Rhodotorula arctica sp. nov., a basidiomycetous yeast from Arctic soil.

Three isolates, typified by Pro94 Y29(T) (=JCM 13290(T) =CBS 9278(T) =DBVPG 7841(T)), that represent a novel species, Rhodotorula arctica sp. nov., were studied. R. arctica differed from its only close relative, Bensingtonia yamatoana, by requiring thiamine and by failing to assimilate maltose and quinate, but strain Pro94 Y29(T) could be most readily identified using the rDNA sequence of the LSU D1/D2 region, which differed from that of B. yamatoana CBS 7423(T) at four positions, and the ITS sequence, which differed at nine positions. One R. arctica isolate, Pro94 Y56 (=JCM 13292 =CBS 9280 =DBVPG Y7843), was unique in requiring either l-arginine or l-citrulline as a source of nitrogen.

Assimilation of volatiles from ripe apples by Sporidiobolus salmonicolor and Tilletiopsis washingtonensis.

Sporidiobolus salmonicolor ATCC 623 and Tilletiopsis washingtonensis NRRL Y-2555 grew on carbon resources provided as volatiles by ripe 'Golden Delicious' apples. This ability was not correlated with the reported natural habitats of the 21 species (26 strains) tested. Ethylene, the major volatile produced, was not utilized but butyl acetate, hexyl acetate and hexyl-2-methyl-butanoate (identified by GC-MS) were. These yeasts also assimilated ethanol, butanol, hexanol (Tilletiopsis excepted), acetate, propionate, butyrate and ethyl acetate at appropriately low concentrations. Ethanol and acetate aside, this is the first report of such assimilations by any yeast.

Simulated in situ competitive ability and survival of a representative soil yeast, Cryptococcus albidus.

Microcosms containing an air-dried autoclaved loamy sand (Eufala A) with low salt and organic content were inoculated with a representative (obligately aerobic, encapsulated) soil yeast, Cryptococcus albidus var. albidus (T) ATCC 10666, singly (for growth rate and survival determinations) and together with the bacterial biota native to Eufala A. The yeast competed successfully with the more rapidly growing bacteria in the presence of added water from 1% (5.7% of field capacity) to 14% (80% of field capacity) but grew for shorter times than when grown alone; times correlated with the lag phase of the bacterial biota. When well-watered (10 and 14%) competition cultures were allowed to dry and used as inoculum for subcultures, the yeast made significant growth only at 1% added water but survived at the higher moisture concentrations. The competitive ability of Cr. albidus confirms the previously reported advantages of the cryptococcal capsule in hydration and desiccation and, together with lengthy survival, suggests that the importance of such yeasts in the biogeochemistry of arid soils has been seriously underestimated.

Urease inhibition by EDTA in the two varieties of Cryptococcus neoformans.

Cryptococcus neoformans var. neoformans (74 isolates) and C. neoformans var. gattii (44 isolates) were used to test urease activity after growth on both yeast extract-glucose-peptone agar (YEPG) and on YEPG supplemented with 100 microM EDTA. Every isolate grown on YEPG agar for 48 h at 30 degrees C produced a positive reaction within 1 h in a modified rapid urease assay at 37 degrees C. However, isolates grown on YEPG with 100 microM EDTA showed a distinct pattern which corresponded to their varietal status. All but 1 of 74 C. neoformans var. neoformans isolates (98.7%) produced a positive reaction within 1 to 4 h, while none of 44 C. neoformans var. gattii isolates produced a positive reaction within the same period. The urease inhibition results and the canavanine-glycine-bromthymol blue agar test results showed 100% correlation among isolates of C. neoformans var. gattii and 98.7% correlation among isolates of C. neoformans var. neoformans. Two representative isolates of C. neoformans var. gattii (serotypes B and C) were further tested for urease during a prolonged incubation period in urea broth. These isolates failed to show a positive reaction even after 11 h of incubation. The uptake of EDTA was negligible in the two varieties. Extracts of cells grown on YEPA agar showed a high level of urease activity in both varieties. Extracts of cells grown on the agar with 100 microM EDTA showed a marked reduction (86%) of urease activity in one isolate of C. neoformans var. gattii but showed only a 30% reduction in one isolate of C. neoformans var. neoformans. Based on these results, the differential effect of EDTA on the two varieties of C. neoformans appeared to be due to greater inhibition of urease synthesis in C. neoformans var. gattii.

Urease testing and yeast taxonomy.

When urease production was assayed by the hydrolysis of [14C]urea, all basidiomycetous yeasts tested, including the Cryptococcus vishniacii complex (previously reported urease negative), produced significant amounts of 14CO2. The Schizosaccharomycetaceae were the only urease-positive ascomycetous yeasts tested. Yarrowia lipolytica was urease negative. The stoichiometry of [14C]urea hydrolysis paralleled by Roberts' rapid urea hydrolysis (RUH) test indicated that causes of anomalous results in conventional urease testing include acidification and alkalinization of the test medium by products of endogenous metabolism and autolysis rather than urease activity. Anomalous results also occurred when cells were grown on media containing the chelating agent ethylenediaminetetraacetic acid (EDTA) prior to RUH. The addition of EDTA to a complex natural medium inhibited urease production in all yeasts reportedly growing at 35 degrees C (and all other yeasts tested), except Filobasidiella (Cr.) neoformans var. neoformans (NIH 12). The RUH test could differentiate at the varietal level: Fil. (Cr.) neoformans var. neoformans was about 10 times more resistant to EDTA in media used for the growth of cells prior to RUH testing than was Fil. neoformans var. bacillispora (Cr. neoformans var. gattii) (NIH 191). Urease production by Fil. neoformans var. bacillispora was specifically restored to half maximal activity by the addition of 22 microM Ni+2 (as NiCl2) to a growth medium containing 0.100 mM EDTA.

Cryptococcus socialis sp. nov. and Cryptococcus consortionis sp. nov., Antarctic basidioblastomycetes.

New yeasts from the Ross Desert (dry valley area) of Antarctica include Cryptococcus socialis sp. nov. and Cryptococcus consortionis sp. nov. Cryptococcus socialis MYSW A801-3aY1 (= ATCC 56685) requires no vitamins, assimilates L-arabinose, cellobiose, D-glucuronate, maltose, melezitose, raffinose, soluble starch, sucrose, and trehalose, and may be distinguished from all other basidioblastomycetes by the combination of amylose production, cellobiose assimilation, and failure to utilize nitrate, D-galactose, myo-inositol, and mannitol. Its guanine-plus-cytosine content is 56 mol%. Cryptococcus consortionis MYSW A801-3aY92 (= ATCC 56686) requires thiamine, assimilates L-arabinose, D-glucuronate, 2-ketogluconate, salicin, succinate, sucrose, trehalose, and D-xylose, and may be distinguished from all other basidioblastomycetes by the combination of amylose production and failure to utilize nitrate, cellobiose, D-galactose, myo-inositol, and mannitol. Its guanine-plus-cytosine content is 56 mol%.

Cryptococcus friedmannii, a new species of yeast from the Antarctic.

Cryptococcus friedmannii Vishniac sp. nov. from an Antarctic cryptoendolithic community is a psychrophilic basidioblastomycete characterized by cream-colored colonies of cells with smooth, layered walls, budding monopolarly, producing amylose and extracellular proteinase, utilizing nitrate and D-alanine (inter alia) as nitrogen sources and L-arabinose, arbutin, cellobiose, D-glucuronate, maltose, melezitose, salicin, soluble starch, trehalose, and D-xylose as carbon sources. This species differs from all other basidiomycetous yeasts in possessing the following combination of characters: amylose production (positive), assimilation of cellobiose (positive), D-galactose (negative), myo-inositol (negative), D-mannitol (negative), and sucrose (negative).

25S ribosomal RNA homologies of basidiomycetous yeasts: taxonomic and phylogenetic implications.

Genera, families, and possibly orders of basidiomycetous yeasts can be defined by 25S rRNA homology and correlated phenotypic characters. The teleomorphic genera Filobasidium, Leucosporidium, and Rhodosporidium have greater than 96 relative binding percent (rb%) intrageneric 25S rRNA homology and significant intergeneric separation from each other and from Filobasidiella. The anamorphic genus Cryptococcus can be defined by morphology (monopolar budding), colony color, and greater than 75 rb% intrageneric homology; Vanrija is heterogeneous. Agaricostilbum (Phragmobasidiomycetes, Auriculariales), Hansenula (Ascomycotera, Endomycota), Tremella (Phragmobasidiomycetes, Tremellales), and Ustilago (Ustomycota, Ustilaginales) appear equally unrelated to the Cryptococcus, Filobasidiella, and Rhodosporidium spp. used as probes. The Filobasidiaceae and Sporidiaceae, Filobasidiales and Sporidiales, form coherent homology groups which appear to have undergone convergent 25S rRNA evolution, since their relatedness is much greater than that indicated by 5S rRNA homology. Ribosomal RNA homologies do not appear to measure evolutionary distance.

Complementary DNA-25S ribosomal RNA hybridization: an improved method for phylogenetic studies.

In a new combination of techniques for ribosomal RNA hybridization, complementary DNA is synthesized on 25S ribosomal RNA fragments generated by mild alkali treatment, by the enzymatic addition of polyadenylic acid tails, hybridization of these tails with oligo deoxyribosylthymine, and reverse transcription in the presence of tritiated TTP. Hybridization reactions are performed in solution. Heteroduplexes are collected on diethylaminoethylcellulose filter discs after treatment with S1 nuclease. The problems presented by secondary rRNA structure are avoided by denaturation before reverse transcription and before hybridization. The high percentage of duplex formation (78-87%), the low standard deviation of relative binding (averaging +/- 1.30% relative binding), and small differences in reciprocal hybridizations (1.71-5.18% relative binding), as well as the elimination of complications resulting from differences in the number of rRNA cistrons in nuclear DNA, make this method preferable to the membrane-filter technique commonly used in phylogenetic classifications based on the homology of large rRNAs.

A fixation method for visualization of yeast ultrastructure in the electron microscope.

A primary fixative containing glutaraldehyde (3%), acrolein (1.5%), and paraformaldehyde (1.5%) buffered in 0.05 M sodium cacodylate at pH 7.2 was applied to the cells Cryptococcus vishniacii for 2 hours on ice. The cells were then treated with a 6% aqueous solution of potassium permanganate for 1 hour at room temperature. This method preserves most of the yeast cell fine structural components including cell walls and membrane, nuclear membrane, mitochondria, endoplasmic reticula, microbodies, vacuoles, nucleoli, and ribosomes. However, it leads to disruption of capsular materials and loss of some of the lipid and glycogen granules.