Streptomyces are a genus of ubiquitous soil bacteria from which the majority of clinically utilized antibiotics derive
. The production of these antibacterial molecules reflects the relentless ...competition Streptomyces engage in with other bacteria, including other Streptomyces species
. Here we show that in addition to small-molecule antibiotics, Streptomyces produce and secrete antibacterial protein complexes that feature a large, degenerate repeat-containing polymorphic toxin protein. A cryo-electron microscopy structure of these particles reveals an extended stalk topped by a ringed crown comprising the toxin repeats scaffolding five lectin-tipped spokes, which led us to name them umbrella particles. Streptomyces coelicolor encodes three umbrella particles with distinct toxin and lectin composition. Notably, supernatant containing these toxins specifically and potently inhibits the growth of select Streptomyces species from among a diverse collection of bacteria screened. For one target, Streptomyces griseus, inhibition relies on a single toxin and that intoxication manifests as rapid cessation of vegetative hyphal growth. Our data show that Streptomyces umbrella particles mediate competition among vegetative mycelia of related species, a function distinct from small-molecule antibiotics, which are produced at the onset of reproductive growth and act broadly
. Sequence analyses suggest that this role of umbrella particles extends beyond Streptomyces, as we identified umbrella loci in nearly 1,000 species across Actinobacteria.
Abstract
Cancer may be recognized as non-self by antibiotics such as minor groove binders, which show self / non-self recognition partially due to preferential DNA sequence recognition and ...distinguish from other non-self bacteria. We learned minor groove binders produced from Streptomyces and synthesized Pyrrole-Imidazole polyamide indol-seco CBI conjugate to alkylate specific sites in the cancer genome.
Although a tremendous amount of studies has been made to directly target oncogenic drivers, such as RAS and MYC, yet no drug is clinically available because of difficulties to develop RAS- or Myc-targeted anti-cancer therapeutics due to the smooth 3D surface topology. One of major limitations of targeting the RAS pathway may be intrinsic or acquired resistance as seen in the other molecular target therapy. New approaches that directly target driver genes may provide a more direct route to helps address unmet medical needs for refractory cancer conquest.
We therefore synthesized several Pyrrole-Imidazole polyamide conjugates, each of which specifically alkylated KRAS codon 12 mutant DNA (G12D or G12V), amplified MYCN or mutated DNA of PI3K E542K mutation. All three conjugates reduced expression of the mutated oncogenic-protein by RNA transcription inhibition and induced cancer cell death at low dose (1 to 50 nM). Low dose tail vein injections of conjugates-targeting KRS or MYCN also demonstrated significant anti-tumor effects on xenograft models of human tumors harboring oncogenic mutated driver with minimum host toxicity, but not in xenografts harboring wild type or non-recognized mutations. We also performed a series of biological searches for toxicities by applying Modified SHIRPA (behavioral and functional analysis of mouse phenotype) to test any pathological phenotypes and examinations of blood and urine in ICR mice. Modified SHIRPA screening, blood chemistry, blood cell analysis and urea tests exhibited no toxicologically significant changes. Additionally, we examine pharmacokinetics of PI polyamide conjugates In vivo using LC-mass and fluorescent imaging of tumor-bearing mice. Intriguingly, 48 hours after the administration the highest fluorescence intensity was observed in the tumor-cell nuclear and almost no fluorescent intensity in all other organs, tissues and cells. These data suggest that sequence-dependent alkylating approach using antibiotic mimics of alkylating PI-polyamide conjugates, may open a new strategy not only targeting point mutation of driver oncogene but also targeting key driver gene in the cancer amplicon. This approach should be used for future ‘ Precision cancer medicine’.
Citation Format: Hiroki Nagase, Kiriko Hiraoka, Takahiro Inoue, Hiroyuki Yoda, Krishnamurthy Sakthisri, Jason Lin, Takayoshi Watanabe, Nobuko Koshikawa, Atsushi Takatori. Mutated cancer cell-specific cell death activity of alkylating Pyrrole-Imidazole polyamide conjugates targeting a variety of oncogenic driver gene mutations. abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3775.
The main aim of the current study is to explore the bioactive potential of Streptomyces sp. VITJS8 isolated from the marine saltern. The cultural, biochemical, and morphological studies were ...performed to acquire the characteristic features of the potent isolate VITJS8. The 16Sr DNA sequencing was performed to investigate the phylogenetic relationship between the Streptomyces genera. The structure of the compound was elucidated by gas chromatography-mass spectrometry (GC-MS), infra-red (IR), and ultra-violet (UV) spectroscopic data analysis. The GC-MS showed the retention time at 22.39 with a single peak indicating the purity of the active compound, and the molecular formula was established as C sub(14)H sub(9)ONCl sub(2 ) based on the peak at m/z 277 M super(+). Furthermore, separated by high-performance liquid chromatography (HPLC), their retention time (t sub(r)) 2.761 was observed with the absorption maxima at 310 nm. The active compound showed effective inhibitory potential against four clinical pathogens at 500 mu g/mL. The antioxidant activity was found effective at the IC sub(50) value of 500 mu g/mL with 90 % inhibition. The 3-(4,5-dimethylthiazol-2-yl)-2,5-ditetrazolium bromide (MTT) assay revealed the cytotoxicity against HepG2 cells at IC sub(50) of 250 mu g/mL. The progression of apoptosis was evidenced by morphological changes by nuclear staining. The DNA fragmentation pattern was observed at 250 mu g/mL concentration. Based on flow cytometric analysis, it was evident that the compound was effective in inhibiting the sub-G0/G1 phase of cell cycle. The in vitro findings were also supported by the binding mode molecular docking studies. The active compound revealed minimum binding energy of -7.84 and showed good affinity towards the active region of topoisomerase-2 alpha that could be considered as a suitable inhibitor. Lastly, we performed 30 ns molecular dynamic simulation analysis using GROMACS to aid in better designing of anticancer drugs. Simulation result of root mean square deviation (RMSD) analysis showed that protein-ligand complex reaches equilibration state around 10 ns that illustrates the docked complex is stable. We propose the possible mechanism of sesquiterpenes to play a significant role in antitumor cascade. Hence, our studies open up a new facet for a potent drug as an anticancer agent.
Laccases (EC 1.10.3.2) are multicopper oxidases that can oxidize a range of substrates, including phenols, aromatic amines, and nonphenolic substrates. To investigate the involvement of the small ...Streptomyces laccases in lignin degradation, we generated acid-precipitable polymeric lignin obtained in the presence of wild-type Streptomyces coelicolor A3(2) (SCWT) and its laccase-less mutant (SCΔLAC) in the presence of Miscanthus x giganteus lignocellulose. The results showed that strain SCΔLAC was inefficient in degrading lignin compared to strain SCWT, thereby supporting the importance of laccase for lignin degradation by S. coelicolor A3(2). We also studied the lignin degradation activity of laccases from S. coelicolor A3(2), Streptomyces lividans TK24, Streptomyces viridosporus T7A, and Amycolatopsis sp. 75iv2 using both lignin model compounds and ethanosolv lignin. All four laccases degraded a phenolic model compound (LM-OH) but were able to oxidize a nonphenolic model compound only in the presence of redox mediators. Their activities are highest at pH 8.0 with a low k rel/K app for LM-OH, suggesting that the enzymes’ natural substrates must be different in shape or chemical nature. Crystal structures of the laccases from S. viridosporus T7A (SVLAC) and Amycolatopsis sp. 75iv2 were determined both with and without bound substrate. This is the first report of a crystal structure for any laccase bound to a nonphenolic β-O-4 lignin model compound. An additional zinc metal binding site in SVLAC was also identified. The ability to oxidize and/or rearrange ethanosolv lignin provides further evidence of the utility of laccase activity for lignin degradation and/or modification.
Background: Bioactive metabolites that inhibit cellular migration are considered to be useful for the suppression of cancer metastasis. Then, we have looked for cancer cell migration inhibitors from ...microorganisms. In one hand, ovarian carcinoma is highly invasive and often metastasizes into liver, lung, and peritoneal cavity.Materials and methods: Cellular migration was assessed by wound healing assay with breast carcinoma MDA-MB-231 cells. We employed about 1500 microbial broths for random screening. From the culture filtrate with positive activity, active principle was isolated by chromatography. The structure was determined mainly by NMR and mass spectroscopy after the purification.Results: After testing the thousands of microbial culture filtrates, we have isolated novel compounds migracin A and B from Streptomyces. Migracin A and B showed similar inhibitory activity on the migration of MDA-MB-231 cells and fibrosarcoma HT-1080 cells. They also inhibited TNF-alpha and TGF-beta-induced migration of lung adenocarcinoma A549 cells. Migracin A inhibited the Matrigel invasion of clear cell ovarian carcinoma ES-2 cells without any toxicity. Since the structure of migracin is related to that of luminacin that inhibits tube formation, we have studied the effect on lumina formation. As a result, migracin A inhibited VEGF-induced limina formation in HUVEC. The mechanism of inhibition is being studied.Conclusions: We have discovered migracins as novel inhibitors of cellular migration and invasion. Migracin inhibited invasion of ovarian carcinoma cells, and is considered to be a candidate of new metastasis inhibitor.
The roles of many sigma factors are unclear in regulatory mechanism of secondary metabolism in Streptomyces. Here, we report the regulation network of a group 3 sigma factor, WhiG sub(ch), from a ...natamycin industrial strain Streptomyces chattanoogensis L10. WhiG sub(ch) regulates the growth and morphological differentiation of S. chattanoogensis L10. The whiG sub(ch) deletion mutant decreased natamycin production by about 30 % and delayed natamycin production more than 24 h by delaying the growth. Overexpression of the whiG sub(ch) gene increased natamycin production in large scale production medium by about 26 %. WhiG sub(ch) upregulated the transcription of natamycin biosynthetic gene cluster and inhibited the expression of migrastatin and jadomycin analog biosynthetic polyketide synthase genes. WhiG sub(ch) positively regulated natamycin biosynthetic gene cluster by directly binding to the promoters of scnC and scnD, which were involved in natamycin biosynthesis, and these binding sites adjacent to translation start codon were determined. Thus, this paper further elucidates the high natamycin yield mechanisms of industrial strains and demonstrates that a valuable improvement in the yield of the target metabolites can be achieved through manipulating the transcription regulators.
Aims
To evaluate the potential of chitinolytic endophytic Actinomycetes isolated from medicinal plants in order to diminish the collar rot infestation induced by Sclerotium rolfsii in chickpea.
...Methods and Results
Sixty‐eight chitinolytic endophytic Actinomycetes were recovered from various medicinal plants and evaluated for their chitinase activity. Among these isolates, 12 were screened for their plant growth promoting abilities and antagonistic potential against Sc. rolfsii. Further, these isolates were validated in vivo for their ability to protect chickpea against Sc. rolfsii infestation under greenhouse conditions. The isolates significantly (P < 0·05) increased the biomass (1·2–2·0 fold) and reduced plant mortality (42–75%) of chickpea. On the basis of 16S rDNA profiling, the selected antagonistic strains were identified as Streptomyces diastaticus, Streptomyces fradiae, Streptomyces olivochromogenes, Streptomyces collinus, Streptomyces ossamyceticus and Streptomyces griseus.
Conclusion
This study is the first report of the isolation of endophytic Actinomycetes from various medicinal plants having antagonistic and plant growth promoting abilities. The isolated species showed potential for controlling collar rot disease on chickpea and could be useful in integrated control against diverse soil borne plant pathogens.
Significance and Impact of the Study
Our investigation suggests that endophytic Actinomycetes associated with medicinal plants can be used as bioinoculants for developing safe, efficacious and environment‐friendly biocontrol strategies in the near future.
Abstract
Despite their importance in the chemical industry, the significance of rare earths in biology has been largely overlooked. Here, it is reported that the rare earth, scandium (Sc), causes ...antibiotic overproduction by 2–25-fold when added at a low concentration (10–100 µM) to cultures of Streptomyces coelicolor A3(2) (actinorhodin producer), Streptomyces antibioticus (actinomycin producer), and Streptomyces griseus (streptomycin producer). Not just for enhancement of antibiotic production, scandium was also effective in activating the dormant ability to produce actinorhodin in Streptomyces lividans. The effects of scandium were exerted at the level of transcription of pathway-specific positive regulatory genes, as demonstrated by marked up-regulation of actII-ORF4 in S. coelicolor cells exposed to this element. The bacterial alarmone, guanosine 5′-diphosphate 3′-diphosphate, was essential for actinorhodin overproduction provoked by scandium.
Cellulases are hydrolases of great importance to industries, especially due to their ability to produce ethanol via hydrolysis of cellulolytic materials. Actinomycetes are the producers of these ...enzymes, particularly the genus Streptomyces sp. The present study is the first report on the production and characterization of cellulolytic complex secreted by Streptomyces capoamus, isolated from the rhizosphere soil of Caatinga. In selecting the microbial producers of cellulolytic complex in qualitative tests, 171x microorganism showed the most expressive enzymatic index. Regarding the production time of the complex, fermentation was done for 7 days, with aliquots being taken every 24 h. Peak production was obtained during 48 h fermentation. It was done at 37 super(o)C and under an agitation of 180 rpm. It was noted also that the 171x micro-organism produced the enzyme in greater quantity. The experiment was done with the most significant actinomycetes (171x), optimal substrate concentration (carboximeticellulose), cultivation temperature and pH of initial output. The results showed that a higher cellulolytic complex was obtained with 2% substrate, 45 degree C temperature and initial pH 4.0. The microorganism was identified at genus level by microculture method; and with molecular identification method, it was identified as S. capoamus UFPEDA-3410. In optimal culture conditions, this strain produced 0.309 U/mL cellulose, a good production for a thermostable endoglucanase stable in a broad range of pH and stable temperature. It has potential applications in a wide range of industries. Industrial processes are generally carried out at elevated temperatures. Therefore enzymes with a high optima temperature and stability are desired for such applications.
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