Hansenula Wingei Research Articles

In vitro and in vivo antifungal activity of natural inhibitors against Penicillium expansum

Penicillium expansum is the causative agent of apple blue mold. The inhibitory effects of the capsaicin derived from Capsicum spp. fruits and yeast Hansenula wingei against P. expansum were evaluated in an in vitro and in in vivo assay using Fuji apples. The minimum inhibitory concentration of capsaicin determined using the broth micro-dilution method was 122.16 µg mL-1. Capsaicin did not reduce blue mold incidence in apples. However, it was able to delay fungal growth in the first 14 days of the in vivo assay. The in vivo effect of the yeast Hansenula wingei AM2(-2), alone and combined with thiabendazole at low dosage (40 µg mL-1), on the incidence of apple diseases caused by P. expansum was also described. H. wingei AM2(-2) combined with a low fungicide dosage (10% of the dosage recommended by the manufacturer) showed the best efficacy (100%) up to 7 days of storage at 21 oC, later showing a non-statistically different decrease (p > 0.05) after 14 (80.45%) and 21 days (72.13%), respectively. These results contribute providing new options for using antifungal agents against Penicillium expansum.

Up-to-dating of complete sequenced DNA data of Hansenula wingei yeast mitochondria

To update sequenced data, we determined the 5' and 3' termini of yeast Hansenula wingei (Pichia canadensis) mitochondrial (mt) large subunit ribosomal RNA (LSU) which is encoded in the mt genome. The 5' end position was mapped downstream from a putative transcription starting site which is homologous to a Saccharomyces cerevisiae mitochondrial promoter sequence. This suggests that the primary transcript of LSU is processed from 5' end and then mature transcript is formed. This processing is different from that of S. cerevisiae mt LSU in which processing on its 5' end does not occur. Based on the sequence data of H. wingei mt LSU, we constructed its secondary structure, and compared it with those of the other fungal organisms. Conserved regions of H. wingei LSU were identified and used for subsequent phylogenetic analysis. In genome structure and gene content, H. wingei mt genome has several characteristics similar to those in filamentous fungi, but the phylogenetic analysis indicates closer kinship to yeast S. cerevisiae. This agrees with previous non-sequencing phylogenies and suggests that extraordinary rearrangements have occurred in yeast mt genomes during divergent evolution.

Yeast mapping reports. Clone bank of the mitochondrial genome of yeast Hansenula wingei

For sequencing, mitochondrial DNA from Hansenula wingei yeast was digested with various restriction enzymes and the resultant DNA fragments were cloned into a pEMBL phasmid vector. Our clone bank consists of 39 overlapping clones which cover the entire 27,694 bp region of the H. wingei mitochondrial genome.

Electrophoretic karyotype of Saprolegnia monoica

The electrophoretic karyotype of Saprolegnia monoica was determined by contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Eight chromosomal bands were separated. The size of these bands, based on migration relative to those of chromosomal DNA of Saccharomyces cerevisiae, Schizosaccharomyces pombe and Hansenula wingei, is estimated to be between 0.9 and 5.8 Mb. The genome size is estimated to be 51 Mb.

Electrophoretic karyotype ofSaprolegnia monoica

The electrophoretic karyotype of Saprolegnia monoica was determined by contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Eight chromosomal bands were separated. The size of these bands, based on migration relative to those of chromosomal DNA of Saccharomyces cerevisiae, Schizosaccharomyces pombe and Hansenula wingei, is estimated to be between 0.9 and 5.8 Mb. The genome size is estimated to be 51 Mb.

The complete mitochondrial DNA sequence of Hansenula wingei reveals new characteristics of yeast mitochondria

The complete 27,694-bp mitochondrial (mt) DNA sequence of Hansenula wingei, which is a typical budding yeast and contains circular mitochondrial DNA, has been determined. The mt sequence contains genes encoding large and small ribosomal RNAs, 25 tRNAs, three subunits of cytochrome c oxidase (subunits 1, 2 and 3), three subunits of ATPase (subunits 6, 8 and 9), apocytochrome b, seven subunits of NADH dehydrogenase (subunits 1, 2, 3, 4, 4L, 5 and 6), and a ribosomal protein, VAR1. The VAR1 gene is considered to be a typical yeast type. This is consistent with data on DNA and the deduced amino-acid sequence homology comparisons of genes ubiquitous in yeast and fungi. However, we have identified seven genes encoding NADH dehydrogenase subunits, which are not found in other yeast mitochondrial genomes, thus placing the H. wingei mitochondrial genome in a unique position. In addition the H. wingei mitochondrial genome also encodes one tRNA pseudogene and one short unidentified ORF. The genome is compact with only two introns both of which contain an ORF. One intron lies in the large rRNA gene while the other is situated in the cytochrome c oxidase subunit-1 gene. The conserved nonanucleotide motif (A/T)TATAAG (T/A)(A/T), which is a transcription start signal in Saccharomyces cerevisiae mitochondria, has also been found in the H. wingei mitochondrial genome. The codon assignments for ATA and CTN in H. wingei mitochondria are different from those in S. cerevisiae mitochondria. These results indicate a unique and novel structure for the H. wingei mitochondrial genome in terms of characteristics which are typical for both yeast and for filamentous fungi. This is the first complete mt DNA sequence report in yeast.

Electrophoretic karyotype of the astaxanthin-producing yeast Phaffia rhodozyma

The electrophoretic karyotype of three different strains of Phaffia rhodozyma was determined by contour-clamped homogeneous electric field (CHEF)-gel electrophoresis. Significant differences in electrophoretic karyotyping patterns were found among the three strains studied. Between nine and 17 bands were observed. The size of these bands, based on their migration relative to the chromosomal DNA of Schizosaccharomyces pombe, Hansenula wingei was estimated to be between 0.48 and 3.1 Mb.

Electrophoretic Karyotype of Intracellular Yeast-like Symbiotes in Rice Planthoppers and Anobiid Beetles

Chromosomal DNA molecules of intracellular yeast-like symbiotes (YLS) of three species of rice planthoppers and two species of anobiid beetles have been separated by pulsed-field gel electrophoresis. Probable chromosome numbers of Nilaparvata lugens, Sogatella furcifera, and Laodelphax striatellus, were 4, 4, and 5, respectively, and tentative genome sizes were 17.3, 17.6, and 20.1 Mbp, respectively, based upon migration of individual chromosome-sized DNA relative to the size standards of Schizosaccharomyces pombe, Hansenula wingei, and Saccharomyces cerevisiae chromosomes. Chromosome numbers of Lasioderma serricorne and Stegobium paniceum were 11 and 15, respectively, and total genome sizes were 20.9 and 15.1 Mbp, respectively. The chromosomes carrying ribosomal RNA genes were identified by Southern blot analysis. Chromosomal organization and the genome size of YLS were similar to those of nonsymbiotic yeasts and fungi.

Separate structural elements within internal transcribed spacer 1 of Saccharomyces cerevisiae precursor ribosomal RNA direct the formation of 17S and 26S rRNA.

Structural features of Internal Transcribed Spacer 1 (ITS1) that direct its removal from Saccharomyces cerevisiae pre-rRNA during processing were identified by an initial phylogenetic approach followed by in vivo mutational analysis of specific structural elements. We found that S. cerevisiae ITS1 can functionally be replaced by the corresponding regions from the yeasts Torulaspora delbrueckii, Kluyveromyces lactis and Hansenula wingei, indicating that structural elements required in cis for processing are evolutionarily conserved. Despite large differences in size, all ITS1 regions conform to the secondary structure proposed by Yeh et al. [Biochemistry 29 (1990) 5911-5918], showing five domains (I-V; 5'-->3') of which three harbour an evolutionarily highly conserved element. Removal of most of domain II, including its highly conserved element, did not affect processing. In contrast, highly conserved nucleotides directly downstream of processing site A2 in domain III play a major role in production of 17S, but not 26S rRNA. Domain IV and V are dispensable for 17S rRNA formation although an alternative, albeit inefficient, processing route to mature 17S rRNA may be mediated by a conserved region in domain IV. Each of these two domains is individually sufficient for efficient production of 26S rRNA, suggesting two independent processing pathways. We conclude that ITS1 is organized into two functionally and structurally distinct halves.

Sulfonate-sulfur assimilation by yeasts resembles that of bacteria.

Three sulfonates were tested for their ability to serve as nutrients for Hansenula wingei, Rhodotorula glutinis, Trigonopsis variabilis and Saccharomyces cerevisiae. Cysteate, taurine and isethionate, under aerobic conditions, could be utilized as sources of sulfur, although in some instances final cell yields were less than those obtained with an equimolar amount of sulfate-sulfur. Sulfonate assimilation by S. cerevisiae resembled that of bacteria (reported earlier by us) in several aspects: first, sulfate-S was used in preference to that of sulfonate, when both were present; second, mutants unable to use sulfate as a source of sulfur because of deficiencies in ATP sulfurylase, adenylylsulfate kinase (APS kinase) or PAPS reductase were able to utilize sulfonates; and third, mutants deficient in sulfite reductase were unable to utilize sulfonates.

Nucleotide sequences and secondary structures of four tRNA genes in mitochondrial DNA of Hansenula wingei yeast.

Nucleotide sequences and secondary structures of four tRNA genes in mitochondrial DNA of Hansenula wingei yeast.

Effect of nitrogen source on the levels of nitrate reductase in the yeast Hansenula anomala

Levels of nitrate reductase (NR) protein in Hansenula anomala and Hansenula wingei were determined using specific antiserum raised against the enzyme from H. anomala. Extracts from nitrate-grown cells contained NR protein, while in those from cells grown on ammonium, glutamine or peptone, no cross-reacting material could be observed. Enzyme activity correlated with the levels of cross-reacting material. When nitrate was used as nitrogen source, NR was always present, even in cultures with ammonium, glutamine or peptone, although in these cases both the levels of activity and protein were lower. NR activity was consistently two to four times higher in cells grown in glucose than in cells grown in ethanol. Nitrate was required for NR induction, and deprivation of nitrate from nitrate-grown cells resulted in a rapid loss of NR activity.

Functional analysis of internal transcribed spacer 2 of Saccharomyces cerevisiae ribosomal DNA

Using the previously described “tagged ribosome” (pORCS) system for in vivo mutational analysis of yeast rDNA, we show that small deletions in the 5′-terminal portion of ITS2 completely block maturation of 26 S rRNA at the level of the 29 S b precursor (5.8 S rRNA-ITS2–26 S rRNA). Various deletions in the 3′-terminal part, although severely reducing the efficiency of processing, still allow some mature 26 S rRNA to be formed. On the other hand, none of the ITS2 deletions affect the production of mature 17 S rRNA. Since all of the deletions severely disturb the recently proposed secondary structure of ITS2, these findings suggest an important role for higher order structure of ITS2 in processing. Analysis of the effect of complete or partial replacement of S. cerevisiae ITS2 with its counterpart sequences from Saccharomyces rosei or Hansenula wingei, points to helix V of the secondary structure model as an important element for correct and efficient processing. Direct mutational analysis shows that disruption of base-pairing in the middle of helix V does not detectably affect 26 S rRNA formation. In contrast, introduction of clustered point mutations at the apical end of helix V that both disrupt base-pairing and change the sequence of the loop, severely reduces processing. Since a mutant containing only point mutations in the sequence of the loop produces normal amounts of mature 26 S rRNA, we conclude that the precise (secondary and/or primary) struture at the lower end of helix V, but excluding the loop, is of crucial importance for efficient removal of ITS2.

Physical mapping and RFLP analysis of mtDNAs from the ascosporogenous yeasts: Saccharomyces exiguus, S. kluyveri and Hansenula wingei.

Mitochondrial DNAs from the ascosporogenous yeasts S. exiguus, S. kluyveri and H. wingei were prepared by a new rapid method without CsCl isopycnic centrifugation. The mtDNA RFLPs were identified and clearly distinguished each species from the other. The physical maps were constructed by single and double digestion with nine restriction endonucleases. The location of rRNA genes was assigned to the maps by Southern hybridization with synthetic consensus probes. The genome sizes of these mtDNAs were estimated to be 45 kb for S. exiguus, 54 kb for S. kluyveri and 27 kb for H. wingei. The mtDNA RFLP analysis indicates a phylogenetic relationship among these yeasts. This indicates that S. cerevisiae is closer to H. wingei than S. kluyveri. However, the derived phylogenetic tree is completely consistent with that which was previously constructed on amino acid replacement in mating pheromones and electrophoretic karyotypes (Yoshida et al., 1989).

Antimicrobial Activity of Sodium Bicarbonate

ABSTRACTSodium bicarbonate (SB) inhibited the growth of bacteria and yeasts in agar media model systems under certain conditions. Escherichia coli, Lactobacillus plantarum, Staphylococcus aureus, and Pseudomonas aeruginosa aerobic plate counts were reduced 10,000‐fold by 0.12M (1% w/v) SB. Saccharomyces cerevisiae and Hansenula wingei were more sensitive; counts were reduced 100,000‐fold by 0.06M SB. Potassium bicarbonate was equally inhibitory, but equimolar sodium chloride had no effect, ruling out osmotic‐ and sodium‐mediated mechanisms of inhibition. The bicarbonate ion was identified as the probable cause of SB‐mediated inhibition although, in some cases, pH elevation played a significant role.

Cell Wall Proteins Liberated by Zymolyase from Several Ascomycetous and Imperfect Yeasts

SUMMARY: Eight yeast species (the ascomycetes Saccharomyces cerevisiae, Zygosaccharomyces rouxii, Hansenula wingei, Saccharomycopsis lipolytica, Schizosaccharomyces pombe and Pichia scolyti, and the imperfect yeasts Candida albicans and Rhodotorula glutinis) have been compared with respect to the proteins solubilized by glucanase treatment of amino-acid-labelled, purified walls. Except for R. glutinis, a significant quantity of radioactively labelled protein material was liberated by this treatment. Among the major protein components solubilized was a material larger than 100 kDa (heterogeneous in size in some species) and a molecule ranging in size from 31.5 to 34 kDa depending on the yeast species. The large material was of glycoprotein nature, with the sugar portion N-glycosidically linked to the protein moiety; in some species, this material did not bind concanavalin A (ConA), indicating the presence of terminal sugar residues different from mannose, glucose or glucosamine. The form of 31.5-34 kDa was present in all the species studied except R. glutinis; it was a mannoprotein, except in Schiz. pombe, which possessed a 31.5 kDa form not sensitive to tunicamycin or endoglycosidase H and not recognized by ConA. Antigenic cross-reactivity was observed between the protein moieties of the 33 kDa form of Sacch. cerevisiae and the species of equivalent size in C. albicans and H. wingei. Similar partial proteolysis patterns were obtained for the 33 kDa form of Sacch. cerevisiae and the 34 kDa forms of C. albicans and P. scolyti.

The Role of Nitrogen Sources in the Regulation of Nitrate Reductase and Nitrite Reductase Levels in the Yeast Hansenula wingei

SUMMARY: The ascomycete yeast Hansenula wingei was able to grow with nitrate, ammonium, glutamate, glutamine or hypoxanthine as sole nitrogen source. Conditions for assay of nitrate reductase (EC 1.6.6.2; NR)and nitritereductase (EC 1.6.6.4; NiR) wereoptimized incell-freeextracts from nitrate-grown cells. NR utilized NADH and NADPH as electron donor and required FAD for maximum activity; NiR was NADPH-specific. Glutamate-grown cells possessed low levels of both enzyme activities and, of the nitrogen sources tested only nitrate was able to induce both enzyme activities above this low basal level of constitutive expression. Ammonium, glutamate and glutamine each prevented nitrate induction of the activities in glutamate-grown cells. Addition of ammonium, glutamate or glutamine to nitrate-grown cells which possessed appreciable levels of NR and NiR caused loss of activity, even if added with nitrate. Loss of both activities under these conditions occurred faster than in cells in which activity loss occurred solely due to nitrate depletion. Both increases and decreases in NR and NiR activity were dependent on protein synthesis.

3'-End formation of transcripts from the yeast rRNA operon.

Deletion analysis of artificial rRNA minigenes transformed into Saccharomyces cerevisiae revealed that a 110 bp long fragment corresponding to positions -36 to +74 relative to the 3'-end of the 26S rRNA gene, is both necessary and sufficient for obtaining transcripts whose 3'-termini are identical to those of 26S and 37S (pre-)rRNA. These termini are produced via processing of longer transcripts because in an rna 82.1 mutant the majority of the minigene transcripts extend further downstream. Since the rna 82.1 mutation inactivates an endonuclease involved in the 3'-processing of 5S pre-rRNA it is concluded that the maturation of 37S- and that of 5S pre-rRNA requires a common factor. Comparison of the spacer sequences between Saccharomyces carlsbergensis, Saccharomyces rosei and Hansenula wingei revealed several conserved sequence blocks within the region between +10 and +55. These conserved sequence tracts, which are part of a longer region showing dyad symmetry, are supposed to be involved in the interaction with the processing component(s). Deletion of the sequences required for the formation of the 3'-ends of 26S rRNA and 37S pre-rRNA revealed a putative terminator for transcription by RNA polymerase I situated at position +210. This site maps within a DNA fragment that also contains the enhancing element for rDNA transcription by RNA polymerase I.

A conserved sequence element is present around the transcription initiation site for RNA polymerase A in Saccharomycetoideae.

To identify DNA elements involved in the initiation of rRNA transcription in yeast we located the start site of the rRNA operon of Kluyveromyces lactis and Hansenula wingei, both members of the Saccharomycetoideae, by S1 nuclease analysis and determined the surrounding nucleotide sequences. Comparison of these sequences with those of Saccharomyces carlsbergensis, S. cerevisiae and S. rosei (all belonging to the same yeast subfamily) reveals an identical sequence at the site of transcription initiation from position +1 to +7 which is part of a larger conserved region extending from position -9 to +23; the conserved heptanucleotide sequence is supposed to constitute an important part of the promoter for yeast RNA polymerase A. The non-transcribed spacers (NTS) upstream of position -9 have diverged strongly with the exception of two short elements around positions -75 and -135. The external transcribed spacer (ETS) downstream of position +23 is largely conserved between K. lactis, S. rosei and S. carlsbergensis except for a divergent region around position +75. On the other hand, the ETS of H. wingei has diverged significantly.

Mating-type-specific cell cycle arrest and shmoo formation by ? pheromone of Saccharomyces cerevisiae in Hansenula wingei

The cell cycle of a (5) mating type cells of Hansenula wingei was arrested in the G1 phase by α pheromone of Saccharomyces cerevisiae but not by α(21) pheromone of H. wingei, although both the α pheromones are known to induce sexual agglutination ability of a mating type cells of H. wingei. Cells of α mating type of H. wingei became shmooed or arrested in the G1 phase in response to neither a pheromone of H. wingei nor α pheromone of S. cerevisiae.