The non-conventional yeast Kluyveromyces marxianus has recently emerged as a promising candidate for many food, environment, and biotechnology applications. This yeast is thermotolerant and has robust growth under many adverse conditions. Here, we show that its ability to grow under potassium-limiting conditions is much better than that of Saccharomyces cerevisiae, suggesting a very efficient and high-affinity potassium uptake system(s) in this species. The K. marxianus genome contains two genes for putative potassium transporters: KmHAK1 and KmTRK1. To characterize the products of the two genes, we constructed single and double knock-out mutants in K. marxianus and also expressed both genes in an S. cerevisiae mutant, that lacks potassium importers. Our results in K. marxianus and S. cerevisiae revealed that both genes encode efficient high-affinity potassium transporters, contributing to potassium homeostasis and maintaining plasma-membrane potential and cytosolic pH. In K. marxianus, the presence of HAK1 supports growth at low K+ much better than that of TRK1, probably because the substrate affinity of KmHak1 is about 10-fold higher than that of KmTrk1, and its expression is induced ~80-fold upon potassium starvation. KmHak1 is crucial for salt stress survival in both K. marxianus and S. cerevisiae. In co-expression experiments with ScTrk1 and ScTrk2, its robustness contributes to an increased tolerance of S. cerevisiae cells to sodium and lithium salt stress.

• Klíčová slova
• Kluyveromyces marxianu, K+–H+ symporter, affinity, potassium, transporter, uniporter,
• MeSH
• draslík * metabolismus   MeSH
• fungální proteiny genetika   metabolismus   MeSH
• genový knockout MeSH
• Kluyveromyces * genetika   metabolismus   růst a vývoj   MeSH
• koncentrace vodíkových iontů MeSH
• membránové potenciály MeSH
• proteiny přenášející kationty * genetika   metabolismus   MeSH
• regulace genové exprese u hub MeSH
• Saccharomyces cerevisiae * genetika   metabolismus   růst a vývoj   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• draslík * MeSH
• fungální proteiny MeSH
• proteiny přenášející kationty * MeSH

Saccharomyces species, which are mostly used in the food and beverage industries, are known to differ in their fermentation efficiency and tolerance of adverse fermentation conditions. However, the basis of their difference has not been fully elucidated, although their genomes have been sequenced and analyzed. Five strains of four Saccharomyces species (S. cerevisiae, S. kudriavzevii, S. bayanus, and S. paradoxus), when grown in parallel in laboratory conditions, exhibit very similar basic physiological parameters such as membrane potential, intracellular pH, and the degree to which they are able to quickly activate their Pma1 H+-ATPase upon glucose addition. On the other hand, they differ in their ability to proliferate in media with a very low concentration of potassium, in their osmotolerance and tolerance to toxic cations and cationic drugs in a growth-medium specific manner, and in their capacity to survive anhydrobiosis. Overall, S. cerevisiae (T73 more than FL100) and S. paradoxus are the most robust, and S. kudriavzevii the most sensitive species. Our results suggest that the difference in stress survival is based on their ability to quickly accommodate their cell size and metabolism to changing environmental conditions and to adjust their portfolio of available detoxifying transporters.

• Klíčová slova
• Intracellular pH, Membrane potential, Saccharomyces, Stress tolerance,
• MeSH
• fermentace MeSH
• fungální proteiny genetika   metabolismus   MeSH
• fyziologický stres MeSH
• glukosa metabolismus   MeSH
• protonové ATPasy genetika   metabolismus   MeSH
• Saccharomyces klasifikace   genetika   růst a vývoj   fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• Názvy látek
• fungální proteiny MeSH
• glukosa MeSH
• protonové ATPasy MeSH

Amino acids are key sources of nitrogen for growth of Candida albicans. In order to detect and take up these amino acids from a broad range of different and changing nitrogen sources inside the host, this fungus must be able to adapt via its expression of genes for amino acid uptake and further metabolism. We analyzed six C. albicans putative general amino acid permeases based on their homology to the Saccharomyces cerevisiae Gap1 general amino acid permease. We generated single- and multiple-deletion strains and found that, based on growth assays and transcriptional or posttranscriptional regulation, Gap2 is the functional orthologue to ScGap1, with broad substrate specificity. Expression analysis showed that expression of all GAP genes is under control of the Csy1 amino acid sensor, which is different from the situation in S. cerevisiae, where the expression of ScGAP1 is not regulated by Ssy1. We show that Gap4 is the functional orthologue of ScSam3, the only S-adenosylmethionine (SAM) transporter in S. cerevisiae, and we report that Gap4 is required for SAM-induced morphogenesis. IMPORTANCECandida albicans is a commensal organism that can thrive in many niches in its human host. The environmental conditions at these different niches differ quite a bit, and this fungus must be able to sense these changes and adapt its metabolism to them. Apart from glucose and other sugars, the uptake of amino acids is very important. This is underscored by the fact that the C. albicans genome encodes 6 orthologues of the Saccharomyces. cerevisiae general amino acid permease Gap1 and many other amino acid transporters. In this work, we characterize these six permeases and we show that C. albicans Gap2 is the functional orthologue of ScGap1 and that C. albicans Gap4 is an orthologue of ScSam3, an S-adenosylmethionine (SAM) transporter. Furthermore, we show that Gap4 is required for SAM-induced morphogenesis, an important virulence factor of C. albicans.

• Klíčová slova
• Candida albicans, GAP1, S-adenosyl methionine, general amino acid permease, morphogenesis,
• Publikační typ
• časopisecké články MeSH

Zygosaccharomyces rouxii is a fructophilic yeast that consumes fructose preferably to glucose. This behavior seems to be related to sugar uptake. In this study, we constructed Z. rouxii single-, double-, and triple-deletion mutants in the UL4 strain background (a ura3 strain derived from CBS 732(T)) by deleting the genes encoding the specific fructose facilitator Z. rouxii Ffz1 (ZrFfz1), the fructose/glucose facilitator ZrFfz2, and/or the fructose symporter ZrFsy1. We analyzed the effects on the growth phenotype, on kinetic parameters of fructose and glucose uptake, and on sugar consumption profiles. No growth phenotype was observed on fructose or glucose upon deletion of FFZ genes. Deletion of ZrFFZ1 drastically reduced fructose transport capacity, increased glucose transport capacity, and eliminated the fructophilic character, while deletion of ZrFFZ2 had almost no effect. The strain in which both FFZ genes were deleted presented even higher consumption of glucose than strain Zrffz1Δ, probably due to a reduced repressing effect of fructose. This study confirms the molecular basis of the Z. rouxii fructophilic character, demonstrating that ZrFfz1 is essential for Z. rouxii fructophilic behavior. The gene is a good candidate to improve the fructose fermentation performance of industrial Saccharomyces cerevisiae strains.

• MeSH
• biologický transport genetika   MeSH
• fermentace genetika   MeSH
• fruktosa metabolismus   MeSH
• fungální proteiny genetika   metabolismus   MeSH
• genový knockdown MeSH
• glukosa metabolismus   MeSH
• proliferace buněk genetika   MeSH
• regulace genové exprese u hub MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• Zygosaccharomyces genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fruktosa MeSH
• fungální proteiny MeSH
• glukosa MeSH

The virulence of Candida species depends on many environmental conditions, including extracellular pH and concentration of alkali metal cations. Tests of the tolerance/sensitivity of four pathogenic Candida species (C. albicans, C. dubliniensis, C. glabrata, and C. parapsilosis) to alkali metal cations under various growth conditions revealed significant differences among these species. Though all of them can be classified as rather osmotolerant yeast species, they exhibit different levels of tolerance to different salts. C. parapsilosis and C. albicans are the most salt-tolerant in general; C. dubliniensis is the least tolerant on rich YPD media and C. glabrata on acidic (pH 3.5) minimal YNB medium. C. dubliniensis is relatively salt-sensitive in spite of its ability to maintain as high intracellular K(+)/Na(+) ratio as its highly salt-tolerant relative C. albicans. On the other hand, C. parapsilosis can grow in the presence of very high external NaCl concentrations in spite of its high intracellular Na(+) concentrations (and thus lower K(+)/Na(+) ratio) and thus resembles salt-tolerant (halophilic) Debaryomyces hansenii.

• MeSH
• Candida albicans patogenita   fyziologie   MeSH
• Candida glabrata patogenita   fyziologie   MeSH
• Candida metabolismus   patogenita   fyziologie   MeSH
• chlorid draselný analýza   farmakologie   MeSH
• chlorid lithný analýza   farmakologie   MeSH
• chlorid sodný analýza   farmakologie   MeSH
• druhová specificita MeSH
• soli MeSH
• tolerance k soli fyziologie   MeSH
• virulence MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• chlorid draselný MeSH
• chlorid lithný MeSH
• chlorid sodný MeSH
• soli MeSH

Cationic amphipathic drugs, such as amiodarone, interact preferentially with lipid membranes to exert their biological effect. In the yeast Saccharomyces cerevisiae, toxic levels of amiodarone trigger a rapid influx of Ca(2+) that can overwhelm cellular homeostasis and lead to cell death. To better understand the mechanistic basis of antifungal activity, we assessed the effect of the drug on membrane potential. We show that low concentrations of amiodarone (0.1-2 microm) elicit an immediate, dose-dependent hyperpolarization of the membrane. At higher doses (>3 microm), hyperpolarization is transient and is followed by depolarization, coincident with influx of Ca(2+) and H(+) and loss in cell viability. Proton and alkali metal cation transporters play reciprocal roles in membrane polarization, depending on the availability of glucose. Diminishment of membrane potential by glucose removal or addition of salts or in pma1, tok1Delta, ena1-4Delta, or nha1Delta mutants protected against drug toxicity, suggesting that initial hyperpolarization was important in the mechanism of antifungal activity. Furthermore, we show that the link between membrane hyperpolarization and drug toxicity is pH-dependent. We propose the existence of pH- and hyperpolarization-activated Ca(2+) channels in yeast, similar to those described in plant root hair and pollen tubes that are critical for cell elongation and growth. Our findings illustrate how membrane-active compounds can be effective microbicidals and may pave the way to developing membrane-selective agents.

• MeSH
• amiodaron farmakologie   MeSH
• fluorescence MeSH
• imunoprecipitace MeSH
• iontový transport MeSH
• lidé MeSH
• membránové proteiny * MeSH
• Saccharomyces cerevisiae účinky léků   fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• amiodaron MeSH
• membránové proteiny * MeSH