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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.