Genomic and Metabolomic Investigation of a Rhizosphere Isolate Streptomyces netropsis WLXQSS-4 Associated with a Traditional Chinese Medicine.

Rhizosphere microorganisms play important ecological roles in promoting herb growth and producing abundant secondary metabolites. Studies on the rhizosphere microbes of traditional Chinese medicines (TCMs) are limited, especially on the genomic and metabolic levels. In this study, we reported the isolation and characterization of a Steptomyces netropsis WLXQSS-4 strain from the rhizospheric soil of Clematis manshurica Rupr. Genomic sequencing revealed an impressive total of 40 predicted biosynthetic gene clusters (BGCs), whereas metabolomic profiling revealed 13 secondary metabolites under current laboratory conditions. Particularly, medium screening activated the production of alloaureothin, whereas brominated and chlorinated pimprinine derivatives were identified through precursor-directed feeding. Moreover, antiproliferative activities against Hela and A549 cancer cell lines were observed for five compounds, of which two also elicited potent growth inhibition in Enterococcus faecalis and Staphylococcus aureus, respectively. Our results demonstrated the robust secondary metabolism of S. netropsis WLXQSS-4, which may serve as a biocontrol agent upon further investigation.

Streptomyces marianii sp. nov., a novel marine actinomycete from southern coast of India.

A novel marine actinomycete strain designated ICN19T was isolated from the subtidal sediment of Chinnamuttam coast of Kanyakumari, India and subjected to polyphasic taxonomic analysis. Neighbour-joining tree based on 16S rRNA gene sequences of validly described type strains had revealed the strain ICN19T formed distinct cluster with Streptomyces wuyuanensis CGMCC 4.7042T, Streptomyces tirandamycinicus HNM0039T and Streptomyces spongiicola HNM0071T. Morphological, physiological and chemotaxonomic characteristics were consistent with those of members of the genus Streptomyces. The strain possessed LL-diaminopimelic acid as the diagnostic diamino acid. The predominant isoprenoid quinone was identified as MK-9(H8) (70%), MK-9(H6) (20%) and MK-9(H2) (2%), with the major cellular fatty acids (>10%) being anteiso-C15:0, C16:0 and iso-C16:0. The main polar lipids were found to be diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol mannosides and three unidentified phospholipids. The dendrogram generated on the basis of MALDI-TOF mass spectra supports the strain differentiated from its neigbours. The genome sequence of strain ICN19T was 9,010,366 bp in size with a total of 7420 protein-coding genes and 98 RNA genes. The genomic G+C content of the novel strain was 71.27 mol%. The DNA-DNA relatedness between strain ICN19T and the reference strains with S. wuyuanensis CGMCC 4.7042T, S. tirandamycinicus HNM0039T and S. spongiicola HNM0071T were 42.8%, 39.5% and 38%, respectively. Based on differences in physiological, biochemical, chemotaxonomic differences and whole-genome characteristics the isolated strain represents a novel species of the genus Streptomyces, for which the name Streptomyces marianii sp. nov. is proposed. Type strain is ICN19T (=MCC 3599T = KCTC 39749T).

Exploration of cryptic organic photosensitive compound as Zincphyrin IV in Streptomyces venezuelae ATCC 15439.

Zincphyrin IV is a potential organic photosensitizer which is of significant interest for applications in biomedicine, materials science, agriculture (as insecticide), and chemistry. Most studies on Zincphyrin are focused on Zincphyrin III while biosynthesis and application of Zincphyrin IV is comparatively less explored. In this study, we explored Zincphyrin IV production in Streptomyces venezuelae ATCC 15439 through combination of morphology engineering and "One strain many compounds" approach. The morphology engineering followed by change in culture medium led to activation of cryptic Zincphyrin IV biosynthetic pathway in S. venezuelae with subsequent detection of Zincphyrin IV. Morphology engineering applied in S. venezuelae increased the biomass from 7.17 to 10.5 mg/mL after 48 h of culture. Moreover, morphology of engineered strain examined by SEM showed reduced branching and fragmentation of mycelia. The distinct change in color of culture broth visually demonstrated the activation of the cryptic biosynthetic pathway in S. venezuelae. The production of Zincphyrin IV was found to be initiated after overexpression ssgA, resulting in the increase in titer from 4.21 to 7.54 µg/mL. Furthermore, Zincphyrin IV demonstrated photodynamic antibacterial activity against Bacillus subtilis and photodynamic anticancer activity against human ovarian carcinoma cell lines.

Streptomyces qaidamensis sp. nov., isolated from sand in the Qaidam Basin, China.

A novel actinobacterial strain, designated S10T, was isolated from a sand sample collected from the Qaidam Basin in Qinghai province, China. The strain S10T exhibited antibacterial activity against MRSA. The taxonomic position of the strain S10T was determined by a polyphasic approach. There were six copies of 16S rDNA in S10T which were not same exactly (MH257693-MH257698). Phylogenetic analysis of 16S rRNA gene sequences indicated the strain belonging to the genus Streptomyces and it showed high sequence similarities with Streptomyces chartreusis NBRC 12753T (99.31%), Streptomyces phaeoluteigriseus DSM 41896T (99.24%), Streptomyces variegatus NRRL B-16380T (99.17%) and Streptomyces flavovariabilis NRRL B-16367T (99.17%) comparing with MH257693, MH257695, MH257696, MH257697, and MH257698. Similarities with Streptomyces kunmingensis NBRC14463T (98.82%), Streptomyces bungoensis DSM 41781T(98.76%), S. chartreusis NBRC 12753T (98.69%) and S. phaeoluteigriseus DSM 41896T (98.62%) with MH257694. Whole-genome average nucleotide identity (ANI) values between strain S10T and S. chartreusis NBRC 12753T, S. phaeoluteigriseus DSM 41896T, S. variegatus NRRL B-16380T, S. flavovariabilis NRRL B-16367T, S. kunmingensis NBRC 14463T, S. bungoensis DSM 41781T were 83.63%, 82.89%, 92.55%, 92.51%, 79.29, and 82.87%, respectively, suggesting that the strain S10T represented a new species. A phylogenetic analysis comparing the S10T genome with those of 336 other sequenced Streptomyces genomes confirmed its relatedness with Streptomyces variegatus NRRL B-16380T and Streptomyces flavovariabilis NRRL B-16367T. Strain S10T contained LL-diaminopimelic acid in the cell wall. The predominant menaquinones were MK-9(H6) and MK-9(H8) and the major fatty acids were iso-C15:0, anteiso-C15:0, iso-C16:0, and anteiso-C17:0. Phospholipids detected were diphosphatidyl glycerol, phosphatidyl ethanolamine, phosphatidyl choline, three unknown phospholipids, an unknown aminophospholipid and an unknown phosphatidyl glycolipid. On the basis of these genotypic and phenotypic data, it is proposed that isolate S10T (=JCM 31184T =CGMCC 4.7315T) should be classified in the genus Streptomyces as Streptomyces qaidamensis sp. nov.

Streptomyces urticae sp. nov., isolated from rhizosphere soil of Urtica urens L.

Two novel Gram-stain positive, spore-forming, aerobic actinomycetes, designated NEAU-PCY-1T and NEAU-PCY-2, were isolated from rhizosphere soil of Urtica urens L. collected from Anshan, Liaoning Province, northeast China. The 16S rRNA gene sequence analysis showed that strains NEAU-PCY-1T and NEAU-PCY-2 exhibited 99.8% similarity with each other and are closely related to Streptomyces abietis DSM 42080T (98.2, 98.3%) and Streptomyces fildesensis DSM 41987T (98.0, 98.1%). Phylogenetic analysis based on the 16S rRNA gene sequences indicated that the two strains formed a cluster with these two closely related species. Moreover, DNA-DNA hybridization results and some phenotypic, physiological and biochemical properties differentiated the two strains from their close relatives in the genus Streptomyces. Based on a polyphasic taxonomy study, strains NEAU-PCY-1T and NEAU-PCY-2 are considered to represent a novel species of the genus Streptomyces, for which the name Streptomyces urticae sp. nov. is proposed, with NEAU-PCY-1T (= DSM 105115T = CCTCC AA 2017015T) as the type strain.

Efficacy of protease inhibitor from marine Streptomyces sp. VITBVK2 against Leishmania donovani - An in vitro study.

In the present study the leishmanicidal effect of potential protease inhibitor producing marine actinobacterial isolate has been investigated against Leishmania donovani, the causative agent of visceral leishmaniasis. Among 89 marine actinobacteria isolated from a salt pan in Kanyakumari, only one isolate (BVK2) showed 97% of protease inhibition activity against trypsin. Moderate to high protease inhibitor activity was shown by isolate BVK2 on proteinase (30%) and chymotrypsin (85%). In optimization study for protease inhibitor production glucose as carbon source and casein as nitrogen source showed the best activity. In the in-vitro Fluorescence-activated cell sorting (FACS) assay, 100 µg/ml of BVK2 extract was active against amastigotes in infected J774A.1 macrophages and showed 87% of parasitic inhibition. The isolate BVK2 showed significant anti-parasitic activity with an IC50 of 27.1 µg/ml after double doses were administered. The potential isolate was identified by molecular 16S rRNA gene sequencing as Streptomyces sp. VITBVK2. The results obtained suggest that the marine actinobacterial extract which have novel metabolites can be considered as a potential source for the development of drugs.

Purification, characterization, cytotoxicity and anticancer activities of L-asparaginase, anti-colon cancer protein, from the newly isolated alkaliphilic Streptomyces fradiae NEAE-82.

L-asparaginase is an important enzyme as therapeutic agents used in combination with other drugs in the treatment of acute lymphoblastic leukemia. A newly isolated actinomycetes strain, Streptomyces sp. NEAE-82, was potentially producing extracellular L-asparaginase, it was identified as Streptomyces fradiae NEAE-82, sequencing product was deposited in the GenBank database under accession number KJ467538. L-asparaginase was purified from the crude enzyme using ammonium sulfate precipitation, dialysis and ion exchange chromatography using DEAE Sepharose CL-6B. Further the kinetic studies of purified enzyme were carried out. The optimum pH, temperature and incubation time for maximum L-asparaginase activity were found to be 8.5, 40 °C and 30 min, respectively. The optimum substrate concentration was found to be 0.06 M. The Km and Vmax of the enzyme were 0.01007 M and 95.08 Uml(-1)min(-1), respectively. The half-life time (T1/2) was 184.91 min at 50 °Ð¡, while being 179.53 min at 60 °Ð¡. The molecular weight of the subunits of L-asparaginase was found to be approximately 53 kDa by SDS-PAGE analysis. The purified L-asparaginase showed a final specific activity of 30.636 U/mg protein and was purified 3.338-fold. The present work for the first time reported more information in the production, purification and characterization of L-asparaginase produced by newly isolated actinomycetes Streptomyces fradiae NEAE-82.

Streptomyces lunalinharesii 235 prevents the formation of a sulfate-reducing bacterial biofilm

ABSTRACT Streptomyces lunalinharesii strain 235 produces an antimicrobial substance that is active against sulfate reducing bacteria, the major bacterial group responsible for biofilm formation and biocorrosion in petroleum reservoirs. The use of this antimicrobial substance for sulfate reducing bacteria control is therefore a promising alternative to chemical biocides. In this study the antimicrobial substance did not interfere with the biofilm stability, but the sulfate reducing bacteria biofilm formation was six-fold smaller in carbon steel coupons treated with the antimicrobial substance when compared to the untreated control. A reduction in the most probable number counts of planktonic cells of sulfate reducing bacteria was observed after treatments with the sub-minimal inhibitory concentration, minimal inhibitory concentration, and supra-minimal inhibitory concentration of the antimicrobial substance. Additionally, when the treated coupons were analyzed by scanning electron microscopy, the biofilm formation was found to be substantially reduced when the supra-minimal inhibitory concentration of the antimicrobial substance was used. The coupons used for the biofilm formation had a small weight loss after antimicrobial substance treatment, but corrosion damage was not observed by scanning electron microscopy. The absence of the dsrA gene fragment in the scraped cell suspension after treatment with the supra-minimal inhibitory concentration of the antimicrobial substance suggests that Desulfovibrio alaskensis was not able to adhere to the coupons. This is the first report on an antimicrobial substance produced by Streptomyces active against sulfate reducing bacteria biofilm formation. The application of antimicrobial substance as a potential biocide for sulfate reducing bacteria growth control could be of great interest to the petroleum industry.

Antitumor compounds from Streptomyces sp. KML-2, isolated from Khewra salt mines, Pakistan.

BACKGROUND: Actinomycetes are gram positive bacteria with high G + C content in their DNA and are capable of producing variety of secondary metabolites. Many of these metabolites possess different biological activities and have the potential to be developed as therapeutic agents. The aim of the present study was to screen actinomycetes inhabiting halophilic environment such as Khewra salt mines present in Pakistan for cytotoxic and antitumor compounds. RESULTS: An actiomycetes strain designated as Streptomyces sp. KML-2 was isolated from a saline soil of Khewra salt mines, Pakistan. The strain Streptomyces sp. KML-2 showed 84 % cytotoxic activity against larvae of Artemia salina. In the screening phase, the strain exhibited significant antitumor activity with IC50 values of 12, 48 and 56 µg/ml against Hela, MDBK and Vero cell lines, respectively. After that extract from 20 l fermentation was used to purify secondary metabolites by several chromatographic techniques. Structure elucidation of isolated compounds revealed that it is highly stable producer of Chromomycin SA (1) and 1-(1H-indol-3-yl)-propane-1,2,3-triol (2). Both of the isolated compounds showed significant antitumor activity against Hela and MCF-7 cancer cell lines (IC50 values 8.9 and 7.8 µg/ml against Hela; 12.6 and 0.97 µg/ml against MCF-7, respectively). The 16S rRNA gene sequence (1437 bp) of the strain confirm its identity (99 %) with Streptomyces griseus. CONCLUSIONS: From this research work we were successful in isolating two potent antitumor compounds, Chromomycin SA and 1-(1H-indol-3-yl)-propane-1,2,3-triol from Streptomyces KML-2 strain, isolated from Khewra salt mine. As such this is the second report which confirms that S. griseus can produce Chromomycin SA without introducing any mutagenesis in its biosynthesizing gene cluster and isolated indole derivative is being reported first time from any member of actinomycetes group with having novel antitumor activity against Hela and MCF-7 cells. Nucleotide sequences: Nucleotide sequence data reported are available in the GenBank database under the accession number: GenBank KJ009562.

Biomedical potential of actinobacterially synthesized selenium nanoparticles with special reference to anti-biofilm, anti-oxidant, wound healing, cytotoxic and anti-viral activities.

Currently, there is an ever-increasing need to develop environmentally benign processes in place of synthetic protocols. As a result, researchers in the field of nanoparticle synthesis are focusing their attention on microbes from rare biological ecosystems. One potential actinobacterium, Streptomyces minutiscleroticus M10A62 isolated from a magnesite mine had the ability to synthesize selenium nanoparticles (SeNPs), extracellularly. Actinobacteria mediated SeNP synthesis were characterized by UV-visible, Fourier transform infrared (FT-IR), X-ray diffraction (XRD), energy dispersive X-ray spectroscopy (EDX) and high resolution transmission electron microscopy (HR-TEM) analysis. The UV-spectral analysis of SeNPs indicated the maximum absorption at 510nm, FT-IR spectral analysis confirms the presence of capping protein, peptide, amine and amide groups. The selenium signals confirm the presence of SeNPs. All the diffraction peaks in the XRD pattern and HR-TEM confirm the size of SeNPs in the range of 10-250nm. Further, the anti-biofilm and antioxidant activity of the SeNPs increased proportionally with rise in concentration, and the test strains reduced to 75% at concentration of 3.2µg. Selenium showed significant anti-proliferative activity against HeLa and HepG2 cell lines. The wound healing activity of SeNPs reveals that 5% selenium oinment heals the excision wound of Wistar rats up to 85% within 18 days compared to the standard ointment. The biosynthesized SeNPs exhibited good antiviral activity against Dengue virus. The present study concludes that extremophilic actinobacterial strain was a novel source for SeNPs with versatile biomedical applications and larger studies are needed to quantify these observed effects of SeNPs.

Investigation on the effects of ϵ-poly-L-lysine on a producing strain Streptomyces ahygroscopicus GIM8, for better understanding its biosynthesis.

ϵ-Poly-L-lysine (ϵ-PL) is an L-lysine homopolymer with strong antimicrobial activity, which is generally produced by Streptomyces strains. ϵ-PL is only produced under acidic conditions in liquid culture, and to improve the current understanding of ϵ-PL biosynthesis, the present study was undertaken to investigate the effects of ϵ-PL on its producer Streptomyces ahygroscopicus GIM8, under acidic and neutral conditions. The results indicated that a neutral pH favored ϵ-PL adsorption onto the cells, whereas minimal adsorption occurred at pH 4.0, the maximum pH for ϵ-PL production. At pH 7.0, small amounts of ϵ-PL caused considerable ATP leakage from the cells, which showed increased membrane permeability. Conversely, ATP leakage was inhibited by ϵ-PL at pH 4.0. Transmission electron microscopy investigation indicated that the cytoplasmic membrane was the primary site of ϵ-PL activity at pH 7.0, and that cell shape was maintained. Metabolic activity profiles revealed that ϵ-PL decreased cellular metabolic activity at a relatively low rate at pH 7.0. However, the toxic effect was significantly enhanced at pH 4.0. Based on these data, a mechanism for the effect of ϵ-PL on ϵ-PL-producing cells under neutral and acidic conditions is proposed. Additionally, acidic conditions may potentially be required for ϵ-PL biosynthesis in liquid culture because low pH can increase membrane permeability and prevent binding of ϵ-PL onto cells, both of which favor the secretion of the ϵ-PL produced by the cells into the broth. This research contributes to the current understanding of ϵ-PL biosynthesis.

Streptomyces lactacystinicus sp. nov. and Streptomyces cyslabdanicus sp. nov., producing lactacystin and cyslabdan, respectively.

Actinomycete strains OM-6519(T) and K04-0144(T) produce the bioactive compounds lactacystin and cyslabdan, respectively. Here, the taxonomic positions of these two strains were determined. The morphological and chemical features of strains OM-6519(T) and K04-0144(T) indicated that they belonged to the genus Streptomyces. Strain OM-6519(T) showed the highest 16S rRNA gene sequence similarities with Streptomyces xanthocidicus NBRC 13469(T) (99.7%), Streptomyces chrysomallus subsp. fumigatus NBRC 15394(T) (99.6%) and Streptomyces aburaviensis NRRL B-2218(T) (99.5%). However, the DNA-DNA relatedness values between strain OM-6519(T) and the three related strains were below 70%. Strain K04-0144(T) showed the highest 16S rRNA gene sequence similarities with Streptomyces corchorusii NBRC 13032(T) (99.4%), Streptomyces olivaceoviridis NBRC 15394(T) (99.4%) and Streptomyces canarius NRRL B-2218(T) (99.3%). However, the DNA-DNA relatedness values between strain K04-0144(T) and the three related strains were also below 70%. Based on morphological, cultural and physiological characteristics and DNA-DNA relatedness data, strains OM-6519(T) and K04-0144(T) should be classified as new species of the genus Streptomyces, for which the names Streptomyces lactacystinicus sp. nov. and Streptomyces cyslabdanicus sp. nov. are proposed. The type strain of S. lactacystinicus is OM-6519(T) (=NBRC 110082(T), DSM 43136(T)). The type strain of S. cyslabdanicus is K04-0144(T) (=NBRC 110081(T), DSM 42135(T)).

Antitumor compounds from Streptomyces sp. KML-2, isolated from Khewra salt mines, Pakistan

BACKGROUND: Actinomycetes are gram positive bacteria with high G + C content in their DNA and are capable of producing variety of secondary metabolites. Many of these metabolites possess different biological activities and have the potential to be developed as therapeutic agents. The aim of the present study was to screen actinomycetes inhabiting halophilic environment such as Khewra salt mines present in Pakistan for cytotoxic and antitumor compounds. RESULTS: An actiomycetes strain designated as Streptomyces sp. KML-2 was isolated from a saline soil of Khewra salt mines, Pakistan. The strain Streptomyces sp. KML-2 showed 84 % cytotoxic activity against larvae of Artemiasalina. In the screening phase, the strain exhibited significant antitumor activity with IC50 values of 12, 48 and 56 µg/ml against Hela, MDBK and Vero cell lines, respectively. After that extract from 20 l fermentation was used to purify secondary metabolites by several chromatographic techniques. Structure elucidation of isolated compounds revealed that it is highly stable producer of Chromomycin SA (1) and 1-(1H-indol-3-yl)-propane-1,2,3-triol (2). Both of the isolated compounds showed significant antitumor activity against Hela and MCF-7 cancer cell lines (IC50 values 8.9 and 7.8 µg/ml against Hela; 12.6 and 0.97 µg/ml against MCF-7, respectively). The 16S rRNA gene sequence (1437 bp) of the strain confirm its identity (99 %) with Streptomyces griseus. CONCLUSIONS: From this research work we were successful in isolating two potent antitumor compounds, Chromomycin SA and 1-(1H-indol-3-yl)-propane-1,2,3-triol from Streptomyces KML-2 strain, isolated from Khewra salt mine. As such this is the second report which confirms that S. griseus can produce Chromomycin SA without introducing any mutagenesis in its biosynthesizing gene cluster and isolated indole derivative is being reported first time from any member of actinomycetes group with having novel antitumor activity against Hela and MCF-7 cells Nucleotide sequences: Nucleotide sequence data reported are available in the GenBank database under the accession number: GenBank KJ009562.

Streptomyces hundungensis sp. nov., a novel actinomycete with antifungal activity and plant growth promoting traits.

A novel actinobacterium MBRL 251(T), isolated from a sample collected from a limestone quarry at Hundung, Manipur, India, was characterized using a polyphasic approach. The 16S ribosomal RNA gene sequence of strain MBRL 251(T) showed closest similarities with Streptomyces xanthochromogenes NRRL B-5410(T) (99.6%) and Streptomyces michiganensis NBRC 12797(T) (99.6%). The DNA relatedness between MBRL 251(T) and S. xanthochromogenes NBRC 12828(T), and S. michiganensis NBRC 12797(T) was 46.6% and 40.7%, respectively. Strain MBRL 251(T) contained LL-diaminopimelic acid as the diagnostic diamino acid, with glucose and xylose as the main cell wall sugars, whereas small amounts of galactose, mannose, rhamnose and ribose were also detected in the whole-cell wall hydrolysates. The major fatty acids identified were anteiso-C15:0 (35.1%), iso-C16:0 (21.1%) and anteiso-C17:1 (13.2%). The predominant menaquinones detected were MK-9(H6) and MK-9(H8), whereas the polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositolmannosides. The G+C content of the genomic DNA was 72.3%. The phenotypic and genotypic data showed that strain MBRL 251(T) merits the recognition as a representative of a novel species of the genus Streptomyces. It is proposed that the isolate should be classified in the genus Streptomyces as a novel species, Streptomyces hundungensis sp. nov. The type strain is MBRL 251(T) (=JCM 17577(T)=KCTC 29124(T)).

Characteristics of cesium accumulation in the filamentous soil bacterium Streptomyces sp. K202.

A filamentous soil bacterium, strain K202, was isolated from soil where an edible mushroom (Boletopsis leucomelas) was growing and identified as belonging to the genus Streptomyces on the basis of its morphological characteristics and the presence of LL-2, 6-diaminopimelic acid. We studied the existence states of Cs and its migration from extracellular to intracellular fluid in the mycelia of Streptomyces sp. K202. The results indicated that Cs accumulated in the cells through at least 2 steps: in the first step, Cs(+) was immediately and non-specifically adsorbed on the negatively charged cell surface, and in the second step, this adsorbed Cs(+) was taken up into the cytoplasm, and a part of the Cs entering the cytoplasm was taken up by an energy-dependent transport system(s). Further, we confirmed that a part of the Cs(+) was taken up into the mycelia competitively with K(+), because K(+) uptake into the intact mycelia of the strain was significantly inhibited by the presence of Cs(+) in the culture media. This suggested that part of the Cs is transported by the potassium transport system. Moreover, (133)Cs-NMR spectra and SEM-EDX spectra of the mycelia that accumulated Cs showed the presence of at least 2 intracellular Cs states: Cs(+) trapped by intercellular materials such as polyphosphate and Cs(+) present in a cytoplasmic pool.

Cyslabdan, a new potentiator of imipenem activity against methicillin-resistant Staphylococcus aureus, produced by Streptomyces sp. K04-0144. I. Taxonomy, fermentation, isolation and structural elucidation.

Cyslabdan, a new potentiator of imipenem activity against methicillin-resistant Staphylococcus aureus, was isolated from the culture broth of Streptomyces sp. K04-0144 by Diaion HP-20 and ODS column chromatographies and preparative HPLC. The structure of cyslabdan was elucidated by spectroscopic analyses including NMR. The compound has a labdane-type diterpene skeleton connecting with an N-acetylcysteine via thioether linkage.

Streptomyces gulbargensis sp. nov., isolated from soil in Karnataka, India.

During the course of screening for industrially important microorganisms, an alkali-tolerant and thermotolerant actinomycete, strain DAS 131T, was isolated from a soil sample collected from the Gulbarga region, Karnataka province, India. The strain was characterized by a polyphasic approach that showed that it belonged to the genus Streptomyces. Growth was observed over a wide pH range (pH 6-12) and at 45 degrees C. The 16S rRNA gene sequence of strain DAS 131T was deposited in the GenBank database under the accession number DQ317411. 16S rRNA gene sequence analysis revealed that strain DAS 131T was most closely related to Streptomyces venezuelae ISP 5230T (AY999739) with a sequence similarity of 99.5% (8 nucleotide differences out of 1,477). Despite this very high sequence similarity, strain DAS 131T was phenetically distinct from S. venezuelae. The DNA relatedness between these strains was 54%, indicating that strain DAS 131T is a distinct genomic species. On the basis of phenetic and genetic analyses, strain DAS 131T is classified as a new species in the genus Streptomyces, for which we propose the name Streptomyces gulbargensis sp. nov.

Characterization of Streptomyces MITKK-103, a newly isolated actinomycin X2-producer.

A new actinomycete strain designated MITKK-103 was isolated from the soil of a flowerpot using a humic acid agar medium. The newly isolated strain was able to produce a large amount of actinomycin X2 even under nonoptimized growing conditions and serves as a promising source of this antibiotic. Actinomycin X2 has higher cytotoxicity toward cultured human leukemia (HL-60) cells than does actinomycin D, and it induces cell death via apoptosis. A nearly complete 16S ribosomal DNA (rDNA) sequence from the isolate was determined and found to have high identity (98.5-100%) with Streptomyces galbus, Streptomyces griseofuscus, and Streptomyces padanus, indicating that MITKK-103 belongs to the genus Streptomyces. The isolate clustered with species belonging to the S. padanus clade in a 16S-rDNA-based phylogenetic tree and showed 75% overall homology to S. padanus ATCC 25646 in DNA-DNA relatedness analysis. Although the growth of the isolate was somewhat different from the three species mentioned, the strain MITKK-103 most closely resembles S. padanus on the basis of the morphological and phenotypic characteristics, phylogenetic analysis, and genotypic data. As such, this is the first report of a strain of S. padanus capable of producing actinomycins.

Anicemycin, a new inhibitor of anchorage-independent growth of tumor cells from Streptomyces sp. TP-A0648.

The anchorage-independence of cells is closely related to their tumorigenicity. In the screening of inhibitors of anchorage-independent growth of tumor cells, anicemycin was isolated from the fermentation broth of an actinomycete strain TP-A0648. The producing strain was isolated from a leaf of Aucuba japonica collected in Toyama, Japan and identified as Streptomyces sp. based on the taxonomic data. The structure of anicemycin was elucidated as a new analog of spicamycin by NMR and MS analysis. Anicemycin inhibited the anchorage-independent growth of the human ovary cancer SKOV-3 cells with an IC50 of 0.015 microM about three times more potently than their anchorage-dependent growth.

Biological control agent of common scab disease by antagonistic strain Bacillus sp. sunhua.

AIMS: To identify an antagonistic strain against Streptomyces scabiei and to characterize the antibiotic agent. The efficacy of the isolated strain in controlling common scab disease was also evaluated. METHODS AND RESULTS: A bacterial strain antagonistic against S. scabiei was isolated from the soil of a potato-cultivating area. This bacterium was identified as a Bacillus species by 16S rRNA gene sequence analysis and was designated Bacillus sp. sunhua. Antibiotics produced by this strain were proven to be stable within a broad pH range and at high temperatures. The culture broth was extracted with ethyl acetate, and then the crude extract was applied to HPLC. Two compounds were isolated and identified as iturin A and macrolactin A by 1H-NMR, 13C-NMR, HMBC, HMQC and mass spectrometer. The culture broth of Bacillus sp. sunhua had a suppressive effect on common scab disease in a pot assay, decreasing the infection rate from 75 to 35%. This strain also suppressed Fusarium oxysporum, the pathogen of potato dry rot disease. CONCLUSIONS: Bacillus sp. sunhua was shown to inhibit S. scabiei effectively. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report demonstrating that macrolactin A and iturin A inhibit S. scabiei. This study demonstrated the possibility of controlling potato scab disease using Bacillus sp. sunhua.