of Pdr5p were prepared at 30°C in YPD medium. Wild-type strain W303/a carrying the vector pYEp13 and wild-type strain W303/a carrying recombinant plasmid pYSTS1 (overexpressing Pdr5p) were grown in YNB minimal medium overnight at 30°C. The OD600s of the ONCs were adjusted to 0.1, 5 μl of the 100 to 10−3 dilutions was spotted on YPD agar plates without terbinafine (A) and with 100 μg of terbinafine per ml (B), and the strains were grown for 2 days at 30°C.
- FIG. 5.
Growth of wild-type S. cerevisiae strain A2 and two mutants containing allelic forms of PDR1 (PDR1-12 in KPHJ1) and PDR3 (PDR3-33 in KPHJ2) causing overexpression of Pdr5p in the presence and absence of terbinafine. The yeast strains were inoculated into 2 ml of YPD medium to an OD600 of 0.005 and were grown at 30°C for 45 h. The medium was supplemented with 20, 50, or 100μ g of terbinafine per ml dissolved in ethanol. The control culture without terbinafine was supplemented with ethanol. Growth was monitored by measuring the OD600.
- FIG. 6.
Effect of replacement of the wild-type ERG1 gene in A2 and the erg1::URA3 disruption in KLN1 with erg1 alleles of the resistant mutants on terbinafine susceptibility. The erg1 alleles for terbinafine resistance of mutants A2M8, H3, H1B, and H2 were integrated into the chromosome of wild-type strain A2 and strain KLN1 with the erg1 disruption at the ERG1 locus by homologous recombination. The resulting transformants (A2T8, A2H3, KLNH1B, and KLNH2, respectively) were grown overnight in YPD medium at 30°C, 5-μl aliquots of the ONCs were adjusted to an OD600 of 0.1, and serial dilutions (100 to 10−3) were spotted on YPD agar plates without terbinafine (A) and with 100 μg of terbinafine per ml (B). The plates were incubated for 2 days at 30°C.
- FIG. 7.
Effects of erg1 alleles expressed from low-copy-number plasmids on terbinafine resistance. S. cerevisiae KLN1 (erg1::URA3) was transformed with the low-copy-number plasmids pML1 carrying the wild-type ERG1 gene, pML2 carrying the erg1 allele for terbinafine resistance of A2M8, and pNS2 carrying erg1 allele NS2 for terbinafine resistance on pRS315. The strains were grown overnight in YPD medium, 5-μl aliquots of the ONCs were diluted to an OD600 of 0.1, and serial dilutions (100 to 10−3) were spotted on YPD plates without terbinafine (A) and with 100 μg of terbinafine per ml (B) and incubated for 2 days at 30°C.
- FIG. 8.
Multiple-sequence alignment of S. cerevisiae Erg1 protein regions carrying the mutations with the corresponding regions of C. albicans, rat, mouse, and human squalene epoxidases. The amino acids conserved in all squalene epoxidases are shaded. Mutations leading to terbinafine resistance are indicated by arrows, and the corresponding amino acid exchanges are shown above the sequence.
Tables
- TABLE 1.
Strains and plasmids used in this study
Strain or plasmid Genotype or description Source Strains E. coli XL1 endA1 hsdR17(hsdR hsdM+) supE44 thi-1 recA1 gyrA96 relA1 Δ(lac) [F′ proAB lacIqZΔM15 Tn10 (Tetr)] Stratagene S. cerevisiae A2 MAT a leu2 his3 can1 A. Hartig S. cerevisiae W303-1B MATα leu2 ura3 his3 ade2 trp1 can1 J. Kolarov S. cerevisiae W303/a MAT a leu2 ura3 his3 ade2 trp1 S. D. Kohlwein S. cerevisiae H1A, H1B, H2, H3, H4, H5, H6, H7, H8 Terbinafine-resistant mutants of W303-1B after MNNG mutagenesis This study S. cerevisiae A2M8 Terbinafine-resistant mutant of A2 after UV mutagenesis Jandrositz et al. (13) S. cerevisiae KLN1 MATα, ERG1::URA3 leu2 ura3 trp1 Landl et al. (21) S. cerevisiae A2T8 A2 with erg1 allele for terbinafine resistance of A2M8 This study S. cerevisiae A2H3 A2 with erg1 allele for terbinafine resistance of H3 This study S. cerevisiae KLNH1B KLN1 with erg1 allele for terbinafine resistance of H1B This study S. cerevisiae KLNH2 KLN1 with erg1 allele for terbinafine resistance of H2 This study S. cerevisiae KPHJ1 A2 MATα PDR1-12::HIS3 pet Wendler et al. (47) S. cerevisiae KPHJ2 A2 MATα PDR3-33::HIS3 pet Wendler et al. (47) Plasmids pYEp13 Multicopy yeast-E. coli shuttle vector A. Hartig pYEp351 Multicopy yeast-E. coli shuttle vector A. Hartig pRS315 Centromere yeast-E. coli shuttle vector K. Kuchler pBIG1 2.3-kb PstI fragment with the wild-type ERG1 gene in pBluescript This study pKL1 3.9-kb SacI fragment with the erg1 allele for terbinafine resistance of A2M8 in pBluescript This study pKL2 pKL1 with the LEU2 cassette in the PstI site This study pKLH3 erg1 allele for terbinafine resistance of mutant H3 in pKL2 This study pAF22 4.8-kb Sau3A fragment with the erg1 allele for terbinafine resistance of A2M8 Jandrositz et al. (13) pML1 2.3-kb PstI fragment with the wild-type ERG1 gene in pRS315 This study pNS1 2.3-kb PstI fragment with the wild-type ERG1 gene in pRS315 (insert in the orientation opposite that in pML1) This study pML2 2.3-kb PstI fragment with the erg1 allele for terbinafine resistance of A2M8 in pRS315 This study pML3 2.3-kb PstI fragment with the erg1 allele ML3 for terbinafine resistance in pRS315 This study pNS2 2.3-kb PstI fragment with erg1 allele NS2 for terbinafine resistance in pRS315 This study pYSTS1 STS1 (PDR5) gene in pYEp13 Bissinger and Kuchler (2), K. Kuchler - TABLE 2.
Terbinafine-resistant mutants with single base pair exchanges leading to amino acid substitutions in the Erg1 protein
Mutant Base pair exchange in ERG1 gene Amino acid substitution in Erg1 protein A2M8, H1A, H5, ML3a C751T L251F H2 C1206A F402L H1B C1260A F420L H3, H4, H6, H7, H8 C1288T P430S NS2a T1298C F433S ↵ a Alleles ML3 and NS2 for terbinafine resistance were isolated after in vitro PCR mutagenesis of the ERG1 gene.
