Streptomyces pacificus sp. nov., a novel spongiicolazolicin-producing actinomycete isolated from a coastal sediment.

A polyphasic approach was used to determine the taxonomic position of a marine actinomycete, designated isolate CWH03T, which we previously reported to produce new linear azole-containing peptides spongiicolazolicins A and B. Strain CWH03T is mesophilic, neutrophilic, and halotolerant streptomycete that forms spiral spore chains on aerial mycelium. Comparative 16S rRNA gene sequencing showed that CWH03T was most closely related to Streptomyces tirandamycinicus HNM0039T (99.7%), Streptomyces spongiicola HNM0071T (99.4%), 'Streptomyces marianii' ICN19T (99.1%) and Streptomyces wuyuanensis CGMCC4.7042T (99.0%). The phylogenetic tree prepared using the 16S rRNA gene, as well as the phylogenomic tree using the genome BLAST distance phylogeny method and 81 core housekeeping genes, respectively, showed that the closest relative of strain CWH03T was S. spongiicola HNM0071T. The average nucleotide identity and digital DNA-DNA hybridization values between strains CWH03T and S. spongiicola HNM0071T were 91.46% and 44.2%, respectively, which were below the thresholds of 96% and 70% for prokaryotic conspecific assignation. The G+C content of the genomic DNA of strain CWH03T was 72.3%. Whole-cell hydrolysates of strain CWH03T contained LL-diaminopimelic acid. The predominant menaquinone was MK-9(H8) (88.3%), and the major fatty acids were iso-C16:0 (28.4%), anteiso-C15:0 (15.0%) and iso-C15:0 (12.9%). The major phospholipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an unidentified phospholipid. Based on data obtained from phenotypic, phylogenetic, genomic, and chemotaxonomic analyses, strain CWH03T represents a novel species of the genus Streptomyces, for which the proposed name is Streptomyces pacificus sp. nov. The type strain is CWH03T ( = NBRC 114659T = TBRC 15780T).

Cellulomonas algicola sp. nov., an actinobacterium isolated from a freshwater alga.

An actinomycete strain, TKZ-21T, was isolated from a freshwater alga (Chetophoraceae) collected from the Takizawa River, Yamanashi, Japan, and examined using a polyphasic taxonomic approach. Cells were Gram-stain positive, aerobic, non-sporulating, motile, and coccoid or short rod-shaped. The strain grew in the presence of 0-4 % (w/v) NaCl, between pH 6-9.4, and over a temperature range of 15-40 °C, with optimum growth at 30 °C. The peptidoglycan type of strain TKZ-21T was A4ß, containing l-ornithine as diagnostic diamino acid and d-glutamic acid as the interpeptide bridge. The predominant menaquinone was MK-9(H4). The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, ninhydrin-positive glycolipid, and unidentified phospholipids. The major cellular fatty acids were anteiso-C15 : 0 and anteiso-C17 : 0, and the DNA G+C content was 75.6 mol%. On the basis of 16S rRNA gene sequence analysis, strain TKZ-21T was closely related to Cellulomonas fimi (98.5 % sequence similarity) and Cellulomonas biazotea (98.3 %). The genome orthoANI value between strain TKZ-21T and C. biazotea and C. fimi were 84.7 and 84.2 %, respectively. On the basis of fatty acid and MALDI-TOF MS profile analysis, phylogenetic analyses, genomic analysis, and phenotypic data, it is proposed that the isolate be classified as a representative of a novel species of the genus Cellulomonas, with the name Cellulomonas algicola sp. nov. The type strain is TKZ-21T (=NBRC 112905T=TBRC 8129T).

Virgisporangium myanmarense sp. nov., a novel motile actinomycete isolated from an anthill soil in Myanmar.

An actinomycete strain, designated MM04-1133T, was isolated from an anthill soil sample collected in Bagan, Myanmar. To establish the taxonomic status of this strain, the isolate was subjected to a polyphasic approach. Strain MM04-1133T was Gram-staining positive, aerobic, motile and formed long and narrow sporangia directly above the surface of the substrate mycelium. Whole-cell hydrolysates of the strain contained 3-OH-diaminopimelic acid, arabinose, glucose, galactose, mannose, rhamnose and xylose. The predominant menaquinones were MK-10(H6) and MK-10(H8). The major cellular fatty acids were iso-C16:0 and anteiso-C17:0. The diagnostic phospholipid was phosphatidylethanolamine. The G+C content of the DNA was 69.1 mol%. Phylogenetic analysis based on 16S rRNA gene sequence revealed that strain MM04-1133T clustered within the genus Virgisporangium, with the sequence exhibiting highest similarity (98.5% identity) with Virgisporangium ochraceum NBRC 16418T. The strain grew in the presence of 0-1% (w/v) NaCl, at pH 5-8 and at 20-40 °C, with optimal growth at 30-37 °C. Based on phylogenetic analysis and chemotaxonomic and phenotypic data, we propose classifying this isolate as a novel species of the genus Virgisporangium, to be designated as Virgisporangium myanmarense sp. nov. The type strain is MM04-1133T (=NBRC 112733T=VTCC 910008T).

Development of intra-strain self-cloning procedure for breeding baker's yeast strains.

Previously reported self-cloning procedures for breeding of industrial yeast strains require DNA from other strains, plasmid DNA, or mutagenesis. Therefore, we aimed to construct a self-cloning baker's yeast strain that exhibits freeze tolerance via an improved self-cloning procedure. We first disrupted the URA3 gene of a prototrophic baker's yeast strain without the use of any marker gene, resulting in a Δura3 homozygous disruptant. Then, the URA3 gene of the parental baker's yeast strain was used as a selection marker to introduce the constitutive TDH3 promoter upstream of the PDE2 gene encoding high-affinity cyclic AMP phosphodiesterase. This self-cloning procedure was performed without using DNA from other Saccharomyces cerevisiae strains, plasmid DNA, or mutagenesis and was therefore designated an intra-strain self-cloning procedure. Using this self-cloning procedure, we succeeded in producing self-cloning baker's yeast strains that harbor the TDH3p-PDE2 gene heterozygously and homozygously, designated TDH3p-PDE2 hetero and TDH3p-PDE2 homo strains, respectively. These self-cloning strains expressed much higher levels of PDE2 mRNA than the parental strain and exhibited higher viability after freeze stress, as well as higher fermentation ability in frozen dough, when compared with the parental strain. The TDH3p-PDE2 homo strain was genetically more stable than the TDH3p-PDE2 hetero strain. These results indicate that both heterozygous and homozygous strains of self-cloning PDE2-overexpressing freeze-tolerant strains of industrial baker's yeast can be prepared using the intra-strain self-cloning procedure, and, from a practical viewpoint, the TDH3p-PDE2 homo strain constructed in this study is preferable to the TDH3p-PDE2 hetero strain for frozen dough baking.

Forced expression of FLO11 confers pellicle-forming ability and furfural tolerance on Saccharomyces cerevisiae in ethanol production.

We constructed a plasmid that expresses FLO11 encoding a cell surface glycoprotein of Saccharomyces cerevisiae under the control of a constitutive promoter. This plasmid conferred pellicle-forming ability on the non-pellicle-forming industrial strain of S. cerevisiae at the air-liquid interface of the glucose-containing liquid medium. The induced pellicle-forming cells exhibited tolerance to furfural, which is a key toxin in lignocellulosic hydrolysates, in ethanol production.

Streptomyces hokutonensis sp. nov., a novel actinomycete isolated from the strawberry root rhizosphere.

A polyphasic approach was used to determine the taxonomic position of actinomycete strain R1-NS-10(T), which was isolated from a sample of strawberry root rhizosphere obtained from Hokuto, Yamanashi, Japan. Strain R1-NS-10(T) was Gram-staining-positive and aerobic, and formed brownish-white aerial mycelia and grayish-brown substrate mycelia on ISP-2 medium. The strain grew in the presence of 0-5% (w/v) NaCl and optimally grew without NaCl. The strain grew at pH 5-8, and the optimum for growth was pH 7. The optimal growth temperature was 30 °C, but the strain grew at 5-37 °C. Whole-cell hydrolysates of strain R1-NS-10(T) contained A2pm, galactose, mannose and rhamnose. The predominant menaquinones were MK-9(H6) and MK-9(H8). The major cellular fatty acids were anteiso-C15:0 and iso-C16:0. Comparative 16S rRNA gene sequence analysis revealed that strain R1-NS-10(T) was most closely related to Streptomyces prunicolor NBRC 13075(T) (99.4%). The draft genome sequences of both strains were determined for characterization of genome sequence-related parameters such as average nucleotide identity (ANI) and the diversity of secondary metabolite biosynthetic gene clusters. DNA-DNA hybridization (DDH) and ANI values for both strains were below the species delineation cutoff, and differences in physiological and biochemical characteristics differentiated strain R1-NS-10(T) from its closest phylogenetic relative. On the basis of these data, we propose that strain R1-NS-10(T) (=NBRC 108812(T)=KCTC 29186(T)) should be classified as the type strain of a novel Streptomyces species named Streptomyces hokutonensis sp. nov.

A new simple method for isolating multistress-tolerant semidominant mutants of Saccharomyces cerevisiae by one-step selection under lethal hydrogen peroxide stress condition.

Tolerance of microorganisms to diverse stresses (i.e., multistress tolerance) is a very useful property with industrial applications. We have developed a simple method for isolating multistress-tolerant semidominant mutants of the budding yeast Saccharomyces cerevisiae by one-step selection under lethal hydrogen peroxide (H(2)O(2)) stress condition, which we named the lethal concentration of H(2)O(2) (LCH) method. This method involves simply isolating colonies after plating of mutagenized S. cerevisiae cells, which are cultivated overnight in liquid media, on agar plates containing a lethal concentration of H(2)O(2) for the wild-type strain. Phenotypic and genetic analyses of the ten strains isolated by this method revealed that two strains exhibiting stress tolerance to H(2)O(2), ethanol, heat shock, salt, organic solvent, freeze-thaw, chronological aging, and high concentrations of glucose possess semidominant and distinct single-gene mutations designated as MLT1-1 (multistress tolerance) and MLT2-1, which are responsible for multistress tolerance. From these results, we expect this method to confer multistress tolerance on industrial yeasts.

Actinoplanes rishiriensis sp. nov., a novel motile actinomycete isolated by rehydration and centrifugation method.

The motile actinomycete strain RI50-RCA114(T) was isolated using rehydration and centrifugation method from a soil sample obtained from Rishiri Island in Japan. The taxonomic status of this organism was established using a polyphasic approach. Cells of strain RI50-RCA114(T) were Gram positive, aerobic, motile and formed irregular sporangia. The strain grew in the presence of 0-2% (w/v) NaCl, between pH 5 and 8, and over a temperature range of 20-37°C, with optimal growth at 30°C. Whole-cell hydrolysates of the strain contained meso-diaminopimelic acid, galactose, glucose and mannose, in addition to one unidentified O-methyl-hexose. The predominant menaquinone was MK-9(H(4)), and the major polar lipids comprised phosphatidylethanolamine, diphosphatidylglycerol and phosphatidyl-N-methylethylethanolamine. The major cellular fatty acids were iso-C(16:0), iso-C(15:0) and anteiso-C(17:0). Comparative 16S ribosomal RNA gene sequence analysis revealed that strain RI50-RCA114(T) had the closest sequence similarity with Actinoplanes globisporus JCM 3186(T) (97.6%). However, DNA-DNA hybridization assays as well as physiological and biochemical analyses differentiated strain RI50-RCA114(T) from its closest phylogenetic relative. On the basis of these data, we propose that strain RI50-RCA114(T) (=NBRC 108556(T)=BCC 49184(T)) be classified as the type strain of a novel Actinoplanes species and named Actinoplanes rishiriensis sp. nov.

Complete genome sequence of the motile actinomycete Actinoplanes missouriensis 431(T) (= NBRC 102363(T)).

Actinoplanes missouriensis Couch 1963 is a well-characterized member of the genus Actinoplanes, which is of morphological interest because its members typically produce sporangia containing motile spores. The sporangiospores are motile by means of flagella and exhibit chemotactic properties. It is of further interest that members of Actinoplanes are prolific sources of novel antibiotics, enzymes, and other bioactive compounds. Here, we describe the features of A. missouriensis 431(T), together with the complete genome sequence and annotation. The 8,773,466 bp genome contains 8,125 protein-coding and 79 RNA genes.

Variation and predicted structure of the flagellin gene in Actinoplanes species.

Members of the genus Actinoplanes are considered to be representative of motile actinomycetes. To infer the flagellar diversity of Actinoplanes species, novel degenerate primers were designed for the flagellin (fliC) gene. The fliC gene of 21 Actinoplanes strains was successfully amplified and classified into two groups based on whether they were large (type I) or small (type II). Comparison of the translated amino acid sequences revealed that this size difference could be attributed to large number of gaps located in the central variable region. However, the C- and N- terminal regions were conserved. Except for a region on the flagellum surface, structural predictions of type I and II flagellins revealed that the two flagellin types were strongly correlated with each other. Phylogenetic analysis of the 115-amino acid N-terminal sequences revealed that the Actinoplanes species formed three clusters, and type II flagellin gene containing three type strains were phylogenetically closely related each other.

Actinomycetospora iriomotensis sp. nov., a novel actinomycete isolated from a lichen sample.

An actinomycete strain, IR73-Li102(T), was isolated from a lichen sample obtained from Iriomote Island, Japan, and subsequently characterized using a polyphasic approach. Comparative 16S rRNA gene sequence analysis revealed that strain IR73-Li102(T) had the highest sequence similarities with Actinomycetospora chiangmaiensis YIM 0006(T) (98.3%), A. corticola 014-5(T) (98.1%) and A. rishiriensis RI109-Li102(T) (98.0%). However, DNA-DNA hybridization assays, as well as physiological and biochemical analyzes, showed that strain IR73-Li102(T) could be clearly differentiated from its closest phylogenetic relatives. The strain contained meso-diaminopimelic acid, and arabinose and galactose were present in whole-cell hydrolysates. The predominant menaquinone was MK-8(H(4)), and the diagnostic phospholipids were phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol and diphosphatidylglycerol. The major cellular fatty acid was iso-C(16:0) (58%). The chemotaxonomic properties of strain IR73-Li102(T) were consistent with those shared by members of the genus Actinomycetospora. On the basis of the phenotypic features and DNA-DNA hybridization data, strain IR73-Li102(T) (= NBRC 106365(T) = KCTC 19783(T)) represents a novel species of the genus Actinomycetospora, for which the name Actinomycetospora iriomotensis sp. nov. is proposed.

FLO11 is essential for pellicle formation by wild pellicle-forming yeasts isolated from contaminated wines.

The Δflo11/Δflo11 disruptant strains constructed from three wild yeast strains, which were independently isolated from pellicles formed on the surface of contaminated stored wines, completely lost their ability to form pellicles. The FLO11 expression level of the pellicle-forming strains was much higher than that of non-pellicle-forming wine strains. Cell surface hydrophobicity of the Δflo11/Δflo11 disruptant strains was as high as that of the parental pellicle-forming strains. These findings indicate that FLO11 is essential for unfavorable pellicle formation of wild pellicle-forming yeasts by mechanisms other than increasing cell surface hydrophobicity.

Nocardioides iriomotensis sp. nov., an actinobacterium isolated from forest soil.

An actinomycete strain, designated IR27-S3(T), was isolated from a forest soil sample collected from Iriomote Island, Okinawa, Japan. Cells of the isolate were Gram-stain-positive, aerobic, non-sporulating, non-motile coccoids or short rods. The strain grew in the presence of 0-7 % (w/v) NaCl, at pH 6-8 and at 12-37 °C, with optimum growth at 30 °C. Chemotaxonomically, the strain contained LL-diaminopimelic acid as the diagnostic diamino acid and MK-8(H4) as the predominant menaquinone. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and an unknown phospholipid. The major cellular fatty acids were iso-C16:0, C17:1 cis-9, C15:0 and iso-C15:0. The DNA G+C content was 73.7 mol%. On the basis of 16S rRNA gene sequence analysis, strain IR27-S3(T) was closely related to Nocardioides mesophilus MSL-22(T) (98.1 % 16S rRNA gene sequence similarity), Marmoricola bigeumensis MSL-05(T) (97.2 %) and Nocardioides jensenii DSM 20641(T) (96.5 %). On the basis of fatty acid analysis, phylogenetic analysis and phenotypic data, the isolate should be classified in a novel species of the genus Nocardioides, for which the name Nocardioides iriomotensis sp. nov. is proposed. The type strain is IR27-S3(T) ( = NBRC 105384(T)  = KACC 14926(T)).

Actinomycetospora rishiriensis sp. nov., isolated from a lichen.

An actinomycete, strain RI109-Li102(T), was isolated from a lichen sample obtained from Rishiri Island in Japan. Cells of strain RI109-Li102(T) were Gram-positive, aerobic and non-motile and formed bud-like spore chains. The isolate grew with 0-3 % (w/v) NaCl, at pH 5-9 and at 10-30 °C (optimum 30 °C). The whole-cell hydrolysate contained meso-diaminopimelic acid, arabinose and galactose. The predominant menaquinone was MK-8(H(4)) and the diagnostic phospholipids were phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol and diphosphatidylglycerol. The major cellular fatty acids were iso-C(16 : 0) and iso-C(16 : 1) H. Comparative 16S rRNA gene sequence analysis revealed that strain RI109-Li102(T) was most closely related to Actinomycetospora corticicola 014-5(T) (99.0 % rRNA gene sequence similarity) and Actinomycetospora chiangmaiensis YIM 0006(T) (98.4 %). However, DNA-DNA hybridization assays, as well as physiological and biochemical analyses, showed that strain RI109-Li102(T) could be differentiated from its closest phylogenetic relatives. It is proposed that strain RI109-Li102(T) ( = NBRC 106356(T) = KCTC 19782(T)) be classified as the type strain of a novel species, with the name Actinomycetospora rishiriensis sp. nov.

FLO11 is the primary factor in flor formation caused by cell surface hydrophobicity in wild-type flor yeast.

Some strains of Saccharomyces cerevisiae form a biofilm called a "flor" on the surface of wine after ethanolic fermentation, but the molecular mechanism of flor formation by the wild-type flor strain involved in wine making is not clear. Previously, we found that expression of the C-terminally truncated form of NRG1 (NRG1(1-470)) on a multicopy plasmid increases the hydrophobicity of the cell surface, conferring flor formation on the non-flor laboratory strain. Here we show that in Ar5-H12, a wild-type flor haploid strain, flor formation is regulated by NRG1(1-470). Moreover, the disruptant of the wild-type flor diploid strain (Deltaflo11/Deltaflo11) show a weak ability to form the flor. The expression of FLO11 is always high in the wild-type flor strain, regardless of carbon source. Thus FLO11 is primary factor for wild-type flor strains. Furthermore, the disruptant (Deltaflo11) shows lower hydrophobicity of cell surface than the wild type. However, the hydrophobicity of the wild-type flor strains grown in ethanol medium was much higher than those grown in glucose medium. These results indicate that cell surface hydrophobicity is closely related to flor formation in wild-type flor yeasts.

Two alleles of the sulfite resistance genes are differentially regulated in Saccharomyces cerevisiae.

The sulfite resistance gene, SSU1-R, is widely distributed in wine yeasts. This gene has an upstream region distinct from that of the allelic gene, SSU1 and SSU1-R is expressed at a much higher level than SSU1. We characterized the promoters of both of these genes by analysis of their activity using the LacZ gene as a reporter. FZF1, the activator gene of SSU1, was shown to regulate SSU1-R expression indirectly. SSU1-R expression was activated under microaerobic conditions, and four 76-bp repeats, present within the SSU1-R promoter region, was essential for high expression. These results indicate that SSU1-R expression is regulated in different manner from that of SSU1. By deletion analysis of the SSU1-R promoter region, we found that at least two of the 76-bp repeats are necessary for promoter activity, and that the number of 76-bp repeats influences the activity. Hence, it was suggested that the number of 76-bp repeats increases in wine yeasts that require strong sulfite resistance.

Nocardia takedensis sp. nov., isolated from moat sediment and scumming activated sludge.

Chemotaxonomic and morphological characterization of two actinomycete strains, MS1-3T and AS4-2, respectively isolated from moat sediment and scumming activated sludge, was carried out. This characterization clearly demonstrated that strains MS1-3T and AS4-2 belong to the genus Nocardia. 16S rRNA gene sequencing studies showed that these isolates are most closely related to Nocardia beijingensis (98.1-98.3 % similarity), Nocardia brasiliensis (97.9-98.0 %) and Nocardia tenerifensis (97.8-97.9 %). However, the results of DNA-DNA hybridizations and physiological and biochemical tests showed that strains MS1-3T and AS4-2 could be differentiated from their closest phylogenetic relatives both genotypically and phenotypically. It is proposed that the two isolates be classified as representatives of a novel species of Nocardia, Nocardia takedensis sp. nov. The type strain is MS1-3T (=NBRC 100417T=DSM 44801T); AS4-2 (=NBRC 100418=DSM 44802) is a reference strain.

FLO11 is essential for flor formation caused by the C-terminal deletion of NRG1 in Saccharomyces cerevisiae.

The flor strains of Saccharomyces cerevisiae form a flor on the surface of wine after alcoholic fermentation. High hydrophobicity of the cell surface is suggested to be important for flor formation by the flor wine yeasts. However, the molecular mechanism of flor formation is not clear. We found that expression of C-terminal deleted NRG1 lacking its two C2H2 zinc finger motifs (NRG1(1-470)) on the multicopy plasmid conferred the ability to form a flor to a non-flor laboratory strain. The cell surface hydrophobicity of NRG1(1-470) was higher than of the non-flor strain. Disruption of the Nrg1p-repressed gene FLO11, which encodes a cell surface glycoprotein that functions as a flocculin or an adhesin, abolished flor formation. Moreover, expression of FLO11 on a multicopy plasmid could also cause flor formation. These results indicate that FLO11 is essential for flor formation by NRG1(1-470). In addition, the results suggest that the C-terminal truncated form of Nrg1p exerts a dominant negative effect on FLO11 repression, resulting in FLO11 expression and, thus, flor formation.

Characterization of Nocardia asteroides isolates from different ecological habitats on the basis of repetitive extragenic palindromic-PCR fingerprinting.

Thirteen isolates of Nocardia asteroides from both soils and aquatic samples (lake and moat sediments, as well as scum from activated sludge), together with a type strain and two known clinical isolates of this species, were characterized by repetitive extragenic palindromic-PCR fingerprinting with the BOX-A1R primer. The resulting DNA fingerprint patterns proved to be strain specific, and cluster analysis distinguished the soil isolates, the aquatic isolates, and the known strains as being in separate groups.