Feedback regulation of glucose transporter gene transcription in Kluyveromyces lactis by glucose uptake.

In the respirofermentative yeast Kluyveromyces lactis, only a single genetic locus encodes glucose transporters that can support fermentative growth. This locus is polymorphic in wild-type isolates carrying either KHT1 and KHT2, two tandemly arranged HXT-like genes, or RAG1, a low-affinity transporter gene that arose by recombination between KHT1 and KHT2. Here we show that KHT1 is a glucose-induced gene encoding a low-affinity transporter very similar to Rag1p. Kht2p has a lower K(m) (3.7 mM) and a more complex regulation. Transcription is high in the absence of glucose, further induced by low glucose concentrations, and repressed at higher glucose concentrations. The response of KHT1 and KHT2 gene regulation to high but not to low concentrations of glucose depends on glucose transport. The function of either Kht1p or Kht2p is sufficient to mediate the characteristic response to high glucose, which is impaired in a kht1 kht2 deletion mutant. Thus, the KHT genes are subject to mutual feedback regulation. Moreover, glucose repression of the endogenous beta-galactosidase (LAC4) promoter and glucose induction of pyruvate decarboxylase were abolished in the kht1 kht2 mutant. These phenotypes could be partially restored by HXT gene family members from Saccharomyces cerevisiae. The results indicate that the specific responses to high but not to low glucose concentrations require a high rate of glucose uptake.

Saccharomyces cerevisiae Elongator mutations confer resistance to the Kluyveromyces lactis zymocin.

Kluyveromyces lactis killer strains secrete a zymocin complex that inhibits proliferation of sensitive yeast genera including Saccharomyces cerevisiae. In search of the putative toxin target (TOT), we used mTn3:: tagging to isolate zymocin-resistant tot mutants from budding yeast. Of these we identified the TOT1, TOT2 and TOT3 genes (isoallelic with ELP1, ELP2 and ELP3, respectively) coding for the histone acetyltransferase (HAT)-associated Elongator complex of RNA polymerase II holoenzyme. Other than the typical elp ts-phenotype, tot phenocopies hypersensitivity towards caffeine and Calcofluor White as well as slow growth and a G(1) cell cycle delay. In addition, TOT4 and TOT5 (isoallelic with KTI12 and IKI1, respectively) code for components that associate with ELONGATOR: Intriguingly, strains lacking non-Elongator HATs (gcn5, hat1, hpa3 and sas3) or non-Elongator transcription elongation factors TFIIS (dst1) and Spt4p (spt4) cannot confer resistance towards the K.lactis zymocin, thus providing evidence that Elongator equals TOT and that Elongator plays an important role in signalling toxicity of the K.lactis zymocin.

Differences in regulation of yeast gluconeogenesis revealed by Cat8p-independent activation of PCK1 and FBP1 genes in Kluyveromyces lactis.

The yeast Kluyveromyces lactis is can utilise a wide range of non-fermentable carbon compounds as sole sources of carbon and energy, and differs from Saccharomyces cerevisiae in being able to carry out oxidative and fermentative metabolism simultaneously. In S. cerevisiae, growth on all non-fermentable carbon sources requires Cat8p, a transcriptional activator that controls the expression of gluconeogenic and glyoxylate cycle genes via CSREs (Carbon Source Responsive Elements). The down-regulation of Cat8p by fermentable carbon sources is the primary factor responsible for the tight repression of gluconeogenesis by glucose in S. cerevisiae. To analyse the regulation of gluconeogenesis in K. lactis, we have cloned and characterised the K. lactis homologue of CAT8 (KlCAT8). The gene was isolated by multicopy suppression of a fog2/klsnf1 mutation, indicating a similar epistatic relationship between KlSNF1 and KlCAT8 as in the case of the S. cerevisiae homologues. KlCAT8 encodes a protein of 1445 amino acids that is 40% identical to ScCat8p. The most highly conserved block is the putative Zn(II)2Cys6 DNA-binding domain, but additional conserved regions shared with members of the zinc-cluster family from Aspergillus define a subfamily of Cat8p-related proteins. KlCAT8 complements the growth defect of a Sccat8 mutant on non-fermentable carbon sources. In K. lactis, deletion of KlCAT8 severely impairs growth on ethanol, acetate and lactate, but not on glycerol. Derepression of enzymes of the glyoxylate cycle--malate synthase and particularly isocitrate lyase--was impaired in a Klcat8 mutant, whereas Northern analysis revealed that derepression of KlFBP1 and KlPCK1 does not require KlCat8p. Taken together, our results indicate that in K. lactis gluconeogenesis is not co-regulated with the glyoxylate cycle, and only the latter is controlled by KlCat8p.

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.

Glucose repression of the Kluyveromyces lactis invertase gene KlINV1 does not require Mig1p.

Kluyveromyces lactis, a budding yeast related to Saccharomyces cerevisiae, can grow on a wider variety of substrates and shows less sensitivity to glucose repression than does Saccharomyces cerevisiae. Many genes that are subject to glucose repression in S. cerevisiae are repressed only weakly or not at all in K. lactis. The molecular basis for this difference is largely unknown. To compare the mechanisms that regulate glucose repression in K. lactis and S. cerevisiae, we decided to clone and analyse an invertase gene from K. lactis. The SUC2 gene, which encodes invertase in S. cerevisiae, is strongly regulated by glucose and serves as a model system for studies on glucose repression. The invertase gene of K. lactis, KlINV1, was isolated by colony hybridization using a conserved region within the inulinase gene of K. marxianus as a probe. Two independent clones obtained were shown to contain the same ORF of 1827 bp. The deduced amino acid sequence is 59% similar to that of the K. marxianus inulinase and shows 49% similarity to ScSuc2p. Gene disruption experiments and low-stringency Southern analysis indicate that KlINV1 is a unique gene in K. lactis. Northern analysis revealed that the transcription of KlINV1 is strongly repressed in the presence of glucose, but, in contrast to the case in S. cerevisiae, repression is independent of KlMig1p.

Regulated phosphorylation of the Gal4p inhibitor Gal80p of Kluyveromyces lactis revealed by mutational analysis.

The yeast Gal80 protein inhibits the transcription activation function of Gal4p by physically interacting with the activation domain (Gal4-AD). Gal80p interaction with Gal1p or Gal3p is required to relieve Gal4p inhibition in response to galactose. Gal80p orthologs of Saccharomyces cerevisiae and Kluyveromyces lactis, ScGal80p and KIGal80p, can also inhibit the heterologous Gal4p variants; however, heterologous Gal3p/Gal1p only regulate ScGal80p but not KIGal80p. To compare KIGal80p and ScGal80p, point mutations known to affect ScGal80p function were introduced at corresponding positions in KIGal80p, and Gal4p regulation in vivo and KIGal80p-binding to Gst-Gal1p and Gst-Gal4-AD in vitro were analysed. The in vitro binding properties of the KIGal80p mutants were similar to those of ScGal80p, but two out of four mutants differed in Gal4p regulation. E. g. KIGAL80s-0(G302R) but not ScGAL80s-0 (G301R) alleviates Gal4p inhibition. Possibly, this difference is related to a role of phosphorylation in the regulation of Gal80p function in K. lactis. Wild-type and mutant forms of KIGal80p are shown to be subject to carbon source regulated phosphorylation whereas no evidence for ScGal80p phosphorylation exists. (Hyper-)phosphorylation of KIGal80p is strongly reduced in galactose-containing medium. This reduction requires KIGal1p but no interaction with KIGal4p. The inhibition deficient KIGal80s-0p (G302R) variant is under-phosphorylated. We thus propose that phosphorylation of Gal80p in Kluyveromyces lactis contributes to the regulation of Gal4p mediated transcription.

Influence of mutations in hexose-transporter genes on glucose repression in Kluyveromyces lactis.

The variability of Kluyveromyces lactis strains in sensitivity to glucose is correlated with genetic differences in Kluyveromyces hexose transporter (KHT) genes. The glucose sensitive strain JA6 was shown to contain an additional gene, KHT2, not found in strains that are less sensitive. KHT2 is tandemly arranged with KHT1 which is identical to the low-affinity transporter gene RAG1, except for the C-terminus. Sequence analysis indicated that most of KHT2 had been lost by a recombination event between KHT1 and KHT2 generating the chimeric gene RAG1. Recombination between KHT1 and KHT2 was also found in mutants of JA6 selected as 2-deoxyglucose resistant colonies. These mutants, like kht1 kht2 double mutants were unable to grow on glucose when respiration was blocked (Rag- phenotype) and glucose repression was strongly reduced. kht1 or kht2 single mutants of JA6 were Rag+ but still an influence of the kht mutations on glucose repression was detectable. Repression was not affected in a Rag- mutant deleted for the phosphoglucose isomerase gene suggesting that the influence of transporter genes on repression is not caused by a reduction of the glycolytic flux. The data rather suggest that sensitivity to glucose repression is dependent on the rate of glucose uptake.

Activation of Gal4p by galactose-dependent interaction of galactokinase and Gal80p.

Yeast galactokinase (Gal1p) is an enzyme and a regulator of transcription. In addition to phosphorylating galactose, Gal1p activates Gal4p, the activator of GAL genes, but the mechanism of this regulation has been unclear. Here, biochemical and genetic evidence is presented to show that Gal1p activates Gal4p by direct interaction with the Gal4p inhibitor Gal80p. Interaction requires galactose, adenosine triphosphate, and the regulatory function of Gal1p. These data indicate that Gal1p-Gal80p complex formation results in the inactivation of Gal80p, thereby transmitting the galactose signal to Gal4p.

Expression of the Arxula adeninivorans glucoamylase gene in Kluyveromyces lactis.

The glucoamylase gene of the yeast Arxula adeninivorans was expressed in Kluyveromyces lactis by using the GAP promoter from Saccharomyces cerevisiae and a multicopy plasmid vector. The transformants secreted 90.1% of the synthesized glucoamylase into the culture medium. The secreted glucoamylase activities are about 20 times higher in comparison to those of Saccharomyces cerevisiae transformants using the same promoter. Secreted glucoamylase possesses identical N-terminal amino acid sequences to those secreted by A. adeninivorans showing that cleavage of the N-terminal signal peptide takes place at the same site. Biochemical characteristics of glucoamylase expressed by K. lactis and A. adeninivorans are very similar.

Regulation of cytochrome c expression in the aerobic respiratory yeast Kluyveromyces lactis.

Transcriptional regulation of the KlCYC1 gene from the aerobic respiratory yeast Kluyveromyces lactis has been studied. The KlCYC1 gene produces two transcripts of different sizes, in contrast with the single transcripts found for CYC1 and CYC7 from Saccharomyces cerevisiae, and for the CYC gene from Schwanniomyces occidentalis. Both KlCYC1 transcripts respond in the same way to the regulatory signals studied here. The transcription of KlCYC1 is regulated by oxygen and this control is mediated by heme. The KlCYC1 gene is also subject to catabolite repression. Heterologous expression in S. cerevisiae mutants reveals that the factors HAP1 and HAP2 take part in the regulatory mechanism.

Nitric oxide destroys zinc-sulfur clusters inducing zinc release from metallothionein and inhibition of the zinc finger-type yeast transcription activator LAC9.

Nitric oxide, generated from S-nitrosocysteine or applied as gas mediates metal ion release from the Zn2+/Cd(2+)-complexing protein metallothionein via oxidation of SH-groups. Time-dependent S-nitrosylation and subsequent disulfide formation of metallothionein are demonstrated. Furthermore, nitric oxide inhibits DNA binding activity of the yeast transcription factor LAC9 containing a zinc finger like DNA binding domain. These results show that nitric oxide interacts with and destroys zinc-sulfur clusters in proteins.

Gal80 proteins of Kluyveromyces lactis and Saccharomyces cerevisiae are highly conserved but contribute differently to glucose repression of the galactose regulon.

We cloned the GAL80 gene encoding the negative regulator of the transcriptional activator Gal4 (Lac9) from the yeast Kluyveromyces lactis. The deduced amino acid sequence of K. lactis GAL80 revealed a strong structural conservation between K. lactis Gal80 and the homologous Saccharomyces cerevisiae protein, with an overall identity of 60% and two conserved blocks with over 80% identical residues. K. lactis gal80 disruption mutants show constitutive expression of the lactose/galactose metabolic genes, confirming that K. lactis Gal80 functions in essentially in the same way as does S. cerevisiae Gal80, blocking activation by the transcriptional activator Lac9 (K. lactis Gal4) in the absence of an inducing sugar. However, in contrast to S. cerevisiae, in which Gal4-dependent activation is strongly inhibited by glucose even in a gal80 mutant, glucose repressibility is almost completely lost in gal80 mutants of K. lactis. Indirect evidence suggests that this difference in phenotype is due to a higher activator concentration in K. lactis which is able to overcome glucose repression. Expression of the K. lactis GAL80 gene is controlled by Lac9. Two high-affinity binding sites in the GAL80 promoter mediate a 70-fold induction by galactose and hence negative autoregulation by Gal80. Gal80 in turn not only controls Lac9 activity but also has a moderate influence on its rate of synthesis. Thus, a feedback control mechanism exists between the positive and negative regulators. By mutating the Lac9 binding sites of the GAL80 promoter, we could show that induction of GAL80 is required to prevent activation of the lactose/galactose regulon in glycerol or glucose plus galactose, whereas the noninduced level of Gal80 is sufficient to completely block Lac9 function in glucose.

Expression of the transcriptional activator LAC9 (KlGAL4) in Kluyveromyces lactis is controlled by autoregulation.

The concentration of the transcriptional activator LAC9 (KlGAL4) of Kluyveromyces lactis is moderately regulated by the carbon source as is the case for GAL4, its homolog in Saccharomyces cerevisiae. Expression of the LAC9 gene is induced about twofold in galactose. This induction is due to autoregulation. The LAC9 gene product binds to a low-affinity binding site in the LAC9 promoter and moderately activates transcription in response to galactose above a basal level. As for the LAC9-controlled metabolic genes, induction of LAC9 is inhibited in the presence of glucose. This inhibition of induction is a prerequisite for glucose repression of the lactose-galactose metabolic pathway. On the other hand, induced LAC9 levels are required for optimal growth on galactose, since mutating the LAC9 binding site in the LAC9 promoter resulted in poor growth and reduced expression of LAC9-controlled genes. Thus, in addition to the GAL80-dependent regulation by protein-protein interaction, the regulation of LAC9 gene expression is an important parameter in determining carbon source control of the LAC-GAL regulon. Although the mode of control is different, the pattern of LAC9 gene regulation resembles that of the S. cerevisiae GAL4 gene, being lower in glucose and glucose-galactose than in galactose.

Sequence of a cytochrome c gene from Kluyveromyces lactis and its upstream region.

The complete sequence of a cytochrome c gene from Kluyveromyces lactis including its upstream region is reported. Sequence of the translated open reading frame is discussed in terms of cytochrome c structural requirements. Putative regulatory signals in the upstream region are described and compared with reported sequences which modulate the expression of respiratory-related yeast genes.

Glucose repression of lactose/galactose metabolism in Kluyveromyces lactis is determined by the concentration of the transcriptional activator LAC9 (K1GAL4) [corrected].

In the budding yeast Kluyveromyces lactis glucose repression of genes involved in lactose and galactose metabolism is primarily mediated by LAC9 (or K1GAL4) the homologue of the well-known Saccharomyces cerevisiae transcriptional activator GAL4. Phenotypic difference in glucose repression existing between natural strains are due to differences in the LAC9 gene (Breunig, 1989, Mol.Gen.Genet. 261, 422-427). Comparison between the LAC9 alleles of repressible and non-repressible strains revealed that the phenotype is a result of differences in LAC9 gene expression. A two-basepair alteration in the LAC9 promoter region produces a promoter-down effect resulting in slightly reduced LAC9 protein levels under all growth conditions tested. In glucose/galactose medium any change in LAC9 expression drastically affects expression of LAC9 controlled genes e.g. those encoding beta-galactosidase or galactokinase revealing a strong dependence of the kinetics of induction on the LAC9 concentration. We propose that in tightly repressible strains the activator concentration drops below a critical threshold that is required for induction to occur. A model is presented to explain how small differences in activator levels are amplified to produce big changes in expression levels of metabolic genes.

Coregulation of the Kluyveromyces lactis lactose permease and beta-galactosidase genes is achieved by interaction of multiple LAC9 binding sites in a 2.6 kbp divergent promoter.

The coregulated genes LAC4 and LAC12 encoding beta-galactosidase and lactose permease, respectively, are responsible for the ability of the milk yeast Kluyveromyces lactis to utilise lactose. They are divergently transcribed and separated by an unusually large intergenic region of 2.6 kbp. Mapping of the upstream border of the beta-galactosidase gene (LAC4) promoter by introduction of mutations at the chromosomal locus showed that LAC4 and LAC12 share the same upstream activation sites (UAS). The UASs represent binding sites for the trans-activator LAC9, a K. lactis homologue of GAL4, conforming to the consensus sequence 5'-CGG(N5)A/T(N5)CCG-3'. Two binding sites are located in front of each of the genes at almost symmetrical positions. beta-galactosidase activity measurements as well as quantitation of LAC4 and LAC12 mRNA levels demonstrated that all four sites are required for full induction. LAC4 proximal and a LAC12 proximal sites cooperate in activating transcription of both genes. These sites are more than 1.7 kbp apart and the distal site is located more than 2.3 kbp upstream of the respective start of transcription. Thus, the distance between interacting sites is larger than in any of the well characterised yeast promoters. The contribution to gene activation differs for individual binding sites and correlates with the relative affinity of LAC9 for these sites in vitro suggesting that LAC9 binding is a rate limiting step for LAC promoter function.

A mutation in the Zn-finger of the GAL4 homolog LAC9 results in glucose repression of its target genes.

The transcriptional activator LAC9, a GAL4 homolog of Kluyveromyces lactis which mediates lactose and galactose-dependent activation of genes involved in the utilization of these sugars can also confer glucose repression to those genes. Here we report on the isolation and characterization of LAC9-2, an allele which encodes a glucose-sensitive activator in contrast to the one previously cloned. A single amino acid exchange of leu-104 to tryptophan is responsible for the glucose-insensitive phenotype. The mutation is located within the Zn-finger-like DNA binding domain which is highly conserved between LAC9 and GAL4. Glucose repression is also eliminated by duplication of the LAC9-2 allele. The data indicate that LAC9 is a limiting factor for beta-galactosidase gene expression under all growth conditions and that glucose reduces the activity of the activator.

Glucose repression of LAC gene expression in yeast is mediated by the transcriptional activator LAC9.

In the yeast Kluyveromyces lactis the beta-galactosidase gene is induced by lactose or galactose. As shown here it can also be repressed by glucose but only in some strains. When the LAC9 gene of a repressible strain is substituted by an allele of a non-repressible strain, the beta-galactosidase gene is no longer glucose repressed. LAC9 codes for a regulatory protein homologous to GAL4 which activates transcription in the presence of the inducer. Since the LAC9 product is also present in the repressed strain and binds to DNA in vitro, as shown by DNA footprinting, glucose repression cannot be caused by repression of LAC9 gene expression. Instead, our results demonstrate that glucose repression is mediated by the LAC9 gene product, and is separable from the ability of LAC9 to activate transcription.

Multicopy plasmids containing the gene for the transcriptional activator LAC9 are not tolerated by K. lactis cells.

The high copy number 2-microns DNA-like Kluyveromyces plasmid pKD1 was extremely unstable in Kluyveromyces lactis when carrying the gene for the regulatory protein LAC9, a transcriptional activator involved in the induction of the LAC and GAL genes. Transformants of a lac9 mutant strain normally contained rearranged plasmids and all were Lac-, indicating that the LAC9 gene was inactive. Lac+ "revertants" could be obtained from Lac- transformants by selection on lactose plates. In some of these, the pKD1-based plasmid was stably maintained by being integrated into the chromosome of the cell; in others, the disrupted chromosomal gene was restored by a gene conversion event. None of the Lac+ revertants had more than one intact LAC9 gene, an indication that LAC9 overexpression affects cell viability.

Functional homology between the yeast regulatory proteins GAL4 and LAC9: LAC9-mediated transcriptional activation in Kluyveromyces lactis involves protein binding to a regulatory sequence homologous to the GAL4 protein-binding site.

As shown previously, the beta-galactosidase gene of Kluyveromyces lactis is transcriptionally regulated via an upstream activation site (UASL) which contains a sequence homologous to the GAL4 protein-binding site in Saccharomyces cerevisiae (M. Ruzzi, K.D. Breunig, A.G. Ficca, and C.P. Hollenberg, Mol. Cell. Biol. 7:991-997, 1987). Here we demonstrate that the region of homology specifically binds a K. lactis regulatory protein. The binding activity was detectable in protein extracts from wild-type cells enriched for DNA-binding proteins by heparin affinity chromatography. These extracts could be used directly for DNase I and exonuclease III protection experiments. A lac9 deletion strain, which fails to induce the beta-galactosidase gene, did not contain the binding factor. The homology of LAC9 protein with GAL4 (J.M. Salmeron and S. A. Johnston, Nucleic Acids Res. 14:7767-7781, 1986) strongly suggests that LAC9 protein binds directly to UASL and plays a role similar to that of GAL4 in regulating transcription.