Life cycle,meiotic recombination and unilateral gene transfer in methanolotrophic yeasts of the genus Pichia

Summary In order to perform strain improvement of the methanol utilising yeasts Pichia pinus and Pichia lindneri their life cycles were elucidated. Both yeasts are monoecious and self-fertile. Karyogamy by autodiploidization is followed by meiosis leading to Mendelian segregation within the offspring. In Pichia pinus crosses via cell or protoplast fusion is feasible as proven by tetrad analysis. This is not the case in Pichia lindneri. The only possiblity to obtain recombinants in this yeast is brought about by a yet unknown mechanism, leading to a unilateral transfer of genetic material, found after cocultivation of mutants. This is revealed by the evaluation of F1 offspring from meiotic divisions after autodiploidisation. Although analysis of nuclear and mitochondrial DNA revealed rather gross differences not only between the species but also within different strains of Pichia pinus, only a limited exchange of genetic material is possible in interstrain crosses.Dedicated to Professor Dr. H. J. Rehm (Münster) on the occasion of his 60th birthday     

Genomic exploration of the hemiascomycetous yeasts: 13. Pichia angusta

As part of a comparative genomics project on 13 hemiascomycetous yeasts, the Pichia angusta type strain was studied using a partial random sequencing strategy. With coverage of 0.5 genome equivalents, about 2500 novel protein-coding genes were identified, probably corresponding to more than half of the P. angusta protein-coding genes, 6% of which do not have homologs in Saccharomyces cerevisiae. Some of them contain one or two introns, on average three times shorter than those in S. cerevisiae. We also identified 28 tRNA genes, a few retrotransposons similar to Ty5 of S. cerevisiae, solo long terminal repeats, the whole ribosomal DNA cluster, and segments of mitochondrial DNA. The P. angusta sequences were deposited in EMBL under the accession numbers AL430961 to AL436044.     

Culturing freshwater, planktonic rotifers on Rhodomonas minuta var. nannoplanctica Skuja and Stichococcus bacillaris Nageli

Eleven freshwater, planktonic rotifer species (Brachionus angularis,Brachionus cafyciflorus, Keratella quadrata, Nolholca squamula,Ascomorpha ecattdis, Synchaeta kitina, Synchaela tremula, Potyarthradolichoptera, Polyanhra vulgarii, Pompholyx sulcata, Filiniabrachiata) were cultured successfully on either Rhodomonas minutavar. nannoplanctica Skuja or Stichococcus bacillaris Nägeli,in a totally synthetic medium. Three of these species had notpreviously been kept in laboratory culture. The method is proposedas a simple technique for the routine batch culture of a varietyof planktonic rotifer species. Some important observations onhandling techniques, incubation temperature, culture vesselsand media are discussed.     

Characterization of microsatellite markers in the interspecific hybrid Phytophthora alni ssp. alni,and cross‐amplification with related taxa

Phytophthora alni ssp. alni is an interspecific hybrid oomycete causing a large‐scale decay of alders throughout Europe. In this study we developed a set of 10 microsatellite markers that shows promise for population studies and for studying hybridization events between the parental species of the hybrid. Moreover, the genotype and the ploidy of the different subspecies of P. alni might be inferred from the quantitative ratio of amplified genome‐specific alleles. Nine primer pairs cross amplified with the related species Phytophthora cambivora and Phytophthora fragariae and yielded distinct alleles.     

条叶车前和小车前的核型报道

本文报道了车前属两种车前的核型。Plantago lessingii为2n＝12,核型属于“2A”,核型公式为K(2n)＝12＝10m+2sm;P. mmuta为2n＝12,核型仍属“2A”型,核型公式K(2n)＝12＝8m+2sm+2st。染色体相对长度组成,前者2n＝2L+4M2+4M1+2S1,后为2n＝6M2+6M1,染色体总长分别为30.60、29.80,由12条染色体组成,还测量了这两种车前的染色体体积。     

Interspecies hybridization and recombination in Saccharomyces wine yeasts

The ascomycetous yeasts traditionally referred to as the Saccharomyces sensu stricto complex are a group of closely related species that are isolated by a postzygotic barrier. They can easily hybridize; and their allodiploid hybrids propagate by mitotic divisions as efficiently as the parental strains, but can barely divide by meiosis, and thus rarely produce viable spores (sterile interspecies hybrids). The postzygotic isolation is not effective in allotetraploids that are able to carry out meiosis and produce viable spores (fertile interspecies hybrids). By application of molecular identification methods, double (Saccharomyces cerevisiae x Saccharomyces uvarum and S. cerevisiae x Saccharomyces kudriavzevii) and triple (S. cerevisiae x S. uvarum x S. kudriavzevii) hybrids were recently identified in yeast populations of fermenting grape must and cider in geographically distinct regions. The genetic analysis of these isolates and laboratory-bred hybrids revealed great variability of hybrid genome structures and demonstrated that the alloploid genome of the zygote can undergo drastic changes during mitotic and meiotic divisions of the hybrid cells. This genome-stabilization process involves loss of chromosomes and genes and recombination between the partner genomes. This article briefly reviews the results of the analysis of interspecies hybrids, proposes a model for the mechanism of genome stabilization and highlights the potential of interspecies hybridization in winemaking.     

Metabolic-flux profiling of the yeasts Saccharomyces cerevisiae and Pichia stipitis

The so far largely uncharacterized central carbon metabolism of the yeast Pichia stipitis was explored in batch and glucose-limited chemostat cultures using metabolic-flux ratio analysis by nuclear magnetic resonance. The concomitantly characterized network of active metabolic pathways was compared to those identified in Saccharomyces cerevisiae, which led to the following conclusions. (i) There is a remarkably low use of the non-oxidative pentose phosphate (PP) pathway for glucose catabolism in S. cerevisiae when compared to P. stipitis batch cultures. (ii) Metabolism of P. stipitis batch cultures is fully respirative, which contrasts with the predominantly respiro-fermentative metabolic state of S. cerevisiae. (iii) Glucose catabolism in chemostat cultures of both yeasts is primarily oxidative. (iv) In both yeasts there is significant in vivo malic enzyme activity during growth on glucose. (v) The amino acid biosynthesis pathways are identical in both yeasts. The present investigation thus demonstrates the power of metabolic-flux ratio analysis for comparative profiling of central carbon metabolism in lower eukaryotes. Although not used for glucose catabolism in batch culture, we demonstrate that the PP pathway in S. cerevisiae has a generally high catabolic capacity by overexpressing the Escherichia coli transhydrogenase UdhA in phosphoglucose isomerase-deficient S. cerevisiae.     

Comparative studies of isozymes in Oryza sativa,O. minuta,and their interspecific derivatives: evidence for homoeology and recombination

Enzyme electrophoresis was used to compare the isozyme phenotypes of Oryza sativa, IR31917 (AA genome), and two O. minuta accessions (Om 101089 and Om101141; BBCC genome) for ten enzyme systems. Between the two species, two systems were monomorphic (isocitrate dehydrogenase and alcohol dehydrogenase) and eight were polymorphic (shikimate dehydrogenase, phosphogluconate dehydrogenase, phosphoglucose isomerase, malate dehydrogenase, glutamate oxaloacetate transaminase, esterase, aminopeptidase, and endopeptidase). Polymorphism between O. minuta accessions was detected for shikimate dehydrogenase and glutamate oxaloacetate. As expected, the quaternary structure of the O. minuta isozymes was comparable to that of O. sativa. Possible allelic relationships with known O. sativa alleles and their genomic designation are discussed. Combined with chromosome data, the interspecific variation was exploited to monitor the relative genetic contribution of the two parents in the IR31917/Om101141 F₁ hybrids and recurrent (IR31917) backcross progenies. The isozyme content of F₁ hybrid reflected its triploid nature (ABC genome composition), while that of the backcross progenies paralleled the duplication of the A genome and the gradual loss of O. minuta chromosomes during the backcrossing process. Evidence is provided for a degree of homoeology between the A, B, and C genomes, and for introgression from O. minuta into O. sativa.     

Hybridization of cells of the same mating type in Saccharomyces yeasts

The problem of mating-type switches in heterothallic yeast cells was investigated. 93% of non-mating hybrids were obtained in a X a crosses. The hybrids obtained in alpha X alpha crosses expressed alpha-mating type predominantly. Hybrids with no major rearrangements or loss of chromosome III were detected among these hybrids. In the selective system for cytoduction in a X a crosses the significant part of all cytoductants were alpha-maters, i.e. those originated through a----alpha switches. In alpha X alpha crosses alpha cytoductants were predominantly obtained either spontaneously or after UV-irradiation, though the frequency of cytoductants after UV-irradiation exceeded the control value several times. So, we developed the method for selection of mating-type "switchers" (a in equilibrium alpha), avoiding the diploid stage, and demonstrated the possibility of hybridization among the alpha-cells without hereditary changes at the MAT locus.     

Stereoselective hydrolysis of dl-menthyl succinate by gel-entrapped Rhodotorula minuta var. texensis cells in organic solvent

Summary dl-Menthyl succinate was successfully hydrolyzed stereoselectively by Rhodotorula minuta var. texensis cells entrapped within photo-crosslinked or polyurethane resin gels in water-saturated n-heptane. The hydrolyzed product was found to be pure l-menthol. The catalytic activity of the immobilized cells, especially those entrapped in urethane polymers, was far more stable than that of the free cells. The half-life of the polyurethaneentrapped cells was estimated to be 55–63 days in the organic solvent.Dedicated to the 65th birthday od Professor Dr. G. Manecke     

Comparative genomics of hemiascomycete yeasts: genes involved in DNA replication, repair, and recombination

Among genes conserved from bacteria to mammals are those involved in replicating and repairing DNA. Following the complete sequencing of four hemiascomycetous yeast species during the course of the Genolevures 2 project, we have studied the conservation of 106 genes involved in replication, repair, and recombination in Candida glabrata, Kluyveromyces lactis, Debaryomyces hansenii, and Yarrowia lipolytica and compared them with their Saccharomyces cerevisiae orthologues. We found that proteins belonging to the replication fork and to the nucleotide excision repair pathway were-on the average-more conserved than proteins involved in the checkpoint response to DNA damage or in meiotic recombination. The meiotic recombination proteins Spo11p and Mre11p-Rad50p, involved in making meiotic double-strand breaks (DSBs), are conserved as is Mus81p, involved in resolving meiotic recombination intermediates. Interestingly, genes found in organisms in which DSB-repair is required for proper synapsis during meiosis are also found in C. glabrata, K. lactis, and D. hansenii but not in Y. lipolytica, suggesting that two modes of meiotic recombination have been selected during evolution of the hemiascomycetous yeasts. In addition, we found that SGS1 and TOP1, respectively, a DEAD/DEAH helicase and a type I topoisomerase, are duplicated in C. glabrata and that SRS2, a helicase involved in homologous recombination, is tandemly duplicated in K. lactis. Phylogenetic analyses show that the duplicated SGS1 gene evolved faster than the original gene, probably leading to a specialization of function of the duplicated copy.     

Indole alkaloids from two cultured cyanobacteria, Westiellopsis sp. and Fischerella muscicola

Chemical investigation of two cultured cyanobacteria, Westiellopsis sp. (SAG strain number 20.93) and Fischerella muscicola (UTEX strain number LB1829) led to the isolation of three hapalindole-type alkaloids, namely hapalindole X (1), deschloro hapalindole I (2), and 13-hydroxy dechlorofontonamide (3), along with ten known indole alkaloids (hapalindoles A, C, G, H, I, J, and U, hapalonamide H, anhydrohapaloxindole A, and fischerindole L) and fischerellins A and B. The structures were determined by a combination of spectroscopic analyses mainly based on 1D and 2D NMR and HRESIMS data. Selected compounds were evaluated for cytotoxicity and exhibited weak to moderate cytotoxicity against HT-29, MCF-7, NCI-H460, SF268, and IMR90 cells. All compounds, except hapalindole C, were evaluated for 20S proteasome inhibition and displayed either weak or no inhibition at 25μg/mL. Selected compounds were also evaluated for antimicrobial activity, and hapalindoles X (1) and A, and hapalonamide H showed potent activity against both Mycobacterium tuberculosis and Candida albicans with MIC values ranging from 0.6 to 2.5μM.     

Energy conversion coupled to cyanide-resistant respiration in the yeasts Pichia membranifaciens and Debaryomyces hansenii

Cyanide-resistant respiration (CRR) is a widespread metabolic pathway among yeasts, that involves a mitochondrial alternative oxidase sensitive to salicylhydroxamic acid (SHAM). The physiological role of this pathway has been obscure. We used the yeasts Debaryomyces hansenii and Pichia membranifaciens to elucidate the involvement of CRR in energy conversion. In both yeasts the adenosine triphosphate (ATP) content was still high in the presence of antimycin A or SHAM, but decreased to low levels when both inhibitors were present simultaneously, indicating that CRR was involved in ATP formation. Also the mitochondrial membrane potential (Delta Psi(m)), monitored by fluorescent dyes, was relatively high in the presence of antimycin A and decreased upon addition of SHAM. In both yeasts the presence of complex I was confirmed by the inhibition of oxygen consumption in isolated mitochondria by rotenone. Comparing in the literature the occurrence of CRR and of complex I among yeasts, we found that CRR and complex I were simultaneously present in 12 out of 13 yeasts, whereas in six out of eight yeasts in which CRR was absent, complex I was also absent. Since three phosphorylating sites are active in the main respiratory chain and only one in CRR, we propose a role for this pathway in the fine adjustment of energy provision to the cell.     

Egg distribution and mortality in the alder aphid, Pterocallis alni

Alder aphid oviparae produced 14 eggs at constant temperature but in the field the average was 6. Favoured oviposition sites were bud axils and bark crevices. Summer pruning of host trees reduced the availability but not the choice of site. Oviparae reared on leaf regrowth of summer pruned alder were smaller and contained fewer eggs than those on mature leaves. Winter pruning resulted in the loss of 41% buds and 25% eggs. Total winter egg mortality was 65%, being greatest in early winter due to insect predation. Spring egg hatch was synchronised with bud burst and both were affected by temperature.

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Distribution des oeufs et mortalité Pterocallis alni

Résumé Les ovipares de P. alni ont pondu 14 oeufs à température constante mais dans la nature la moyenne n'était que de 6. La taille estivale a réduit les disponibilités, mais n'a pas modifié le choix des lieux de ponte. Les ovipares élevés sur feuilles de repousses estivales d'aulnes taillént plus petits et contenaient moins d'oeufs que ceux élevés sur feuilles adultes. La taille hivernale a provoqué la perte de 41% des bourgeons et 25% des oeufs. La mortalité hivernale totale des oeufs a été de 65%, la partie la plus importante, due à la prédation par les insectes, ayant eu lieu au début de l'hiver. L'éclosion des oeufs au printemps était synchronisée avec l'éclatement des bourgeons, tous les deux étant conditionnés par la température.

     

Distribution of aspartate aminotransferase activity in yeasts, and purification and characterization of mitochondrial and cytosolic isoenzymes from Rhodotorula minuta [corrected

The distribution of aspartate aminotransferase activity in yeasts was determined. The number of species of the enzyme in each yeast was determined by zymogram analysis. All the yeasts, except for the genus Saccharomyces, showed two or three activity bands on a zymogram. From among the strains, Rhodotorula minuta [corrected] and Torulopsis candida were selected for examination of the existence of yeast mitochondrial isoenzymes, because these strains showed two clear activity bands on the zymogram and contained a high amount of the enzyme. Only one aspartate aminotransferase was purified from T. candida: the component in the minor band on the zymogram was not an isoenzyme of aspartate aminotransferase. On the other hand, two aspartate aminotransferases were purified to homogeneity from R. minuta [corrected]. The components in the main and minor activity bands on the zymogram were identified as the mitochondrial and cytosolic isoenzymes, respectively, in a cell-fractionation experiment. The enzymatic properties of these isoenzymes were determined. The yeast mitochondrial isoenzyme resembled the animal mitochondrial isoenzymes in molecular weight (subunits and native form), absorption spectrum, and substrate specificity. The amino acid composition was closely similar to that of pig mitochondrial isoenzyme. Rabbit antibody against the yeast mitochondrial isoenzyme, however, did not form a precipitin band with the pig mitochondrial isoenzyme.     

Some aspects on the morphology and ecology of Rhodomonas minuta var. nannoplanctica and R. lens (Cryptophyceae) in two Greek lakes

Significant morphological and ecological differences between Rhodomonas minuta var. nannoplanctica and R. lens were observed during the study of phytoplankton in two Greek lakes. The shape and size characteristics support the validity of these species, so the fusion of closely related species of Rhodomonas proposed by Javornicky is not accepted in this study. Apart from the morphological differences between Rhodomonas minuta var. nannoplanctica and R. lens contrasting ecological requirements were noticed. A broader distribution of Rhodomonas minuta var. nannoplanctica in relation to temperature and pH and a preference for lower nutrient concentrations than R. lens were found. The two taxa may coexist by shifting their niches at different times in Lake Volvi and by vertical niche separation in Lake Vegoritis.