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Teruhiko Beppu

Publications and source records attributed to Teruhiko Beppu.

At least 19 recordsLinked to original sources
PubMed2006

Effective cellulose production by a coculture of Gluconacetobacter xylinus and Lactobacillus mali.

A microbial colony that contained a marked amount of cellulose was isolated from vineyard soil. The colony was formed by the associated growth of two bacterial strains: a cellulose-producing acetic acid bacterium (st-60-12) and a lactic acid bacterium (st-20). The 16S rDNA-based taxonomy indicated that st-60-12 belonged to Gluconacetobacter xylinus and st-20 was closely related to Lactobacillus mali. Cocultivation of the two organisms in corn steep liquor/sucrose liquid medium resulted in a threefold higher cellulose yield when compared to the st-60-12 monoculture. A similar enhancement was observed in a coculture with various L. mali strains but not with other Lactobacillus spp. The enhancement of cellulose production was most remarkable when sucrose was supplied as the substrate. L. mali mutants for exocellular polysaccharide (EPS) production were defective in promoting cellulose production, but the addition of EPS to the monoculture of st-60-12 did not affect cellulose productivity. Scanning electron microscopic observation of the coculture revealed frequent association between the st-60-12 and L. mali cells. These results indicate that cell-cell interaction assisted by the EPS-producing L. mali promotes cellulose production in st-60-12.

Cellulose↗
PubMed2006

The CarA/LitR-family transcriptional regulator: Its possible role as a photosensor and wide distribution in non-phototrophic bacteria.

LitR is a MerR-family regulator that controls light-induced carotenogenesis in Streptomyces coelicolor. The introduction of litR conferred photo-dependent transcription activities on Streptomyces griseus, which does not perform photo-responsive carotenogenesis. LitR discovers homologs encoded at illumination-related loci of various non-phototrophic bacterial genomes. Probably, LitR acts not only as a transcriptional regulator but also a photosensor.

Bacterial Proteins↗
PubMed2006

Proteins encoded by the conservon of Streptomyces coelicolor A3(2) comprise a membrane-associated heterocomplex that resembles eukaryotic G protein-coupled regulatory system.

Streptomyces coelicolor A3(2) retains unique conserved operons termed conservons. Here, one of the conservons (cvn9), which encodes five proteins (A9-E9), was characterized. Mutants for cvnA9 and cvnAlO conditionally overproduced actinorhodin and performed precocious aerial growth, while a cvnE9 mutant showed the parental phenotype. Transcription of bidG, adpA and bldN was upregulated in the cvnA9 mutant. A9-D9 were detected in the insoluble fraction of cell-free extract of S. coelicolor by Western analysis. Biochemical analyses revealed that A9 has ATP-hydrolysing and adenine nucleotide-binding activities; D9 has GTP-hydrolysing and guanine nucleotide-binding activities; and E9 shows a typical spectrum similar to cytochrome P450. The comprehensive interaction assays demonstrated the occurrence of specific interactions between A9 and B9, A9 and C9, B9 and B9, B9 and D9, and C9 and D9. A9 associated with and dissociated from B9 (and C9) when ATP and ATP-gamma-S were supplied in the reaction respectively. Similarly, D9 associated with and dissociated from B9 (and C9) when GTP and GTP-gamma-S were supplied respectively. A9 and B9 were also shown for the occurrence as homocomplexes. Probably, Cvn9 proteins comprise a membrane-associated heterocomplex resembling the eukaryotic G-protein-coupled receptor system, which may serve as a signal transducer that connects to the bld cascade.

Adenosine Triphosphate↗
PubMed2006

Isolation and characterization of a new Clostridium sp. that performs effective cellulosic waste digestion in a thermophilic methanogenic bioreactor.

A methanogenic bioreactor that utilized wastepaper was developed and operated at 55 degrees C. Microbial community structure analysis showed the presence of a group of clostridia that specifically occurred during the period of high fermentation efficiency. To isolate the effective cellulose digester, the sludge that exhibited high fermentation efficiency was inoculated into a synthetic medium that contained cellulose powder as the sole carbon source and was successively cultivated. A comprehensive 16S rRNA gene sequencing study revealed that the enriched culture contained various clostridia that had diverse phylogenetic positions. The microorganisms were further enriched by successive cultivation with filter paper as the substrate, as well as the bait carrier. A resultant isolate, strain EBR45 (= Clostridium sp. strain NBRC101661), was a new member of the order Clostridiales phylogenetically and physiologically related to Clostridium thermocellum and Clostridium straminisolvens. Specific PCR-based monitoring demonstrated that strain EBR45 specifically occurred during the high fermentation efficiency period in the original methanogenic sludge. Strain EBR45 effectively digested office paper in its pure cultivation system with a synthetic medium.

Bioreactors↗
PubMed2006

CO2 supply induces the growth of Symbiobacterium thermophilum, a syntrophic bacterium.

Symbiobacterium thermophilum is a unique syntrophic bacterium that exhibits marked growth only in coculture with a cognate Bacillus sp. In this study, we found that the bacterium is capable of marked mono-growth when supplied with CO2 or bicarbonate. The evidence suggests that the genetic defect for carbonic anhydrase in this bacterium is a reason for the syntrophic property based on CO2 requirement.

Actinobacteria↗
PubMed2005

Genetic control for light-induced carotenoid production in non-phototrophic bacteria.

Carotenoids are naturally occurring yellow or orange pigments that serve as a protectant against photo-oxidative damages. Among the wide variety of producers, the prokaryotes generate a broad spectrum of carotenoids with diverse chemical structures that are expected to have a high potential in biotechnological applications. Bacterial carotenogenesis occurs in a constitutive or light-induced manner, which suggests the diversity of the regulatory mechanism. The mechanism for light-induced carotenoid production in non-phototrophic bacteria has been studied in detail in Myxococcus xanthus, a Gram-negative gliding bacterium. The complicated mechanism involves the activation of an extracytoplasmic function (ECF) sigma factor (CarQ), which leads to the sequestration of a MerR family transcriptional regulator (CarA) that represses the expression of the carotenoid biosynthesis genes in the dark. Recently, we identified another regulatory mechanism for light-induced carotenogenesis in Streptomyces coelicolor A3(2), a Gram-positive soil bacterium. In this organism, the transcription of the carotenoid biosynthesis gene cluster is specified by LitS, a photo-inducible ECF sigma factor. The evidence indicates that the photo-dependent transcription of litS is mediated by LitR, a MerR family transcriptional regulator. In addition, it is suggested that the conformational alteration of LitR upon receiving the illumination signal determines its binding to DNA. The carboxy-terminal domain of LitR contains a possible binding site for Vitamin B12, which may serve as a capturing apparatus for the illumination signal.

Bacterial Proteins↗
PubMed2005

A putative dual-specific protein phosphatase encoded by YVH1 controls growth, filamentation and virulence in Candida albicans.

In response to stimulants, such as serum, the yeast cells of the opportunistic fungal pathogen Candida albicans form germ tubes, which develop into hyphae. Yvh1p, one of the 29 protein phosphatases encoded in the C. albicans genome, has 45% identity with the dual-specific phosphatase Yvh1p of the model yeast Saccharomyces cerevisiae. In this study, Yvh1p expression was not observed during the initial step of germ tube formation, although Yvh1p was expressed constitutively in cell cycle progression of yeast or hyphal cells. In an attempt to analyse the function of Yvh1p phosphatase, the complete ORFs of both alleles were deleted by replacement with hph200-URA3-hph200 and ARG4. Although YVH1 has nine single-nucleotide polymorphisms in its coding sequence, both YVH1 alleles were able to complement the YVH1 gene disruptant. The vegetative growth of Deltayvh1 was significantly slower than the wild-type. The hyphal growth of Deltayvh1 on agar, or in a liquid medium, was also slower than the wild-type because of the delay in nuclear division and septum formation, although germ tube formation was similar between the wild-type and the disruptant. Despite the slow hyphal growth, the expression of several hypha-specific genes in Deltayvh1 was not delayed or repressed compared with that of the wild-type. Infection studies using mouse models revealed that the virulence of Deltayvh1 was less than that of the wild-type. Thus, YVH1 contributes to normal vegetative yeast or hyphal cell cycle progression and pathogenicity, but not to germ tube formation.

Candida albicans↗
PubMed2005

Desferrioxamine E produced by Streptomyces griseus stimulates growth and development of Streptomyces tanashiensis.

The authors previously reported that interspecific stimulatory events between Streptomyces species for antibiotic production and/or morphological differentiation mediated by putative diffusible metabolites take place at a high frequency. This paper reports the isolation and characterization of a substance produced by Streptomyces griseus that stimulates the growth and development of Streptomyces tanashiensis. The substance was purified from the culture supernatant of S. griseus by using anion-exchange chromatography, gel filtration chromatography and reverse-phase HPLC. FAB-MS and NMR analyses of the purified preparation indicated the substance to be desferrioxamine E (synonym: nocardamine), a siderophore that is widely produced by Streptomyces species and related organisms. Similar stimulatory effects on the growth and development of S. tanashiensis were exerted by desferrioxamine E produced by another actinomycete strain, but not by other siderophores tested, including ferrichrome and nocobactin and free ferric ion. An exogenous supply of desferrioxamine E stimulated secondary metabolite formation and/or morphological differentiation in various actinomycete strains. Disruption of the desferrioxamine biosynthesis gene cluster in Streptomyces coelicolor A3(2) abolished the production of desferrioxamine E and the activity to stimulate the growth and differentiation of S. tanashiensis. The S. coelicolor mutant showed impaired growth and development on Bennett's/glucose agar medium, but it was rescued by the exogenous supply of desferrioxamine E. These results indicate that desferrioxamines play an important role in streptomycete physiology. Similar to several pathogenic bacteria and fungi, S. tanashiensis may be defective in the production of siderophores; however, it can utilize the siderophores excreted by other organisms.

Chromatography, High Pressure Liquid↗
PubMed2005

Dual transcriptional control of amfTSBA, which regulates the onset of cellular differentiation in Streptomyces griseus.

The amf gene cluster encodes a probable secretion system for a peptidic morphogen, AmfS, which induces aerial mycelium formation in Streptomyces griseus. Here we examined the transcriptional control mechanism for the promoter preceding amfT (PamfT) directing the transcription of the amfTSBA operon. High-resolution S1 analysis mapped a transcriptional start point at 31 nucleotides upstream of the translational start codon of amfT. Low-resolution analysis showed that PamfT is developmentally regulated in the wild type and completely abolished in an amfR mutant. The -35 region of PamfT contained the consensus sequence for the binding of BldD, a pleiotropic negative regulator for morphological and physiological development in Streptomyces coelicolor A3(2). The cloned bldD locus of S. griseus showed high sequence similarity to the S. coelicolor counterpart. Transcription of bldD occurred constitutively in both the wild type and an A-factor-deficient mutant of S. griseus, which suggests that the regulatory role of BldD is independent of A-factor. The gel retardation assay revealed that purified BldD and AmfR recombinant proteins specifically bind PamfT. Overproduction of BldD in the wild-type cell conferred a bald phenotype (defective in aerial growth and streptomycin production) and caused marked repression of PamfT activity. An amfT-depleted mutant also showed a bald phenotype but PamfT activity was not affected. Both the bldD-overproducing wild-type strain and the amfT mutant were unable to induce aerial growth of an amfS mutant in a cross-feeding assay, which indicates that these strains are defective in the production of an active AmfS peptide. The results overall suggests that two independent regulators, AmfR and BldD, control PamfT activity via direct binding to determine the transcriptional level of the amf operon responsible for the production and secretion of AmfS peptide, which induces the erection of aerial hyphae in S. griseus.

Bacterial Proteins↗
PubMed2005

Light-induced carotenogenesis in Streptomyces coelicolor A3(2): identification of an extracytoplasmic function sigma factor that directs photodependent transcription of the carotenoid biosynthesis gene cluster.

Carotenoids are produced by a variety of organisms, but the mechanisms that regulate gene expression leading to carotenoid biosynthesis have been characterized for only a few organisms. In this study, we found that Streptomyces coelicolor A3(2), a gram-positive filamentous bacterium, produces carotenoids under blue light induction. The carotenoid fraction isolated from the cell extract contained multiple compounds, including isorenieratene and beta-carotene. The carotenoid biosynthesis gene cluster of S. coelicolor consists of two convergent operons, crtEIBV and crtYTU, as previously shown for Streptomyces griseus. The crtEIBV null mutant completely lost its ability to produce carotenoids. The crt gene cluster is flanked by a regulatory region that consists of two divergent operons, litRQ and litSAB. The lit (light-induced transcription) genes encode a MerR-type transcriptional regulator (LitR), a possible oxidoreductase (LitQ), an extracytoplasmic function sigma factor (sigmaLitS), a putative lipoprotein (LitA), and a putative anti-sigma factor (LitB). S1 protection assay revealed that the promoters preceding crtE (PcrtE), crtY (PcrtY), litR (PlitR), and litS (PlitS) are activated upon illumination. A litS mutant lost both the ability to produce carotenoids and the activities of PcrtE, PcrtY, and PlitS, which suggested that sigmaLitS directs light-induced transcription from these promoters. An RNA polymerase holocomplex containing purified sigmaLitS recombinant protein generated specific PcrtE and PcrtY transcripts in an in vitro runoff transcriptional assay. A litR mutant that had an insertion of the kanamycin resistance gene was defective both in the ability to produce carotenoids and in all of the light-dependent promoter activities. Overexpression of litS resulted in constitutive carotenoid production in both the wild type and the litR mutant. These results indicate that sigmaLitS acts as a light-induced sigma factor that directs transcription of the crt biosynthesis gene cluster, whose activity is controlled by an unknown LitR function. This is the first report to describe light-inducible gene expression in Streptomyces.

Amino Acid Sequence↗
PubMed2004

Genome sequence of Symbiobacterium thermophilum, an uncultivable bacterium that depends on microbial commensalism.

Symbiobacterium thermophilum is an uncultivable bacterium isolated from compost that depends on microbial commensalism. The 16S ribosomal DNA-based phylogeny suggests that this bacterium belongs to an unknown taxon in the Gram-positive bacterial cluster. Here, we describe the 3.57 Mb genome sequence of S.thermophilum. The genome consists of 3338 protein-coding sequences, out of which 2082 have functional assignments. Despite the high G + C content (68.7%), the genome is closest to that of Firmicutes, a phylum consisting of low G + C Gram-positive bacteria. This provides evidence for the presence of an undefined category in the Gram-positive bacterial group. The presence of both spo and related genes and microscopic observation indicate that S.thermophilum is the first high G + C organism that forms endospores. The S.thermophilum genome is also characterized by the widespread insertion of class C group II introns, which are oriented in the same direction as chromosomal replication. The genome has many membrane transporters, a number of which are involved in the uptake of peptides and amino acids. The genes involved in primary metabolism are largely identified, except those that code several biosynthetic enzymes and carbonic anhydrase. The organism also has a variety of respiratory systems including Nap nitrate reductase, which has been found only in Gram-negative bacteria. Overall, these features suggest that S.thermophilum is adaptable to and thus lives in various environments, such that its growth requirement could be a substance or a physiological condition that is generally available in the natural environment rather than a highly specific substance that is present only in a limited niche. The genomic information from S.thermophilum offers new insights into microbial diversity and evolutionary sciences, and provides a framework for characterizing the molecular basis underlying microbial commensalism.

Actinobacteria↗
PubMed2004

Accumulation of allo-isocitric acid by a Penicillium strain.

The occurrence of L(S)-allo-isocitric acid in nature, as a new fungal product, was first found in the culture medium of Pen. purpurogenum Stoll var. rubri-sclerotium Thom. No. 1148. The acid was obtained as its gamma-lactone in a crystalline form. Some chemical and physical characters were described. The acid was the only one detectable acid accumulated by its fungus in the given condition, and the yield of the acid by weight exceeded about 70% of the consumed glucose.

Fermentation↗
PubMed2003

Involvement of sigma(H) and related sigma factors in glucose-dependent initiation of morphological and physiological development of Streptomyces griseus.

We cloned and characterized sigH encoding a stress-response sigma factor, sigma(H), of Streptomyces griseus. Nucleotide sequencing of the sigH gene cluster revealed an identical gene organization as in the orthologous region of Streptomyces coelicolor A3(2). Transcriptional analysis by S1 nuclease mapping showed the presence of three tandem promoters (P1-P3) that direct transcription of sigH. The activity of P1 was markedly reduced in a sigH-depleted mutant, suggesting its dependence on sigma(H). P1 was induced by addition of 0.7 M NaCl, and P2 was induced by heat shock at 45 degrees C or addition of 4% ethanol. The sigH mutant of S. griseus showed conditional defect in aerial mycelium formation and streptomycin production depending on high concentration of glucose (>2%). Meanwhile, the wild-type strain of S. griseus introduced with rshA encoding a probable anti-sigma factor for sigma(H) on a high-copy-number plasmid was unable to perform development on media containing 1% glucose while it showed wild-type phenotype on media containing 1% maltose. RshA inhibited the sigma(H)-dependent in vitro run-off transcription at P1, which confirmed its role as a negative regulator for sigma(H). Analysis of RshA-sigma interaction by an Escherichia coli two-hybrid system showed specific interaction of RshA with sigma(H) and two related sigma factors, sigma(L) and sigma(F). We speculate that the high copy number of rshA represses morphological and physiological development of S. griseus through simultaneous inactivation of sigma(H) and related stress-response sigma factors, which play an essential role in the onset of cellular differentiation and antibiotic production on glucose media.

Amino Acid Sequence↗
PubMed2003

Cloning of the conserved regulatory operon by its aerial mycelium-inducing activity in an amfR mutant of Streptomyces griseus.

We report cloning and characterization of a 2.8 kb DNA fragment that suppressed the aerial mycelium-deficient phenotype of an amfR mutant of Streptomyces griseus when it was introduced on a high-copy-number plasmid. Nucleotide sequencing revealed that the cloned DNA fragment contained a part of a regulatory operon homologous to one of the conserved operons identified in the genome of Streptomyces coelicolor A3(2). The operon appeared to consist of 5 CDSs (rarA-E; restoration of aerial mycelium formation in an amfR mutant): rarA encoded a membrane protein with weak similarity to the histidine kinase of the two-component regulatory system; rarB and rarC products did not show marked similarity to other proteins with known function; rarD encoded a G-protein carrying two GTP-binding consensus sequences conserved in the eukaryotic Ras-like proteins; rarE product showed end-to-end homology to cytochrome P450. The 2.8 kb fragment contained a 5'-end incomplete rarA and complete rarB-D in the downstream from the promoter region of mel operon of the vector plasmid. Subcloning showed that the region containing rarA only is sufficient for the aerial mycelium-inducing activity. The truncation of rarA at its 5' terminus was essential for the restoration activity, which implied that the mutated rarA product causes unusual signaling that directs the onset of morphogenesis without amfR function. Inactivation of both rarA in Streptomyces griseus and cvnD9, a rarD ortholog in S. coelicolor resulted in precocious and glucose-resistant formation of aerial mycelium and secondary metabolites, which suggested that the operon negatively regulates the onset of differentiation. S1 nuclease protection analysis showed that the transcriptional activity of the promoter preceding rarA is developmentally regulated in an amfR- and glucose-dependent manner.

Amino Acid Sequence↗
PubMed2003

Characterization of a functional bacterial homologue of sodium-dependent neurotransmitter transporters.

The tnaT gene of Symbiobacterium thermophilum encodes a protein homologous to sodium-dependent neurotransmitter transporters. Expression of the tnaT gene product in Escherichia coli conferred the ability to accumulate tryptophan from the medium and the ability to grow on tryptophan as a sole source of carbon. Transport was Na(+)-dependent and highly selective. The K(m) for tryptophan was approximately 145 nm, and tryptophan transport was unchanged in the presence of 100 microM concentrations of other amino acids. Tryptamine and serotonin were weak inhibitors with K(I) values of 200 and 440 microM, respectively. By using a T7 promoter-based system, TnaT with an N-terminal His(6) tag was expressed at high levels in the membrane and was purified to near-homogeneity in high yield.

Actinobacteria↗
PubMed2003

Enzymological characterization of EpoA, a laccase-like phenol oxidase produced by Streptomyces griseus.

Laccase is an enzyme that catalyzes the oxidation of phenolic compounds by coupling the reduction of oxygen to water. While many laccases have been identified in plant and fungal species, enzymes of prokaryotic origin are poorly known. Here we report the enzymological characterization of EpoA, a laccase-like extracytoplasmic phenol oxidase produced by Streptomyces griseus. EpoA was expressed and purified with an Escherichia coli host-vector system as a recombinant protein fused with a C-terminal histidine-tag (rEpoA). Physicochemical analyses showed that rEpoA comprises a stable homotrimer containing all three types of copper (types 1-3). Various known laccase substrates were oxidized by rEpoA, while neither syringaldazine nor guaiacol served as substrates. Among the substrates examined, rEpoA most effectively oxidized N,N-dimethyl-p-phenylenediamine sulphate with a Km value of 0.42 mM. Several metal chelators caused marked inhibition of rEpoA activity, implying the presence of a metal center essential for the oxidase activity. The pH and temperature optima of rEpoA were 6.5 and 40 degrees C, respectively. The enzyme retained 40% activity after preincubation at 70 degrees C for 60 min. EpoA-like activities were detected in cell extracts of 8/40 environmental actinomycetes strains, which suggests that similar oxidases are widely distributed among this group of bacteria.

Amino Acid Sequence↗
PubMed2002

A novel extracytoplasmic phenol oxidase of Streptomyces: its possible involvement in the onset of morphogenesis.

Exogenous addition of copper stimulates cellular differentiation in Streptomyces spp. Several lines of evidence suggested a parallel correlation between the stimulatory effect of copper and phenol-oxidizing enzyme activities in Streptomyces griseus. Here a novel extracytoplasmic phenol oxidase (EpoA) associated with cellular development of this organism was identified and characterized. EpoA activity, examined by an in-gel stain procedure with N,N'-dimethyl-p-phenylenediamine sulfate as a substrate, was repressed by glucose and induced by copper supplied in the medium. The enzyme activity was abolished and markedly reduced in the mutants forA-factor biosynthesis and amfR, respectively, which suggested that the activity of the enzyme depends on those essential regulators for morphogenesis in S. griseus. EpoA protein was purified to homogeneity and the N-terminal amino acid sequence was determined. A homologous sequence identified in the genomic database of Streptomyces coelicolorA3(2) was used as a probe to clone the complete epoA gene of S. griseus. The deduced amino acid sequence of EpoA revealed that the mature protein with a molecular mass of 34 kDa was preceded by a signal peptide consisting of 34 aa, consistent with EpoA being a secreted enzyme. EpoA was predicted to be a laccase-type oxidase by not only the sequence similarity, but its substrate selectivity, oxidizing not tyrosine but dihydroxyphenylalanine (DOPA) to generate melanin pigment. Introduction of epoA on a plasmid partially restored both the EpoA activity and aerial mycelium productivity in an A-factor-deficient mutant. Exogenous supplementation of a substance synthesized by purified EpoA from DOPA stimulated cellular differentiation in S. griseus and several other species. Ultrafiltration indicated that the molecular mass of the putative stimulant synthesized by EpoA is between 500 and 1000 Da.

4-Butyrolactone↗