Papers by Hiroshi Kinoshita
BarA of Streptomyces virginiae is a specific receptor protein for a member of butyrolactone autor... more BarA of Streptomyces virginiae is a specific receptor protein for a member of butyrolactone autoregulators which binds to an upstream region of target genes to control transcription, leading to the production of the antibiotic virginiamycin M 1 and S. BarA-binding DNA sequences (BarA-responsive elements [BAREs]), to which BarA binds for transcriptional control, were restricted to 26 to 29-nucleotide (nt) sequences on barA and barB upstream regions by the surface plasmon resonance technique, gel shift assay, and DNase I footprint analysis. Two BAREs (BARE-1 and BARE-2) on the barB upstream region were located 57 to 29 bp (BARE-1) and 268 to 241 bp (BARE-2) upstream from the barB translational start codon. The BARE located on the barA upstream region (BARE-3) was found 101 to 76 bp upstream of the barA start codon. High-resolution S1 nuclease mapping analysis revealed that BARE-1 covered the barB transcription start site and BARE-3 covered an autoregulator-dependent transcription start site of the barA gene. Deletion and mutation analysis of BARE-2 demonstrated that at least a 19-nt sequence was required for sufficient BarA binding, and A or T residues at the edge as well as internal conserved nucleotides were indispensable. The identified binding sequences for autoregulator receptor proteins were found to be highly conserved among Streptomyces species.
Molecular Microbiology, 2005
Gamma-butyrolactone signalling molecules are produced by many Streptomyces species, and several h... more Gamma-butyrolactone signalling molecules are produced by many Streptomyces species, and several have been shown to regulate antibiotic production. In Streptomyces coelicolor A3(2) at least one g g g g -butyrolactone (SCB1) has been shown to stimulate antibiotic production, and genes encoding proteins that are involved in its synthesis ( scbA ) and binding ( scbR ) have been characterized. Expression of these genes is autoregulated by a complex mechanism involving the g g g g -butyrolactone. In this study, additional genes influenced by ScbR were identified by DNA microarray analysis, and included a cryptic cluster of genes for a hypothetical type I polyketide. Further analysis of this gene cluster revealed that the pathway-specific regulatory gene, kasO , is a direct target for regulation by ScbR. Gel retardation and DNase I footprinting analyses identified two potential binding sites for ScbR, one at ----3 to ----35 nt and the other at ----222 to ----244 nt upstream of the kasO transcriptional start site. Addition of SCB1 eliminated the DNA binding activity of ScbR at both sites. The expression of kasO was growth phase regulated in the parent (maximal during transition phase), undetectable in a scbA null mutant, and constitutively expressed in a scbR null mutant. Addition of SCB1 to the scbA mutant restored the expression of kasO , indicating that ScbR represses kasO until transition phase, when presumably SCB1 accumulates in sufficient quantity to relieve kasO repression. Expression of the cryptic antibiotic gene cluster was undetectable in a kasO deletion mutant. This is the first report with comprehensive in vivo and in vitro data to show that a g g g gbutyrolactone-binding protein directly regulates a secondary metabolite pathway-specific regulatory gene in Streptomyces .
Journal of Natural Products, 2008
Analysis of fermentation broth of the entomopathogenic fungus Metarhizium anisopliae has led to i... more Analysis of fermentation broth of the entomopathogenic fungus Metarhizium anisopliae has led to isolation of aurovertin D (1) and three new aurovertin-type metabolites, aurovertin F (2), aurovertin G (3), and aurovertin H (4). Their structures were determined on the basis of spectroscopic analyses and chemical conversions.
Journal of Bioscience and Bioengineering, 2014
A total of 412 strains belonging to 14 genera of clavicipitoid entomopathogenic fungi (EPF) were ... more A total of 412 strains belonging to 14 genera of clavicipitoid entomopathogenic fungi (EPF) were screened for activities against two economically important plant pathogenic oomycetes, Phytophthora sojae and Aphanomyces cochlioides. To identify the antioomycete compounds produced by EPF, the extracts of 13 highly active EPF strains were characterized in detail by high performance liquid chromatography with diode array detection and high-resolution mass spectrometric detection and antioomycete assay. The antioomycete activity of several Metarhizium extracts was associated with previously isolated aurovertins, fungerin, N-(methyl-3-oxodec-6-enoyl)-2-pyrroline, and N-(methyl-3-oxodecanoyl)-2-pyrroline. The depsipeptide beauvericin was confirmed to be one of the active principles of three strains of Isaria tenuipes, which strongly inhibited mycelial growth of both P. sojae and A. cochlioides. Two known bioactive metabolites, paecilosetin and aranorosinol A, together with a novel and potent antioomycete compound, farinomalein, were isolated from the extracts of Isaria farinosa and all compounds were confirmed to have antioomycete activity. Identification of 8 antioomycete compounds from 13 clavicipitioid EPF demonstrated a new potential use of EPF as a source of compounds for the control of soil-borne plant pathogenic oomycetes.
Journal of Bioscience and Bioengineering, 2008
Entomopathogenic fungi are unique owing to their versatile ability to produce many bioactive comp... more Entomopathogenic fungi are unique owing to their versatile ability to produce many bioactive compounds and from the dependence of their morphological differentiation on the presence of insect-derived materials. An entomopathogenic fungus, Metarhizium anisopliae HF293, was found to show insect-material-dependent production of antibacterial compounds, which were purified to homogeneity from 10-d culture broth when the production reached maximum. Two compounds were isolated: the major compound was determined to be helvolic acid and the minor one was a novel derivative of helvolic acid (1,2-dihydrohelvolic acid). Discovery of a novel bioactive compound indicated that insect-derived material would be a useful factor for enhancing the diversity of compounds produced by entomopathogenic fungi.
Journal of Bacteriology, 2002
The visA gene of Streptomyces virginiae has been thought to be a part of the virginiamycin S (VS)... more The visA gene of Streptomyces virginiae has been thought to be a part of the virginiamycin S (VS) biosynthetic gene cluster based on its location in the middle of genes that encode enzymes highly similar to those participating in the biosynthesis of streptogramin-type antibiotics. Heterologous expression of the visA gene was achieved in Escherichia coli by an N-terminal fusion with thioredoxin (TrxA), and the intact recombinant VisA protein (rVisA) was purified after cleavage with enterokinase to remove the TrxA moiety. The purified rVisA showed clear L-lysine 2-aminotransferase activity with an optimum pH of around 8.0 and an optimum temperature at 35°C, with 2-oxohexanoate as the best amino acceptor, indicating that VisA converts L-lysine into ⌬ 1 -piperidine 2-carboxylic acid. A visA deletion mutant of S. virginiae was created by homologous recombination, and the in vivo function of the visA gene was studied by phenotypic comparison between the wild type and the visA deletion mutant. No differences in growth in liquid media or in morphological behavior on solid media were observed, indicating that visA is not involved in primary metabolism or morphological differentiation. However, the visA mutant failed to produce VS while maintaining the production of virginiamycin M 1 at a level comparable to that of the parental wild-type strain, demonstrating that visA is essential to VS biosynthesis. These results, together with the observed recovery of the defect in VS production by the external addition of 3-hydroxypicolinic acid (3-HPA), a starter molecule in VS biosynthesis, suggest that VisA is the first enzyme of the VS biosynthetic pathway and that it supplies 3-HPA from L-lysine.
Journal of Bacteriology, 2001
A gene designated varR (for virginiae antibiotic resistance regulator) was identified in Streptom... more A gene designated varR (for virginiae antibiotic resistance regulator) was identified in Streptomyces virginiae 89 bp downstream of a varS gene encoding a virginiamycin S (VS)-specific transporter. The deduced varR product showed high homology to repressors of the TetR family with a conserved helix-turn-helix DNA binding motif. Purified recombinant VarR protein was present as a dimer in vitro and showed clear DNA binding activity toward the varS promoter region. This binding was abolished by the presence of VS, suggesting that VarR regulates transcription of varS in a VS-dependent manner. Northern blot analysis revealed that varR was cotranscribed with upstream varS as a 2.4-kb transcript and that VS acted as an inducer of bicistronic transcription. Deletion analysis of the varS promoter region clarified two adjacent VarR binding sites in the varS promoter.
Journal of Bacteriology, 2004
␥-Butyrolactone autoregulator receptors of the genus Streptomyces have a common activity as DNA-b... more ␥-Butyrolactone autoregulator receptors of the genus Streptomyces have a common activity as DNA-binding transcriptional repressors, controlling secondary metabolism and/or morphological differentiation. A gene encoding a ␥-butyrolactone autoregulator receptor was cloned from a bafilomycin B 1 producer, Kitasatospora setae, for the first time from a non-Streptomyces genus of actinomycetes, and its function was evaluated by in vitro and in vivo analyses. The gene fragment was initially cloned by PCR with primers designed from two highly conserved regions of Streptomyces autoregulator receptors (BarA, FarA, ScbR, and ArpA), followed by genomic Southern hybridization yielding a 7-kb BamHI fragment on which a 654-bp receptor gene (ksbA) was identified. The recombinant KsbA protein demonstrated clear binding activity toward 3 H-labeled autoregulators, especially toward [ 3 H]SCB1, confirming that ksbA encodes a real autoregulator receptor of K. setae. To clarify the in vivo function of ksbA, a ksbA-disrupted strain was constructed by means of homologous recombination after introducing a ksbA disruption construct via transconjugation from Escherichia coli. No difference in morphology was found between the wild-type strain and the ksbA disruptants. However, the ksbA disruptants started producing bafilomycin 18 h earlier than the wild-type strain and showed a 2.4-fold-higher accumulation of bafilomycin. The phenotype was restored to the original wild-type phenotype by complementation with intact ksbA, indicating that the autoregulator receptor protein of K. setae acts as a primary negative regulator of the biosynthesis of bafilomycin but plays no role in cytodifferentiation of K. setae. This indicates that, unlike the A-factor receptor of Streptomyces griseus, the autoregulator receptor (ksbA) of K. setae belongs to a family of autoregulator receptors which control secondary metabolism but play no role in morphological differentiation.
The Journal of Antibiotics, 2014
Verlamelin and its new derivative (verlamelin B) were isolated from fermentation broth of entomop... more Verlamelin and its new derivative (verlamelin B) were isolated from fermentation broth of entomopathogenic fungus Lecanicillium sp. HF627. As the structural elucidation of verlamelin so far was only preliminary, we studied and determined the absolute structure of these two compounds to be cyclo(5S-hydroxytetradecanoic acid-D-alloThr/Ser-D-Ala-L-Pro-L-Gln-D-Tyr-L-Val). This is the first study that precisely analyzed the structure of verlamelin.
The Journal of Antibiotics, 2011
Gene, 2002
Streptomyces virginiae produces g-butyrolactone autoregulators (virginiae butanolide, VB), which ... more Streptomyces virginiae produces g-butyrolactone autoregulators (virginiae butanolide, VB), which control the biosynthesis of virginiamycin M 1 and S. A 6.3-kb region downstream of the virginiamycin S (VS)-resistance operon in S. virginiae was sequenced, and four plausible open reading frames (ORFs) (visA, 1,260 bp; visB, 1,656 bp; visC, 888 bp; visD, 1209 bp) were identified. Homology analysis revealed significant similarities with enzymes involved in the biosynthesis of cyclopeptolide antibiotics: VisA (53% identity, 65% similarity) to l-lysine 2-aminotransferase (NikC) of nikkomycin D biosynthesis, VisB (66% identity, 72% similarity) to 3-hydroxypicolinic acid:AMP ligase of pristinamycin I biosynthesis, VisC (48% identity, 59% similarity) to lysine cyclodeaminase of ascomycin biosynthesis, and VisD (43% identity, 56% similarity) to erythromycin C-22 hydroxylase of erythromycin biosynthesis. Northern blotting as well as high-resolution S1 analysis of the ORFs revealed that they were transcribed as two bicistronic transcripts, namely 3.0-kb visB-visA and another 2.7-kb visC-visD transcript, with promoters locating upstream of visB and visC, respectively. Transcription of the two operons was observed only 1 h after the VB production, which was 2 h before the virginiamycin production. Furthermore, prompt induction of the transcription was observed as a result of external VB addition, suggesting that the expression of the two operons was under the control of VB. q
Biotechnology Letters, 2006
To enhance the variety of genetic tools and thus to promote molecular genetic study, aureobasidin... more To enhance the variety of genetic tools and thus to promote molecular genetic study, aureobasidin A and its resistance gene were adopted as a new marker system together with the incorporation of the Gateway system to facilitate the introduction of long heterologous DNA fragments into Monascus purpureus. The minimum inhibitory concentration of aureobasidin A against Monascus was 0.05 microg/ml and a transformation efficiency of 17 colonies/microg DNA was obtained by the protoplast-PEG method with the vector pAUR316, containing the aureobasidin A resistance gene. Southern analysis of the transformants confirmed that pAUR316 exists as an independent vector, demonstrating that the AMA1 sequence acts as the autonomous replication sequence in M. purpureus. Through the use of the Gateway system, a polyketide synthase gene (7.8 kbp) responsible for citrinin biosynthesis was introduced. As a result, the transformants showed 1.5-fold higher production of citrinin than the wild-type strain.
Archives of Microbiology, 2004
An effective transformation procedure for Kitasatospora setae was established based on transconju... more An effective transformation procedure for Kitasatospora setae was established based on transconjugation from Escherichia coli ET12567 (pUZ8002) using a phi C31-derived integration vector, pSET152, containing oriT and attP fragments. While no transconjugation was observed under the standard transconjugation conditions for Streptomyces species, sufficient transconjugation (>1 x 10(-6)) was achieved on ISP4 medium containing 30 mM MgCl(2) using a 25- to 125-fold excess of E. coli donor cells. In addition, the sequence and location of the chromosomal integration site attB of K. setae was identified for the first time in genera of non- Streptomyces actinomycetes. K. setae contains a single phi C31 attB site. Similar to the case of Streptomyces species, the attB site of K. setae is present within an ORF encoding a pirin-homolog, but the K. setae-attB sequence deviates slightly from the consensus sequence of Streptomyces attB sequences.
Archives of Microbiology, 2008
Virginiae butanolide (VB) is a member of the gamma-butyrolactone autoregulators and triggers the ... more Virginiae butanolide (VB) is a member of the gamma-butyrolactone autoregulators and triggers the production of streptogramin antibiotics virginiamycin M1 and S in Streptomyces virginiae. A VB biosynthetic gene (barS2) was localized in a 10-kb regulatory island which controls the virginiamycin biosynthesis/resistance of S. virginiae, and analyzed by gene disruption/complementation. The barS2 gene is flanked by barS1, another VB biosynthetic gene catalyzing stereospecific reduction of an A-factor-type precursor into a VB-type compound, and barX encoding a pleiotropic regulator for virginiamycin biosynthesis. The deduced product of barS2 possessed moderate similarity to a putative dehydrogenase of Streptomyces venezuelae, encoded by jadW2 located in similar gene arrangement to that in the regulatory island of S. virginiae. A barS2-disruptant (strain IC152), created by means of homologous recombination, showed no differences in growth in liquid medium or morphology on solid medium compared to a wild-type strain, suggesting that BarS2 does not play any role in primary metabolism or morphological differentiation of S. virginiae. In contrast, no initiation of virginiamycin production or VB production was detected with the strain IC152 until 18 h of cultivation, at which time full production of virginiamycin occurs in the wild-type strain. The delayed virginiamycin production of the strain IC152 was fully restored to the level of the wild-type strain either by the exogenous addition of VB or by complementation of the intact barS2 gene, indicating that the lack of VB production at the initiation phase of virginiamycin production is the sole reason for the defect of virginiamycin production, and the barS2 gene is of primary importance for VB biosynthesis in S. virginiae.
Applied Microbiology and Biotechnology, 2010
Based on comparative amino-acid sequence alignment of geranylgeranyl diphosphate (GGPP) synthase ... more Based on comparative amino-acid sequence alignment of geranylgeranyl diphosphate (GGPP) synthase from filamentous fungi, degenerated oligonucleotide primers were designed for searching GGPP synthase gene (s) in entomopathogenic fungi. Polymerase chain reaction with the designed primers amplified GGPP synthase homologues from five representative entomopathogenic fungi: Metarhizium anisopliae, Beauveria bassiana, Verticillium lecanii, Paecilomyces farinosus, and Nomuraea rileyi. Sequence comparison of the amplified of GGPP synthase homologue fragments revealed that M. anisopliae and B. bassiana have at least two different types of the GGPP synthase gene homologues. The first type (designated as ggs1), which is highly conserved among the five strains, has a unique Ser-rich region, SSXSSVSGSSS (X refers to L, A, V, or S), and is constitutively expressed throughout growth. In contrast, the second type of GGPP synthase gene homologue (ggs2) was discovered only in some strains, and genes of this type possessed high similarity to each other but showed relatively weak similarity to the ggs1 genes, with no detectable transcription under the cultivation conditions applied in this experiment. The ggs1 cloned from M. anisopliae, which encoded a putative protein of 359 amino acid residues, was heterologously expressed in E. coli. The recombinant protein showed activity to synthesize GGPP from farnesyl diphosphate and isopentenyl diphosphate. These results strongly suggested that the ggs1 gene encodes a GGPP synthase involved in primary metabolism.
Applied Microbiology and Biotechnology, 2010
A gene (ggs2) having high similarity to the geranylgeranyl diphosphate synthase (GGPP synthase) g... more A gene (ggs2) having high similarity to the geranylgeranyl diphosphate synthase (GGPP synthase) gene was cloned from Metarhizium anisopliae NAFF635007. The ggs2 gene (1,239-bp open reading frame with no intron) encoded a protein of 412 amino acids, and the transcription occurred only after late log-phase during the growth. Gene disruption of ggs2, performed to clarify the function in M. anisopliae, resulted in decreased GGPP synthase activity together with a slight delay of sporulation. An high performance liquid chromatography (HPLC) comparison of compound profiles between the wild-type strain and the disruptant revealed that a compound was abolished by the ggs2 disruption. Purification and structural elucidation by 1 H-NMR and mass spectrometry analyses revealed that the lost compound is helvolic acid. Furthermore, the pathogenicity assay against two species of insect larvae revealed that the ggs2-disruptant possessed much weaker toxicity than the wild-type strain. Based on these results, it was concluded that ggs2 encodes the GGPP synthase influencing the biosynthesis of secondary metabolites in various species, including helvolic acid in M. anisopliae. To the best of our knowledge, this is the first report to identify a GGPP synthase gene related to secondary metabolism in entomopathogenic fungi.
Applied and Environmental Microbiology, 2005
Citrinin produced by Aspergillus, Penicillium, and Monascus species is a polyketide compound that... more Citrinin produced by Aspergillus, Penicillium, and Monascus species is a polyketide compound that has nephrotoxic activity in mammals and is bactericidal toward gram-positive bacteria. To avoid the risk of citrinin contamination in other fermentation products produced by Monascus purpureus, knowledge of the citrinin biosynthetic genes is needed so that citrinin-nonproducing strains can be generated. We cloned a polyketide synthase (PKS) gene from M. purpureus with degenerate primers designed to amplify the conserved region of a ketosynthase domain of a fungal PKS. A 13-kb genomic DNA fragment was identified that contained a full-length PKS gene (pksCT) of 7,838 bp with a single 56-bp intron. pksCT encodes a 2,593-amino-acid protein that contains putative domains for ketosynthase, acyltransferase, acyl carrier protein (ACP), and a rare methyltransferase. There was no obvious thioesterase domain, which usually is downstream of the ACP domain in multi-aromatic-ring PKSs. pksCT transcription was correlated with citrinin production, suggesting that the pksCT gene product was involved in citrinin biosynthesis. Homologous recombination between the wild-type allele and a truncated disruption construct resulted in a pksCT-disrupted strain of M. purpureus. The disruptant did not produce citrinin, but a pksCT revertant generated by successive endogenous recombination events in the pksCT disruptant restored citrinin production, indicating that pksCT encoded the PKS responsible for citrinin biosynthesis in M. purpureus.
The Journal of Antibiotics, 2014
As a result of the continued screening for new metabolites produced by endophytic fungi from Thai... more As a result of the continued screening for new metabolites produced by endophytic fungi from Thai medicinal plants, two new triene fatty acid amides, bipolamides A (1) and B (2), were discovered from the endophytic fungus Bipolaris sp. MU34. The structures of all of the isolated compounds were elucidated on the basis of the spectroscopic data of NMR and MS. An antimicrobial assay revealed that bipolamide B (2) had moderate antifungal activity against Cladosporium cladosporioides FERMS-9, Cladosporium cucumerinum NBRC 6370, Saccharomyces cerevisiae ATCC 9804, Aspergillus niger ATCC 6275 and Rhisopus oryzae ATCC 10404, with Minimum inhibitory concentration (MIC) values of 16, 32, 32, 64 and 64 μg ml(-1), respectively.
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Papers by Hiroshi Kinoshita