Streptomyces Netropsis Research Articles

Natural Products Biosynthesis by Streptomyces netropsis IMV Ac-5025 under Exogenous Sterol Action.

Streptomycetes are known as producers of bioactive substances, particularly antibiotics. Streptomyces netropsis IMV Ac-5025 simultaneously produces different classes of antibiotics, including polyene compounds, phytohormones, and sterols, but the metabolic pathways involved in their biosynthesis are largely understudied. The aim of this work was to explore the biosynthesis of polyene antibiotics, sterols, and phytohormones when the producer is cultivated in a nutrient medium supplemented with exogenous β-sitosterol. Gas chromatography and high-performance liquid chromatography were applied to analyze the spectrum of bioactive compounds. The obtained results demonstrated not only an increase in the accumulation of biomass but also polyene antibiotics, intracellular sterols, auxins, and cytokinins, when cultivating S. netropsis IMV Ac-5025 in a liquid medium with the addition of β-sitosterol. The amount of biomass raised 1.5-2-fold, whilst the sum of polyene antibiotics increased 4.5-fold, sterols' sum (ergosterol, cholesterol, stigmasterol, β-sitosterol, and 24-epibrassinolide) by 2.9-fold, auxins' sum (indole-3-acetic acid, indole-3-acetic acid hydrazide, indole-3-carbinol, indole-3-butyric acid, indole-3-carboxaldehyde, and indole-3-carboxylic acid) by 6-fold, and cytokinins' sum (zeatin, isopentyladenine, zeatin riboside, and isopentenyladenosine) by 11-fold. Thus, we put forward the hypothesis that β-sitosterol plays a regulatory role in the network of biosynthetic reactions of S. netropsis IMV Ac-5025.

Keratinases from Streptomyces netropsis and Bacillus subtilis and Their Potential Use in the Chicken Feather Degrading

Feathers are the most prevalent agricultural waste generated by chicken farms, polluting the environment and wasting protein resources as a result of the accumulation of large amounts of feathers. Therefore, keratinase-producing microorganisms represent a promising potential technique for the degradation of feather waste. Streptomyces netropsis A-ICA and Bacillus subtilis ALICA, previously isolated from the rhizosphere of desert plants (Larrea tridentata and Prosopis juliflora) respectively, were assessed for their feather-degradation ability. Keratinase activity was optimized using various parameters, including incubation time, pH, temperature, and feather concentration. The maximum keratinase activity of S. netropsis A-ICA and B. subtilis ALICA (113.6 ± 5.1 and 135.6 ± 4.1 U/mL) was obtained at the 5th and 3rd day of incubation with initial pH of 7.0 and 7.5 at 25 and 30 °C, and 1% (w/v) of chicken feather, respectively. Under the optimized conditions, the concentration of soluble protein in the feather hydrolysate reached 423.3 ± 25 and 565.3 ± 7.7 µg/mL, with feathers weight loss of 84 ± 2 and 86± 1.5% by S. netropsis A-ICA and B. subtilis ALICA, respectively. The highest disulphide bond reductase activity reached 10.7 ± 0.4 and 10.96 ± 1.1 U/mL, after five and three days of inoculation with S. netropsis A-ICA and B. subtilis ALICA, respectively. Furthermore, the antioxidant activity of feather protein hydrolysate obtained by S. netropsis A-ICA and B. subtilis ALICA was evaluated using DPPH radical-scavenging activity, which exhibited a significant antioxidant potential with an IC50 value of 0.8 and 0.6 mg/mL. The 3D models of detected keratinases in both strains showed high similarity with subtilisin family. Further, the docking results clarified the importance of GSG and VVVFTP domains in B. subtilis and beta-keratin, respectively. The present study revealed the keratinolytic potential of S. netropsis A-ICA and B. subtilis ALICA in chicken feather degradation, which have potential application value and may be exploited as supplementary protein and antioxidant in animal feed formulations.

Exploitation of Soybean Oil Acid Degumming Waste: Biocatalytic Synthesis of High Value Phospholipids

AbstractThe acid degumming waste of the seeds oil refining industry is currently disposed of, but, instead, it could be exploited as an important source for the preparation of many products. In this work, the waste coming from the soybean oil refining step was first recovered and treated, allowing the isolation of a fraction enriched of phospholipids (PLs). Then, the latter was transformed, via an enzymatic reaction catalysed by phospholipase D (from Streptomyces netropsis), into more valuable products: polar head modified PLs‐enriched mixtures containing phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylethanolamine (PE) and phosphatidylhydroxybutyrate (PB). In the following we show how biocatalysis can be exploited for the production of value‐added PLs, to be used as functional food and nutraceutical ingredients, from a renewable feedstock. This alternative to the industrial usual disposal procedures should impart to the whole process a greater value in terms of carbon recycling, in agreement with the paradigms of bioeconomy for a wiser reuse of renewable resources in a circular economy perspective.

Biosynthesis of Polyene Antibiotics and Phytohormones by Streptomyces netropsis IMV Ac-5025 under the Action of Exogenous Isopentenyladenosine

Streptomyces are active producers of a wide range of metabolites with multidirectional biological activity. Streptomyces netropsis IMV Ac-5025 synthesizes a polyene antibiotic complex in which two fractions were identified: heptaene candidine and a new tetraene fraction of unknown structure. The influence of secondary metabolites on the polyene antibiotics biosynthesis by soil streptomycetes is insufficiently explored. The aim of this work was to research the effect of exogenous isopentenyladenosine on the biosynthesis of polyene antibiotics and cytokinins by S. netropsis IMV Ac-5025. Methods. The strain was cultured in submerged cultivation condition in organic (soy) and synthetic (starch-ammonia) liquid nutrient media. The studies of biomass accumulation (gravimetric method), glucose consumption (glucose oxidase method), pH changes of culture media (ionometric method), biosynthesis of polyene antibiotics, and phytohormones (quantitative and qualitative thin layer chromatography spectrodensitometric method) were conducted. The results were analyzed in Statisticav.6.0 program. Results. It was found that polyene antibiotics are synthesized after the first day of cultivation, which indicates their role in the metabolism of streptomycetes. The biggest amount of the polyene antibiotics was accumulated in the stationary phase of producer growth (on the 7th day). It was found the decrease of polyene antibiotics and cytokinins accumulation in the producer’s biomass with the increase of exogenous cytokinin concentration from 25 ng/mL to 500 ng/mL. The bioproduction of the tetraene fraction was suppressed to a greater extent – up to 92% in the synthetic and up to 23% – in organic nutrient media. However, the amount of producer biomass increased under the action of the exogenous substance that confirming the positive effect of exogenous cytokinin on cell division of S. netropsis IMV Ac-5025. Exogenous isopentenyladenosine reduced the accumulation of endogenous cytokinins in streptomycetes biomass. Conclusions. The obtained results indicate an indirect metabolic relationship between the biosynthesis of polyene antibiotics and cytokinins in soil streptomycetes and provide a basis for the regulation of the biotechnological process for bioproduct formation with the appropriate quantitative composition of its components.

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.

A novel streptomyces rhizobacteria from desert soil with diverse anti-fungal properties

The Baja California desert harsher environment harbors a limited biodiversity due to extreme conditions like low moisture of sandy soils and high soil temperature. In the present study, actinobacteria from soil of Baja California desert was isolated and their properties as plant growth promoter and antifungal activity were evaluated. Streptomyces netropsis strain A-ICA was isolated and identified based on 16 S rDNA sequences from rhizosphere of native plant Larrea tridentata. The cell suspension and the cell-free supernatant of its culture showed significant antifungal activity against Macrophomina phaseolina, Fusarium oxysporum, Fusarium solani, Fusarium equiseti, Botrytis cinerea, Alternaria alternata and Colletotrichum gloeosporioides. Additionally, the results showed the potential of this strain for production of phytohormones (indol acetic acid and giberrelins) and exhibited highest concentration of soluble-P with the biggest drop in pH. Overall, the results of this study indicate that potential of S. netropsis strain A-ICA isolated from Baja California desert as a significant plant growth promoter and biocontrol agent to reduce growth of phytopathogens.

Synthesis of liponucleotides using bacterial phospholipase D

A series of conjugates of pharmacologically promising modified nucleosides with phospholipid was synthesized. Some liponucleotides were originally produced. Soybean lecithin served as the donor of phosphatidyl residue. Phospholipase D from strain Streptomyces netropsis BIM B-428D was engaged as biocatalyst in transphosphatidylation reaction. The yield of liponucleotide synthesis reaction varied in the range 44–95 mol% depending on the acceptor of phosphatidyl residue. 65 mg (about 68 µmoles) of pure phosphatidylclofarabine was recovered by chromatography on silica gel column, resulting in 68 mol% yield calculated from the amount of modified nucleoside supplied into the reaction mixture.

RNA‐dependent chaperone (chaperna) as an engineered pro‐region for the folding of recombinant microbial transglutaminase

Transglutaminase (TGase) induces the cross-linking of proteins by catalyzing an acyl transfer reaction. TGase is a zymogen, activated by the removal of its pro-region. Because the pro-region is crucial for folding and inhibition of the TGase activity, the recombinant expression of the mature TGase (mTGase) without the pro-region, usually results in inactive inclusion bodies or low protein yield. Here, Streptomyces netropsis TGase was fused with Escherichia coli lysyl-tRNA synthetase (LysRS), as a module with chaperoning activity in an RNA dependent manner (chaperna). The TGase activity from purified fusion protein induced via the removal of LysRS by tev protease in vitro. Moreover, active mTGase was produced in E. coli via an intracellular cleavage system, wherein LysRS-mTGase was cleaved by the coexpressed tev protease in vivo. The results suggest that LysRS essentially mimics pro-region, which exerts a dual function-folding of TGase into active conformation and keeping it as dormant state-in an RNA-dependent manner. Thus, trans-acting RNAs, prompt the cis-acting chaperone function of LysRS, while being mechanistically similar to the intramolecular chaperone function of the pro-region. These results could be implemented and extended for the folding of "difficult-to-express" recombinant proteins, by harnessing the chaperna function.

Update of the list of QPS-recommended biological agents intentionally added to food or feed as notified to EFSA 6: suitability of taxonomic units notified to EFSA until March 2017.

The qualified presumption of safety (QPS) concept was developed to provide a harmonised generic pre‐evaluation to support safety risk assessments of biological agents performed by EFSA's scientific Panels. The identity, body of knowledge, safety concerns and antimicrobial resistance of valid taxonomic units were assessed. Safety concerns identified for a taxonomic unit are, where possible and reasonable in number, reflected as ‘qualifications’ which should be assessed at the strain level by the EFSA's scientific Panels. No new information was found that would change the previously recommended QPS taxonomic units and their qualifications. Between the end of September 2016 and March 2017, the QPS notification list was updated with 87 applications for market authorisation. From these, 32 biological agents already had a QPS status, and 37 were not included in the evaluation as they are filamentous fungi or enterococci. Streptomyces species (Streptomyces cinnamonensis, Streptomyces mobaraensis and Streptomyces violaceoruber), Bacillus circulans (three notifications) and Escherichia coli (seven notifications) were re‐confirmed not suitable for QPS. Streptomyces rubiginosus and Streptomyces netropsis, not evaluated within the previous mandate, were also not recommended for QPS. Streptomyces spp. and E. coli will be excluded from further QPS evaluations within the current QPS mandate. Hyphomicrobium denitrificans, which has never been evaluated before, was not recommended for the QPS list and for Pseudomonas amyloderamosa, the QPS assessment was not applicable because it is not a validated species. Lactobacillus animalis was a new taxonomic unit recommended to have the QPS status.

Идентификация и антагонистические свойства почвенного стрептомицета Streptomyces sp. 100

Determination of the taxonomic status of the soil streptomycete Streptomyces sp. 100 and study of its antagonistic properties against phytopathogenic and opportunistic human microorganisms. For the identification of the strain a set of conventional methods morphological and cultural, physiological and biochemical characteristics of the producer, as well as molecular genetic analysis of 16S rRNA gene were used. Streptomycete was cultivated on agar nutrient and liquid soy medium until the stationary phase of growth. The antagonistic activity of the strain was studied by agar diffusion method. The study of morphological and cultural properties showed that Streptomyces sp. 100 formed the colonies with irregular edges protruding from the depressed center, straight sporophores were short, gathered in whorls; spores were oval, smooth shell dispute. Growing on agar medium (pH 6.8-7.4, temperature 28 °C, microaerophilic conditions) this strain formed mycelium of various colors: the air white, white-yellow, white-brown or substrate tan, cream and yellow, creamy carmine, yellow-brown. A soluble pigment was yellow and yellow-brown, while melanoid pigment was not detected. The morphological, cultural, physiological, biochemical and molecular genetic characteristics of the soil streptomycete let to identify it as Streptomyces netropsis (Finlay et al., 1951) IMV Ac-5025 (UCM Ac-2186) that is an active antagonist IMV Ac-5025 against plant pathogens. Growing on a surface of agaric nutrient media it inhibits phytopathogenic bacteria (Xanthomonas axonopodis pv. glycines 8609, Pseudomonas savastanoi pv. glycinea 8571, P. syringae pv. coronafaciens 9030) and fungi (Alternaria alternata 16814, Fusarium оxysporum 54201) zone of growth inhibition were 20 - 32 mm and 16 - 30 mm respectively. The supernatant of culture medium and the ethanol extract of biomass inhibited the growth of pathogenic bacteria and fungi. The most sensitive to action of a supernatant of cultural liquid were P. syringae pv. atrofaciens 7886 and Clavibacter michiganensis ssp. michiganensis 102, growth inhibition zones - 42 and 30 mm respectively. It should be noted that in the majority of cases the supernatant of cultural liquid suppressed growth of phytopathogenic bacteria in comparison with biomass extract more actively. At the same time only biomass extract inhibited the growth of P. syringae pv. coronafaciens 9060, P. corrugatа 9070, X. anoxopodis pv. glycines 9075, X. anoxopodis pv. glycines 8609 and Pantoea agglomerans 8490. Tolerant to metabolites of S. netropsis IMV Ac-5025 were P. syringae pv. atrofaciens 8291 and X. visicatoriae 7790. The extract of biomass S. netropsis IMV Ac-5025 inhibited growth of all studied strains of phytopathogenic fungi (A. alternata 16814, A. culmorum 00790, F. оxysporum 54201, F. tricinetum 00795, F. oxysporum n.33, Cladosporium herbarum 16863, Cochliobolus spicifas 16860, Nigrospora oryzae 16864). The supernatant of the cultural liquid also showed the oppressing action on fungi, except for Cladosporium herbarum 16863 and Cochliobolus spicifas 16860. The strain was almost ineffective against opportunistic human microorganisms (Escherichia coli ATCC 25922, Bacillus pumilus NCTC 8241, Staphylococcus aureus FDA 209P et al.). The lack of action of Streptomyces netropsis IMV Ac-5025 on the opportunistic human microorganisms and the active antagonism of phytopathogens, both, define potential its application for plant protection.

Meloidogyne incognita에 살선충활성을 보이는 신규 Streptomyces netropsis의 살선충 특성 규명

Meloidogyne incognita에 살선충활성을 보이는 신규 Streptomyces netropsis의 살선충 특성 규명

Crucial factor for increasing the conjugation frequency in Streptomyces netropsis SD-07 and other strains.

Streptomyces netropsis SD-07, the producer of novel polyene macrolide antifungal antibiotics, was isolated from soil. For the investigation of the functions of its biosynthesis genes and regulation mechanisms, a genetic operating system is necessary. In this study, we successfully transferred the plasmid DNA of pSET152 from the methylation deficient donor, Escherichia coli ET12567/pSET152/pUZ8002, to S. netropsis SD-07 by conjugation and evaluated the crucial factors influencing the conjugation frequency. Ca(2+) ions in presence the conjugation media may increase the conjugation frequency by 1000-10 000 times than Ca(2+) ions absence in the same conjugation media, and 10-100 time higher than Mg(2+) ions. Similar results (increasing the conjugation frequency by 10-100 times when media containing 60 mM CaCl2 ) were also obtained from the conjugation between E. coli ET12567 and Streptomyces coelicolor, S. lavendulae, S. venezuelae, despite their conjugation media were different (MS, CM, GS). So, CaCl2 concentration is a crucial factor for increasing the conjugation frequency, and the suitable concentration may probably be 60 mM. In addition, synthetic medium containing a small amount of organic nitrogen source may benefit increasing the conjugation frequency. These findings could be valuable for the development of a practical method for achieving conjugation in other Streptomyces spp.

Taxonomic re-evaluation of whorl-forming Streptomyces (formerly Streptoverticillium) species by using phenotypes, DNA-DNA hybridization and sequences of gyrB, and proposal of Streptomyces luteireticuli (ex Katoh and Arai 1957) corrig., sp. nov., nom. rev.

The taxonomic status of 64 strains of whorl-forming Streptomyces (formerly Streptoverticillium) species was re-evaluated and strains were reclassified on the basis of their phenotypes, DNA-DNA hybridization data and partial sequences of gyrB, the structural gene of the B subunit of DNA gyrase. These strains, which consisted of 46 species and eight subspecies with validly published names and 13 species whose names have not been validly published [including 10 strains examined by the International Streptomyces Project (ISP)], were divided into two groups, namely typical and atypical whorl-forming Streptomyces species, based on their phenotypes and gyrB gene sequences. The typical whorl-forming species (59 strains) were divided into six major clusters of three or more species, seven minor clusters of two species and five single-member clusters, based on the threshold value of 97 % gyrB sequence similarity. Major clusters were typified by Streptomyces abikoensis, Streptomyces cinnamoneus, Streptomyces distallicus, Streptomyces griseocarneus, Streptomyces hiroshimensis and Streptomyces netropsis. Phenotypically, members of each cluster resembled each other closely except for the S. distallicus cluster, which was divided phenotypically into the S. distallicus and Streptomyces stramineus subclusters, and the S. netropsis cluster, which was divided into the S. netropsis and Streptomyces eurocidicus subclusters. Strains in each minor cluster closely resembled each other phenotypically. DNA-DNA relatedness between the representative species and others in each major cluster and/or subcluster, and between strains in the minor clusters, was >70 %, indicating that the major clusters and/or subclusters and the minor clusters each comprise a single species. It was concluded that 59 strains of typical whorl-forming Streptomyces species consisted of the following 18 species, including subjective synonym(s): S. abikoensis, Streptomyces ardus, Streptomyces blastmyceticus, S. cinnamoneus, S. eurocidicus, S. griseocarneus, S. hiroshimensis, Streptomyces lilacinus, 'Streptomyces luteoreticuli', Streptomyces luteosporeus, Streptomyces mashuensis, Streptomyces mobaraensis, Streptomyces morookaense, S. netropsis, Streptomyces orinoci, S. stramineus, Streptomyces thioluteus and Streptomyces viridiflavus.

Expression of the SpcN Gene from Streptomyces Flavopersicus in Escherichia Coli and Determination of the Gene Function

ABSTRACTThe gene spcN from Straptomyces flavopersicus cloned and sequenced earlier was further haracterized. E. coli expression vector from the pET system was used for overexpression of the protein and characterization of the gene product. SpcN gene was derived as Ndel-HindIII fragment from the plasmid pJOE2686 where it has been cloned earlier. After few subcloning steps the spcN gene was inserted into pET15b as a Ndel-Xhol fragment. The vector containing the target gene was transformed into the expression host E. coli BL21(pLysS). After the induction of the lac promoter by addition of IPTG the expression of the target gene was assessed by analysis of total cell protein on an SDS-polyacrylamide gel followed by Coomassie blue staining. A 30–36 kDa protein was identified which was further purified using His-Tag System and Thrombin cleavage. An enzymic assay confirmed that the gene product of spcN is an ATP-dependent amynoglycoside phosphotransferase.

A spectinomycin resistance determinant from the spectinomycin producer Streptomyces flavopersicus.

The spectinomycin (sp) resistance determinant from Streptomyces flavopersicus was cloned into Streptomyces lividans using the plasmid vector pIJ699. A plasmid, pDGL15, with a 3.65 kb insert from S. flavopersicus conferring resistance to Sp was isolated. DNA sequence analysis of the 3651 1 bp DNA insert revealed four open reading frames (ORFs). The amino acid sequence deduced from one ORF (SpcN) showed a high degree of similarity to an aminoglycoside phosphotransferase (StrN) and from a second one (SpcR) to a regulatory protein (StrR) of the streptomycin biosynthesis gene cluster from S. griseus. The two other ORFs were incomplete and the deduced amino acid sequences showed similarities to an amidinotransferase encoded in the streptomycin biosynthesis gene cluster of S. griseus and to the transposase of IS112, respectively. Expression of the spcN gene in E. coli under the control of tac promoter conferred Sp resistance to the cells. An enzymic assay confirmed that the gene product of spcN is an ATP-dependent aminoglycoside phosphotransferase which phosphorylates Sp and actinamine, the aminocyclitol moiety of Sp.

Studies on the possibility for genetic recombination in Streptomyces flavopersicus.

Genetic recombination has been shown to occur by conventional crosses in the spectinomycin-producing strain Streptomyces flavopersicus. Five auxotrophic markers were arranged on the rudimentary genetic map of S. flavopersicus by haploid recombinant analysis.