Streptomyces Rishiriensis Research Articles

Elucidation of the structure and mechanism of CouO, a small molecule C-methyltransferase fromStreptomyces rishiriensis

Elucidation of the structure and mechanism of CouO, a small molecule C-methyltransferase from<i>Streptomyces rishiriensis</i>

ISOLATION AND CHARACTERIZATION OF STREPTOMYCES RISHIRIENSIS (VY31) WITH ANTIBIOTIC ACTIVITY AGAINST VARIOUS PATHOGENIC MICROORGANISMS

Actinomycete strain VY31 was isolated from agriculture soil of region Východná, Slovakia. Morphological, physiological and biochemical studies indicated that this isolate belongs to the genus Streptomyces. The 16S rRNA sequence data supported the assignment of the isolate to the genus Streptomyces rishiriensis (sequence similarity 97%). Tested isolate was able to produce melanin dark pigment and exopigments on ISP6, ISP7 and SSM+T cultivating media. The optimal pH range was from 6-8 and optimal temperature at 30 °C. The strain exhibited salt tolerance up to 5 % and utilized the carbon sources such as glucose, arabinose, xylose, inositol, mannose, fructose, rhamnose and rafinose. Using ApiZym® stripes, the highest production of enzymes was determined for phosphatase alkaline, leucinearylamidase, valinearylamidase, phosphatase acid, naphtol-AS-BI-phosphohydrolase, galactosidase and glucosidase (&gt;40 nmol). According to ApiCoryne® results, positive reaction was confirmed in case of esculin, alkaline phosphatase, and this strain was also able to hydrolyze gelatine. Minimum Inhibitory Concentration (MIC) of the purified extract of isolate was evaluated against Gram-positive bacteria Staphylococcus aureus and Enterococcus faecium, Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa and against yeast Candida albicans. On the basis of MIC results, strain VY31 had noticeable antibacterial activity against Staphylococcus aures N315 (MRSA) from collection database of University Hospital in Hamburg, Germany. This isolate could be used in the development of new antibiotics for pharmaceutical purposes.

Community Analysis of Root- and Tuber-Associated Bacteria in Field-Grown Potato Plants Harboring Different Resistance Levels against Common Scab.

Eight genotypes of potato plants with different resistance levels against common scab were grown in a field infested with Streptomyces turgidiscabies. DNA was extracted from the roots, tubers, and rhizosphere soils of each of the eight genotypes at the flowering stage, and the quantity of S. turgidiscabies genomic DNA was assessed by real-time PCR using a TaqMan probe. The results obtained showed that the different potato genotypes had significant impacts on the population levels of S. turgidiscabies between resistant and susceptible genotypes in the tubers, but not in the roots or rhizosphere soils. Clone analyses of 16S rRNA gene libraries from the eight potato genotypes identified three phyla (Proteobacteria, Firmicutes, and Actinobacteria) as dominant taxa in root and tuber clone libraries, while a clustering analysis identified 391 operational taxonomic units (OTUs) at the species level. Eleven OTUs closely related to Aquicella siphonis, Arthrobacter nicotinovorans, Streptomyces rishiriensis, Rhodococcus baikonurensis, Rhizobium radiobacter, Rhizobium etli, Phyllobacterium myrsinacearum, Paenibacillus pabuli, Paenibacillus alginolyticus, and Bacillus halmapalus were detected in the root or tuber libraries of all the potato genotypes examined. Furthermore, an abundance of OTUs related to Aquicella and Rhodococcus was observed in the rhizospheres of resistant and susceptible potato genotypes, respectively. Based on this ecological information, an efficient survey may be conducted for biological agents from the potato rhizosphere.

Mass spectrometric pathway monitoring of secondary metabolites: systematic analysis of culture extracts of Streptomyces species

Streptomyces spheroides, Streptomyces rishiriensis, and Streptomyces roseochromogenes are producers of the aminocoumarin-type antibiotics novobiocin, coumermycin A1, and clorobiocin, respectively, all of which are bacterial gyrase inhibitors. In an attempt to develop a general analytical method for pathway monitoring of secondary metabolites from culture extracts of these strains, we used superior mass spectrometric methods. The aim was to develop and apply a technique for the rapid analysis of Streptomyces culture extracts with respect to those substances, thereby providing a method for screening extracts of genetically modified strains for new pharmaceutically active antibiotics with improved pharmacological effects. The combination of full scan mass spectrometry (MS), parent ion scan MS, product ion scan MS, and in-source collision-induced fragmentation prior to product ion scans (pseudo-MS3 scan), using characteristic fragmentation of the central aminocoumarin unit, was employed for the detection and structural interpretation of expected and new intermediates. We were able to show the applicability of this methodology to the three culture extracts, where the main intermediates could be found, and to demonstrate its use for interpretation of secondary metabolite biosynthesis. Some new compounds were discovered, including bis-carbamoylated novobiocin, hydroxylated clorobiocin, and several structurally and not yet fully elucidated coumermycin derivatives or precursors.

Identification of a topoisomerase IV in actinobacteria: purification and characterization of ParYR and GyrBR from the coumermycin A1 producer Streptomyces rishiriensis DSM 40489.

The biosynthetic gene clusters of the gyrase inhibitors coumermycin A(1) and clorobiocin contain two different resistance genes (gyrB(R) and parY(R)). Both genes code for B subunits of type II topoisomerases. The authors have now overexpressed and purified the encoded proteins, as well as the corresponding A subunits GyrA and ParX. Expression was carried out in Streptomyces lividans in the form of hexahistidine fusion proteins, allowing purification by nickel affinity chromatography. The complex of GyrA and GyrB(R) was found to catalyse ATP-dependent supercoiling of DNA, i.e. to function as a gyrase, whereas the complex of ParX and ParY(R) catalysed ATP-dependent decatenation and relaxation, i.e. the functions of topoisomerase IV (topo IV). This is believed to represent the first topo IV identified in the class of actinobacteria, and the first demonstration of the formation of a topo IV as a resistance mechanism of an antibiotic producer.

An unusual amide synthetase (CouL) from the coumermycin A1 biosynthetic gene cluster from Streptomyces rishiriensis DSM 40489.

The aminocoumarin antibiotic coumermycin A1 produced by Streptomyces rishiriensis DSM 40489 contains two amide bonds. The biosynthetic gene cluster of coumermycin contains a putative amide synthetase gene, couL, encoding a protein of 529 amino acids. CouL was overexpressed as hexahistidine fusion protein in Escherichia coli and purified by metal affinity chromatography, resulting in a nearly homogenous protein. CouL catalysed the formation of both amide bonds of coumermycin A1, i.e. between the central 3-methylpyrrole-2,4-dicarboxylic acid and two aminocoumarin moieties. Gel exclusion chromatography showed that the enzyme is active as a monomer. The activity was strictly dependent on the presence of ATP and Mn2+ or Mg2+. The apparent Km values were determined as 26 micro m for the 3-methylpyrrole-2,4-dicarboxylic acid and 44 micro m for the aminocoumarin moiety, respectively. Several analogues of the pyrrole dicarboxylic acid were accepted as substrates. In contrast, pyridine carboxylic acids were not accepted. 3-Dimethylallyl-4-hydroxybenzoic acid, the acyl component in novobiocin biosynthesis, was well accepted, despite its structural difference from the genuine acyl substrate of CouL.

Structures of rishirilides A and B, alpha 2-macroglobulin inhibitors produced by Streptomyces rishiriensis OFR-1056.

Structures of rishirilides A and B, alpha 2-macroglobulin inhibitors produced by Streptomyces rishiriensis OFR-1056.

Potentiation of mitomycin C, 6-mercaptopurine, bleomycin, cis-diamminedichloroplatinum and 5-fluorouracil by mycotrienins and mycotrienols.

Mycotrienins I and II and mycotrienols I and II are ansamycin antibiotics containing a triene structure, isolated from Streptomyces rishiriensis. An amphotericin B-resistant clone (AMBR-1) derived from cultured Chinese hamster V79 cells was cross-resistant to mycotrienins I and II, but not to mycotrienol I or II. These four ansamycin antibiotics were found to potentiate significantly the action of some anti-cancer agents including 5-fluorouracil, cis-diamminedichloroplatinum, bleomycin, mitomycin C and 6-mercaptopurine against cultured V79 cells. The action of adriamycin was not potentiated. These four ansamycin antibiotics showed a synergism spectrum similar to that of smaller polyene antibiotics.

Isolation of metallo-proteinase inhibitor (FMPI) producing microorganism.

A screening test was carried out for the purpose of isolating a microorganism which produced a specific proteinase inhibitor for mold metallo-proteinase. A potent strain identified as Streptomyces rishiriensis was isolated, and this inhibitor was named fungal metallo-proteinase inhibitor (FMPI). The strain was aerobically cultured at 25°C for 26–30 hr in shaking flasks with a medium consisting of 1 % meat extract, 1 % polypepton and 0.3% NaCl (about 100 mg/liter). FMPI showed execellent inhibitory activity against the metallo-proteinase of Aspergillus oryzae, and moderate activity against other microbials. FMPI has a low molecular weight and is stable only at alkaline region (pH > 11).

Influence of Cobalt on Fermentative Methylation

Streptomyces rishiriensis produces at least five closely related antibiotics. Strain selection yielded a culture producing only the most active component, coumermycin A. Hydrolysis of this antibiotic by barium hydroxide yielded both 5-methyl-pyrrole-2-carboxylic acid and pyrrole-2-carboxylic acid, which could be separated by paper chromatography. Coumermycin A was thus shown to be two fractions, designated A(1) and A(2) depending upon the nature of the pyrrole carboxylic acid portion. The addition of cobalt to the fermentation medium at a level as low as 0.01 mug/ml shifted the fermentation exclusively to the production of coumermycin A(1). Other ions were ineffective, except nickel, whose activity could be explained by the presence of contaminating cobalt.