Identification of novel secreted fatty acids that regulate nitrogen catabolite repression in fission yeast.

Uptake of poor nitrogen sources such as branched-chain amino acids is repressed in the presence of high-quality nitrogen sources such as NH4(+) and glutamate (Glu), which is called nitrogen catabolite repression. Amino acid auxotrophic mutants of the fission yeast Schizosaccharomyces pombe were unable to grow on minimal medium containing NH4Cl or Glu even when adequate amounts of required amino acids were supplied. However, growth of these mutant cells was recovered in the vicinity of colonies of the prototrophic strain, suggesting that the prototrophic cells secrete some substances that can restore uptake of amino acids by an unknown mechanism. We identified the novel fatty acids, 10(R)-acetoxy-8(Z)-octadecenoic acid and 10(R)-hydroxy-8(Z)-octadecenoic acid, as secreted active substances, referred to as Nitrogen Signaling Factors (NSFs). Synthetic NSFs were also able to shift nitrogen source utilization from high-quality to poor nitrogen sources to allow adaptive growth of the fission yeast amino acid auxotrophic mutants in the presence of high-quality nitrogen sources. Finally, we demonstrated that the Agp3 amino acid transporter was involved in the adaptive growth. The data highlight a novel intra-species communication system for adaptation to environmental nutritional conditions in fission yeast.

Draft Genome Sequence of the Archiascomycetous Yeast Saitoella complicata.

The draft genome sequence of the archiasomycetous yeast Saitoella complicata was determined. The assembly of newly and previously sequenced data sets resulted in 104 contigs (total of 14.1 Mbp; N 50, 239 kbp). On the newly assembled genome, a total of 6,933 protein-coding sequences (7,119 transcripts, including alternative splicing forms) were identified.

The early diverging ascomycetous budding yeast Saitoella complicata has three histone deacetylases belonging to the Clr6, Hos2, and Rpd3 lineages.

We sequenced the genomic DNA and the transcribed RNA of the ascomycetous budding yeast Saitoella complicata, which belongs to the earliest lineage (Taphrinomycotina) of ascomycetes. We found 3 protein-coding regions similar to Clr6 of Schizosaccharomyces (a member of Taphrinomycotina). Clr6 has a structure similar to that of Rpd3 and Hos2 of Saccharomyces. These proteins belong to the class 1 histone deacetylase (HDAC) family. The phylogenetic tree showed that the Clr6, Hos2, and Rpd3 lineages are separated in fungal HDACs. Basidiomycetes have 3 proteins belonging to the Clr6, Hos2, and Rpd3 lineages. On the other hand, whereas ascomycetes except for Schizosaccharomyces have the Hos2 and Rpd3 homologs, and lack the Clr6 homolog, Schizosaccharomyces has the Clr6 and Hos2 homologs, and lacks the Rpd3 homolog. Interestingly, Pneumocystis and Saitoella have 3 proteins belonging to the Clr6, Hos2, and Rpd3 lineages. Thus, these fungi are the first ascomycete found to possess all 3 types. Our findings indicated that Taphrinomycotina has conserved the Clr6 protein, suggesting that the ancestor of Dikarya (ascomycetes and basidiomycetes) had 3 proteins belonging to the Clr6, Hos2, and Rpd3 lineages. During ascomycete evolution, Pezizomycotina and Saccharomycotina appear to have lost their Clr6 homologs and Schizosaccharomyces to have lost its Rpd3 homolog.

The SAGA histone acetyltransferase complex regulates leucine uptake through the Agp3 permease in fission yeast.

Metabolic responses of unicellular organisms are mostly acute, transient, and cell-autonomous. Regulation of nutrient uptake in yeast is one such rapid response. High quality nitrogen sources such as NH(4)(+) inhibit uptake of poor nitrogen sources, such as amino acids. Both transcriptional and posttranscriptional mechanisms operate in nutrient uptake regulation; however, many components of this system remain uncharacterized in the fission yeast, Schizosaccharomyces pombe. Here, we demonstrate that the Spt-Ada-Gcn acetyltransferase (SAGA) complex modulates leucine uptake. Initially, we noticed that a branched-chain amino acid auxotroph exhibits a peculiar adaptive growth phenotype on solid minimal media containing certain nitrogen sources. In fact, the growth of many auxotrophic strains is inhibited by excess NH(4)Cl, possibly through nitrogen-mediated uptake inhibition of the corresponding nutrients. Surprisingly, DNA microarray analysis revealed that the transcriptional reprogramming during the adaptation of the branched-chain amino acid auxotroph was highly correlated with reprogramming observed in deletions of the SAGA histone acetyltransferase module genes. Deletion of gcn5(+) increased leucine uptake in the prototrophic background and rendered the leucine auxotroph resistant to NH(4)Cl. Deletion of tra1(+) caused the opposite phenotypes. The increase in leucine uptake in the gcn5Δ mutant was dependent on an amino acid permease gene, SPCC965.11c(+). The closest budding yeast homolog of this permease is a relatively nonspecific amino acid permease AGP3, which functions in poor nutrient conditions. Our analysis identified the regulation of nutrient uptake as a physiological function for the SAGA complex, providing a potential link between cellular metabolism and chromatin regulation.

Isolation of a novel bacterium, Blautia glucerasei sp. nov., hydrolyzing plant glucosylceramide to ceramide.

A bacterial strain that is capable of hydrolyzing plant glucosylceramide (GluCer) was newly isolated from dog feces. The novel strain, designated as strain HFTH-1(T), hydrolyzed plant GluCer with a variety of chemical structures, but did not hydrolyze glucosylsphingosine, lactosylceramide, or monosialoganglioside GM(3), indicating that strain HFTH-1(T) produces GluCer-specific glucosylceramidase. Strain HFTH-1(T) was Gram-positive, anaerobic, oval-spore-forming, rod-shaped, lecithinase-negative, and lipase-negative. It fermented a wide variety of carbohydrates and produced mainly acetate, formate, and lactate from glucose. The G + C content of its DNA was 40.7 mol%. The phylogenetic analysis of 16S rRNA sequence revealed that strain HFTH-1(T) is placed in the clostridial rRNA cluster XIVa, with Ruminococcus obeum as the nearest relative. Pairwise comparison revealed approximately 5.0% sequence divergence between strain HFTH-1(T) and the type strain of R. obeum. On the basis of its phenotypic characteristics and phylogenetic divergence, it is proposed that the hitherto unknown rod-shaped bacterial strain HFTH-1(T) (= DSM 22028(T) = NBRC 104932(T)) should be placed in the genus Blautia as a novel species, Blautia glucerasei sp. nov, the only currently known isolate of the species.

A novel yeast cell-based screen identifies flavone as a tankyrase inhibitor.

The telomere-associated protein tankyrase 1 is a poly(ADP-ribose) polymerase and is considered to be a promising target for cancer therapy, especially for BRCA-associated cancers. However, an efficient assay system for inhibitor screening has not been established, mainly due to the difficulty of efficient preparation of the enzyme and its substrate. Here, we report a cell-based assay system for detecting inhibitory activity against tankyrase 1. We found that overexpression of the human tankyrase 1 gene causes a growth defect in the fission yeast Schizosaccharomyces pombe. Chemicals that restore the growth defect phenotype can be identified as potential tankyrase 1 inhibitors. We performed a high-throughput screen using this system, and identified flavone as a compound that restores the growth of yeast cells overexpressing tankyrase 1. Indeed, flavone inhibited poly(ADP-ribosyl)ation of proteins caused by overexpression of tankyrase 1 in yeast cells. This system allows rapid identification of inhibitory activity against tankyrase 1 and is amenable to high-throughput screening using robotics.

Dipodascus tetrasporeus sp. nov., an ascosporogenous yeast isolated from deep-sea sediments in the Japan Trench.

Dipodascus tetrasporeus sp. nov. is described as a novel yeast species in the family Dipodascaceae to accommodate an isolate recovered from sediments collected on the deep-sea floor in the north-western Pacific Ocean. In the clade comprising the genera Dipodascus, Galactomyces and Geotrichum, this is the only species that forms asci that bear four ascospores. The ascospore is surrounded by an irregular exosporium wall, similar to what is observed in the genus Galactomyces, but they are released by rupture, which is characteristic of Dipodascus and not Galactomyces. D. tetrasporeus is remarkably divergent (>10 % difference) in its D1/D2 26S rDNA sequence from any other known species. Although maximum-likelihood analysis of combined 18S rDNA and D1/D2 26S rDNA sequences cannot elucidate a reliable position for this species, it was placed among Geotrichum carabidarum, Geotrichum cucujoidarum, Geotrichum fermentans and Geotrichum histeridarum, which also have morphological and physiological affinity with the species. The species is homothallic. The type strain of Dipodascus tetrasporeus sp. nov. is strain SY-277T (=NBRC 103136T =CBS 10071T).

ORFeome cloning and global analysis of protein localization in the fission yeast Schizosaccharomyces pombe.

Cloning of the entire set of an organism's protein-coding open reading frames (ORFs), or 'ORFeome', is a means of connecting the genome to downstream 'omics' applications. Here we report a proteome-scale study of the fission yeast Schizosaccharomyces pombe based on cloning of the ORFeome. Taking advantage of a recombination-based cloning system, we obtained 4,910 ORFs in a form that is readily usable in various analyses. First, we evaluated ORF prediction in the fission yeast genome project by expressing each ORF tagged at the 3' terminus. Next, we determined the localization of 4,431 proteins, corresponding to approximately 90% of the fission yeast proteome, by tagging each ORF with the yellow fluorescent protein. Furthermore, using leptomycin B, an inhibitor of the nuclear export protein Crm1, we identified 285 proteins whose localization is regulated by Crm1.

Phylogenetic relationship within the Erythrobasidium clade: molecular phylogenies, secondary structure, and intron positions inferred from partial sequences of ribosomal RNA and elongation factor-1alpha genes.

Phylogenetic relationships within the Erythrobasidium clade as a lineage of the urediniomycetous yeasts were examined using partial regions of 18S rDNA, 5.8S rDNA, 26S rDNA, internal transcribed spacers (ITSs), and elongation factor (EF)-1alpha. Combined data analysis of all segments successfully yielded a reliable phylogeny and confirmed the cohesion of species characterized by Q-10(H2) as a major ubiquinone. Differences in secondary structure predicted for a variable region in 26S rDNA corresponded to major divergences in the phylogenetic tree based on the primary sequence. The common presence of a shortened helix in this region was considered to be evidence of monophyly for species with Q-10(H2), Sakaguchia dacryoides, Rhodotorula lactosa, and Rhodotorula lamellibrachiae, although it was not as strongly supported by the combined data tree. The information on intron positions in the EF-1alpha gene had potential usefulness in the phylogenetic inference between closely related species.

Rhodotorula pacifica sp. nov., a novel yeast species from sediment collected on the deep-sea floor of the north-west Pacific Ocean.

A novel species of the genus Rhodotorula was isolated from sediments collected on the deep-sea floor in the north-west Pacific Ocean. Strains SY-96T, isolated from the Yap Trench, and SY-246, isolated from the Iheya Ridge, had almost identical nucleotide sequences for their internal transcribed spacers and their 5.8S rDNA. Their physiological characteristics were also almost identical. The strains were assumed to be related to Rhodotorula mucilaginosa and Rhodotorula dairenensis based on sequence similarities in the D1/D2 region of the 26S rDNA. The low DNA-DNA relatedness and sequence similarity between strain SY-96T and related species revealed that strains SY-96T and SY-246 represent a hitherto unknown species. As ballistoconidia and sexual reproduction were not observed in strains SY-96T and SY-246, these strains are described as Rhodotorula pacifica sp. nov. The type strain is SY-96T (= JCM 10908T = CBS 10070T).

Cryptococcus surugaensis sp. nov., a novel yeast species from sediment collected on the deep-sea floor of Suruga Bay.

A novel species of the genus Cryptococcus was isolated from sediment collected on the deep-sea floor of Suruga Bay, Japan. Nucleotide sequences of 18S rDNA, internal transcribed spacers, 5.8S rDNA and the D1/D2 region of 26S rDNA of strain SY-260(T) suggested affinities to a phylogenetic lineage that includes Cryptococcus luteolus. Comparisons of the rDNA sequences of each region clarified that strain SY-260(T) is related distantly to Bullera coprosmaensis and Bullera oryzae, but is distinct at the species level. As ballistoconidia and sexual reproduction were not observed in strain SY-260(T), this strain is described as Cryptococcus surugaensis sp. nov. (type strain, SY-260(T)=JCM 11903(T)=CBS 9426(T)).

Rhodotorula benthica sp. nov. and Rhodotorula calyptogenae sp. nov., novel yeast species from animals collected from the deep-sea floor, and Rhodotorula lysiniphila sp. nov., which is related phylogenetically.

Three novel species of the genus Rhodotorula are described. Rhodotorula benthica sp. nov. (type strain JCM 10901(T) = SY-91(T)) and Rhodotorula calyptogenae sp. nov. (type strain JCM 10899(T) = SY-86(T)) were respectively isolated from the tubeworm Lamellibrachia sp. and the giant white clam Calyptogena sp., collected from the deep-sea floor of the Pacific Ocean off Japan. Rhodotorula lysiniphila sp. nov. (type strain JCM 5951(T)) is proposed for strains isolated previously in Japan and Pakistan. The three species were placed phylogenetically into a species complex comprising Rhodotorula laryngis, Rhodotorula minuta, Rhodotorula pallida and Rhodotorula slooffiae. R. minuta and R. slooffiae are closely related in both the D1/D2 region of the 26S rDNA and the internal transcribed spacer and 5.8S rDNA regions. R. benthica and R. laryngis were closer to R. pallida based on the D1/D2 region. Other relationships were not clear.

Bannoa hahajimensis gen. nov., sp. nov., and three related anamorphs, Sporobolomyces bischofiae sp. nov., Sporobolomyces ogasawarensis sp. nov. and sporobolomyces syzygii sp. nov., yeasts isolated from plants in Japan.

Fourteen ballistoconidium-forming yeast strains were isolated from leaves of plants collected in the Ogasawara Islands, which are isolated islands in the Pacific Ocean, about 1,000 km south of the Japanese mainland, in the subtropical zone. The 14 isolates were characterized by the absence of xylose in whole-cell hydrolysates, the presence of Q-10(H2) as the major ubiquinone isoprenologue, G+C contents of 47.6-52.0 mol%, the inability to ferment sugars or to assimilate nitrate and positive Diazonium blue B and urease reactions. They formed a phylogenetically coherent cluster within the Erythrobasidium lineage in the Urediniomycetes of the Basidiomycota based on 18S rDNA sequences. Analyses of the nucleotide sequences of internal transcribed spacer regions and DNA complementarity showed that four genospecies were recognized among the 14 isolates. A mating reaction was observed in one of the four genospecies, which produced one-celled basidia on dikaryotic hyphae with clamp connections. On the basis of the morphological, physiological, chemotaxonomic and phylogenetic analyses, a new teleomorphic genus, Bannoa, is proposed, in which one novel species is described, Bannoa hahajimensis gen. nov., sp. nov. (type strain OK-248(T) = JCM 10336T = CBS 9039(T)). The other three anamorphic genospecies are described as Sporobolomyces bischofiae sp. nov. (type strain OK-257T = JCM 10338T =CBS 9041T), Sporobolomyces ogasawarensis sp. nov. (type strain OK-14T = JCM 10326T = CBS 9038T) and Sporobolomyces syzygii sp. nov. (type strain OK-227T = JCM 10337T = CBS 9040T.

Kockovaella barringtoniae sp. nov., a new basidiomycetous yeast species isolated from a plant leaf collected in a tropical rain forest in Thailand.

One yeast strain characterized by the proliferation of non-ballistosporous stalked conidia, budding cells and ballistoconidia, the presence of xylose in whole-cell hydrolysates, the presence of Q-10 as the major ubiquinone isoprenologue, the inability to ferment sugars and positive diazonium blue B (DBB) and urease reactions was isolated from a plant sample collected in a tropical rain forest in Thailand. The isolate clustered with Kockovaella species in the 18S rDNA-based tree. On the basis of the morphological, biochemical and molecular phylogenetic characteristics, the isolate was assigned to the genus Kockovaella. DNA complementarity experiments showed that the isolate was genetically distinct from known species of the genus Kockovaella. The isolate is described as Kockovaella barringtoniae sp. nov. The type strain is strain TY-278T (= JCM 10998T = TISTR 5770T).

Systematic study of basidiomycetous yeasts - evaluation of the ITS regions of rDNA to delimit species of the genus Rhodosporidium.

Nucleotide sequences of internal transcribed spacer (ITS) regions were determined to establish the guidelines for species identification in the genus Rhodosporidium. Forty-two strains of nine species of the genus Rhodosporidium were used for ITS (ITS1 and ITS2) analysis. Intraspecific length polymorphisms and sequence variations were observed within R. azoricum, R. diobovatum, R. paludigenum, R. sphaerocarpum and R. toruloides, while no variation was observed within R. babjevae and R. kratochvilovae. Based on comparison of the levels of intraspecific and interspecific sequence similarity, strains with identical sequences were considered to represent a single species and strains with 92% or lower similarity of ITS sequences were considered to be distinct species in the genus Rhodosporidium.

Genetic relatedness among species in Aspergillus section Clavati as measured by electrophoretic comparison of enzymes, DNA base composition, and DNA-DNA hybridization.

Aspergillus subgenus Clavati has four recognized species: A. clavatus (the type species), A. clavatonanicus, A. giganteus, and A. longivesica. These species are strictly anamorphic (mitotic) and defined by the morphological species concept. However, their genealogical relationships remain uncertain. In this study, we examined the genetic relatedness among the four species in this section, using electrophoretic comparison of enzymes, DNA base composition, and DNA-DNA hybridization. In a dendrogram based on the calculated similarity values of four enzymes, 10 strains in section Clavati, 3 strains in the xerophilic species, a strain in section Ornati, and a strain in section Cremei were separated into nine major clusters at a 60% similarity level. A. longivesica JCM 10186(T) had Q-10 in our analysis, but Kuraishi et al. (1990) reported A. longivesica JCM 1720(T) had Q-9 (49%) and Q-10 (46%). The G+C contents of the four species of section Clavati ranged from 48 to 50 mol%. The degree of the intraspecific reassociation among the DNAs from the strains of these species ranged from 77 to 99%, whereas the degrees of interspecific relative binding among strains of the four species ranged from 30 to 59%. Our data from enzyme patterns and DNA relatedness support the validity of the three species in section Clavati, except for A. longivesica.