Four novel yeasts from decaying organic matter: Blastobotrys robertii sp. nov., Candida cretensis sp. nov., Candida scorzettiae sp. nov. and Candida vadensis sp. nov.

Four novel yeast species are described, two from decaying mushrooms, viz. Candida cretensis and Candida vadensis, and two from rotten wood, viz. Blastobotrys robertii and Candida scorzettiae. Accession numbers for the CBS and ARS Culture Collections, and GenBank accession numbers for the D1/D2 domains of the large subunit of ribosomal DNA are: B. robertii CBS 10106(T), NRRL Y-27775, DQ839395; C. cretensis CBS 9453(T), NRRL Y-27777, AY4998861 and DQ839393; C. scorzettiae CBS 10107(T), NRRL Y-27665, DQ839394; C. vadensis CBS 9454(T), NRRL Y-27778, AY498863 and DQ839396. The GenBank accession number for the ITS region of C. cretensis is AY498862 and that for C. vadensis is AY498864. C. cretensis was the only species of the four that displayed fermentative activity. All four type strains grew on n-hexadecane. C. scorzettiae is the only one of the new species that assimilates some phenolic compounds, viz. 3-hydroxy derivatives of benzoic, phenylacetic and cinnamic acids, but not the corresponding 4-hydroxy acids. This is indicative of an operative gentisate pathway.

Utilization of hexamethylenetetramine (urotropine) by bacteria and yeasts.

A slow growing bacterial population able to utilize hexamethylelenetetramine (urotropine) as sole source of carbon, nitrogen and energy was isolated from soil. From this crude enrichment culture two bacteria were isolated and identified as Brevundimonas diminuta and a Phyllobacterium sp. by sequencing of 16S ribosomal DNA. These bacteria also grew on urotropine but at a lower rate than the enrichment culture. Addition of glucose to the latter resulted in growth of some yeasts that overgrew the bacteria. Assimilation of urotropine as sole nitrogen source is very common among yeasts, 46 out of 60 species tested showed this characteristic.

Polysaccharides and phenolic compounds as substrate for yeasts isolated from rotten wood and description of Cryptococcus fagi sp.nov.

Pieces of rotten wood collected in the forest were screened for the presence of yeasts. In spring time 3 tree species were sampled, followed by 9 species in summer. Yeast strains were identified by traditional methods. Identifications were confirmed by sequencing of ribosomal DNA in case of doubt. In total 14 yeast species of ascomycetous affiliation and 6 anamorphic basidiomycetous yeasts were isolated and identified. Most species were represented by only one strain, but Candida bertae by two and Trichosporon porosum by six strains, all from different wood samples. Three strains represented novel species, one of which is described as Cryptococcus fagi Middelhoven et Scorzetti. The type strain is CBS 9964 (JCM 13614). All strains were tested for growth on several polysaccharides as sole carbon source. Only some of these polymers supported growth of ascomycetous yeasts. Basidiomycetous yeasts assimilated soluble starch, pullulan, dextran, xylan, polygalacturonate, galactomannan and tannic acid or at least some of these. Cryptococcus podzolicus and T. porosum were the most active in this respect. None of the isolated strains grew on carboxymethyl cellulose, colloidal chitin, arabinogalactan and gum xanthan. Phenolic compounds were assimilated by several strains, belonging to the Trichosporonales and the Microbotryum and Stephanoascus/Blastobotrys clades, but not by members of the Tremellales (Cryptococcus musci excepted) and the Debaryomyces/Lodderomyces clade. Most of the ascomycetes assimilated n-hexadecane.

Cryptococcus allantoinivorans sp.nov., an anamorphic basidiomycetous yeast (Tremellales) physiologically resembling other species of the Cryptococcus laurentii complex that degrade polysaccharides and C2 compounds.

A novel Cryptococcus species is proposed to accommodate a yeast strain (CBS 9604) able to assimilate allantoin as sole carbon source, a characteristic very uncommon among yeasts. By traditional methods, the strain could not be distinguished from Cryptococcus laurentii, but nucleotide sequences of the D1D2 region of the large subunit (26S) and of the ITS region of ribosomal DNA showed relationship to the Bulleromyces clade of the genus Cryptococcus (order Tremellales) with some Tremella spp. as the closest relatives. A traditional morphological and physiological description of the strain is given. Data on the assimilation of some C2 compounds and polysaccharides are provided and compared with those of other type strains of novel species of the C. laurentii complex.

Systematics of the anamorphic basidiomycetous yeast genus Trichosporon Behrend with the description of five novel species: Trichosporon vadense, T. smithiae, T. dehoogii, T. scarabaeorum and T. gamsii.

Phylogenetic trees of the anamorphic basidiomycetous yeast genus Trichosporon Behrend, based on molecular sequence analysis of the internal transcribed spacer region and the D1/D2 region of the large subunit of ribosomal (26S) DNA, are presented. This study includes three novel species from soils, Trichosporon vadense sp. nov. (type strain, CBS 8901(T)), Trichosporon smithiae sp. nov. (type strain, CBS 8370(T)) and Trichosporon gamsii sp. nov. (type strain, CBS 8245(T)), one novel species from an insect, Trichosporon scarabaeorum sp. nov. (type strain, CBS 5601(T)) and one species of unknown origin, Trichosporon dehoogii sp. nov. (type strain, CBS 8686(T)). The phylogenetic positions and physiological characteristics that distinguish the new taxa from related species, based partly on growth tests that are not traditionally used in yeast taxonomy (uric acid, ethylamine, L-4-hydroxyproline, tyramine and L-phenylalanine as sources of carbon and nitrogen, and polygalacturonate, quinate, 4-ethylphenol, phloroglucinol, 2,3-dihydroxybenzoate and orcinol as sole carbon sources), are discussed. Assimilation of L-rhamnose and erythritol and maximum growth temperature were also used to delineate species.

Trichosporon wieringae sp.nov., an anamorphic basidiomycetous yeast from soil, and assimilation of some phenolic compounds, polysaccharides and other non-conventional carbon sources by saprophytic Trichosporon species.

A morphological and physiological description of an anamorphic basidiomycetous yeast species isolated from soil, named Trichosporon wieringae, is presented. The phylogenetic position within the genus, based on nuclear base sequencing of the D1/D2 region of the large subunit of rDNA and of the ITS region reveals close relationship to the Porosum clade of the genus Trichosporon Behrend, T. gamsii being the closest relative. The ability of non-pathogenic Trichosporon species to assimilate uric acid, ethylamine, l-4-hydroxyproline, some aromatic compounds and polysaccharides as sole sources of carbon and energy was studied. An identification key to these species, based on growth tests on these non-conventional carbon sources, is proposed. The type strain of T. wieringae is CBS 8903 (JCM 12201).

Cryptococcus haglerorum, sp. nov., an anamorphic basidiomycetous yeast isolated from nests of the leaf-cutting ant Atta sexdens.

A yeast strain (CBS 8902) was isolated from the nest of a leaf-cutting ant and was shown to be related to Cryptococcus humicola. Sequencing of the D1/D2 region of the 26S ribosomal DNA and physiological characterization revealed a separate taxonomic position. A novel species named Cryptococcus haglerorum is proposed to accommodate strain CBS 8902 that assimilates n-hexadecane and several benzene compounds. Physiological characteristics distinguishing the novel species from some other members of the C. humicola complex are presented. The phylogenetic relationship of these strains to species of the genus Trichosporon Behrend is discussed.

Relation between phylogeny and physiology in some ascomycetous yeasts.

The question of whether yeasts with similar physiological properties are closely related has been examined using recently published phylogenetic analyses of 26S domain D1/D2 rDNA nucleotide sequences from all currently recognized ascomycetous yeasts. When apparently unique metabolic pathways are examined, some relationships between physiology and rDNA phylogeny are evident. Most Candida and Pichia species that are able to assimilate methanol as the sole carbon source are in a clade delimited by C. nanospora and C. boidinii. Exceptions are P. capsulata and P. pastoris which are phylogenetically separated from the other methanol-assimilating yeasts. Yeasts subject to the petite mutation, resulting in respiratory deficiency, belong to three different clades, viz, a Saccharomyces clade delimited by S. cerevisiae and S. rosinii, the Dekkera/Brettanomyces clade, and some Schizosaccharomyces species ('Archiascomycete' clade). However, petite mutants were also found in Zygosaccharomyces fermentati and some other more distantly related species. Yeasts able to assimilate n-hexadecane, uric acid or amines as sole carbon source are broadly distributed over the ascomycetous phylogenetic tree. However, species that assimilate adenine as sole carbon source are closely related. Most of these species also assimilated glycine, uric acid, n-hexadecane, putrescine and branched-chain aliphatic compounds such as isobutanol, leucine and isoleucine. Among the Saccharomycetales, species utilizing all or the great majority of these eight compounds are in the Stephanoascus/Arxula/Blastobotrys clade. Candida blankii, which is distantly related to this clade, proved to be an exception and assimilated six of eight of these compounds.

Identification of yeasts present in sour fermented foods and fodders.

This paper deals with rapid methods for identification of 50 yeast species frequently isolated from foods and fodders that underwent a lactic acid fermentation. However, many yeast species present in olive brine, alpechin, and other olive products were not treated. The methods required for identification include light microscopy, physiological growth tests (ID32C system of BioMérieux), assimilation of nitrate and of ethylamine as sole nitrogen sources, vitamin requirement, and maximum growth temperature. An identification key to treated yeast species is provided. In another table characteristics of all yeast species treated are listed.

Novel pathway for alcoholic fermentation of delta-gluconolactone in the yeast Saccharomyces bulderi.

Under anaerobic conditions, the yeast Saccharomyces bulderi rapidly ferments delta-gluconolactone to ethanol and carbon dioxide. We propose that a novel pathway for delta-gluconolactone fermentation operates in this yeast. In this pathway, delta-gluconolactone is first reduced to glucose via an NADPH-dependent glucose dehydrogenase (EC 1.1.1.47). After phosphorylation, half of the glucose is metabolized via the pentose phosphate pathway, yielding the NADPH required for the glucose-dehydrogenase reaction. The remaining half of the glucose is dissimilated via glycolysis. Involvement of this novel pathway in delta-gluconolactone fermentation in S. bulderi is supported by several experimental observations. (i) Fermentation of delta-gluconolactone and gluconate occurred only at low pH values, at which a substantial fraction of the substrate is present as delta-gluconolactone. Unlike gluconate, the latter compound is a substrate for glucose dehydrogenase. (ii) High activities of an NADP(+)-dependent glucose dehydrogenase were detected in cell extracts of anaerobic, delta-gluconolactone-grown cultures, but activity of this enzyme was not detected in glucose-grown cells. Gluconate kinase activity in cell extracts was negligible. (iii) During anaerobic growth on delta-gluconolactone, CO(2) production exceeded ethanol production by 35%, indicating that pyruvate decarboxylation was not the sole source of CO(2). (iv) Levels of the pentose phosphate pathway enzymes were 10-fold higher in delta-gluconolactone-grown anaerobic cultures than in glucose-grown cultures, consistent with the proposed involvement of this pathway as a primary dissimilatory route in delta-gluconolactone metabolism.