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2.
J Biosci Bioeng ; 124(6): 611-617, 2017 Dec.
Article in English | MEDLINE | ID: mdl-28728974

ABSTRACT

The genomes of actinomycetes encode many cryptic novel/useful bioactive compounds, but access to these cryptic secondary metabolites remains limited. Streptomyces avermitilis predominantly produces three polyketide antibiotics (avermectin, filipin, and oligomycin) but has the potential to produce more secondary metabolites based on the number of cryptic biosynthetic gene clusters. Here, we extensively investigated the metabolite profiles of a gene disruptant of AvaR3 (an autoregulator receptor homologue), which is involved in the pleiotropic regulation of antibiotic production and cell morphology. Unlike the wild-type strain, the avaR3 mutant accumulated compound 3 in the culture. The chemical structure of compound 3 was elucidated on the basis of various spectroscopic analyses, and was identified as phthoxazolin A, a cellulose synthesis inhibitor. Bioassays demonstrated that compound 3 exerts growth inhibitory activity against a broad range of plant pathogenic oomycetes. Moreover, unlike avermectin production, phthoxazolin A (3) production was negatively controlled by avenolide, a new type of autoregulator in streptomycetes, through the function of AvaR3. These results suggest that the genetic manipulation of autoregulator receptor homologues would be a valuable tool for the discovery of cryptic bioactive compounds.


Subject(s)
Bacterial Proteins/genetics , Fatty Alcohols/metabolism , Oxazoles/metabolism , Polyunsaturated Alkamides/metabolism , Streptomyces/genetics , Streptomyces/metabolism , Anti-Bacterial Agents/biosynthesis , Anti-Bacterial Agents/metabolism , Bacterial Proteins/metabolism , Biological Assay , Fatty Alcohols/chemistry , Fatty Alcohols/isolation & purification , Fatty Alcohols/pharmacology , Gene Expression Regulation, Bacterial , Ivermectin/analogs & derivatives , Ivermectin/chemistry , Ivermectin/metabolism , Multigene Family , Oomycetes/drug effects , Oxazoles/chemistry , Oxazoles/isolation & purification , Oxazoles/pharmacology , Polyunsaturated Alkamides/chemistry , Polyunsaturated Alkamides/isolation & purification , Polyunsaturated Alkamides/pharmacology , Secondary Metabolism/genetics , Streptomyces/cytology
3.
Appl Microbiol Biotechnol ; 100(22): 9581-9591, 2016 Nov.
Article in English | MEDLINE | ID: mdl-27541747

ABSTRACT

Streptomyces hormones, sometimes called as autoregulators, are important signaling molecules to trigger secondary metabolism across many Streptomyces species. We recently identified a butenolide-type autoregulator (termed avenolide) as a new class of Streptomyces hormone from Streptomyces avermitilis that produces important anthelmintic agent avermectin. Avenolide triggers the production of avermectin with minimum effective concentration of nanomolar. Here, we describe the characterization of avaR1 encoding an avenolide receptor in the regulation of avermectin production and avenolide biosynthesis. The disruption of avaR1 resulted in transcriptional derepression of avenolide biosynthetic gene with an increase in avenolide production, with no change in the avermectin production profile. Moreover, the avaR1 mutant showed increased transcription of avaR1. Together with clear DNA-binding capacity of AvaR1 toward avaR1 upstream region, it suggests that AvaR1 negatively controls the expression of avaR1 through the direct binding to the promoter region of avaR1. These findings revealed that the avenolide receptor AvaR1 functions as a transcriptional repressor for avenolide biosynthesis and its own synthesis.


Subject(s)
4-Butyrolactone/analogs & derivatives , Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Ivermectin/analogs & derivatives , Receptors, Cell Surface/metabolism , Repressor Proteins/metabolism , Streptomyces/metabolism , 4-Butyrolactone/metabolism , Bacterial Proteins/genetics , Gene Knockout Techniques , Ivermectin/metabolism , Receptors, Cell Surface/genetics , Repressor Proteins/genetics , Streptomyces/genetics , Transcription, Genetic
4.
Biol Open ; 5(6): 709-19, 2016 Jun 15.
Article in English | MEDLINE | ID: mdl-27170256

ABSTRACT

Direct reprogramming is a promising, simple and low-cost approach to generate target cells from somatic cells without using induced pluripotent stem cells. Recently, peripheral blood mononuclear cells (PBMCs) have attracted considerable attention as a somatic cell source for reprogramming. As a cell source, PBMCs have an advantage over dermal fibroblasts with respect to the ease of collecting tissues. Based on our studies involving generation of photosensitive photoreceptor cells from human iris cells and human dermal fibroblasts by transduction of photoreceptor-related transcription factors via retrovirus vectors, we transduced these transcription factors into PBMCs via Sendai virus vectors. We found that retinal disease-related genes were efficiently detected in CRX-transduced cells, most of which are crucial to photoreceptor functions. In functional studies, a light-induced inward current was detected in some CRX-transduced cells. Moreover, by modification of the culture conditions including additional transduction of RAX1 and NEUROD1, we found a greater variety of retinal disease-related genes than that observed in CRX-transduced PBMCs. These data suggest that CRX acts as a master control gene for reprogramming PBMCs into photoreceptor-like cells and that our induced photoreceptor-like cells might contribute to individualized drug screening and disease modeling of inherited retinal degeneration.

5.
Appl Environ Microbiol ; 80(16): 5028-36, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24907338

ABSTRACT

Mycosporines and mycosporine-like amino acids (MAAs), including shinorine (mycosporine-glycine-serine) and porphyra-334 (mycosporine-glycine-threonine), are UV-absorbing compounds produced by cyanobacteria, fungi, and marine micro- and macroalgae. These MAAs have the ability to protect these organisms from damage by environmental UV radiation. Although no reports have described the production of MAAs and the corresponding genes involved in MAA biosynthesis from Gram-positive bacteria to date, genome mining of the Gram-positive bacterial database revealed that two microorganisms belonging to the order Actinomycetales, Actinosynnema mirum DSM 43827 and Pseudonocardia sp. strain P1, possess a gene cluster homologous to the biosynthetic gene clusters identified from cyanobacteria. When the two strains were grown in liquid culture, Pseudonocardia sp. accumulated a very small amount of MAA-like compound in a medium-dependent manner, whereas A. mirum did not produce MAAs under any culture conditions, indicating that the biosynthetic gene cluster of A. mirum was in a cryptic state in this microorganism. In order to characterize these biosynthetic gene clusters, each biosynthetic gene cluster was heterologously expressed in an engineered host, Streptomyces avermitilis SUKA22. Since the resultant transformants carrying the entire biosynthetic gene cluster controlled by an alternative promoter produced mainly shinorine, this is the first confirmation of a biosynthetic gene cluster for MAA from Gram-positive bacteria. Furthermore, S. avermitilis SUKA22 transformants carrying the biosynthetic gene cluster for MAA of A. mirum accumulated not only shinorine and porphyra-334 but also a novel MAA. Structure elucidation revealed that the novel MAA is mycosporine-glycine-alanine, which substitutes l-alanine for the l-serine of shinorine.


Subject(s)
Actinomycetales/genetics , Amino Acids/biosynthesis , Bacterial Proteins/genetics , Gene Expression , Glycine/analogs & derivatives , Multigene Family , Streptomyces/metabolism , Actinomycetales/metabolism , Bacterial Proteins/metabolism , Cyclohexanones , Cyclohexylamines , Glycine/biosynthesis , Streptomyces/genetics
6.
Genes Cells ; 19(3): 198-208, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24456169

ABSTRACT

Redirecting differentiation of somatic cells by over-expression of transcription factors is a promising approach for regenerative medicine, elucidation of pathogenesis and development of new therapies. We have previously defined a transcription factor combination, that is, CRX, RAX and NEUROD, that can generate photosensitive photoreceptor cells from human iris cells. Here, we show that human dermal fibroblasts are differentiated to photoreceptor cells by the same transcription factor combination as human iris cells. Transduction of a combination of the CRX, RAX and NEUROD genes up-regulated expression of the photoreceptor-specific genes, recoverin, blue opsin and PDE6C, in all three strains of human dermal fibroblasts that were tested. Additional OTX2 gene transduction increased up-regulation of the photoreceptor-specific genes blue opsin, recoverin, S-antigen, CNGB3 and PDE6C. Global gene expression data by microarray analysis further showed that photoreceptor-related functional genes were significantly increased in induced photoreceptor cells. Functional analysis, that is, patch-clamp recordings, clearly revealed that induced photoreceptor cells from fibroblasts responded to light. Both the NRL gene and the NR2E3 gene were endogenously up-regulated in induced photoreceptor cells, implying that exogenous CRX, RAX, OTX2 and NEUROD, but not NRL, are sufficient to generate rod photoreceptor cells.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/genetics , Eye Proteins/genetics , Fibroblasts/cytology , Homeodomain Proteins/genetics , Otx Transcription Factors/genetics , Photoreceptor Cells, Vertebrate/cytology , Trans-Activators/genetics , Transcription Factors/genetics , Cell Differentiation , Dermis/cytology , Dermis/metabolism , Fibroblasts/metabolism , Humans , Iris/cytology , Iris/metabolism , Photoreceptor Cells, Vertebrate/metabolism , Retina/cytology , Retina/metabolism , Transduction, Genetic
7.
J Antibiot (Tokyo) ; 64(12): 781-7, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21989459

ABSTRACT

The first total synthesis of extracellular factor, "Avenolide", in Streptomyces avermitilis has been achieved using a convergent approach. The stereogenic centers in two key segments were installed using Sharpless epoxidation and dihydroxylation. This synthetic study allowed the determination of the absolute configuration of avenolide as 4S,10R, and yielded important information on its structure-activity relationship.


Subject(s)
4-Butyrolactone/analogs & derivatives , Streptomyces/metabolism , 4-Butyrolactone/biosynthesis , 4-Butyrolactone/chemical synthesis , 4-Butyrolactone/chemistry , 4-Butyrolactone/metabolism , Magnetic Resonance Spectroscopy , Spectrometry, Mass, Fast Atom Bombardment , Spectrophotometry, Infrared , Stereoisomerism , Streptomyces/chemistry , Structure-Activity Relationship
8.
Proc Natl Acad Sci U S A ; 108(39): 16410-5, 2011 Sep 27.
Article in English | MEDLINE | ID: mdl-21930904

ABSTRACT

Gram-positive bacteria of the genus Streptomyces are industrially important microorganisms, producing >70% of commercially important antibiotics. The production of these compounds is often regulated by low-molecular-weight bacterial hormones called autoregulators. Although 60% of Streptomyces strains may use γ-butyrolactone-type molecules as autoregulators and some use furan-type molecules, little is known about the signaling molecules used to regulate antibiotic production in many other members of this genus. Here, we purified a signaling molecule (avenolide) from Streptomyces avermitilis--the producer of the important anthelmintic agent avermectin with annual world sales of $850 million--and determined its structure, including stereochemistry, by spectroscopic analysis and chemical synthesis as (4S,10R)-10-hydroxy-10-methyl-9-oxo-dodec-2-en-1,4-olide, a class of Streptomyces autoregulator. Avenolide is essential for eliciting avermectin production and is effective at nanomolar concentrations with a minimum effective concentration of 4 nM. The aco gene of S. avermitilis, which encodes an acyl-CoA oxidase, is required for avenolide biosynthesis, and homologs are also present in Streptomyces fradiae, Streptomyces ghanaensis, and Streptomyces griseoauranticus, suggesting that butenolide-type autoregulators may represent a widespread and another class of Streptomyces autoregulator involved in regulating antibiotic production.


Subject(s)
Anthelmintics/metabolism , Hormones/metabolism , Ivermectin/analogs & derivatives , Streptomyces/metabolism , Culture Media , Hormones/chemistry , Ivermectin/metabolism , Ligands , Mass Spectrometry , Nuclear Magnetic Resonance, Biomolecular , Solvents
9.
Microbiology (Reading) ; 157(Pt 8): 2266-2275, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21622528

ABSTRACT

The γ-butyrolactone autoregulator receptor has been shown to control secondary metabolism and/or morphological differentiation across many Streptomyces species. Streptomyces avermitilis produces an important anthelmintic agent (avermectin) and two further polyketide antibiotics, filipin and oligomycin. Genomic analysis of S. avermitilis revealed that this micro-organism has the clustered putative autoregulator receptor genes distant from the antibiotic biosynthetic gene clusters. Here, we describe the characterization of avaR3, one of the clustered receptor genes, which encodes a protein containing an extra stretch of amino acid residues that has not been found in the family of autoregulator receptors. Disruption of avaR3 resulted in markedly decreased production of avermectins, with delayed expression of avermectin biosynthetic genes, suggesting that AvaR3 positively controls the avermectin biosynthetic genes. Moreover, the disruption caused increased production of filipin without any changes in the transcriptional profile of the filipin biosynthetic genes, suggesting that filipin production is indirectly controlled by AvaR3. The avaR3 disruptant displayed fragmented growth in liquid culture and conditional morphological defects on solid medium. These findings demonstrated that AvaR3 acts as a global regulator that controls antibiotic production and cell morphology.


Subject(s)
Anti-Bacterial Agents/biosynthesis , Bacterial Proteins/metabolism , Filipin/biosynthesis , Gene Expression Regulation, Bacterial , Ivermectin/analogs & derivatives , Streptomyces/cytology , Streptomyces/metabolism , Bacterial Proteins/genetics , Biosynthetic Pathways/genetics , Culture Media/chemistry , Gene Expression Profiling , Gene Knockout Techniques , Genes, Bacterial , Ivermectin/metabolism , Multigene Family , Mutagenesis, Insertional , Streptomyces/growth & development
10.
Proc Natl Acad Sci U S A ; 105(35): 12921-6, 2008 Sep 02.
Article in English | MEDLINE | ID: mdl-18728192

ABSTRACT

Membrane fusion is an essential step in the encounter of two nuclei from sex cells-sperm and egg-in fertilization. However, aside from the involvement of two molecules, CD9 and Izumo, the mechanism of fusion remains unclear. Here, we show that sperm-egg fusion is mediated by vesicles containing CD9 that are released from the egg and interact with sperm. We demonstrate that the CD9(-/-) eggs, which have a defective sperm-fusing ability, have impaired release of CD9-containing vesicles. We investigate the fusion-facilitating activity of CD9-containing vesicles by examining the fusion of sperm to CD9(-/-) eggs with the aid of exogenous CD9-containing vesicles. Moreover, we show, by examining the fusion of sperm to CD9(-/-) eggs, that hamster eggs have a similar fusing ability as mouse eggs. The CD9-containing vesicle release from unfertilized eggs provides insight into the mechanism required for fusion with sperm.


Subject(s)
Antigens, CD/metabolism , Membrane Glycoproteins/metabolism , Ovum/metabolism , Secretory Vesicles/metabolism , Sperm-Ovum Interactions/physiology , Spermatozoa/cytology , Animals , Female , Fertilization , Green Fluorescent Proteins/metabolism , Male , Membrane Glycoproteins/deficiency , Mice , Ovum/cytology , Ovum/ultrastructure , Recombinant Fusion Proteins/metabolism , Secretory Vesicles/ultrastructure , Tetraspanin 29
11.
Mol Reprod Dev ; 75(1): 150-5, 2008 Jan.
Article in English | MEDLINE | ID: mdl-17290409

ABSTRACT

Tetraspanin CD81 is closely homologous in amino acid sequence with CD9. CD9 is well known to be involved in sperm-egg fusion, and CD81 has also been reported to be involved in membrane fusion events. However, the function of CD81 as well as that of CD9 in membrane fusion remains unclear. Here, we report that disruption of the mouse CD81 gene led to a reduction in the fecundity of female mice, and CD81-/- eggs had impaired ability to fuse with sperm. Furthermore, we demonstrated that when CD81-/- eggs were incubated with sperm, some of the sperm that penetrated into the perivitelline space of CD81-/- eggs had not yet undergone the acrosome reaction, indicating that the impaired fusibility of CD81-/- eggs may be in part caused by failure of the acrosome reaction of sperm. In addition, we showed that CD81 was highly expressed in granulosa cells, somatic cells that surround oocytes. Our observations suggest that there is an interaction between sperm and CD81 on somatic cells surrounding eggs before the direct interaction of sperm and eggs. Our results may provide new clues for clarifying the cellular mechanism of the acrosome reaction, which is required for sperm-egg fusion.


Subject(s)
Acrosome Reaction/genetics , Antigens, CD/physiology , Infertility, Female/genetics , Ovarian Follicle/growth & development , Spermatozoa/metabolism , Animals , Antigens, CD/analysis , Antigens, CD/genetics , Antigens, CD/metabolism , Female , Fertility/genetics , Male , Membrane Glycoproteins/metabolism , Mice , Mice, Mutant Strains , Ovarian Follicle/chemistry , Ovarian Follicle/metabolism , Tetraspanin 28 , Tetraspanin 29
12.
Biochem Biophys Res Commun ; 322(2): 601-8, 2004 Sep 17.
Article in English | MEDLINE | ID: mdl-15325272

ABSTRACT

BRAF-HDAC complex (BHC) has been shown to contain six components, including BHC80, and to mediate REST-dependent transcriptional repression of neuron-specific genes in non-neuronal cells. In this study, we have examined the functional role(s) of BHC80 in mouse tissues and human cultured cells. Two isoforms of mouse BHC80 were predominantly present in the central nervous system and spermatogenic cells. Human cultured cells also contained two isoforms of BHC80. Immunohistochemical analysis showed the presence of mouse BHC80 in the nucleus of neuronal cells in the hippocampus and cerebellum. The C-terminal region of human BHC80 containing PHD zinc-finger domain was capable of binding directly to each of five other components of BHC, and of organizing BHC mediating transcriptional repression. Moreover, two isoforms of human BHC80 were distinguished from each other by reduced binding to HDAC1 and HDAC2, despite the presence of the PHD finger domain. These results suggest that BHC80 presumably serves as a scaffold protein in BHC in neuronal as well as non-neuronal cells. A possible role of BHC80 in spermatogenesis is also suggested.


Subject(s)
Gene Expression Regulation/physiology , Histone Deacetylases/metabolism , Neurons/metabolism , Proto-Oncogene Proteins c-raf/metabolism , Alternative Splicing/physiology , Animals , Brain/metabolism , Histone Deacetylases/genetics , Humans , Male , Mice , Protein Isoforms , Proto-Oncogene Proteins c-raf/genetics , Spermatogenesis/physiology , Testis/metabolism
13.
Science ; 298(5600): 1999-2002, 2002 Dec 06.
Article in English | MEDLINE | ID: mdl-12471261

ABSTRACT

Spermatogenesis is a highly specialized process of cellular differentiation to produce spermatozoa. This differentiation process accompanies morphological changes that are controlled by a number of genes expressed in a stage-specific manner during spermatogenesis. Here we show that in mice, the absence of a testis-specific, cytoplasmic polyadenylate [poly(A)] polymerase, TPAP, results in the arrest of spermiogenesis. TPAP-deficient mice display impaired expression of haploid-specific genes that are required for the morphogenesis of germ cells. The TPAP deficiency also causes incomplete elongation of poly(A) tails of particular transcription factor messenger RNAs. Although the overall cellular level of the transcription factor TAF10 is unaffected, TAF10 is insufficiently transported into the nucleus of germ cells. We propose that TPAP governs germ cell morphogenesis by modulating specific transcription factors at posttranscriptional and posttranslational levels.


Subject(s)
Polynucleotide Adenylyltransferase/metabolism , RNA, Messenger/metabolism , Spermatogenesis , Spermatozoa/physiology , Testis/enzymology , Animals , Apoptosis , Cell Cycle Proteins , Cytoplasm/enzymology , DNA-Binding Proteins , Female , Gene Expression Regulation, Developmental , Gene Targeting , In Situ Nick-End Labeling , Male , Mice , Mice, Inbred C57BL , Mutation , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Organ Size , Poly A/metabolism , Polynucleotide Adenylyltransferase/genetics , Protein Biosynthesis , Spermatids/physiology , Spermatocytes/physiology , Testis/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, Genetic , mRNA Cleavage and Polyadenylation Factors/genetics , mRNA Cleavage and Polyadenylation Factors/metabolism
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