Streptomyces Purpurascens Research Articles

Isolation and Characterization of Antagonistic Activity of Some Streptomycetes and their Specific Actinophages from Soil.

<b>Background and Objective:</b> Phages specific to actinomycetes are common, active in the soil and gladly detected. Soil streptomycetes are having antibiosis activities against numerous bacteria, fungi and plant viruses. Thus, this study was designed to isolate, purify and characterize some streptomycetes active against some microorganisms from soil followed by isolation of their specific phages. <b>Materials and Methods:</b> Antagonistic activities of these streptomycetes isolates were tested against <i>Bacillus subtilis</i>,<i> Pseudomonas</i> sp., <i>Serratia</i> sp. and <i>Aspergillus niger</i>. To confirm their biological characterization of the streptomycetes isolates under investigation, the 16SrRNA gene was also used. The presence of specific lysate actinophages in the soil samples were tested by spot test technique and then propagated and purified for further characterization. The morphology of the purified actinophages was determined by electron microscopy. <b>Results:</b> The five selected <i>Streptomyces</i> isolates having effective antagonistic activity were biologically and molecularly identified as <i>Streptomyces sclerogranulatus </i>(QQ06), <i>Streptomyces mutabilis </i>(QQ07), <i>Streptomyces heilongjiangensis </i>(QQ08), <i>Streptomyces sparsus </i>(QQ09) and <i>Streptomyces purpurascens </i>(QQ10) strains. Electron micrographs showed the presence of filamentous virus-like particles with lengths of 21.4×928.57, 25×750, 21.4×857.14, 21.4×885.7 and 21.4×857.14 nm specific to <i>Streptomyces</i> strains QQ06, QQ07, QQ08, QQ09 and QQ10, respectively and belong to the family Inoviridae. <b>Conclusion:</b> Phage of Inoviridae was considered as the first time against streptomycetes isolates, therefore, additional and advanced studies should be carried out at the level of molecular characterization.

Streptomyces cupreus sp. nov., an antimicrobial producing actinobacterium isolated from Himalayan soil.

A novel actinobacterium, designated Streptomyces sp strain PSKA01T, was isolated from a soil sample of Kashmir Himalaya, India (latitude:34˚-01'N; longitude:74˚-47'E; altitude: 5328 ft). The taxonomic position of this strain was revealed by a polyphasic approach. Comparative analysis of the 16S rRNA gene sequences indicated that PSKA01T was closely related to the members of the genus Streptomyces, with the highest similarity to Streptomyces venetus CMU-AB225T (99.3%), Streptomyces levis NBRC 15423T (99.3%), Streptomyces misionensis DSM 40306T (99.2%), Streptomyces carpinensis NRRL B-16921T (99.2%), Streptomyces purpurascens NBRC 13077T (99.2%), and Streptomyces phaeoluteichromatogenes NRRL 5799T (99.2%). A phylogenomic tree based on genome sequences of the isolated strains, conferred by Type Strain Genome Server (TYGS), separated them from rest of the Streptomyces. The cell wall contained LL-diaminopimelic acid and the whole-cell hydrolysates were xylose and rhamnose. Major fatty acid methyl esters (FAME) were observed as 16:0 iso and 17:0 anteiso. In addition to the differences in phenotypic characters, the average nucleotide identity (ANI), average amino acid identity (AAI), digital DNA-DNA hybridization (dDDH) values, tetranucleotide frequency correlation coefficient (TETRA), and multilocus sequence analysis (MLSA) between PSKA01T and closely related type strains were considerably lower than the recommended threshold value; therefore, this isolate represents a novel Streptomyces species, for which the name Streptomyces cupreus sp. nov. is proposed. The type strain is PSKA01T (= JCM 33752T = MCC 4182T).

Novel aromatic polyketides from soil Streptomyces spp.: purification, characterization and bioactivity studies

Aromatic polyketides are important therapeutic compounds which include front line antibiotics and anticancer drugs. Since most of the aromatic polyketides are known to be produced by soil dwelling Streptomyces, 54 Streptomyces strains were isolated from the soil samples. Five isolates, R1, B1, R3, R5 and Y8 were found to be potent aromatic polyketide producers and were identified by 16S rRNA gene sequencing as Streptomyces spectabilis, Streptomyces olivaceus, Streptomyces purpurascens, Streptomyces coeruleorubidus and Streptomyces lavendofoliae respectively. Their sequences have been deposited in the GenBank under the accession numbers KF468818, KF681280, KF395224, KF527511 and KF681281 respectively. The Streptomyces strains were cultivated in the media following critically optimised culture conditions. The resulting broth extracts were fractionated on a silica gel column and preparative TLC to obtain pure compounds. The pure compounds were tested for bioactivity and the most potent compound from each isolate was identified by UV-Vis, IR and NMR spectroscopic methods. Isolated S. spectabilis (R1), yielded one potent compound identified as dihydrodaunomycin with an MIC of 4µg/ml against Bacillus cereus and an IC50 value of 24µM against HeLa. S. olivaceus (B1), yielded a comparatively less potent compound, elloramycin. S. purpurascens (R3) yielded three compounds, rhodomycin, epelmycin and obelmycin. The most potent compound was rhodomycin with an MIC of 2µg/ml against B. cereus and IC50 of 15µM against HeLa. S. coeruleorubidus (R5), yielded daunomycin showing an IC50 of 10µM and also exhibiting antimetastatic properties against HeLa. S. lavendofoliae (Y8), yielded a novel aclacinomycin analogue with IC50 value of 2.9µM and potent antimetastatic properties at 1µM concentration against HeLa. The study focuses on the characterization of aromatic polyketides from soil Streptomyces spp., which can serve as potential candidates for development of chemotherapeutic drugs in future.

Streptomyces jeddahensis sp. nov., an oleaginous bacterium isolated from desert soil.

A novel strain, G25T, was isolated from desert soil collected near Jeddah in Saudi Arabia. The strain could accumulate nearly 65 % of its cell dry weight as fatty acids, grow on a broad range of carbon sources and tolerate temperatures of up to 50 °C. With respect to to its 16S rRNA gene sequence, G25T is most closely related to Streptomyces massasporeus DSM 40035T, Streptomyces hawaiiensis DSM 40042T, Streptomyces indiaensis DSM 43803T, Streptomyces luteogriseus DSM 40483T and Streptomyces purpurascens DSM 40310T. Conventional DNA-DNA hybridization (DDH) values ranged from 18.7 to 46.9 % when G25T was compared with these reference strains. Furthermore, digital DDH values between the draft genome sequence of G25T and the genome sequences of other species of the genus Streptomyces were also significantly below the threshold of 70 %. The DNA G+C content of the draft genome sequence, consisting of 8.46 Mbp, was 70.3 %. The prevalent cellular fatty acids of G25T comprised anteiso-C15 : 0, iso-C15 : 0, C16 : 0 and iso-C16 : 0. The predominant menaquinones were MK-9(H6), MK-9(H8) and MK-9(H4). The polar lipids profile contained diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol and phosphatidylinositol mannosides as well as unidentified phospholipids and phosphoaminolipids. The cell wall contained ll-diaminopimelic acid. Whole-cell sugars were predominantly glucose with small traces of ribose and mannose. The results of the polyphasic approach confirmed that this isolate represents a novel species of the genus Streptomyces, for which the name Streptomyces jeddahensis sp. nov. is proposed. The type strain of this species is G25T (=DSM 101878T =LMG 29545T =NCCB 100603T).

Optimization of Culture Conditions for Production of Bioactive Metabolites by <i>Streptomyces</i> spp. Isolated from Soil

The current work was carried out under a screening program targeted at isolation of bioactive Streptomyces species from soil samples. A total of 54 Streptomyces species were isolated from soil samples, out of which 4 isolates were found to be promising. These isolates were identified as Streptomyces spectabilis, Streptomyces purpurascens, Streptomyces coeruleorubidus and Streptomyces lavendofoliae and their sequences have been deposited in the GenBank. The influence of culture conditions including, incubation time, incubation temperature, initial pH and different carbon and nitrogen sources on growth and bioactive compound formation was investigated. Isolate R1, identified as Streptomyces spectabilis, showed maximum bioactive metabolite production with cellobiose and peptone as the carbon and nitrogen sources, on the 5th day at pH 5 at 30℃. The optimum conditions for production by isolate R3, identified as Streptomyces purpurascens, were observed to be starch and casein as the carbon and nitrogen sources, pH 7, temperature 30℃ and an incubation period of eight days. For isolate R5, identified as Streptomyces coeruleorubidus, maximal production resulted on the sixth day at pH 6 and temperature of 35℃ with mannitol and JBM. Isolate Y8, identified as Streptomyces lavendofoliae, was found to produce high levels of bioactive metabolites in the medium supplemented with starch and peptone on the 10th day at pH 7 and at an incubation temperature of 30℃. The four strains tested here behaved differently, each one requiring specific conditions for maximum growth as well as bioactive metabolite production.

Effect of mechanical grinding of Sphagnum on the structure and physiological state of bacterial communities

The microcosm method was used to demonstrate an increase in bacterial numbers and drastic changes in the taxonomic structure of saprotrophic bacteria as a result of mechanical grinding of Sphagnum moss. Ekkrisotrophic agrobacteria predominant in untreated moss were replaced by hydrolytic bacteria. Molecular biological approaches revealed such specific hydrolytic bacteria as Janthinobacterium agaricum and Streptomyces purpurascens among the dominant taxa. The application of kinetic technique for determination of the physiological state of bacteria in situ revealed higher functional diversity of hydrolytic bacteria in ground moss than in untreated samples. A considerable decrease of the C/N ratio in ground samples of living Sphagnum incubated using the microcosm technique indicated decomposition of this substrate.

Rhodomycin analogues from Streptomyces purpurascens: isolation, characterization and biological activities

During a screening program for bioactive natural products, a potential Streptomyces sp was isolated from soil. On the basis of biochemical, cultural, physiological and 16S rRNA gene analysis, it was identified as Streptomyces purpurascens. The isolate was grown in liquid medium and the crude antibiotic complex was obtained by ethyl acetate extraction. Seven purified fractions were obtained by preparative Thin Layer Chromatography (TLC). Acid hydrolysis of each fraction and subsequent TLC led to the identification of aglycones and sugars indicating these compounds to be Rhodomycin and its analogues. The identity of these compounds was established on the basis of UV-visible and FT-IR spectra and comparison with published data. The compounds were active against Gram-positive bacteria. Compound E, identified as Rhodomycin B, was found to be the most potent compound with an MIC of 2 μg/ml against Bacillus subtilis. Compounds A and F identified as α2-Rhodomycin II and Obelmycin respectively, and Compound E exhibited an IC50 of 8.8 μg/ml against HeLa cell line but no cytotoxicity was found against L929.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-2-93) contains supplementary material, which is available to authorized users.

Crystal Structure of Aclacinomycin-10-Hydroxylase, a S-Adenosyl-l-Methionine-dependent Methyltransferase Homolog Involved in Anthracycline Biosynthesis in Streptomyces purpurascens

Anthracyclines are aromatic polyketide antibiotics, and several of these compounds are widely used as anti-tumor drugs in chemotherapy. Aclacinomycin-10-hydroxylase (RdmB) is one of the tailoring enzymes that modify the polyketide backbone in the biosynthesis of these metabolites. RdmB, a S-adenosyl-l-methionine-dependent methyltransferase homolog, catalyses the hydroxylation of 15-demethoxy-ε-rhodomycin to β-rhodomycin, one step in rhodomycin biosynthesis in Streptomyces purpurascens. The crystal structure of RdmB, determined by multiwavelength anomalous diffraction to 2.1Å resolution, reveals that the enzyme subunit has a fold similar to methyltransferases and binds S-adenosyl-l-methionine. The N-terminal domain, which consists almost exclusively of α-helices, is involved in dimerization. The C-terminal domain contains a typical α/β nucleotide-binding fold, which binds S-adenosyl-l-methionine, and several of the residues interacting with the cofactor are conserved in O-methyltransferases. Adjacent to the S-adenosyl-l-methionine molecule there is a large cleft extending to the enzyme surface of sufficient size to bind the substrate. Analysis of the putative substrate-binding pocket suggests that there is no enzymatic group in proximity of the substrate 15-demethoxy-ε-rhodomycin, which could assist in proton abstraction and thus facilitate methyl transfer. The lack of a suitably positioned catalytic base might thus be one of the features responsible for the inability of the enzyme to act as a methyltransferase.

Crystal Structure of Aclacinomycin Methylesterase with Bound Product Analogues: IMPLICATIONS FOR ANTHRACYCLINE RECOGNITION AND MECHANISM

Aclacinomycin methylesterase (RdmC) is one of the tailoring enzymes that modify the aklavinone skeleton in the biosynthesis of anthracyclines in Streptomyces species. The crystal structures of this enzyme from Streptomyces purpurascens in complex with the product analogues 10-decarboxymethylaclacinomycin T and 10-decarboxymethylaclacinomycin A were determined to nominal resolutions of 1.45 and 1.95 A, respectively. RdmC is built up of two domains. The larger alpha/beta domain shows the common alpha/beta hydrolase fold, whereas the smaller domain is alpha-helical. The active site and substrate binding pocket are located at the interface between the two domains. Decarboxymethylaclacinomycin T and decarboxymethylaclacinomycin A bind close to the catalytic triad (Ser102-His276-Asp248) in a hydrophobic pocket, with the sugar moieties located at the surface of the enzyme. The binding of the ligands is dominated by hydrophobic interactions, and specificity appears to be controlled mainly by the shape of the binding pocket rather than through specific hydrogen bonds. Mechanistic key features consistent with the structure of complexes of RdmC with product analogues are Ser102 acting as nucleophile and transition state stabilization by an oxyanion hole formed by the backbone amides of residues Gly32 and Met103.

Crystallization and preliminary X-ray diffraction studies of aclacinomycin-10-methyl esterase and aclacinomycin-10-hydroxylase from Streptomyces purpurascens.

Two enzymes participating in the biosynthesis of anthracyclines in Streptomyces purpurascens, aclacinomycin-10-methyl esterase (RdmC) and aclacinomycin-10-hydroxylase (RdmB), have been crystallized. RdmB is a S-adenosyl-methionine-dependent hydroxylase and RdmC hydrolyses the carboxymethyl group of the aglycone skeleton of aclacinomycin. Crystals of RdmB obtained in the presence of S-adenosyl-L-methionine were orthorhombic, space group C222(1), with unit-cell parameters a = 63.2, b = 92.2, c = 115.3 A; diffraction data were collected to 2.1 A resolution. RdmC was crystallized as a complex with the substrate, aclacinomycin T. These crystals diffracted to 1.45 A resolution and belonged to space group P2(1), with unit-cell parameters a = 38.2, b = 84.7, c = 44.3 A, beta = 99.9 degrees.

Characterization of aklavinone-11-hydroxylase from Streptomyces purpurascens

Aklavinone-11-hydroxylase (RdmE) is a FAD monooxygenase participating in the biosynthesis of daunorubicin, doxorubicin and rhodomycins. The rdmE gene encodes an enzyme of 535 amino acids. The sequence of the Streptomyces purpurascens enzyme is similar to other Streptomyces aromatic polyketide hydroxylases. We overexpressed the gene in Streptomyces lividans and purified aklavinone-11-hydroxylase to apparent homogeneity with four chromatographic steps utilizing a kinetic photometric enzyme assay. The enzyme is active as the monomer with a molecular mass of 60 kDa; it hydroxylates aklavinone and other anthracyclinones. Aklavinone-11-hydroxylase can use both NADH and NADPH as coenzyme but it is slowly inactivated in the presence of NADH. The apparent K m for NADPH is 2 mM and for aklavinone 10 μM. The enzyme is inactivated in the presence of phenylglyoxal and 2,3-butanedione. NADPH protects against inactivation of aklavinone-11-hydroxylase by phenylglyoxal.

Nucleotide sequences and expression of genes from Streptomyces purpurascens that cause the production of new anthracyclines in Streptomyces galilaeus

Six open reading frames, rdmA to rdmF, in a 6,077-bp segment of Streptomyces purpurascens DNA which caused the production of hybrid anthracyclines were identified. The minimal fragment that produced anthracyclines modified at the 10th position contained rdmB to rdmD; rdmE is the gene for aklavinone-11-hydroxylase. RdmC is similar to a putative open reading frame in the daunorubicin biosynthetic cluster of Streptomyces peucetius and is likely to participate in the removal of the side chain at the 10th position.

TAN-1323 C and D, new concanamycin-group antibiotics; detection of the angiostatic activity with a wide range of macrolide antibiotics.

We detected potent angiostatic activity in a MeOH extract from the mycelia of microbial strain S-45628 in the chick chorioallantoic membrane (CAM) assay. The producer was taxonomically characterized as Streptomyces purpurascens. Active principles designated TAN-1323 A-D were isolated and determined to be 18-membered macrolide antibiotics; components C and D are new members of this group, while components A and B are identical to concanamycins C and A, respectively. When tested in the CAM assay, components B and D gave huge avascular zones at the extremely low doses of 10-100 ng/disk, although components A and C showed far weaker activity due to their preferential tissue-damaging effect on the CAM. The discovery that these 18-membered macrolide antibiotics are angiostatic substances prompted us to examine other types of macrolide antibiotics, leading to the discovery that 16-membered macrolide antibiotics such as bafilomycin C1, tylosin and leucomycin also show angiostatic activity on the CAM. Thus, angiostatic potential is widely distributed among macrolide antibiotics. The mechanism of action of these macrolide antibiotics is also discussed.

Hybrid anthracycline antibiotics: production of new anthracyclines by cloned genes from Streptomyces purpurascens in Streptomyces galilaeus.

A DNA segment cloned from Streptomyces purpurascens ATCC 25489 close to a region that hybridized to a probe containing part of the actinorhodin polyketide synthase caused S. galilaeus ATCC 31615 to produce new anthracyclines. When transformed with certain sub-clones of this segment, the host produced glycosides of epsilon-rhodomycinone, beta-rhodomycinone, 10-demethoxycarbonylaklavinone and 11-deoxy-beta-rhodomycinone in addition to those of aklavinone, the natural anthracyclines of S. galilaeus. The first two compounds are S. purpurascens products and the other two are novel compounds that conceptually are structural hybrids between S. galilaeus and S. purpurascens products. Three glycosides of one of the novel aglycones, 11-deoxy-beta-rhodomycinone, were purified and found to possess cytotoxic activity against L1210 mouse leukaemia cells. Separate regions of the cloned S. purpurascens DNA are responsible for modification of the S. galilaeus host product at the 10- and 11-positions.

7-Hydroxyguanine, a novel antimetabolite from a strain of Streptomyces purpurascens. I. Taxonomy of producing organism, fermentation, isolation and biological activity.

Strain A-347, an actinomycete isolated from a soil sample, was found to produce a new antimetabolite, 7-hydroxyguanine. The aerial mycelium formed spiral spore chains with spiny spore surface. The chemical composition of strain A-347 indicated that it was an actinomycete of cell wall Type I. From its morphological, cultural, physiological characteristics and direct comparison with the type culture, this strain was classified as Streptomyces purpurascens. 7-Hydroxyguanine was purified from the broth filtrate by ion exchange chromatography and crystallized from hot 2 M NH4OH. 7-Hydroxyguanine inhibited the growth of experimental tumors such as L1210 leukemia.

Influence of ergot alkaloids on growth of streptomyces purpurascens and production of its secondary metabolites.

Different ergot alkaloids, in equimolar amounts, were added to submerged cultures of rhodomycins producing Streptomyces purpurascens, strain CBS 311.55. Influence of the alkaloids on growth and production of rhodomycins was measured by determination of mycelia weight and content of rhodomycinones. The production of rhodomycins is inhibited almost totally by as little as 82.5 µg/ml agroclavine, whereas the growth of the bacteria is stopped totally by 165 µg/ml. The remaining alkaloids show different effects.

Transformation of 12‐deoxycardenolids with Streptomyces purpurascens

AbstractStreptomyces purpurascens transforms primary and secondary 12‐deoxycardenolids into digoxin, 7β‐hydroxy‐digitoxin and 7β‐hydroxy‐digoxin, respectively. A metabolic pathway is proposed for cardenolid transformations and the enzymes participating in it are investigated. The substrate concentration in the medium can be increased by using a water‐miscible solvent of electron donor character. The newly developed procedure yields 0.5 g/l−1 of digoxin in a five‐day fermentation.

An .ALPHA.-L-arabinofuranosidase from Streptomyces purpurascens IFO 3389.

By screening 46 strains of Actinomycetes for their ability to hydrolyze arabinan, 16 strains were found to have α-L-arabinofuranosidase activity, and Streptomyces purpurascens IFO 3389 was selected as the most promising of the sixteen. An α-L-arabinofuranosidase [EC 3.2.1.55] has been highly purified from the culture fluid of this organism grown on beet arabinan as the carbon source. The molecular weight of the native enzyme was determined to be 495, 000 by gel filtration and that of the subunit to be 62, 000 by SDS polyacrylamide gel electrophoresis. The pI value was 3.9. The purified enzyme was active on ρ -nitrophenyl α-L-arabinofuranoside and arabino-oligomers, and inactive on arabinan, arabinoxylan and arabinogalactan. The optimum pH was 6.5. The enzyme was inhibited by Hg 2+, Ag + and L-arabino-γ-lactone. The values of Km and Vmax for ρ-nitrophenyl α-L-arabinofuranoside were determined to be 8.2 × 10-5 M and 89.3 μmol per min per mg of protein, respectively.

Transformation of 12-deoxycardenolids withStreptomyces purpurascens

Streptomyces purpurascens transforms primary and secondary 12-deoxycardenolids into digoxin, 7 beta-hydroxy-digitoxin and 7 beta-hydroxy-digoxin, respectively. A metabolic pathway is proposed for cardenolid transformations and the enzymes participating in it are investigated. The substrate concentration in the medium can be increased by using a water-miscible solvent of electron donor character. The newly developed procedure yields 0.5 g/1(-1) of digoxin in a five-day fermentation.

Isolation of an antibiotic, S-583-B, related to rhodomycin A and B.

From a streptomyces strain identified as Streptomyces purpurascens, an anthracycline antibiotic named S-583-B was isolated together with rhodomycin A and B. The chromophore of the antibiotic S-583-B was proved to be identical or closely similar to β-rhodomycinone, but the sugar moiety was found to consist of several sugar components including an aminosugar found to be rhodosamine.