Bioactive properties of streptomyces may affect the dominance of Tricholoma matsutake in shiro

Tricholoma matsutake is known to be the dominant fungal species in matsutake fruitbody neighboring (shiro) soil. To understand the mechanisms behind matsutake dominance, we studied the bacterial communities in matsutake dominant shiro soil and non-shiro soil, isolated the strains of Streptomyces from matsutake mycorrhizal root tips both from shiro soil and from the Pinus densiflora seedlings cultivated in shiro soil. Further, we investigated three Streptomyces spp. for their ability to inhibit fungal growth and Pinus densiflora seedling root elongation as well as two strains for their antifungal and antioxidative properties. Our results showed that Actinobacteria was the most abundant phylum in shiro soil. However, the differences in the Actinobacterial community composition (phylum or order level) between shiro and non-shiro soils were not significant, as indicated by PERMANOVA analyses. A genus belonging to Actinobacteria, Streptomyces, was present on the matsutake mycorrhizas, although in minority. The two antifungal assays revealed that the broths of three Streptomyces spp. had either inhibitory, neutral or promoting effects on the growth of different forest soil fungi as well as on the root elongation of the seedlings. The extracts of two strains, including one isolated from the P. densiflora seedlings, inhibited the growth of either pathogenic or ectomycorrhizal fungi. The effect depended on the medium used to cultivate the strains, but not the solvent used for the extraction. Two Streptomyces spp. showed antioxidant activity in one out of three assays used, in a ferric reducing antioxidant power assay. The observed properties seem to have several functions in matsutake shiro soil and they may contribute to the protection of the shiro area for T. matsutake dominance.Peer reviewe

Effects of carbon ion beam-induced mutagenesis for the screening of RED production-deficient mutants of Streptomyces coelicolor JCM4020.

Streptomyces lividans TK23 interacts with mycolic acid-containing bacteria (MACB), such as Tsukamurella pulmonis TP-B0596, and this direct cell contact activates its secondary metabolism (e.g., the production of undecylprodigiosin: RED). Here, we employed carbon (12C5+) ion beam-induced mutagenesis to investigate the signature of induced point mutations and further identify the gene(s) responsible for the production of secondary metabolites induced by T. pulmonis. We irradiated spores of the Streptomyces coelicolor strain JCM4020 with carbon ions to generate a mutant library. We screened the RED production-deficient mutants of S. coelicolor by mixing them with T. pulmonis TP-B0596 on agar plates, identifying the red/white phenotype of the growing colonies. Through this process, we selected 59 RED-deficient mutants from around 152,000 tested spores. We resequenced the genomes of 16 mutants and identified 44 point mutations, which revealed the signatures induced by 12C5+-irradiation. Via gene complementation experiments, we also revealed that two genes-glutamate synthase (gltB) and elongation factor G (fusA)-are responsible for the reduced production of RED

Characterization of TetR-type Repressor Which Cause the Delayed Contact-dependent RED Production in Streptomyces coelicolor

Actinomycetes, filamentous Gram-positive bacteria, are a major source of bioactive natural products which can be drug candidates. We have studied about the bacterial interaction induced production of secondary metabolites by actinomycetes. Tsukamurella pulmonis TP-B0596 (Tp) had been shown to possess ability to induce production of secondary metabolites by Streptomyces species, which are not detected or poorly produced in a mono-culture. Until now, 7 classes, total 29 new compounds had been isolated from the co-culture with various actinomycetes and Tp. Object of this study is to elucidate the gene(s) which are involved in the response for activation of secondary metabolism within actinomycetes. Elucidation of the mechanism can lead to the fundamental understanding of bacterial interaction and secondary metabolism, as well as application for genetic tools to discover novel bioactive natural products from untapped gene matters. Here we employed forward-genetic study using mutagenesis by heavy ion beam to investigated the gene(s) responsible for the responsive production of secondary metabolism induced by Tp. Through this screening, 118 mutants from around 152,000 tested spores were obtained. We further tested the phenotype of the 118 mutants by growth on minimum medium and formation of aerial mycelia, and finally selected 59 mutants. We then re-sequenced the genome of 26 mutants and further gene complementation study revealed that molybdopterin biosynthetic enzyme (moeA), and tetR-like transcriptional regulator are also responsible for the phenotype differences. We speculate that the TetR-like protein is a repressor for the expression of adjacent ABC transporter, thus efflux of the unknown small molecule is prevented and accumulated in the cell, which caused stress to cell that can lead to the undecylprodigiosin production

Identification of Genes Responsible for the Contact-dependent RED Response in Streptomyces coelicolor

Actinomycetes, filamentous Gram-positive bacteria, are a major source of bioactive natural products which can be drug candidates. We are studying about the bacterial-bacterial interaction induced secondary metabolism by filamentous actinomycetes e.g. Streptomyces species. Mycolic acid-containing bacteria, Tsukamurella pulmonis TP-B0596 (Tp) possess ability to induce production of secondary metabolites in a range of filamentous actinomycetes, which are not produced in their single culture. Object of this study is to elucidate the gene(s) which are involved in the response for activation of secondary metabolism within filamentous actinomycetes. Elucidation of the mechanism may lead for development of versatile genetic tools to discover novel bioactive natural products. S. lividans TK23 and S.s coelicolor JCM4020 were observed to produce red pigment compound (RED) in response to direct cell-cell contact interaction of Tp. We investigated the gene(s) responsible for the activation of RED which was induced by Tp using S. coelicolor JCM4020 as a model to explore the molecular basis of the response mechanism. We employed carbon ion beams (12C5+, 220 MeV), accelerated by the AVF cyclotron at TIARA, to induce mutagenesis on spores to generate mutant library of S. coelicolor. We screened the RED production deficient mutants of S. coelicolor by mixing them with Tp on agar plates. Using red / white color phenotype of the colonies as indication, we obtained 59 mutants which were deficient in induced RED production by Tp, but were normal in their apparent growth and morphological development. We re-sequenced the genome of 16 mutants using MiSeq and identified mutational points existed in CDS

Chojalactones A–C, Cytotoxic Butanolides Isolated from <i>Streptomyces</i> sp. Cultivated with Mycolic Acid Containing Bacterium

The soil-derived bacterium, <i>Streptomyces</i> sp. CJ-5, was cocultured with the mycolic acid-containing bacterium <i>Tsukamurella pulmonis</i> TP-B0596. The combined culture method significantly enhanced the production of the secondary metabolites in <i>Streptomyces</i> sp. CJ-5, leading to the isolation of three novel butanolide chojalactones A–C (<b>1</b>–<b>3</b>), with unusual γ-butyrolactone scaffolds. The complete structures, including the absolute configurations of <b>1</b>–<b>3</b>, were determined based on spectroscopic data and total syntheses. In methylthiazole tetrazolium (MTT) assays, <b>1</b> and <b>2</b> showed moderate cytotoxicity against P388 cells