Quantification of different yeasts associated with the bark beetle,Ips typographus,during its attack on a spruce tree

There were different amounts and types of yeasts associated with individuals ofIps typographus spruce bark beetles during different phases of their attack on a healthy spruce tree. The yeasts were isolated on Sabouraud agar medium in order to identify them and estimate their numbers.Hansenula holstii andCandida diddensii type yeasts were most frequently isolated. The increase in number of these two yeast types probably accounted for most of the total yeast increase found during the later attack phases of the bark beetles. Lesser amounts ofHansenula capsulata, Pichia pinus, Candida nitratophila, and twoCryptococcus type yeasts were also found.     

Microbial assimilation of alkyl nitro compounds and formation of nitrite

66 representative strains of bacteria, yeasts and fungi were tested for their ability to grow in a semidefined medium containing 0.5% nitroethane as a nitrogen source. About half of them were found capable of growing in the medium. Hansenula beijerinckii, Candida utilis, and Penicillium chrysogenum were most active in assimilating nitroethane. 2-Nitropropane inhibited growth of most of the microorganisms tested in a medium containing 0.2% peptone and 0.2% glycerol. Hansenula mrakii was found to grow rapidly in the nitroethane-peptone medium after a lag phase. Nitrite was accumulated in the culture fluid after the phase of logarithmic multiplication, and increased with increase of the growth, followed by a decline after the maximum growth. The alkyl nitro compounds were oxidatively denitrified to form nitrite by the crude enzyme from Hansenula mrakii. Nitroethane was generally a poor substrate, but was the best inducer to produce the nitro compounds oxidizing enzyme. 2-Nitropropane and nitroethane were enzymatically oxidized to and acetone and acetaldehyde, respectively, which were isolated as 2,4-dinitrophenylhydrazones and identified. Nitrite formed was found to be reduced into ammonia by the intact cells and also the crude enzyme.     

Isolation of the Melanization and Reddish Coloration Hormone (MRCH) of the Armyworm,Leucania separata,from the Silkworm,Bombyx mori

Two types of glycerol dehydrogenase (GDH) were found on DEAE-cellulose column chromatography of cell-free extracts of methylotrophic yeasts. One type, designated as GDH I, showed only the reductive activity which was detected in the reaction system containing dihydroxyacetone and NADH, at pH 6.0. The other type, designated as GDH II, showed the oxidative activity which was detected in the system containing glycerol and NAD ⁺, at pH 9.0, together with the reductive activity.

Candida boidinii No. 2201, which possesses the phosphorylative pathway for glycerol dissimilation, had only GDH I when grown on glycerol or methanol as the carbon source. Hansenula ofunaensis, which has the oxidative pathway, had both GDH I and GDH II when grown on glycerol, but only GDH I when grown on methanol. Hansenula polymorpha Dl-1, which has both pathways, had both GDH I and GDH II when grown on glycerol or methanol.     

Methanol metabolism in yeasts: Regulation of the synthesis of catabolic enzymes

The regulation of the synthesis of four dissimilatory enzymes involved in methanol metabolism, namely alcohol oxidase, formaldehyde dehydrogenase, formate dehydrogenase and catalase was investigated in the yeasts Hansenula polymorpha and Kloeckera sp. 2201. Enzyme profiles in cell-free extracts of the two organisms grown under glucose limitation at various dilution rates, suggested that the synthesis of these enzymes is controlled by derepression — represion rather than by induction — repression. Except for alcohol oxidase, the extent to which catabolite repression of the catabolic enzymes was relieved at low dilution rates was similar in both organisms. In Hansenula polymorpha the level of alcohol oxidase in the cells gradually increased with decreasing dilution rate, whilst in Kloeckera sp. 2201 derepression of alcohol oxidase synthesis was only observed at dilution rates below 0.10 h^–1 and occurred to a much smaller extent than in Hansenula polymorpha.Derepression of alcohol oxidase and catalase in cells of Hansenula polymorpha was accompanied by synthesis of peroxisomes. Moreover, peroxisomes were degraded with a concurrent loss of alcohol oxidase and catalase activities when excess glucose was introduced into the culture. This process of catabolite inactivation of peroxisomal enzymes did not affect cytoplasmic formaldehyde dehydrogenase.     

A Novel Class of Fungicides: α-Benzylidene-γ-valerolactones

Enzyme activities involved in the initial step of glycerol metabolism were determined in cells of methylotrophic yeasts grown on glycerol, methanol or glucose. In Candida boidinii (Kloeckera sp.) No. 2201, the activities of glycerol kinase and dihydroxyacetone kinase were detected in cells grown on glycerol and methanol, respectively. The activity of NAD⁺-linked glycerol dehydrogenase of Hansenula polymorpha dl-1 was induced by glycerol and methanol, while that of Hansenula ofunaensis was induced by glycerol. The enzymes of both strains were subject to catabolite repression by glucose.

The yeasts tested were divided into three groups as to the glycerol dissimilation patterns. Strains of the genera Candida, Saccharomyces, Pichia and Torulopsis had the phosphorylative pathway, in which glycerol is first phosphorylated. H. ofunaensis had the oxidative pathway, in which glycerol is first oxidized. H. polymorpha dl-1 had both the phosphorylative and oxidative pathways.     

Search for Yeast Producers of Brassylic and Sebacic Fatty Acids

Yeast cultures belonging to the genera Candida, Torulopsis, Saccharomyces, Debaryomyces, Hansenula, Pichia, and Yarrowia, capable of synthesizing brassylic and sebacic fatty acids, were screened. Overall about 200 cultures grown in media containing decane or tridecane as a sole source of carbon were tested. On the medium with tridecane, yeasts synthesized insignificant amounts of brassylic acid. Sebacic acid was produced more intensively in the medium with n-decane. The culture Candida tropicalis, displaying the highest ability to synthesize sebacic acid, was selected.     

Enzymatic preparation of [1, 3-13C]dihydroxyacetone phosphate from [13C]methanol and hydroxypyruvate using the methanol-assimilating system of methylotrophic yeasts

Dihydroxyacetoone synthase (EC 2.2.1.3), which is a key enzyme of the C₁-compound-assimilating pathway in yeasts, catalyzes transketolation between formaldehyde and hydroxypyruvate, leading to the formation of dihydroxyacetone and CO₂. When [¹³C]formaldehyde was used as a substrate with dihydroxyacytone synthase from Candida boidinii 2201, ¹³C was confirmed to be incorporated to the C-1 and C-3 positions of dihydroxyacetone, and the ¹³C content of each carbon (atoms/100 atoms) was estimated to be 50%. [¹³C]Methanol was also useful for the enrichment of dihydroxyacetone with ¹³C, when alcohol oxidase from a methylotrophic yeast was added for the conversion of methanol to formaldehyde. A fed-batch reaction with periodic addition of the substrates was required for the accumalation of ¹³C-labelled dihydroxyacetone at a higher concentration, because the enzyme system was relatively susceptible to the C donor, formaldehyde or methanol. The optimum conditions for the production gave 160mM (14.4 mg/ml) dihydroxyacetone for 180 min; the molar yield relative to methanol added was 80%. Diyhdroxyacetone kinase (EC 2.7.1.29) from methanol-grown Hansenula polymorpha CBS 4732 was a suitable enzyme for the phosphorylation of dihydroxyacytone. The phosphorylation system, comprising of dihydroxyacetone kinase, adenylate kinase, and ATP, could be coupled with the system for dihydroxyacetone production. A fed-batch reaction afforded 185 mM [1, 3-¹³C]dihydroxyacetone phosphate from [¹³C]methanol; the molar yield of the ester relative to methanol added was 92.5%     

Yeasts in some Netherlands soils

Summary In 12 Netherlands soil samples, in two successive analyses carried out in the autumn of 1952, the author has isolated up to 105 cultures of yeasts to be classified under the following species:Kloeckera apiculata, Torulopsis inconspicua, Torulopsis pulcherrima, Candida Guilliermondii, Rhodotorula mucilaginosa, Aureobasidium pullulans, Hansenula saturnus, Hansenula californica, Saccharomyces mangini, Saccharomyces ellipsoideus andPichia membranefaciens. In all the soils examined the quantity of yeasts found has been very small. The results obtained do not offer evidence for any relationship between manurial treatment and species and number of yeasts occurring. Sporeforming yeasts were less numerous than non-sporeforming ones.

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Riassunto Da 12 terreni Olandesi, in due successive analisi, eseguite nell'autunno 1952, l'A. riferisce di avere isolato ben 105 colture pure di lieviti da riportare alle seguenti specie:Kloeckera apiculata, Torulopsis inconspicua, Torulopsis pulcherrima, Candida Guilliermondii, Rhodotorula mucilaginosa, Aureobasidium pullulans, Hansenula saturnus, Hansenula californica, Saccharomyces mangini, Saccharomyces ellipsoideus ePichia membranefaciens. In tutti i terreni esaminati la quantità di lieviti riscontrati è stata molto esigua; non è stata posta in evidenza alcuna influenza in rapporto alle diverse concimazioni ed è stata rilvata la scarsissima diffusione delle specie sporigene su quelle incapaci di formare spore.

     

Butyrate Activation in Rhodopseudomonas palustris No. 82

Halotolerant killer yeasts which showed killer activity in the presence of NaCl were isolated from fermented foods, such as miso, soy sauce and salted vegetables, and identified as Debaryomyces hansenii, Hansenula anomala, Candida naeodendra and Pichia farinosa. The killer strains of C. naeodendra and P. farinosa were found here for the first time. Seventy-six percent of the salted vegetable samples contained killer yeasts, mainly D. hansenii. On the other hand, killer strains were isolated from 3 of 18 samples of miso and soy sauce. The killer spectra against the standard killer strains, K1 ~ K10, were different from those of other killer strains reported previously.     

Yeasts from U.S.A. soils

Summary Investigations of yeasts from 38 U.S.A. soils samples show the occurrence of 22 different species (16 sporogenous and 6 asporogenous).The most widespread species were Pichia fermentans and Hansenula anomala. The other isolated species were Saccharomyces ellipsoideus, Torulaspora delbrueckii, Pichia membranefaciens, Saccharomyces smittii, Saccharomyces carlsbergensis, Saccharomyces uvarum, Torulaspora rosei, Zygosaccharomyces rouxii, Zygosaccharomyces n. sp., Hansenula saturnus, Hansenula californica, Hansenula n. sp., Hansenula suaveolens, Debaryomyces n. sp., Torulopsis glabrata, Torulopsis n. sp. Candida tropicalis, Candida robusta, Rhodotorula glutinis, Trichosporon cutaneum and Trichosporon cutaneum var. multisporus.     

Growth of Natural Yeast Flora during the Fermentation of Inoculated Wines

The growth of yeasts that occur naturally in grape juice was quantitatively examined during the fermentation of four wines that had been inoculated with Saccharomyces cerevisiae. Although S. cerevisiae dominated the wine fermentations, there was significant growth of the natural species Kloeckera apiculata, Candida stellata, Candida colliculosa, Candida pulcherrima, and Hansenula anomala.     

Continuous measurement of ethanol production by aerobic yeast suspensions with an enzyme electrode

Summary An alcohol electrode was constructed which consisted of an oxygen probe onto which alcohol oxidase was immobilized. This enzyme electrode was used, in combination with a reference oxygen electrode, to study the short-term kinetics of alcoholic fermentation by aerobic yeast suspensions after pulsing with glucose. The results demonstrate that this device is an excellent tool in obtaining quantitative data on the short-term expression of the Crabtree effect in yeasts.Samples from aerobic glucose-limited chemostat cultures of Saccharomyces cerevisiae not producing ethanol, immediately (within 2 min) exhibited aerobic alcoholic fermentation after being pulsed with excess glucose. With chemostat-grown Candida utilis, however, ethanol production was not detectable even at high sugar concentrations. The Crabtree effect in S. cerevisiae was studied in more detail with commercial baker's yeast. Ethanol formation occurred only at initial glucose concentrations exceeding 150 mg·l^-1, and the rate of alcoholic fermentation increased with increasing glucose concentrations up to 1,000 mg·l^-1 glucose.Similar experiments with batch cultures of certain non-fermentative yeasts revealed that these organisms are capable of alcoholic fermentation. Thus, even under fully aerobic conditions, Hansenula nonfermentans and Candida buffonii produced ethanol after being pulsed with glucose. In C. buffonii ethanol formation was already apparent at very low glucose concentrations (10 mg·l^-1) and alcoholic fermentation even proceeded at a higher rate than in S. cerevisiae. With Rhodotorula rubra, however, the rate of ethanol formation was below the detection limit, i.e., less than 0.1 mmol·g cells^-1·h^-1.     

Two new species of the genus Candida in the Zygoascus clade, Candida lundiana sp. nov. and Candida suthepensis sp. nov., isolated from raw honey in Thailand

During a survey of yeasts associated with raw honey collected in Thailand, two strains of the Zygoascus clade were isolated from the Asian cavity-nesting honeybee Apis cerana and the stingless bee Homotrigona fimbriata. Phylogeny based on 26S rDNA D1/D2 sequences placed these yeasts as members of a clade including Candida bituminiphila, Candida patagonica and Candida polysorbophila. The strains of the two novel species, CBS 12271^T and CBS 12270^T, respectively, could be unquestionably distinguished from their relatives by rDNA sequences and other taxonomic characteristics. Therefore, the novel anamorphic species, Candida lundiana sp. nov. (type strain CBS 12271^T = JCM 16823^T) and Candida suthepensis sp. nov. (type strain CBS 12270^T = JCM 16822^T) are described.     

Strain differentiation of pathogenic yeasts by the killer system

High sensitivity rates to the activity of killer toxins produced by 25 species of yeasts belonging to the genera Candida, Hansenula, Pichia, Rhodotorula, Saccharomyces and Trichosporon have been observed among 112 yeast isolates (25 Cryptococcus neoformans, 29 C. glabrata, 16 C. parapsilosis, 20 C. pseudotropicalis and 22 C. tropicalis). The highest sensitivity has been observed among the C. parapsilosis isolates, the lowest in C. glabrata strains. Genera Pichia and Hansenula proved to have the greatest killer activity. A killer system, formerly used for differentiating C. albicans isolates within the species, proved to be valid as epidemiological marker when applied to 112 strains of pathogenic yeasts.     

Activities of the enzymes of formaldehyde catabolism in recombinant strains of <Emphasis Type="Italic">Hansenula polymorpha</Emphasis>

Activities of the enzymes of formaldehyde (FA) catabolism in recombinant strains of the methylotrophic yeast Hansenula polymorpha overproducing NAD⁺- and glutathione-dependent formaldehyde dehydrogenase (FADH) were studied under different cultivation conditions and at elevated FA content. Southern dot-blot analysis confirmed the presence of six to eight copies of the target FLD1 gene in stable recombinant clones of H. polymorpha. Under certain cultivation conditions, the transformants resistant to elevated FA concentrations were shown to produce FADH and other bioanalytically important enzymes: formate dehydrogenase, alcohol dehydrogenase, alcohol oxidase, and formaldehyde reductase. The optimal cultivation conditions for recombinants were determined, resulting in maximum synthesis of FADH: methanol as a carbon source, methylamine as a nitrogen source, FA as an inducer, temperature of 37°C, and cells in the early exponential phase of growth.     

Selenium Tolerance of Yeasts

Selenium tolerance of yeasts widely varies: the growth of some yeasts can be inhibited by a selenium concentration as low as 10^–4 M, whereas others can grow in the presence of 10^–1 M selenium. Homogeneous yeast taxa are characterized by a certain level of selenium tolerance, and heterogeneous taxa show a variable level of tolerance to selenium. In general, ascomycetous yeasts are more tolerant to selenium than basidiomycetous yeasts. Among the ascomycetous yeasts, the genera Dekkera and Schizosaccharomyces exhibited the lowest and the species Candida maltosa, Hanseniaspora valbyensis, Kluyveromyces marxianus, and Yarrowia lipolytica the highest tolerance to selenium. Among the basidiomycetous yeasts, the genera Bullera, Cryptococcusand Holtermannia showed the lowest and the species Cryptococcus curvatus, Cr. humicola, and Trichosporon spp. the highest tolerance to selenium. The selenium tolerance of yeasts depends on the composition of the growth medium, in particular, on the presence of sulfate, sulfur-containing amino acids, and glutamine in the medium.     

S-formylglutathione: The substrate for formate dehydrogenase in methanol-utilizing yeasts

Formaldehyde dehydrogenase and formate dehydrogenase were purified 45- and 16-fold, respectively, from Hansenula polymorpha grown on methanol. Formaldehyde dehydrogenase was strictly dependent on NAD and glutathione for activity. The K _mvalues of the enzyme were found to be 0.18 mM for glutathione, 0.21 mM for formaldehyde and 0.15 mM for NAD. The enzyme catalyzed the glutathine-dependent oxidation of formaldehyde to S-formylglutathione. The reaction was shown to be reversible: at pH 8.0 a K _mof 1 mM for S-formylglutathione was estimated for the reduction of the thiol ester with NADH. The enzyme did not catalyze the reduction of formate with NADH. The NAD-dependent formate dehydrogenase of H. polymorpha showed a low affinity for formate (K _mof 40 mM) but a relatively high affinity for S-formylglutathione (K _mof 1.1 mM). The K _mvalues of formate dehydrogenase in cell-free extracts of methanol-grown Candida boidinii and Pichia pinus for S-formylglutathione were also an order of magnitude lower than those for formate. It is concluded that S-formylglutathione rather than free formate is an intermediate in the oxidation of methanol by yeasts.     

Identification and properties of yeasts associated with the aerobic deterioration of wheat and alfalfa silages

Populations of fungi in aerobically deteriorating wheat and alfalfa silages were identified as: Endomycopsis burtonii, E. selenospora, Hansenula canadensis, Candida tenuis and C. silvicola. The yeasts recovered were similar for both silages, but H. canadensis was recovered only in wheat silages. All of these yeasts could utilize lactic acid aerobically, but not anaerobically. Only Endomycopsis spp. could utilize propionic acid aerobically and none of the yeasts utilized this acid anaerobically. However, all yeasts grew in complete media supplemented with propionate. Therefore, while lactic and propionic acids may contribute to stability under anaerobic conditions, they are much less less effective after the silage is exposed to air.     

Relative Incidence of Ascomycetous Yeasts in Arctic Coastal Environments

Previous studies of fungi in polar environments have revealed a prevalence of basidiomycetous yeasts in soil and in subglacial environments of polythermal glaciers. Ascomycetous yeasts have rarely been reported from extremely cold natural environments, even though they are known contaminants of frozen foods. Using media with low water activity, we have isolated various yeast species from the subglacial ice of four glaciers from the coastal Arctic environment of Kongsfjorden, Spitzbergen, including Debaryomyces hansenii and Pichia guillermondii, with counts reaching 10⁴ CFU L⁻¹. Together with the basidiomycetes Cryptococcus liquefaciens and Rhodotorula mucilaginosa, these yeasts represent the stable core of the subglacial yeast communities. Other glacial ascomycetous species isolated included Candida parapsilosis and a putative new species that resembles Candida pseudorugosa. The archiascomycete Protomyces inouyei has seldom been detected anywhere in the world but was here recovered from ice in a glacier cave. The glacier meltwater contained only D. hansenii, whereas the seawater contained D. hansenii, Debaryomyces maramus, Pichia guilliermondii, what appears to represent a novel species resembling Candida galli and Metschnikowia bicuspidata. Only P. guilliermondii was isolated from sea ice, while snow/ice in the fjord tidal zone included C. parapsilosis, D. hansenii, P. guilliermondii and Metschnikowia zobellii. All of these isolated strains were characterized as psychrotolerant and xero/halotolerant, with the exception of P. inouyei.     

Oxidation of methanol,formaldehyde and formate by catalase purified from methanol-grown Hansenula polymorpha

Catalase has been partially purified from cell-free extracts of methanol-grown Hansenula polymorpha and its peroxidative properties were studied. It was shown that the enzyme is capable of oxidizing methanol, formaldehyde and formate in the presence of hydrogen peroxide. The physiological significance of these reactions in the transduction of energy from the oxidation of methanol in yeasts is discussed.