Cdc42p controls yeast-cell shape and virulence of Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis is characterized by a multiple budding phenotype and a polymorphic cell growth, leading to the formation of cells with extreme variations in shape and size. Since Cdc42 is a pivotal molecule in establishing and maintaining polarized growth for diverse cell types, as well as during pathogenesis of certain fungi, we evaluated its role during cell growth and virulence of the yeast-form of P. brasiliensis. We used antisense technology to knock-down PbCDC42's expression in P. brasiliensis yeast cells, promoting a decrease in cell size and more homogenous cell growth, altering the typical polymorphism of wild-type cells. Reduced expression levels also lead to increased phagocytosis and decreased virulence in a mouse model of infection. We provide genetic evidences underlying Pbcdc42p as an important protein during host-pathogen interaction and the relevance of the polymorphic nature and cell size in the pathogenesis of P. brasiliensis.

Post-transcriptional control of the Saccharomyces cerevisiae proteome by 14-3-3 proteins.

UNLABELLED: 14-3-3 proteins form a family of conserved eukaryotic proteins binding to over 200 different proteins involved in nearly all cellular processes. The yeast Saccharomyces cerevisiae has two genes encoding 14-3-3 proteins, BMH1 and BMH2. As 14-3-3 proteins are essential in most S. cerevisiae strains, we constructed a bmh mutant with suboptimal 14-3-3 protein activity. Here, we report the effect of these bmh mutations on the proteome as determined by two-dimensional gel electrophoresis and mass spectrometry. We identified 26 proteins of which the levels increased by more than 2.0-fold and 51 proteins of which the levels decreased by more than 2.0-fold in the bmh mutant compared with those of the wild-type strain. For only 9 of these proteins, a more than 2.0-fold alteration was found at the transcriptional level. The levels of many proteins involved in gluconeogenesis, including Fba1, Eno1, Eno2, Tpi1, Pck1, Mdh2, Tdh2, Tdh3, and Gpm1, increased in the mutant, whereas the levels of several proteins involved in amino acid biosynthesis and translation and heat shock proteins were lower. Our studies indicate that 14-3-3 proteins control the S. cerevisiae proteome at the post-transcriptional level, in agreement with the binding of 14-3-3 proteins to proteins involved in protein synthesis and degradation. In addition, our studies suggest a key role in the regulation of carbohydrate metabolism by 14-3-3 proteins. KEYWORDS: 14-3-3 proteins * Saccharomyces cerevisiae * proteome * gluconeogenesis * BMH1 * BMH2.

Attachment of MAL32-encoded maltase on the outside of yeast cells improves maltotriose utilization.

The fermentation of maltotriose, the second most abundant fermentable sugar in wort, is often incomplete during high-gravity brewing. Poor maltotriose consumption is due to environmental stress conditions during high-gravity fermentation and especially to a low uptake of this sugar by some industrial strains. In this study we investigated whether the use of strains with an alpha-glucosidase attached to the outside of the cell might be a possible way to reduce residual maltotriose. To this end, the N-terminal leader sequence of Kre1 and the carboxy-terminal anchoring domain of either Cwp2 or Flo1 were used to target maltase encoded by MAL32 to the cell surface. We showed that Mal32 displayed on the cell surface of Saccharomyces cerevisiae laboratory strains was capable of hydrolysis of alpha-1,4-linkages, and that it increased the ability of a strain lacking a functional maltose permease to grow on maltotriose. Moreover, the enzyme was also expressed and found to be active in an industrial strain. These data show that expressing a suitable maltase on the cell surface might provide a means of modifying yeast for more complete maltotriose utilization in brewing and other fermentation applications.

Maltotriose utilization in lager yeast strains: MTT1 encodes a maltotriose transporter.

Maltotriose is the second most abundant fermentable sugar in wort and, due to incomplete fermentation, residual maltotriose in beer causes both quality and economic problems in the brewing industry. To identify genes that might improve utilization of maltotriose, we developed a library containing genomic DNA from four lager strains and a laboratory Saccharomyces cerevisiae strain and isolated transformants that could grow on YP/2% maltotriose in the presence of 3 mg/l of the respiratory inhibitor antimycin A. In this way we found a gene which shared 74% similarity with MPH2 and MPH3, 62% similarity with AGT1 and 91% similarity with MAL61 and MAL31, all encoding known maltose transporters. Moreover, the gene shared an even higher similarity (98%) with the uncharacterized Saccharomyces pastorianus mty1 gene (M. Salema-Oom, unpublished; NCBI Accession No. AJ491328). Therefore, we named the gene MTT1 (mty1-like transporter). We showed that the gene was present in four different lager strains but was absent from the laboratory strain CEN.PK113-7D. The ORF in the plasmid isolated from the library lacks 66 base pairs from the 3'-end of MTT1 but instead contains 54 bp of the vector. We named this ORF MTT1alt (NCBI Accession No. DQ010174). 14C-Maltose and repurified 14C-maltotriose were used to show that MTT1 and, especially, MTT1alt, encode maltose transporters for which the ratio between activities to maltotriose and maltose is higher than for most known maltose transporters. Introduction of MTT1 or MTT1alt into lager strain A15 raised maltotriose uptake by about 17% or 105%, respectively.

Transcriptional regulation of YML083c under aerobic and anaerobic conditions.

YML083c and DAN1 were among the Saccharomyces cerevisiae ORFs that displayed the strongest increase in transcript abundance during anaerobic growth compared to aerobic growth, as determined by oligonucleotide microarrays. We here report that transcription of YML083c is regulated by at least three different factors. First, repression under aerobic conditions depends on the presence of heme. Second, deletion analysis of the 5'-flanking region of YML083c and DAN1 revealed two regions responsible for anaerobic induction. Each of these regions conferred anoxia-regulated expression to the heterologous, minimal, CYC1-lacZ reporter. Mutations in the AAACGA subelement, common to the positive acting regions of YML083c and DAN1, almost completely abolished the ability to drive anaerobic expression of the reporter gene. This subelement is similar to the AR1 site, which is involved in anaerobic induction of the DAN/TIR genes. Activation through the AR1 site depends on Upc2. Indeed, transcription from the YML083c promoter was decreased in an upc2 null mutant. Third, expression of Sut1 under aerobic conditions enhanced transcription of YML083c, suggesting that aerobic repression of YML083c is promoted by the general Tup1-Ssn6 co-repressor complex. However, despite the presence of a sequence that matches the consensus for binding of Rox1, YML083c is not controlled by Rox1, since deletion or replacement of the putative binding site did not cause aerobic derepression. Moreover, YML083c expression was undetectable in aerobically grown cells of a rox1 null mutant.

The acetyl co-enzyme A synthetase genes of Kluyveromyces lactis.

Two Kluyveromyces lactis genes encoding acetyl co-enzyme A synthetase isoenzymes were isolated. One we named KlACS1, as it has high similarity to the ACS1 gene of Saccharomyces cerevisiae. The other gene, KlACS2, showed more similarity to S. cerevisiae ACS2 than to KlACS1 or ScACS1. This suggests that divergence of the two isogenes occurred before the evolutionary separation of the species and that the different functions have been conserved. In line with this idea is the regulation of transcription of the genes. The mode of regulation appeared to be maintained between ScACS1 and KlACS1 and between ScACS2 and KlACS2. The KlACS1 transcript was absent in glucose-grown cells, whereas transcription levels in ethanol- and acetate-grown cells were high. Disruption of the KlACS1 gene did not result in growth defects on glucose or ethanol. The growth rate on acetate, however, was reduced by a factor of two. KlACS2 was expressed at similar levels during growth on glucose and acetate, whereas expression on ethanol was slightly higher. A null mutant in this gene showed a reduced growth rate on all three carbon sources. Taken together, these data suggest that KlACS2 is used during growth on glucose and that KlACS1 is most dominant during growth on acetate. Strains in which both ACS genes are deleted could only be retrieved when a plasmid containing the ACS2 gene was present, suggesting that the double mutant is lethal. Tetrad analysis confirmed that non-viable spores with a deduced Klacs1Klacs2 genotype germinated but could not divide further. It therefore appears that, as in S. cerevisiae, the pyruvate dehydrogenase bypass formed by the enzymes pyruvate decarboxylase, acetaldehyde dehydrogenase and acetyl co-enzyme A synthetase is essential for growth. These results are in apparent contradiction with the growth on glucose of a strain with a disruption in the only structural pyruvate decarboxylase gene of K. lactis. Residual enzyme activity might, however, account for this discrepancy, or Acs fulfils an additional as yet unknown function, separate from its enzymatic activity.

Two mechanisms for oxidation of cytosolic NADPH by Kluyveromyces lactis mitochondria.

Null mutations in the structural gene encoding phosphoglucose isomerase completely abolish activity of this glycolytic enzyme in Kluyveromyces lactis and Saccharomyces cerevisiae. In S. cerevisiae, the pgi1 null mutation abolishes growth on glucose, whereas K.lactis rag2 null mutants still grow on glucose. It has been proposed that, in the latter case, growth on glucose is made possible by an ability of K. lactis mitochondria to oxidize cytosolic NADPH. This would allow for a re-routing of glucose dissimilation via the pentose-phosphate pathway. Consistent with this hypothesis, mitochondria of S. cerevisiae cannot oxidize NADPH. In the present study, the ability of K. lactis mitochondria to oxidize cytosolic NADPH was experimentally investigated. Respiration-competent mitochondria were isolated from aerobic, glucose-limited chemostat cultures of the wild-type K. lactis strain CBS 2359 and from an isogenic rag2Delta strain. Oxygen-uptake experiments confirmed the presence of a mitochondrial NADPH dehydrogenase in K.lactis. This activity was ca. 2.5-fold higher in the rag2Delta mutant than in the wild-type strain. In contrast to mitochondria from wild-type K. lactis, mitochondria from the rag2Delta mutant exhibited high rates of ethanol-dependent oxygen uptake. Subcellular fractionation studies demonstrated that, in the rag2Delta mutant, a mitochondrial alcohol dehydrogenase was present and that activity of a cytosolic NADPH-dependent 'acetaldehyde reductase' was also increased. These observations indicate that two mechanisms may participate in mitochondrial oxidation of cytosolic NADPH by K. lactis mitochondria: (a) direct oxidation of cytosolic NADPH by a mitochondrial NADPH dehydrogenase; and (b) a two-compartment transhydrogenase cycle involving NADP(+)- and NAD(+)-dependent alcohol dehydrogenases.

14-3-3 Proteins are essential for regulation of RTG3-dependent transcription in Saccharomyces cerevisiae.

14-3-3 proteins comprise a family of highly conserved proteins that bind more than 60 different, mostly phosphorylated, proteins. The yeast Saccharomyces cerevisiae has two genes, BMH1 and BMH2, encoding 14-3-3 proteins. Disruption of both genes together is lethal. In this study we constructed a mutant with a single, temperature-sensitive bmh allele. Recessive mutations in SIN4 and RTG3 can suppress the temperature-sensitive phenotype of this mutant. These genes encode a global transcriptional regulator and a basic helix-loop-helix transcription factor, respectively. The yeast 14-3-3 proteins were shown to bind to the Rtg3 protein. Overexpression of RTG3 is lethal even in wild-type cells. These genetic and biochemical data are consistent with a model in which the 14-3-3 proteins are required to keep the Rtg3 protein in an inactive state, which is (one of) the essential function(s) of the 14-3-3 proteins.

14-3-3 proteins: key regulators of cell division, signalling and apoptosis.

The 14-3-3 proteins constitute a family of conserved proteins present in all eukaryotic organisms so far investigated. These proteins have attracted interest because they are involved in important cellular processes such as signal transduction, cell-cycle control, apoptosis, stress response and malignant transformation and because at least 100 different binding partners for the 14-3-3 proteins have been reported. Although the exact function of 14-3-3 proteins is still unknown, they are known to (1) act as adaptor molecules stimulating protein-protein interactions, (2) regulate the subcellular localisation of proteins and (3) activate or inhibit enzymes. In this review, we discuss the role of the 14-3-3 proteins in three cellular processes: cell cycle control, signal transduction and apoptosis. These processes are regulated by the 14-3-3 proteins at multiple steps. The 14-3-3 proteins have an overall inhibitory effect on cell cycle progression and apoptosis, whereas in signal transduction they may act as stimulatory or inhibitory factors. This article contains supplementary material which may be viewed at the BioEssays website at http://www.interscience.wiley.com/jpages/0265-9247/Suppmat/23/v23_10.936.

Red fluorescent protein (DsRed) as a reporter in Saccharomyces cerevisiae.

We describe the utilization of a red fluorescent protein (DsRed) as an in vivo marker for Saccharomyces cerevisiae. Clones expressing red and/or green fluorescent proteins with both cytoplasmic and nuclear localization were obtained. A series of vectors are now available which can be used to create amino-terminal (N-terminal) and carboxyl-terminal (C-terminal) fusions with the DsRed protein.

Plasmids with the Cre-recombinase and the dominant nat marker, suitable for use in prototrophic strains of Saccharomyces cerevisiae and Kluyveromyces lactis.

Two plasmids are described which can be used to remove the "loxP-markerMX-loxP" cassettes in strains lacking the ura3 mutation. Both contain the Cre-recombinase under control of the GAL1 promoter and the natMX cassette with the dominant marker nat, which gives yeasts resistance to the antibiotic ClonNat. pNatCre contains ARSH and CEN6 for maintenance in Saccharomyces cerevisiae. pKlNatCre has a Kluyveromyces lactis replication origin and centromere in addition.

Construction of a genomic library of the food spoilage yeast Zygosaccharomyces bailii and isolation of the beta-isopropylmalate dehydrogenase gene (ZbLEU2).

A genomic library of the yeast Zygosaccharomyces bailii ISA 1307 was constructed in pRS316, a shuttle vector for Saccharomyces cerevisiae and Escherichia coli. The library has an average insert size of 6 kb and covers the genome more than 20 times assuming a genome size similar to that of S. cerevisiae. This new tool has been successfully used, by us and others, to isolate Z. bailii genes. One example is the beta-isopropylmalate dehydrogenase gene (ZbLEU2) of Z. bailii, which was cloned by complementation of a leu2 mutation in S. cerevisiae. An open reading frame encoding a protein with a molecular mass of 38.7 kDa was found. The nucleotide sequence of ZbLEU2 and the deduced amino acid sequence showed a significant degree of identity to those of beta-isopropylmalate dehydrogenases from several other yeast species. The sequence of ZbLEU2 has been deposited in the EMBL data library under accession number AJ292544.

Isolation and partial characterization of the Kluyveromyces lactis homologue of SKP1.

The SKP1 gene of Kluyveromyces lactis was isolated as a suppressor of a lethal temperature-sensitive mutation in the Saccharomyces cerevisiae CTF13 gene (Chromosome Transmission Factor 13). KlSKP1 was localized at chromosome V, adjacent to KlPAS3. A similar arrangement of the two genes is present in S. cerevisiae. Disruption of the KISKP1 gene was lethal, whereas overexpression of KlSKP1 lead to a decreased growth rate, to swollen and chain-forming cells with an increased DNA content, and to decreased plasmid stability. In both yeasts, promoter constructs lacking most of the purported binding sequence showed increased transcription levels of KlSKP1 in comparison to constructs with the entire promoter.

Isolation and characterization of KIUBP2, a ubiquitin hydrolase gene of Kluyveromyces lactis that can suppress a ts-mutation in CBF2, a gene encoding a centromeric protein of Saccharomyces cerevisiae.

The Kluyveromyces lactis UBP2 gene was isolated as a suppressor of a temperature-sensitive mutation in CBF2, a gene coding for a centromere-binding protein of Saccharomyces cerevisiae. The UBP genes are hydrolases than can cleave a ubiquitin moiety from a protein substrate. KlUBP2 is not essential for growth since a disruption of the KlUBP2 gene had little effect, except for a slight decrease in the growth rate. The stability of centromere-containing plasmids was not influenced either. In addition to KlUBP2, five S. cerevisiae genes involved in the ubiquitination pathway could suppress the ts-mutation in the CBF2 gene, namely UBA1, UBA2, UBP1, UBP2 and YUH1, although YUH1 was the only one that could do this like KlUBP2 from a single-copy plasmid. Surprisingly, these genes encode proteins with antagonistic activity as two, UBA1 and UBA2, are ubiquitin-activating enzymes whereas the other three are de-ubiquitinating hydrolases.

Regulation of pyruvate metabolism in chemostat cultures of Kluyveromyces lactis CBS 2359.

Regulation of currently identified genes involved in pyruvate metabolism of Kluyveromyces lactis strain CBS 2359 was studied in glucose-limited, ethanol-limited and acetate-limited chemostat cultures and during a glucose pulse added to a glucose-limited steady-state culture. Enzyme activity levels of the pyruvate dehydrogenase complex, pyruvate decarboxylase, alcohol dehydrogenase, acetyl-CoA synthetase and glucose-6-phosphate dehydrogenase were determined in all steady-state cultures. In addition, the mRNA levels of KlADH1-4, KlACS1, KlACS2, KlPDA1, KlPDC1 and RAG1 were monitored under steady-state conditions and during glucose pulses. In K. lactis, as in Saccharomyces cerevisiae, enzymes involved in glucose utilization (glucose-6-phosphate dehydrogenase, pyruvate dehydrogenase, pyruvate decarboxylase) showed the highest expression levels on glucose, whereas enzymes required for ethanol or acetate consumption (alcohol dehydrogenase, acetyl-CoA synthetase) showed the highest enzyme activities on ethanol. In cases where mRNA levels were determined, these corresponded well with the corresponding enzyme activities, suggesting that regulation is mostly achieved at the transcriptional level. Surprisingly, the activity of the K. lactis pyruvate dehydrogenase complex appeared to be regulated at the level of KlPDA1 transcription. The conclusions from the steady-state cultures were corroborated by glucose pulse experiments. Overall, expression of the enzymes of pyruvate metabolism in the Crabtree-negative yeast K. lactis appeared to be regulated in the same way as in Crabtree-positive S. cerevisiae, with one notable exception: the PDA1 gene encoding the E1alpha subunit of the pyruvate dehydrogenase complex is expressed constitutively in S. cerevisiae.

Genome-wide transcriptional analysis of aerobic and anaerobic chemostat cultures of Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae is unique among eukaryotes in exhibiting fast growth in both the presence and the complete absence of oxygen. Genome-wide transcriptional adaptation to aerobiosis and anaerobiosis was studied in assays using DNA microarrays. This technique was combined with chemostat cultivation, which allows controlled variation of a single growth parameter under defined conditions and at a fixed specific growth rate. Of the 6,171 open reading frames investigated, 5,738 (93%) yielded detectable transcript levels under either aerobic or anaerobic conditions; 140 genes showed a >3-fold-higher transcription level under anaerobic conditions. Under aerobic conditions, transcript levels of 219 genes were >3-fold higher than under anaerobic conditions.

T-DNA from Agrobacterium tumefaciens as an efficient tool for gene targeting in Kluyveromyces lactis.

The soil bacterium Agrobacterium tumefaciens can transfer a part of its tumour-inducing (Ti) plasmid, the T-DNA, to plant cells. The virulence (vir) genes, also located on the Ti plasmid, encode proteins involved in the transport of T-DNA into the plant cell. Once in the plant nucleus, T-DNA is able to integrate into the plant genome by an illegitimate recombination mechanism. The host range of A. tumefaciens is not restricted to plant species. A. tumefaciens is also able to transfer T-DNA to the yeast Saccharomyces cerevisiae. In this paper we demonstrate transfer of T-DNA from A. tumefaciens to the yeast Kluyveromyces lactis. Furthermore, we found that T-DNA serves as an ideal substrate for gene targeting in K. lactis. We have studied the efficiency of gene targeting at the K. lactis TRP1 locus using either direct DNA transfer (electroporation) or T-DNA transfer from Agrobacterium. We found that gene targeting using T-DNA was at least ten times more efficient than using linear double-stranded DNA introduced by electroporation. Therefore, the outcome of gene targeting experiments in some organisms may depend strongly upon the DNA substrate used.

Transient mRNA responses in chemostat cultures as a method of defining putative regulatory elements: application to genes involved in Saccharomyces cerevisiae acetyl-coenzyme A metabolism.

To identify common regulatory sequences in the promoters of genes, transcription of 31 genes of Saccharomyces cerevisiae was analysed during the transient response to a glucose pulse in a chemostat culture. mRNA levels were monitored during the subsequent excess glucose, ethanol and acetate phases, while other conditions were kept constant. This setup allowed a direct comparison between regulation by glucose, ethanol and acetate. Genes with identical regulation patterns were grouped to identify regulatory elements in the promoters. In respect to regulation on glucose four classes were identified: no transcription under any of the conditions tested, no difference in regulation on glucose, induced on glucose and repressed on glucose. In addition, genes were found that were repressed or induced on ethanol or acetate. Sequence alignment of genes with similar regulation patterns revealed five new, putative regulatory promoter elements. (i) The glucose-inducible fermentation genes PDC1 and ADH1 share the sequence ATACCTTCSTT. (ii) Acetate-repression might be mediated by the decamer CCCGAG RGGA, present in the promoters of ACS2 and ACR1. (iii) A specific element (CCWTTSRNCCG) for the glyoxylate cycle was present in seven genes studied: CIT2, ICL1, MLS1, MDH2, CAT2, ACR1 and ACH1. These genes were derepressed on ethanol or acetate. (iv) The sequence ACGTSCRGAATGA was found in the promoters of the partially ethanol-repressed genes ACS1 and YAT1. (v) Ethanol induction, as seen for ACS2, ADH3 and MDH1, might be mediated via the sequence CGGSGCCGRAG.

Overproduction of acetyl-coenzyme A synthetase isoenzymes in respiring Saccharomyces cerevisiae cells does not reduce acetate production after exposure to glucose excess.

To investigate whether the production of acetate which occurs after exposure of respiring Saccharomyces cerevisiae cells to excess glucose can be reduced by overproduction of acetyl-CoA synthetase (ACS, EC 6.2.1.1), the ACS1 and ACS2 genes were introduced on multi-copy plasmids. For each isoenzyme, the level in glucose-limited chemostat cultures was increased by 3-6-fold, relative to an isogenic reference strain. However, ACS overproduction did not result in a reduced production of acetate after a glucose pulse (100 mmol l-1) to these cultures. This indicates that a limited capacity of ACS is not the sole cause of acetate accumulation in S. cerevisiae.