Crystallization and preliminary crystallographic studies of the butyrolactone autoregulator receptor protein (BarA) from Streptomyces virginiae.

The Streptomyces butyrolactone autoregulator receptor protein (BarA) is a DNA-binding protein that regulates the biosynthesis of the antibiotic virginiamycin. In this study, BarA from S. virginiae was overexpressed in Escherichia coli, purified and crystallized. Crystals of purified protein have been grown that diffracted to beyond 3.0 A resolution at 100 K using synchrotron radiation. The protein crystals belonged to the hexagonal space group P6(5)22, with unit-cell parameters a = b = 128.0, c = 286.2 A. With four molecules per asymmetric unit, the crystal volume per unit protein mass (V(M)) was 3.2 A(3) Da(-1) and the solvent content was 62%.

Isolation and characterization of bamA genes, homologues of the gamma-butyrolactone autoregulator-receptor gene in Amycolatopsis mediterranei, a rifamycin producer.

Four genes (bamA1, bamA2, bamA3 and bamA4) encoding homologues of the gamma-butyrolactone autoregulator receptor of Streptomyces were found and cloned from Amycolatopsis mediterranei, a typical non-Streptomyces actinomycetes and a producer of rifamycin, one of the major anti-tuberculosis drugs in clinical treatment. Transcriptional analysis demonstrated that bamA1 and bamA2 are transcribed in a growth-dependent manner, while bamA3 and bamA4 are constitutively transcribed during growth. Binding assays using (3)H-labeled autoregulator analogues as ligands confirmed that all of the recombinant BamA proteins expressed in Escherichia coli have clear binding activity toward several types of Streptomyces autoregulators. The ligand specificity of the recombinant BamA1 protein was identical to that of the crude cell-free lysates of A. mediterranei reported in our previous work. These results suggest that A. mediterranei, which is phylogenetically situated in a distal clade from the genus Streptomyces as non-Streptomyces actinomycetes, has an autoregulator-mediated signaling system.

In vivo functions of the gamma-butyrolactone autoregulator receptor in Streptomyces ambofaciens producing spiramycin.

A gene encoding a gamma-butyrolactone autoregulator receptor was cloned in to E. coli from Streptomyces ambofaciens producing spiramycin, a macrolide antibiotic used in both veterinary medicine and human medicine. A 714-bp intact receptor gene (saaR) was obtained by PCR and genomic Southern hybridization with the 100-bp PCR product as a probe. To clarify the in vivo function of saaR, a saaR-disrupted strain was constructed by means of homologous recombination, and phenotypes were compared with those of the wild-type strain. The number of saaR-disruptant spores was 4-fold less than that of the wild-type strain. In addition, saaR deletion from the S. ambofaciens chromosome resulted in complete loss of spiramycin production suggesting that saaR is a rare positive regulator, controlling both spiramycin biosynthesis and sporulation.

Methanolic extracts of Plocamium telfairiae induce cytotoxicity and caspase-dependent apoptosis in HT-29 human colon carcinoma cells.

Natural marine products have recently become the focus of increased research interest, due to their potential pharmacological activities. Therefore, we have screened 50 varieties of marine seaweed and determined that the methanolic extracts from Plocamium telfairiae (PTE) exhibited a cytotoxic effect against HT-29 human colon carcinoma cells. In this study, we report on the cytotoxic activity and mechanism of PTE-induced apoptosis in HT-29 cells. The treatment of HT-29 cells with various PTE concentrations resulted in the inhibition of growth and the induction of apoptosis in a dose-dependent manner, as determined by the results of a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay, a lactate dehydrogenase release assay, a morphological assay, and a colony formation assay. Interestingly, we also detected apoptotic bodies on Hoechst staining and attempted to determine whether the PTE-induced apoptosis involved the caspase pathway, using a caspase colorimetric assay. The activation of caspases-8, -9, -3, and -7 and the specific proteolytic cleavage of poly(ADP-ribose) polymerase were detected over the course of apoptosis induction. Our results showed that PTE may function as a chemopreventive and/or chemotherapeutic agent in colon carcinoma cells via the reduction of cell viability and the induction of apoptosis.

Induction of extracellular matrix synthesis in normal human fibroblasts by anthraquinone isolated from Morinda citrifolia (Noni) fruit.

In previous studies we found that Morinda citrifolia (Noni) fruit extract up-regulated biosynthesis of type I collagen and glycosaminoglycans in primary cultures of normal human fibroblasts. The objective of this study was to identify the active ingredients in Noni fruit extract. An active single compound having a type I collagen-stimulating effect was isolated and identified as 1,4-dihydroxy-2-methoxy-7-methylanthraquinone by nuclear magnetic resonance, infrared, and mass analysis. It was revealed that anthraquinone showed significantly increased elaboration of procollagen type I C-terminal peptide and glycosaminoglycans and reduced expression of the collagenase matrix metalloproteinase-1 dose-dependently in human dermal fibroblasts. Furthermore, in a clinical trial, a nano-emulsion containing anthraquinone predominantly increased the dermal type I procollagen in nude mouse skin. These results suggest that anthraquinone derived from Noni extract is a good candidate for use as a new anti-wrinkle agent due to its strong induction of biosynthetic activity of extracellular matrix components.

Identification of a novel unpaired histidine sensor kinase affecting secondary metabolism and morphological differentiation in Streptomyces acidiscabies ATCC 49003.

Two-component systems (TCSs) are an important signaling transduction pathway that adapt to changing environments. Commonly, a TCS comprises a sensor kinase that is usually an integral membrane histidine sensor kinase and a response regulator that mediates the cellular responses. Presently, however, we cloned a novel sensor kinase gene (tcsK) that is not adjacent to its cognate response regulator from Streptomyces acidiscabies that produces two secondary metabolites, thaxtomin A and WS5995B, and identified its functional involvement in the production of secondary metabolites and morphological differentiation. The elevated expression and disruption of the tcsK gene enhanced 7.1-fold and almost abolished WS5995B production in S. acidiscabies, respectively, but did not affect the production of thaxtomin A. In addition, spore formation of S. acidiscabies was decreased 120-fold by the disruption of tcsK, and the actinorhodin production of Streptomyces lividans TK24 was increased 5.7-fold by the high expression of tcsK. These results indicate that the novel unpaired tcsK gene may be related to the control of secondary metabolite production and spore formation in actinomycetes.

Enhanced glutamic acid production of Brevibacterium sp. with temperature shift-up cultivation.

For cells of Brevibacterium sp. under conditions of biotin limitation, the efflux of metabolites through the cell membrane was affected by temperature. After the temperature shift-up from 30 degrees C to 38 degrees C, both the specific production rate of glutamic acid and the excretion of intracellular materials, such as glucose-6-phosphate and nucleotides, were increased simultaneously. For the production of glutamic acid, not only the yield but also the specific production rate was increased by the temperature shift-up.

Enhanced productivity of human lysozyme by pH-controlled batch fermentation of recombinant Saccharomyces cerevisiae.

Human lysozyme (HLY) was successfully produced by Saccharomyces cerevisiae with a plasmid (pHK501) containing a synthetic HLY gene connected with a native secretion-signal sequence under control of the promoter for the glyceraldehydes-3-phosphate dehydrogenase gene. For large-scale production, batch fermentation was adopted with 1 l SD (Leu(-)) minimal medium in a 2-l jar fermentor. While pH-uncontrolled fermentation gave 42.3 units/ml of HLY over a period of 72-h cultivation, HLY production increased to 74.5 units/ml by performing pH-controlled fermentation, although a longer fermentation time was needed for maximal HLY production. A pH-shift strategy was designed in this study, resulting in higher HLY production and a shorter fermentation time.

Cloning of metK from Actinoplanes teichomyceticus ATCC31121 and effect of its high expression on antibiotic production.

A metK gene encoding S-adenosyl-L-methionine synthetase was cloned from the non-Streptomyces actinomycetes, Actinoplanes teichomyceticus ATCC31121. In order to evaluate the effect of the metK expression on antibiotic production in actinomycetes, an expression vector harboring the metK gene was constructed and introduced into Streptomyces lividans TK24 and A. teichomyceticus, and the antibiotic production of the exconjugants was assessed. As a result, it was determined that the expression of metK induced 17-fold and 2.2-fold increases in actinorhodin production from S. lividans TK24 and teicoplanin production from A. teichomyceticus, respectively, compared with the control strains.

Application of conjugation using phiC31 att/int system for Actinoplanes teichomyceticus, a producer of teicoplanin.

Actinoplanes teichomyceticus produces teicoplanin, which is a glycopeptide antibiotic for Gram-positive pathogenic bacteria and methicillin-resistant Staphylococcus aureus (MRSA). For a molecular genetic study of A. teichomyceticus, an effective transformation method using the conjugal transfer of DNA from E. coli to spores of A. teichomyceticus was established for the first time, based on the bacteriophage varphiC31 att/int system, in the genus of Actinoplanes. The high frequency of transconjugation was obtained on MS medium containing 40 mM MgCl(2), using 1.25 x 10(8) E. coli donor cells and 10(5) spores without a heat treatment. In addition, by cloning and sequencing the attB site A. teichomyceticus was shown to contain a single attB site within an ORF coding for a pirin homolog. Also, its attB site sequence showed high homology to that of Streptomyces lividans, unlike the case of Kitasatospora setae despite being a non-Streptomyces actinomycete, which seems to be closely related to the high transconjugation frequency of A. teichomyceticus.

Conjugal transfer using the bacteriophage phiC31 att/int system and properties of the attB site in Streptomyces ambofaciens.

To facilitate molecular genetic studies of Streptomyces ambofaciens that produces spiramycin, a commercially important macrolide antibiotic used in human medicine against Gram-positive pathogenic bacteria, the conditions for the conjugal transfer of DNA from E. coli to S. ambofaciens were established using a bacteriophage phiC31 att/int system. The transconjugation efficiency of S. ambofaciens varied with the medium used; the highest frequency was obtained on AS-1 medium containing 10 mM MgCl(2) without heat treatment of the spores. In addition, by cloning and sequencing the attB site, we identified that S. ambofaciens contains a single attB site within an ORF coding for a pirin homolog, and its attB site sequence shows 100% nt identity to the sequence of S. coelicolor and S. lividans, which have the highest efficiency in transconjugation using the phiC31 att/int system.

Functional analysis of a BarX homologue (SngA) as a pleiotropic regulator in Streptomyces natalensis.

The prior sequencing of the upstream region of the gamma-butyrolactone autoregulator receptor gene (sngR) in Streptomyces natalensis revealed the presence of a 972-bp gene encoding a BarX homologue (SngA), which acts as a pleiotropic regulator controlling secondary metabolism and morphological differentiation. In this study, we investigated the in vivo function of SngA in S. natalensis, by comparing the natamycin production, morphology, and transcription of genes related to natamycin biosynthesis in a wild-type strain and a sngA-deleted mutant. The disruption of sngA resulted in a decrease in natamycin production, and in the induction of pigment production that had not been previously observed from S. natalensis. On the other hand, the insertion of the intact sngA with its own promoter, into the wild-type strain, resulted in a 1.7-fold increase in natamycin production. Spore formation decreased in comparison to that of the wild-type strain when the sngA-deleted mutant was grown on YEME agar, MS medium, and ISP4 medium. All phenotypes were restored to the original wild-type phenotypes upon complementation with the intact sngA, suggesting that SngA has pleiotropic functions in controlling both morphological differentiation and secondary metabolite production.

Culture Conditions and Characterizations of a New Phytase-Producing Fungal Isolate, Aspergillus sp. L117.

A novel fungal strain Aspergillus sp. L117 that produced acid-stable and thermostable phytase was isolated on basis of the clearing zone on PSM plate and the ability of Na-phytate hydrolysis. The phytase of isolate showed a 3-fold higher activity than that of A. ficuun NRRL3135. The Aspergillus sp. L117 produced maximal level of phytase at initial pH of 5.0 and 30℃. The optimal pH and temperature for phytase activity were 5.5 and 50℃, respectively. The phytase showed totally stable activity after 20 min of exposure between 30 and 90℃, and even at 100℃. The highest level of residual phytase activity was obtained at pH 5.5, and still retained the stability at the broadest pH ranges (2.0 to 7.0) of all the aforementioned phytases. Storage stability of phytase was preserved over 96% of initial activities for 60 days at 4, -20, and -70℃ and to retain even 70% of the initial activity at room temperature.

Cloning and in vivo functional analysis by disruption of a gene encoding the gamma-butyrolactone autoregulator receptor from Streptomyces natalensis.

A gene encoding a gamma-butyrolactone autoregulator receptor, which has a common activity as DNA-binding transcriptional repressors controlling secondary metabolism and/or morphological differentiation in Streptomyces, was cloned from a natamycin producer, Streptomyces natalensis. PCR using the primers designed for the two highly conserved regions of Streptomyces autoregulator receptors (BarA, FarA, ScbR, and ArpA) gave a 102-bp band. The sequence of this band had a high similarity to the expected region of a receptor gene. By genomic Southern hybridization with the 102-bp insert as a probe, a 687-bp intact receptor gene (sngR) was obtained from S. natalensis. To clarify the in vivo function of sngR, a sngR-disrupted strain was constructed, and the phenotypes were compared with those of the wild-type strain. The sngR-disruptants started natamycin production 6 h earlier and showed a 4.6-fold higher production of natamycin than the wild-type strain. In addition, the sporulation began earlier and the number of spores was tenfold more abundant than that of the wild-type strain. All the phenotypes were restored back to the original phenotypes of the wild-type strain by complementation with the intact sngR, indicating that the autoregulator receptor protein of S. natalensis acts as a primary negative regulator both on the biosynthesis of natamycin and sporulation.

Cloning and functional analysis by gene disruption of a gene encoding a gamma-butyrolactone autoregulator receptor from Kitasatospora setae.

Gamma-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 gamma-butyrolactone autoregulator receptor was cloned from a bafilomycin B1 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 3H-labeled autoregulators, especially toward [3H]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.

Intergeneric conjugal transfer of plasmid DNA from Escherichia coli to Kitasatospora setae, a bafilomycin B1 producer.

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.

Gamma-butyrolactone autoregulators and receptor proteins in non- Streptomyces actinomycetes producing commercially important secondary metabolites.

The presence of gamma-butyrolactone autoregulators and their receptor proteins were investigated in five representative strains of non- Streptomyces actinomycetes producing commercially important secondary metabolites. Ethyl acetate extracts of culture were assayed using wild-type S. virginiae for virginiae butanolide, S. lavendulae FRI-5 for IM-2, and S. griseus HH1 for A-factor. Actinoplanes teichomyceticus and Amycolatopsis mediterranei were shown to produce autoregulators. Corresponding autoregulator-binding activities were found in the crude cell-free lysates of these strains, using the binding assay with tritium-labeled autoregulator analogues as ligands, which suggests that non- Streptomyces actinomycetes might have autoregulator-dependent signaling cascades.

Cloning and characterization of a gene encoding the gamma-butyrolactone autoregulator receptor from Streptomyces clavuligerus.

With primers designed for the conserved region of the gamma-butyrolactone autoregulator receptor proteins from Streptomyces species, PCR using the Streptomyces clavuligerus genome DNA as a template gave a clear band of 100 bp, the sequence of which revealed high similarity to the expected region of a receptor gene. By Southern blot and colony hybridization with the 100-bp insert as a probe, plasmid pSCA, harboring a 4.2 kb- SalI fragment, was obtained. Sequence analysis on the insert revealed a 702-bp ORF encoding a protein with a moderate similarity (identity, 33-43%; similarity, 51-62%) to known gamma-butyrolactone autoregulator receptor proteins from Streptomyces sp. The ORF was named scaR ( S. clavuligerus autoregulator receptor). The scaR/pET-3d plasmid was constructed for overexpression of the recombinant ScaR protein (rScaR) in Escherichia coli, and the rScaR protein was purified to homogeneity by DEAE-ion-exchange HPLC. The molecular mass of the purified rScaR protein was determined to be 27 kDa as determined by SDS-PAGE, and 54 kDa by gel filtration HPLC under nondenatured conditions at a low protein concentration, indicating that the majority of the native ScaR is present in the form of a dimer, although rScaR tended to aggregate into a higher molecular form of 230 kDa at a high protein concentration. A binding assay with tritium-labeled autoregulators indicated that IM-2 type compounds with a long C2 side chain were the most effective ligands for rScaR, demonstrating for the first time that the beta-lactam producer S. clavuligerus contains a gene for the gamma-butyrolactone autoregulator receptor.