Saccharomyces cerevisiae pell and crd1 mutants deficient in the biosynthesis of mitochondrial phosphatidylglycerol (PG) and cardiolipin (CL) as well as Kluyveromyces lactis mutants impaired in the ...respiratory chain function (RCF) containing dysfunctional mitochondria show altered sensitivity to metabolic inhibitors. The S. cerevisiae pell mutant displayed increased sensitivity to cycloheximide, chloramphenicol, oligomycin and the cell-wall perturbing agents caffeine, caspofungin and hygromycin. On the other hand, the pel1 mutant was less sensitive to fluconazole, similarly as the K. lactis mutants impaired in the function of mitochondrial cytochromes. Mitochondrial dysfunction resulting either from the absence of PG and CL or impairment of the RCF presumably renders the cells more resistant to fluconazole. The increased tolerance of K. lactis respiratory chain mutants to amphotericin B, caffeine and hygromycin is probably related to a modification of the cell wall.
Decreased susceptibility of K. lactis mutants impaired in the function of cytochrome c, cytochrome c1 and cytochrome-c oxidase to fluconazole, bifonazole and amphotericin B in comparison with the ...isogenic wild-type strain was observed. Flow cytometry with rhodamine 6G did not show any changes in the accumulation of the dye in the mutant cells compared with the corresponding wild-type strain. Sterol analysis showed similar overall amount of sterols in both wild-type and mutant cells. Taking into account the increased amphotericin B resistance and significantly diminished susceptibility of mutant cells to lyticase digestion, the cell wall structure and/or composition may probably be responsible for the observed changes in the susceptibility of mutants to the antifungal compounds used.
The available genomic sequences of five closely related hemiascomycetous yeast species (Kluyveromyces lactis, Kluyveromyces waltii, Candida glabrata, Ashbya (Eremothecium) gossypii with Saccharomyces ...cerevisiae as a reference) were analysed to identify multidrug resistance (MDR) transport proteins belonging to the ATP-binding cassette (ABC) and major facilitator superfamilies (MFS), respectively. The phylogenetic trees clearly demonstrate that a similar set of gene (sub)families already existed in the common ancestor of all five fungal species studied. However, striking differences exist between the two superfamilies with respect to the evolution of the various subfamilies. Within the ABC superfamily all six half-size transporters with six transmembrane-spanning domains (TMs) and most full-size transporters with 12 TMs have one and only one gene per genome. An exception is the PDR family, in which gene duplications and deletions have occurred independently in individual genomes. Among the MFS transporters, the DHA2 family (TC 2.A.1.3) is more variable between species than the DHA1 family (TC 2.A.1.2). Conserved gene order relationships allow to trace the evolution of most (sub)families, for which the Kluyveromyces lactis genome can serve as an optimal scaffold. Cross-species sequence alignment of orthologous upstream gene sequences led to the identification of conserved sequence motifs ("phylogenetic footprints"). Almost half of them match known sequence motifs for the MDR regulators described in S. cerevisiae. The biological significance of those and of the novel predicted motifs awaits to be confirmed experimentally.
The resistance of Saccharomyces cerevisiae to three structurally different strobilurin fungicides, strobilurin A, kresoximethyl and azoxystrobin was investigated in this study. This resistance is ...under the control of both mitochondrial cob gene and the PDR network of nuclear genes involved in multidrug resistance. The mucidin-resistant muc1 and muc2 mutants of S. cerevisiae containing point mutations in mtDNA were found to be cross-resistant to kresoximethyl and azoxystrobin. Cross-resistance to all three strobilurin fungicides was also observed in yeast transformants containing gain-of-function mutations in the nuclear PDR3 gene. Nuclear mutants containing disrupted chromosomal copies of the PDR1 and PDR3 genes or the PDR5 gene alone were hypersensitive to kresoxim-methyl, azoxystrobin and strobilurin A. The results indicate that resistance to strobilurin fungicides, differing in chemical structure and specific activity, can be caused by the same molecular mechanism involving changes in the structure of apocytochrome b and/or increased efflux of strobilurins from fungal cells.
In yeast the resistance to kresoxim-methyl and azoxystrobin, like the resistance to strobilurin A (mucidin) is under the control of both mitochondrialcob gene and the PDR network of nuclear genes ...involved in multidrug resistance. The mucidin-resistantmucl (G137R) andmuc2 (L275S) mutants ofSaccharomyces cerevisiae containing point mutations in mtDNA were found to be cross-resistant to kresoximmethyl and azoxystrobin. Cross-resistance to all three strobilurin fungicides was also observed in yeast transformants containing gain-of-function mutations in the nuclearPDR3 gene. On the other hand, nuclear mutants containing disrupted chromosomal copies of thePDR1 andPDR3 genes or thePDR5 gene alone werehypersensitive to kresoxim-methyl, axoxystrobin and strobilurin A. The frequencies of spontaneous mutants selected for resistance either to kresoxim-methyl, azoxystrobin or strobilurin A were similar and resulted from mutations both in mitochondrial and nuclear genes. The results indicate that resistance to strobilurin fungicides, differing in chemical structure and specific activity, can be caused by the same molecular mechanism involving changes in the structure of apocytochromeb and/or increased efflux of strobilurins from fungal cells.
Decreased susceptibility ofK. lactis mutants impaired in the function of cytochromec, cytochromec1 and cytochrome-c oxidase to fluconazole, bifonazole and amphotericin B in comparison with the ...isogenic wild-type strain was observed. Flow cytometry with rhodamine 6G did not show any changes in the accumulation of the dye in the mutant cells compared with the corresponding wild-type strain. Sterol analysis showed similar overall amount of sterols in both wild-type and mutant cells. Taking into account the increased amphotericin B resistance and significantly diminished susceptibility of mutant cells to lyticase digestion, the cell wall structure and/or composition may probably be responsible for the observed changes in the susceptibility of mutants to the antifungal compounds used.
Sequence analysis of a DNA fragment containing the KlCOX18 gene originating from chromosome II of the yeast Kluyveromyces lactis revealed the presence of an adjacent open reading frame (ORF) for a ...protein exhibiting 78.4% identity with the Saccharomyces cerevisiae Spt4p. Based on the identical length (102 aa) and the conservation of the zinc-finger motif found in Spt4p we named this ORF KlSPT4. When expressed in S. cerevisiae the KlSPT4 gene complemented all spt4 mutant phenotypes. It is proposed that KlSpt4p, like its S. cerevisiae counterpart is a protein involved in the establishment or maintenance of the chromatin structure that influences the expression of many yeast genes.
The b-Zip transcription factor Yap1p plays an important role in oxidative stress response and multidrug resistance in
Saccharomyces cerevisiae. We have previously demonstrated that the
KNQ1 gene, ...encoding a multidrug transporter of the major facilitator superfamily in
Kluyveromyces lactis and containing two potential Yap1p response elements in its promoter, is a putative transcriptional target of
KlYap1p, the structural and functional homologue of
ScYap1p. In this work, we provide evidence that
KlYAP1 controls the expression of the
KNQ1 gene. Using a
P
KNQ1
-
gusA fusion construct we showed that the expression of
KNQ1 is induced upon cell treatment with the oxidizing agents H
2O
2 and menadione and that this induction is mediated by
KlYap1p. These results were confirmed by Northern-blot analysis showing that the expression of
KNQ1 is responsive to hydrogen peroxide and dependent on the presence of
KlYap1p. The role of
KlYAP1 in the control of
KNQ1 expression was further demonstrated by EMSA experiments and drug resistance assays. These results clearly demonstrate the involvement of the
KlYap1p transcription factor in the control of
KNQ1 gene expression.
A nuclear mutant of Saccharomyces cerevisiae deficient in mitochondrial fumarase has been identified through the in vitro biochemical assays of enzyme activity after visual selection due to an ...increased acidification ability of its colonies. Cells of the fumarase-deficient mutant fermenting glucose accumulated extracellular fumaric acid. This accumulation was observed only in growing cultures and required functional mitochondrial electron transport from succinate dehydrogenase to oxygen.
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