Showing papers in "Current Genetics in 2003"

Transcriptional control of nonfermentative metabolism in the yeast Saccharomyces cerevisiae.

Abstract: Although sugars are clearly the preferred carbon sources of the yeast Saccharomyces cerevisiae, nonfermentable substrates such as ethanol, glycerol, lactate, acetate or oleate can also be used for the generation of energy and cellular biomass. Several regulatory networks of glucose repression (carbon catabolite repression) are involved in the coordinate biosynthesis of enzymes required for the utilization of nonfermentable substrates. Positively and negatively acting complexes of pleiotropic regulatory proteins have been characterized. The Snf1 (Cat1) protein kinase complex, together with its regulatory subunit Snf4 (Cat3) and alternative β-subunits Sip1, Sip2 or Gal83, plays an outstanding role for the derepression of structural genes which are repressed in the presence of a high glucose concentration. One molecular function of the Snf1 complex is deactivation by phosphorylation of the general glucose repressor Mig1. In addition to regulation of alternative sugar fermentation, Mig1 also influences activators of respiration and gluconeogenesis, although to a lesser extent. Snf1 is also required for conversion of specific regulatory factors into transcriptional activators. This review summarizes regulatory cis-acting elements of structural genes of the nonfermentative metabolism, together with the corresponding DNA-binding proteins (Hap2-5, Rtg1-3, Cat8, Sip4, Adr1, Oaf1, Pip2), and describes the molecular interactions among general regulators and pathway-specific factors. In addition to the influence of the carbon source at the transcriptional level, mechanisms of post-transcriptional control such as glucose-regulated stability of mRNA are also discussed briefly.

Mating, conidiation and pathogenicity of Fusarium graminearum, the main causal agent of the head-blight disease of wheat, are regulated by the MAP kinase gpmk1

Abstract: To date, only very little is known about the molecular infection mechanisms of the head-blight pathogen of wheat, Fusarium graminearum (teleomorph Gibberella zeae). Here, we report on the isolation and characterization of the Fus3/Pmk1 mitogen-activated protein kinase homologue Gpmk1 from F. graminearum. Disruption of the gpmk1 gene in F. graminearum results in mutants that are reduced in conidial production, are sexually sterile and are fully apathogenic. This leads to the conclusion that gpmk1 is responsible for signal transduction processes taking place during the most important developmental stages in the life cycle of this fungal pathogen. Thus, Delta gpmk1 mutants are a useful tool to find other important genes involved in plant-infection mechanisms. Previously, only the trichothecene biosynthesis pathway was identified as a virulence factor in F. graminearum. Hence, Gpmk1 is now the second pathogenicity trait to be known in this important plant pathogen.

An improved transformation protocol for the human fungal pathogen Candida albicans.

Abstract: Commonly used protocols for the transformation of the dimorphic human fungal pathogen Candida albicans rely on established methods for the yeast Saccharomyces cerevisiae. With respect to transformation efficiency, however, there is a great difference between these two organisms when using the lithium acetate procedure. Here we present a modified version of this protocol for use with C. albicans. Among the different parameters tested, two turned out to be particularly relevant and, when combined, resulted in an up to 10-fold increase in transformation efficiency (400-500 integrative transformants) compared with previous protocols: first, adjusting the heat shock applied to the cells to 44 degrees C for C. albicans instead of 42 degrees C for S. cerevisiae and, second, treating C. albicans cells with lithium acetate in an overnight incubation instead of for 30 min as used for S. cerevisiae. With these modifications, the lithium acetate procedure becomes a very efficient and reliable tool for C. albicans transformation.

Adaptation to environmental pH in Candida albicans and its relation to pathogenesis

Abstract: For microorganisms that grow over a wide range of extracellular pH, systems must have evolved to sense and respond appropriately. The human opportunistic pathogen Candida albicans colonizes and infects anatomical sites of diverse pH, including the oral and gastro-intestinal tracts and the vaginal cavity. The ability to sense and respond to neutral-alkaline environments is governed by signal transduction pathways, one of which culminates in the activation of the transcription factor, Rim101p. The RIM101/pacC pathway, which governs pH responses and differentiation, has been the focus of study in both Saccharomyces cerevisiae and Aspergillus nidulans. This pathway has been identified in C. albicans and governs pH responses, dimorphism, and pathogenesis. Although C. albicans and S. cerevisiae are related fungi, it is becoming apparent that there are unique aspects of the pH response and the role the RIM101 pathway plays in this response in C. albicans.

Regulation of phosphate acquisition in Saccharomyces cerevisiae

Abstract: Membrane transport systems active in cellular inorganic phosphate (P(i)) acquisition play a key role in maintaining cellular P(i) homeostasis, independent of whether the cell is a unicellular microorganism or is contained in the tissue of a higher eukaryotic organism. Since unicellular eukaryotes such as yeast interact directly with the nutritious environment, regulation of P(i) transport is maintained solely by transduction of nutrient signals across the plasma membrane. The individual yeast cell thus recognizes nutrients that can act as both signals and sustenance. The present review provides an overview of P(i) acquisition via the plasma membrane P(i) transporters of Saccharomyces cerevisiae and the regulation of internal P(i) stores under the prevailing P(i) status.

Comparison of different transformation methods for Aspergillus giganteus

Abstract: Four different transformation methods were tested and compared in an attempt to facilitate the genetic transformation of Aspergillus giganteus, the producer of an antifungal protein (AFP). The fungus was transformed to hygromycin B resistance, using the hph gene of Escherichia coli by protoplast transformation, electroporation, biolistic transformation, and Agrobacterium tumefaciens-mediated transformation. Electroporation and biolistic transformation were found to be inappropriate for transforming A. giganteus, due to a low transformation yield. The conventional transformation technique based on protoplasts yielded up to 55 transformants in 10(8) protoplasts/microg DNA and was enhanced to 140-fold by A. tumefaciens-mediated transfer of its T-DNA. Here, the germination time prior to cocultivation and the fungus:bacterium ratio were found to alter the transformation efficiency. Southern blot analysis revealed that the A. giganteus transformants contained a randomly integrated single T-DNA copy, whereas multiple integration events were frequent in transformants obtained by the protoplast method.

Loss of function of the Fusarium oxysporum SNF1 gene reduces virulence on cabbage and Arabidopsis

Abstract: Fusarium oxysporum pathogenicity is believed to require the activity of cell wall-degrading enzymes. Production of these enzymes in fungi is subject to carbon catabolite repression, a process that in yeast is mostly controlled by the SNF1 (sucrose non-fermenting 1) gene. To elucidate the role of cell wall-degrading enzymes in F. oxysporum pathogenicity, we cloned and disrupted its SNF1 homologue (FoSNF1). The fosnf1 mutants had a reduced expression of several genes encoding cell wall-degrading enzymes and grew poorly on certain carbon sources. Infection assays on Arabidopsis thaliana and Brassica oleracea revealed that progression of wilt symptoms in plants infected by fosnf1 mutants was considerably delayed, in comparison with those infected by a wild-type strain. In conclusion, mutations in FoSNF1 prevent F. oxysporum from properly derepressing the production of cell wall-degrading enzymes, compromise the utilization of certain carbon sources, and reduce its virulence on A. thaliana and B. oleracea.

The Nag1 N-acetylglucosaminidase of Trichoderma atroviride is essential for chitinase induction by chitin and of major relevance to biocontrol.

Abstract: The nag1 gene of the mycoparasitic fungus Trichoderma atroviride encodes a 73-kDa N-acetyl-β-d-glucosaminidase, which is secreted into the medium and partially bound to the cell wall. To elucidate the role of this enzyme in chitinase induction and biocontrol, a nag1-disruption mutant was prepared. It displayed only 4% of the original N-acetyl-β-d-glucosaminidase activity, indicating that the nag1 gene product accounts for the majority of this activity in T. atroviride. The nag1-disruption strain was indistinguishable from the parent strain in growth and morphology, but exhibited delayed autolysis. Northern analysis showed that colloidal chitin disruption does not induce ech42 gene transcription in the nag1-disruption strain. Enzyme activities capable of hydrolysing p-nitrophenyl-N,N′-diacetylchitobioside and p-nitrophenyl-N,N′-diacetylchitotriose were also absent from the nag1-disruption strain under the same conditions. Retransformation of the T. atroviride nag1-disruption strain with the nag1 gene essentially led to the parent-type behaviour in all these experiments. However, addition of N-acetyl-β-d-glucosaminidase to the medium of the nag1-disruption strain did not rescue the mutant phenotype. The disruption-nag1 strain showed 30% reduced ability to protect beans against infection by Rhizoctonia solani and Sclerotinia sclerotiorum. The data indicate that nag1 is essential for triggering chitinase gene expression in T. atroviride and that its functional impairment reduces biocontrol by T. atroviride by a significant extent.

Nucleo-cytoplasmic partitioning of proteins in plants: implications for the regulation of environmental and developmental signalling

Abstract: Considerable progress has been made in the past few years in characterising Arabidopsis nuclear transport receptors and in elucidating plant signal transduction pathways that employ nucleo-cytoplasmic partitioning of a member of the signal transduction chain. This review briefly introduces the major principles of nuclear transport of macromolecules across the nuclear envelope and the proteins involved, as they have been described in vertebrates and yeast. Proteins of the plant nuclear transport machinery that have been identified to date are discussed, the focus being on Importin β-like nuclear transport receptors. Finally, the importance of nucleo-cytoplasmic partitioning as a regulatory tool for signalling is highlighted, and different plant signal transduction pathways that make use of this regulatory potential are presented.

Versatile EGFP reporter plasmids for cellular localization of recombinant gene products in filamentous fungi

Abstract: The recent development of variants of the green fluorescent protein (GFP) with altered codon composition facilitated the efficient expression of this reporter protein in a number of fungal species. In this report, we describe the construction and application of a series of plasmids, which support the expression of an enhanced gfp (egfp) gene in filamentous fungi and assist the study of diverse developmental processes. Included were a promoterless egfp vector for monitoring the expression of cloned promoters/enhancers in fungal cells and vectors for creating translation fusions to the N-terminus of EGFP. The vectors were further modified by introducing a variant hygromycin B phosphotransferase (hph) gene, lacking the commonly found NcoI site. Instead, this site, which contained an ATG start codon, was placed in front of the egfp gene and thus was made suitable for the cloning of translational fusions. The applicability of these vectors is demonstrated by analyzing transcription regulation and protein localization and secretion in two ascomycetes, Acremonium chrysogenum and Sordaria macrospora. In the latter, the heterologous egfp gene is stably inherited during meiotic divisions, as can easily be seen from fluorescent ascospores.

Characterization of spontaneous mutants of Magnaporthe grisea expressing stable resistance to the Qo-inhibiting fungicide azoxystrobin.

Abstract: The class of Qo-inhibiting fungicides (QoIs) act as respiration inhibitors by binding to the Qo center of cytochrome b. The longevity of these fungicides has been challenged by the selection of fungal sub-populations resisting high doses of QoI fungicides, with a G143A amino acid exchange in the cytochrome b target site identified as the most common cause of resistance. In contrast, the mechanism of alternative respiration, as another mechanism of fungal QoI resistance, has thus far not been affiliated with practical resistance. In the present study, azoxystrobin-resistant mutants of Magnaporthe grisea were generated and characterized. Emergence of these spontaneous mutants was facilitated when resting melanized mycelia were allowed to escape full inhibition by azoxystrobin. This escape was related to the intactness of alternative respiration, indicating that residual expression of this rescue mechanism was involved in the spontaneous emergence of target-site mutants. The two mutants characterized resisted high doses of the QoI, azoxystrobin, with resistance factors exceeding 1,000. Two different mutations of the cytochrome b gene were identified as exchanges of guanine, leading to a G143A or a G143S amino acid exchange. Resistance of both target-site mutants remained stable during four consecutive disease cycles in the absence of azoxystrobin. Several parameters tested to measure fitness penalties inherent to the mutational changes revealed that the G143A mutant was not compromised. In contrast, the conidia production of the G143S mutant was significantly lower under both saprophytic and pathogenic conditions of reproduction.

Mitotic recombination in Saccharomyces cerevisiae.

Abstract: Mitotic homologous recombination (HR) is an important mechanism for the repair of double-strand breaks and errors occurring during DNA replication It is likely that the recombinational repair of DNA lesions occurs preferentially by sister chromatid exchanges that have no genetic consequences However, most genetically detectable HR events occur between homologous DNA sequences located at allelic positions in homologous chromosomes, or between DNA repeats located at ectopic positions in either the same, homologous or heterologous chromosomes Mitotic recombination may occur by multiple mechanisms, including double-strand break repair, synthesis-dependent strand annealing, break-induced replication and single-strand annealing The occurrence of one recombination mechanism versus another depends on different elements, including the position of the homologous partner, the initiation event, the length of homology of the recombinant molecules and the genotype The genetics and molecular biology of the yeast Saccharomyces cerevisiae have proved essential for the understanding of mitotic recombination mechanisms in eukaryotes Here, we review recent genetic yeast data that contribute to our understanding of the different mechanisms of mitotic recombination and the in vivo role of the recombination proteins

Repression of transcription by Rgt1 in the absence of glucose requires Std1 and Mth1.

Abstract: In the yeast Saccharomyces cerevisiae, glucose induces expression of the hexose transporter (HXT) genes by inhibiting the repressor function of the transcription factor Rgt1. We have previously shown that Rgt1 binds to the HXT gene promoters only in the absence of glucose. In the presence of glucose, Rgt1 becomes phosphorylated and is unable to bind to the HXT promoters and repress their transcription. We report that Rgt1 interacts with Std1 and Mth1 in a yeast two-hybrid assay and co-immunoprecipitates with both proteins in vivo only when glucose is absent. In addition, we demonstrate that repression of HXT gene expression by Rgt1 is abolished in the std1 mth1 double mutant. While Rgt1 is normally phosphorylated only in the presence of high concentrations of glucose, it is constitutively modified in the std1 mth1 double mutant. Based on these data, we conclude that, in the absence of glucose, Rgt1 associates with Std1 and Mth1 to repress HXT gene expression.

Agrobacterium-mediated transformation of Botrytis cinerea, simple purification of monokaryotic transformants and rapid conidia-based identification of the transfer-DNA host genomic DNA flanking sequences.

Abstract: The Agrobacterium tumefaciens-mediated transfer of foreign DNA to the phytopathogenic fungus Botrytis cinerea was investigated. Fifteen stable transformants per 10(6) conidia were consistently produced. Monokaryons were purified in a single step and their molecular analysis demonstrated the random integration of predominantly single or tandem copies of the foreign DNA into their genome. Thermal asymmetric interlaced PCR performed directly on conidia led to the rapid identification of the genomic DNA sequences that flanked the integration sites of the transfer-DNA. Transcriptional fusions of green fluorescent protein and beta-glucuronidase-encoding genes to the promoter of the secreted proteolytic enzyme ACP1 were realised to validate the system. We provide herein observations of B. cinerea hyphae producing green fluorescent protein or beta-glucuronidase under growth conditions similar to those known to induce transcription of the acp1 gene.

Mitochondrial genome diversity: evolution of the molecular architecture and replication strategy.

Abstract: Mitochondrial genomes in organisms from diverse phylogenetic groups vary in both size and molecular form. Although the types of mitochondrial genome appear very dissimilar, several lines of evidence argue that they do not differ radically. This would imply that interconversion between different types of mitochondrial genome might have occurred via relatively simple mechanisms. We exemplify this scenario on patterns accompanying evolution of mitochondrial telomeres. We propose that mitochondrial telomeres are derived from mobile elements (transposons or plasmids) that invaded mitochondria, integrated into circular or polydisperse linear mitochondrial DNAs (mtDNAs) and subsequently enabled precise resolution of the linear genophore. Simply, the selfish elements generated a problem - how to maintain the ends of a linear DNA - and, at the same time, made themselves essential by providing its solution. This scenario implies that insertion or deletion of such resolution elements may represent relatively simple routes for interconversion between different forms of the mitochondrial genome.

Characterisation and expression analysis of a nitrate transporter and nitrite reductase genes, two members of a gene cluster for nitrate assimilation from the symbiotic basidiomycete Hebeloma cylindrosporum

Abstract: Symbiotic ectomycorrhizal fungi contribute to the nitrogen nutrition of their host-plants but little information is available on the molecular control of their nitrogen metabolism. We cloned and characterised genes encoding a nitrite reductase and a nitrate transporter in the ectomycorrhizal basidiomycete Hebeloma cylindrosporum. These two genes are divergently transcribed and linked to a previously cloned nitrate reductase gene, thus demonstrating that nitrate assimilation gene clusters occur in homobasidiomycetes. The nitrate transporter polypeptide (NRT2) is characterised by 12 transmembrane domains and presents both a long putative intracellular loop and a short C-terminal tail, two structural features which distinguish fungal high-affinity transporters from their plant homologues. In different wild-type genetic backgrounds, transcription of the two genes was repressed by ammonium and was strongly stimulated not only in the presence of nitrate but also in the presence of organic nitrogen sources or under nitrogen deficiency.

The role of adaptor protein Ste50-dependent regulation of the MAPKKK Ste11 in multiple signalling pathways of yeast.

Abstract: In Saccharomyces cerevisiae, Ste50 functions in cell signalling between the activated G protein and the mitogen-activated protein kinase (MAPK) kinase kinase (MAPKKK) Ste11. ScSte50 is an essential component of three MAPK-mediated signalling pathways, which control the mating response, invasive/filamentous growth and osmotolerance (HOG pathway), respectively. ScSte50 signalling may also contribute to cell wall integrity in vegetative cells. The protein contains a sterile alpha motif (SAM) and a putative Ras-associated domain (RAD), which are essential for signal transduction. Ste50 and Ste11 interact constitutively via their SAM regions. Ste50 interacts weakly and probably transiently with the pheromone receptor-bound heterotrimeric G protein G(alpha beta gamma), and with the small G proteins Cdc42, Ras1 and Ras2. It is specifically the RAD region of Ste50 that mediates the interactions with Cdc42 and Ras. Homologues of ScSTE50 are also found in other fungi, like S. kluyveri, Hansenula polymorpha, Candida albicans and Neurospora crassa. In this review, the role of Ste50 as an adaptor that links the G protein-associated Cdc42-Ste20 kinase complex to the effector kinase Ste11 and thus modulates signal transduction, especially in the pheromone-response pathway of S. cerevisiae, is discussed.

Chloroplast RNA-binding proteins

Abstract: Chloroplast gene expression is regulated by nucleus-encoded factors, which mainly act at the post-transcriptional level. Plastid RNA-binding proteins (RBPs) represent good candidates for mediating these functions. The picture emerging from recent analyses is that of a great number of differentially regulated RBPs, which are organized in distinct, spatially separated supramolecular complexes. This reflects the complexity of the regulatory network that underlies the intracellular communication system between the nucleus and the chloroplast.

Characterisation of the mating-type locus of the plant pathogenic ascomycete Leptosphaeria maculans

Abstract: The nucleotide sequences of regions containing the mating-type locus of the plant-pathogenic ascomycete Leptosphaeria maculans are described. The MAT1-1 gene is 1,368 bp, encoding a predicted protein of 441 amino acids, with a 45-bp intron. The MAT1-2 gene is 1,246 bp, encoding a predicted protein of 397 amino acids, with a 55-bp intron. This latter gene is 334 bp downstream of a small open reading frame (32 amino acids) with four amino acids in identical positions to those in the high mobility group binding domain of the MAT1–2 genes. The DNA lyase and anaphase promoting complex genes are 3′ of the MAT gene, whilst a gene denoted ORF1 in Cochliobolus heterostrophus and the GTPase activating protein are present 5′ of MAT. The transcriptional patterns of genes within and flanking the L. maculans MAT locus are determined. The MAT transcripts are about twice the length of the gene. The ORF1 transcript is 1.2 kb in the MAT1-1 isolate and 1.0 kb in the MAT1-2 isolate; and probes cross-hybridise weakly. A mating-type PCR assay with three nucleotide primers is developed for L. maculans.

PCR-based Methods Facilitate Targeted Gene Manipulations and Cloning Procedures

Abstract: Genome sequencing of a large number of organisms has provided a wealth of previously uncharacterized genes. Rapid functional analysis of these genes relies on efficient methods for targeted gene disruption. Gene replacement requires homologous recombination at the target locus. The efficiency of homologous recombination largely depends on the size of the flanking homology regions provided with the disruption cassette. Therefore, the ratio of targeted versus random integration into the genome governs the choice of tools applicable in any organism. PCR-based methods for gene disruption were first reported in Saccharomyces cerevisiae. Over the past years, additional tools have been developed for epitope- or green fluorescent protein-tagging of genes and for promoter exchanges. The attractiveness of these tools led to the generation of PCR modules for use in a wide variety of bacterial and fungal species. The high capacity of in vivo recombination of Sac. cerevisiae and Escherichia coli may also be used for heterologous DNA manipulations. This facilitates the generation of disruption cassettes for organisms that cannot be transformed with very short flanks of target homology regions. Furthermore, laborious cloning procedures, e.g. the generation of point mutations or the deletion of internal domains of genes, can be simplified by using these organisms as workhorses which will advance the general genetic toolkit.

The G protein β subunit FGB1 regulates development and pathogenicity in Fusarium oxysporum

Abstract: The cloning of fgb1, the gene encoding a heterotrimeric G protein β subunit FGB1 in Fusarium oxysporum, was performed by standard PCR techniques to evaluate the role of G protein signaling in this fungus. The full-length open reading frame spanned 1,077 nucleotides and the deduced primary structure of the protein (359 amino acid residues) showed high identity with Gβ subunits from other organisms. Disruption of fgb1 led to decreased intracellular cAMP levels, reduced pathogenicity, and alterations in physiological characteristics, including heat resistance, colony morphology, conidia formation and germination frequency. We previously showed that most of these alterations (except germination frequency) were also observed in the disruptants of fga1, the gene for Gα subunit FGA1 in F. oxysporum. These results suggest that FGA1 and FGB1 have partially overlapping functions in the regulation of development and pathogenicity in F. oxysporum.

Cryptosporidium parvum Cpn60 targets a relict organelle.

Abstract: Chaperonin 60 (Cpn60) is a well-established marker protein for eukaryotic mitochondria and plastids. In order to determine whether the small double-membrane-bounded organelle posterior to the nucleus in the apicomplexan Cryptosporidium parvum is a mitochondrion, the Cpn60 gene of C. parvum sporozoites ( CpCpn60) was analyzed and antibodies were generated for localization of the peptide. Sequence and phylogenetic analyses indicated that CpCpn60 is a mitochondrial isotype and that antibodies against it localize to the rough endoplasmic reticulum-enveloped remnant organelle of C. parvum sporozoites. These data show this organelle is of mitochondrial origin.

Genetic conservation versus variability in mitochondria: the architecture of the mitochondrial genome in the petite-negative yeast Schizosaccharomyces pombe.

Abstract: The great amount of molecular information and the many molecular genetic techniques available make Schizosaccharomyces pombe an ideal model eukaryote, complementary to the budding yeast Saccharomyces cerevisiae In particular, mechanisms involved in mitochiondrial (mt) biogenesis in fission yeast are more similar to higher eukaryotes than to budding yeast In this review, recent findings on mt morphogenesis, DNA replication and gene expression in this model organism are summarised A second aspect is the organisation of the mt genome in fission yeast On the one hand, fission yeast has a strong tendency to maintain mtDNA intact; and, on the other hand, the mt genomes of naturally occurring strains show a great variability Therefore, the molecular mechanisms behind the susceptibility to mutations in the mtDNA and the mechanisms that promote sequence variations during the evolution of the genome in fission yeast mitochondria are discussed

Phylogenetic analysis reveals five independent transfers of the chloroplast gene rbcL to the mitochondrial genome in angiosperms

Abstract: We used the chloroplast gene rbcL as a model to study the frequency and relative timing of transfer of chloroplast sequences to the mitochondrial genome. Southern blot survey of 20 mitochondrial DNAs confirmed three previously reported groups of plants containing rbcL in their mitochondrion, while PCR studies identified a new mitochondrial rbcL. Published and newly determined mitochondrial and chloroplast rbcL sequences were used to reconstruct rbcL phylogeny. The results imply five or six separate interorganellar transfers of rbcL among the angiosperms examined, and hundreds of successful transfers across all flowering plants. By taxonomic criteria, the crucifer transfer is the most ancient, two separate transfers within the grass family are of intermediate ancestry, and the morning-glory transfer is most recent. All five mitochondrial copies of rbcL examined exhibit insertion and/or deletion events that disrupt the reading frame (three are grossly truncated); and all are elevated in the proportion of nonsynonymous substitutions, providing clear evidence that these sequences are pseudogenes.

Malo-ethanolic fermentation in Saccharomyces and Schizosaccharomyces.

Abstract: Yeast species are divided into the K(+) or K(−) groups, based on their ability or inability to metabolise tricarboxylic acid (TCA) cycle intermediates as sole carbon or energy source. The K(−) group of yeasts includes strains of Saccharomyces, Schizosaccharomyces pombe and Zygosaccharomyces bailii, which is capable of utilising TCA cycle intermediates only in the presence of glucose or other assimilable carbon sources. Although grouped together, these yeasts have significant differences in their abilities to degrade malic acid. Typically, strains of Saccharomyces are regarded as inefficient metabolisers of extracellular malic acid, whereas strains of Sch. pombe and Z. bailii can effectively degrade high concentrations of malic acid. The ability of a yeast strain to degrade extracellular malic acid is dependent on both the efficient transport of the dicarboxylic acid and the efficacy of the intracellular malic enzyme. The malic enzyme converts malic acid into pyruvic acid, which is further metabolised to ethanol and carbon dioxide under fermentative conditions via the so-called malo-ethanolic (ME) pathway. This review focuses on the enzymes involved in the ME pathway in Sch. pombe and Saccharomyces species, with specific emphasis on the malate transporter and the intracellular malic enzyme.

Protective mechanisms against the antitumor agent bleomycin: lessons from Saccharomyces cerevisiae

Abstract: Bleomycin is a small glycopeptide antibiotic used in combination therapy for the treatment of a few types of human cancer. The antitumor effect of bleomycin is most likely caused by its ability to bind to DNA and induce the formation of toxic DNA lesions via a free radical reactive (Fe.bleomycin) complex. However, the chemotherapeutic potential of bleomycin is limited, as it causes pulmonary fibrosis and tumor resistance at high doses. The chemical structure and modes of action of bleomycin have been extensively studied and these provide a foundation towards improving the therapeutic value of the drug. This review provides a first account of the current state of knowledge of the cellular processes that can allow the yeast Saccharomyces cerevisiae to evade the lethal effects of bleomycin. This model organism is likely to provide rapid clues in our understanding of bleomycin resistance in tumor cells.

The npgA/ cfwA gene encodes a putative 4'-phosphopantetheinyl transferase which is essential for penicillin biosynthesis in Aspergillus nidulans.

Abstract: Non-ribosomal peptide synthetases, polyketides and fatty acid synthetases have a modular organisation of multi-enzymatic activities. In all of them, the acyl or peptidyl carrier proteins have 4'-phosphopantetheine (P-pant) as an essential prosthetic group. This is added by 4'-phosphopantetheinyl transferases (PPTases) that derive the P-pant group from coenzyme A. While many PPTases of varying specificity have now been isolated from a number of bacteria, a filamentous fungal PPTase has yet to be characterised. Through database searching of the Aspergillus fumigatus genome sequence against Sfp from Bacillus subtilis, we identified a unique sequence which appears to encode a PPTase, as deduced from conserved residues considered important in PPTases. The PPTase candidate was used to search the NCBI data base and an unexpected homologue in A. nidulans was identified as npgA. Mutations in this gene (cfwA/npgA) were identified previously as leading to defects in growth and pigmentation. To test whether the temperature-sensitive cfwA2 mutation impairs penicillin biosynthesis, which is dependent on the δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine synthetase, bioassays with B. calidolactis were set up at permissive and non-permissive temperatures. The cfwA2 mutant did not produce penicillin at the non-permissive temperature. Since no other PPTase homologue has been detected in the A. fumigatus genome to date, the data suggest that a single enzyme may be able to transfer the cofactor to a broad range of enzymes with acyl or peptidyl carrier protein domains.

4′-Phosphopantetheinyl transferase-encoding npgA is essential for siderophore biosynthesis in Aspergillus nidulans

Abstract: Aspergillus nidulans produces two major siderophores: it excretes triacetylfusarinine C to capture iron and contains ferricrocin as an intracellular iron-storage compound. Siderophore biosynthesis involves the enzymatic activity of nonribosomal peptide synthetases (NRPS). NRPS contain 4′-phosphopantetheine as an essential prosthetic group, which is attached by 4′-phosphopantetheinyl transferases. A. nidulans appears to possess at least one gene, npgA, encoding such an enzyme. Using a strain carrying a temperature-sensitive allele, cfwA2, we showed that NpgA is essential for biosynthesis of both the peptide bond-containing ferricrocin and the ester bond-containing triacetylfusarinene C. The cfwA2 strain was found to be iron-starved at the restrictive temperature during iron-replete conditions, consistent with the siderophore system being the major iron-uptake system—as we recently demonstrated. Northern analysis indicated that, in contrast to other genes which are involved in siderophore biosynthesis and uptake, expression of npgA is not controlled by the GATA-transcription factor SreA. It was shown previously that NpgA is required for biosynthesis of penicillin, pigment, and potentially lysine via the α-aminoadipate pathway. Supplementation with lysine plus triacetylfusarinine C restored normal growth of the cfwA2 strain at the restrictive temperature, suggesting that the growth defect of the mutant is mainly due to impaired biosynthesis of siderophores and lysine.

High-efficiency electroporation by freezing intact yeast cells with addition of calcium.

Abstract: We developed a novel freezing method to generate competent cells of Schizosaccharomyces pombe and Saccharomyces cerevisiae prior to electroporation Freezing the intact cells in sorbitol with the addition of calcium at −80 °C allowed us to improve the transformation efficiency after freezing and thawing The optimum concentration of CaCl2 was found to be 5–10 mM The addition of other cations had no effect on the efficiency, while the addition of calcium meant that a broad concentration of sorbitol (06–25 M) could be used, independent of strain Moreover, increasing the cell concentration to 2×109 cells/ml during an applied electric pulse further increased the efficiency after freezing and resulted in a wide range of electric field strength (90–115 kV/cm) Therefore, there was no need to optimize both the concentration of cryoprotectant and the electric field strength of the applied pulse This procedure for electroporation allows the frozen competent cells to be stored long-term without any significant loss of efficiency

Evolution, biochemistry and genetics of protein kinase C in fungi

Abstract: From yeast to humans, protein kinase C is an enzyme of central importance in signal transduction processes. In baker's yeast, Saccharomyces cerevisiae, a single gene encoding Pkc1p has been identified. Mutant analyses revealed that Pkc1p function is essential to ensure cellular integrity through regulation of a specific MAP kinase cascade. Due to the involvement of different defective mammalian isozymes in various diseases and the model character of simple eukaryotes, increasing attention has been paid to the structure and regulation of the enzymes of fungal origin. This review summarizes the knowledge gathered so far.