An approach to the hypoxic and oxidative stress responses in Kluyveromyces lactis by analysis of mRNA levels

Genome duplication, after the divergence of Saccharomyces cerevisiae from Kluyveromyces lactis along evolution, has been proposed as a mechanism of yeast evolution from strict aerobics, such as Candida albicans, to facultatives/fermentatives, such as S. cerevisiae. This feature, together with the preponderance of respiration and the use of the pentose phosphate pathway in glucose utilization, makes K. lactis a model yeast for studies related to carbon and oxygen metabolism. In this work, and based on the knowledge of the sequence of the genome of K. lactis, obtained by the Génolevures project, we have constructed DNA arrays from K. lactis including a limited amount of selected probes. They are related to the aerobiosis-hypoxia adaptation and to the oxidative stress response, and have been used to test changes in mRNA levels in response to hypoxia and oxidative stress generated by H(2)O(2). The study was carried out in both wild-type and rag2 mutant K. lactis strains in which glycolysis is blocked at the phosphoglucose isomerase step. This approach is the first analysis carried out in K. lactis for the majority of the genes selected.     

Why does Kluyveromyces lactis not grow under anaerobic conditions? Comparison of essential anaerobic genes of Saccharomyces cerevisiae with the Kluyveromyces lactis genome

Although some yeast species, e.g. Saccharomyces cerevisiae, can grow under anaerobic conditions, Kluyveromyces lactis cannot. In a systematic study, we have determined which S. cerevisiae genes are required for growth without oxygen. This has been done by using the yeast deletion library. Both aerobically essential and nonessential genes have been tested for their necessity for anaerobic growth. Upon comparison of the K. lactis genome with the genes found to be anaerobically important in S. cerevisiae, which yielded 20 genes that are missing in K. lactis, we hypothesize that lack of import of sterols might be one of the more important reasons that K. lactis cannot grow in the absence of oxygen.     

Phenotypic expression of Kluyveromyces lactis killer toxin against Saccharomyces spp.

The secretion of killer toxins by some strains of yeasts is a phenomenon of significant industrial importance. The activity of a recently discovered Kluyveromyces lactis killer strain against a sensitive Saccharomyces cerevisiae strain was determined on peptone-yeast extract-nutrient agar plates containing as the carbon source glucose, fructose, galactose, maltose, or glycerol at pH 4.5 or 6.5. Enhanced activity (50 to 90% increase) was found at pH 6.5, particularly on the plates containing galactose, maltose, or glycerol, although production of the toxin in liquid medium was not significantly different with either glucose or galactose as the carbon source. Results indicated that the action of the K. lactis toxin was not mediated by catabolite repression in the sensitive strain. Sensitivities of different haploid and polyploid Saccharomyces yeasts to the two different killer yeasts S. cerevisiae (RNA-plasmid-coded toxin) and K. lactis (DNA-plasmid-coded toxin) were tested. Three industrial polyploid yeasts sensitive to the S. cerevisiae killer yeast were resistant to the K. lactis killer yeast. The S. cerevisiae killer strain itself, however, was sensitive to the K. lactis killer yeast.     

Phenotypic expression of Kluyveromyces lactis killer toxin against Saccharomyces spp

The secretion of killer toxins by some strains of yeasts is a phenomenon of significant industrial importance. The activity of a recently discovered Kluyveromyces lactis killer strain against a sensitive Saccharomyces cerevisiae strain was determined on peptone-yeast extract-nutrient agar plates containing as the carbon source glucose, fructose, galactose, maltose, or glycerol at pH 4.5 or 6.5. Enhanced activity (50 to 90% increase) was found at pH 6.5, particularly on the plates containing galactose, maltose, or glycerol, although production of the toxin in liquid medium was not significantly different with either glucose or galactose as the carbon source. Results indicated that the action of the K. lactis toxin was not mediated by catabolite repression in the sensitive strain. Sensitivities of different haploid and polyploid Saccharomyces yeasts to the two different killer yeasts S. cerevisiae (RNA-plasmid-coded toxin) and K. lactis (DNA-plasmid-coded toxin) were tested. Three industrial polyploid yeasts sensitive to the S. cerevisiae killer yeast were resistant to the K. lactis killer yeast. The S. cerevisiae killer strain itself, however, was sensitive to the K. lactis killer yeast.     

Genetics and Molecular Physiology of the Yeast Kluyveromyces lactis

With the recent development of powerful molecular genetic tools, Kluyveromyces lactis has become an excellent alternative yeast model organism for studying the relationships between genetics and physiology. In particular, comparative yeast research has been providing insights into the strikingly different physiological strategies that are reflected by dominance of respiration over fermentation in K. lactis versus Saccharomyces cerevisiae. Other than S. cerevisiae, whose physiology is exceptionally affected by the so-called glucose effect, K. lactis is adapted to aerobiosis and its respiratory system does not underlie glucose repression. As a consequence, K. lactis has been successfully established in biomass-directed industrial applications and large-scale expression of biotechnically relevant gene products. In addition, K. lactis maintains species-specific phenomena such as the “DNA-killer system,” analyses of which are promising to extend our knowledge about microbial competition and the fundamentals of plasmid biology.     

Dynamic analysis of the KlGAL regulatory system in Kluyveromyces lactis: a comparative study with Saccharomyces cerevisiae

The GAL regulatory system is highly conserved in yeast species of Saccharomyces cerevisiae and Kluyveromyces lactis. While the GAL system is a well studied system in S. cerevisiae, the dynamic behavior of the KlGAL system in K. lactis has not been characterized. Here, we have characterized the GAL system in yeast K. lactis by developing a dynamic model and comparing its performance to its not-so-distant cousin S. cerevisiae. The present analysis demonstrates the significance of the autoregulatory feedbacks due to KlGal4p, KlGal80p, KlGal1p and Lac12p on the dynamic performance of the KlGAL switch. The model predicts the experimentally observed absence of bistability in the wild type strain of K. lactis, unlike the short term memory of preculturing conditions observed in S. cerevisiae. The performance of the GAL switch is distinct for the two yeast species although they share similarities in the molecular components. The analysis suggests that the whole genome duplication of S. cerevisiae, which resulted in a dedicated inducer protein, Gal3p, may be responsible for the high sensitivity of the system to galactose concentrations. On the other hand, K. lactis uses a bifunctional protein as an inducer in addition to its galactokinase activity, which restricts its regulatory role and hence higher galactose levels in the medium are needed to trigger the GAL system. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11693-011-9082-7) contains supplementary material, which is available to authorized users.     

Heterologous complementation of the Klaac null mutation of Kluyveromyces lactis by the Saccharomyces cerevisiae AAC3 gene encoding the ADP/ATP carrier

The KlAAC gene, encoding the ADP/ATP carrier, has been assumed to be a single gene in Kluyveromyces lactis, an aerobic, petite-negative yeast species. The Klaac null mutation, which causes a respiratory-deficient phenotype, was fully complemented by AAC2, the Saccharomyces cerevisiae major gene for the ADP/ATP carrier and also by AAC1, a gene that is poorly expressed in S. cerevisiae. In this study, we demonstrate that the Klaac null mutation is partially complemented by the ScAAC3 gene, encoding the hypoxic ADP/ATP carrier isoform, whose expression in S. cerevisiae is prevented by oxygen. Once introduced into K. lactis, the AAC3 gene was expressed both under aerobic and under partial anaerobic conditions but did not support the growth of K. lactis under strict anaerobic conditions.     

Ability for anaerobic growth is not sufficient for development of the petite phenotype in Saccharomyces kluyveri

Saccharomyces cerevisiae is a petite-phenotype-positive ("petite-positive") yeast, which can successfully grow in the absence of oxygen. On the other hand, Kluyveromyces lactis as well as many other yeasts are petite negative and cannot grow anaerobically. In this paper, we show that Saccharomyces kluyveri can grow under anaerobic conditions, but while it can generate respiration-deficient mutants, it cannot generate true petite mutants. From a phylogenetic point of view, S. kluyveri is apparently more closely related to S. cerevisiae than to K. lactis. These observations suggest that the progenitor of the modern Saccharomyces and Kluyveromyces yeasts, as well as other related genera, was a petite-negative and aerobic yeast. Upon separation of the K. lactis and S. kluyveri-S. cerevisiae lineages, the latter developed the ability to grow anaerobically. However, while the S. kluyveri lineage has remained petite negative, the lineage leading to the modern Saccharomyces sensu stricto and sensu lato yeasts has developed the petite-positive characteristic.     

Milk and sugar: regulation of cell wall synthesis in the milk yeast Kluyveromyces lactis

The milk yeast Kluyveromyces lactis is an alternative model yeast to the well established Saccharomyces cerevisiae. The cell wall of these fungi consists of polysaccharides (i.e. long chains of β-1,3- and β-1,6-linked sugar chains and some chitin) and mannoproteins, both of which are continually adapted to environmental conditions in terms of their abundance and organization. This implies the need to perceive signals at the cell surface and to transform them into a proper cellular response. The signal transduction cascade involved in this process is generally referred to as the cell wall integrity (CWI) pathway. CWI signaling and cell wall composition have been extensively studied in the Baker's yeast S. cerevisiae and are also of interest in other yeast species with commercial potential, such as K. lactis. We here summarize the results obtained in the past years on CWI signaling in K. lactis and use a comparative approach to the findings obtained in S. cerevisiae to highlight special adaptations to their natural environments.     

Analysis of the deubiquitinating enzymes of the yeast Saccharomyces cerevisiae

Attachment of proteins to ubiquitin is reversed by specialized proteases called deubiquitinating enzymes (Dubs), which are also essential for ubiquitin precursor processing. In the genome of Saccharomyces cerevisiae, 17 potential DUB genes can be discerned. We have now constructed strains deleted for each of these genes. Surprisingly, given the essential nature of the ubiquitin system, none of the mutants is lethal or strongly growth defective under standard conditions, although a number have detectable abnormalities. Including results from this study, 14 of the 17 Dubs have now been shown to have ubiquitin-cleaving activity. The most extensively characterized yeast Dub is Doa4, which is required for both ubiquitin homeostasis and proteasome-dependent proteolysis. To help determine what distinguishes Doa4 functionally from other Dubs, we have cloned a DOA4 ortholog from the yeast Kluyveromyces lactis. The K. lactis protein is 42% identical to Doa4, but unexpectedly the K. lactis gene is slightly closer in nucleotide sequence to UBP5, which cannot substitute for DOA4 even in high dosage. The data suggest that the DOA4 locus underwent a duplication after the divergence of K. lactis and S. cerevisiae. This information will facilitate fine-structure analysis of the Doa4 protein to help delineate its key functional elements.