Streptomyces Atratus Research Articles

Genome-Driven Discovery of Antiviral Atralabdans A-C from the Soil-Dwelling Streptomyces atratus.

Heterologous expression of an atr terpenoid gene cluster derived from Streptomyces atratus Gö66 in S. albus J1074 led to the discovery of three novel labdane diterpenoids featuring an unprecedented 6/6/5-fused tricyclic skeleton, designated as atralabdans A-C (1-3), along with a known compound, labdanmycin A. Compounds 1-3 were identified through extensive spectroscopic analysis, including NMR calculations with DP4+ probability analysis, and a comparative assessment of experimental and theoretical electronic circular dichroism (ECD) spectra. A plausible biosynthetic pathway for these compounds was proposed. Compounds 1-3 exhibited inhibitory activity against the human neurotropic coxsackievirus B3 (CVB3); 1 was the most potent, surpassing the positive control ribavirin with a higher therapeutic index.

Efficient ilamycins production utilizing Enteromorpha prolifera by metabolically engineered Streptomyces atratus

With the invasion of green tides and the increase of urban green areas worldwide, multimillion tons of Enteromorpha need to be reutilized. In this study, Enteromorpha prolifera powder is considered a promising biomass resource for the production of commercial chemical products production. Ilamycins, novel cyclic heptapeptides with significant anti-TB activities, are isolated from Streptomyces atratus SCSIO ZH16, a deep-sea-derived strain. Using EP powder as a nitrogen source, the production of ilamycins reached 709.97 mg/L through optimization of the nitrogen source using the engineered strain S. atratus SCSIO ZH16 ΔR. After mutant strain constructions and tests, strain S. atratus SCSIO ZH16 ΔR::bldD EP powder achieved a higher production titer of ilamycins. Furthermore, the production titer of ilamycins and ilamycin E reached 1561.77 mg/L and 745.44 mg/L, respectively, in a 5 L bioreactor. This study suggests that E. prolifera is a promising and eco-friendly nitrogen source for the production of ilamycins.

Genome Resource of Streptomyces atratus PY-1, a Broad-Spectrum Antimicrobial Strain in Particular Antagonistic Against Plasmopara viticola.

Streptomyces atratus PY-1 exhibited promising antimicrobial properties; in particular, it is highly inhibitory to Plasmopara viticola, which causes downy mildew of grape. It is very necessary to carry out systematic and in-depth research on the PY-1 strain for the improvement, application, and promotion of biocontrol agents. The PY-1 genome was fully sequenced and assembled. We present the draft genome sequence of PY-1, with a size of 9, 254, and 781 bp. Preliminary analysis on the PY-1 genome sequence shows that at least 35 gene clusters are involved in the biosynthesis of polyketides, terpenes, and nonribosomally synthesized peptides.

Production of copropophyrin III, biliverdin and bilirubin by the rufomycin producer, Streptomyces atratus.

Heme is best known for its role as a versatile prosthetic group in prokaryotic and eukaryotic proteins with diverse biological functions including gas and electron transport, as well as a wide array of redox chemistry. However, free heme and related tetrapyrroles also have important roles in the cell. In several bacterial strains, heme biosynthetic precursors and degradation products have been proposed to function as signaling molecules, ion chelators, antioxidants and photoprotectants. While the uptake and degradation of heme by bacterial pathogens is well studied, less is understood about the physiological role of these processes and their products in non-pathogenic bacteria. Streptomyces are slow growing soil bacteria known for their extraordinary capacity to produce complex secondary metabolites, particularly many clinically used antibiotics. Here we report the unambiguous identification of three tetrapyrrole metabolites from heme metabolism, coproporphyrin III, biliverdin and bilirubin, in culture extracts of the rufomycin antibiotic producing Streptomyces atratus DSM41673. We propose that biliverdin and bilirubin may combat oxidative stress induced by nitric oxide production during rufomycin biosynthesis, and indicate the genes involved in their production. This is, to our knowledge, the first report of the production of all three of these tetrapyrroles by a Streptomycete.

Rhizospheric Actinomycetes Revealed Antifungal and Plant-Growth-Promoting Activities under Controlled Environment.

Actinomycetes has large habitats and can be isolated from terrestrial soil, rhizospheres of plant roots, and marine sediments. Actinomycetes produce several bioactive secondary metabolites with antibacterial, antifungal, and antiviral properties. In this study, some Actinomycetes strains were isolated from the rhizosphere zone of four different plant species: rosemary, acacia, strawberry, and olive. The antagonistic activity of all isolates was screened in vitro against Escherichia coli and Bacillus megaterium. Isolates with the strongest bioactivity potential were selected and molecularly identified as Streptomyces sp., Streptomyces atratus, and Arthrobacter humicola. The growth-promoting activity of the selected Actinomycetes isolates was in vivo evaluated on tomato plants and for disease control against Sclerotinia sclerotiorum. The results demonstrated that all bacterized plants with the studied Actinomycetes isolates were able to promote the tomato seedlings’ growth, showing high values of ecophysiological parameters. In particular, the bacterized seedlings with Streptomyces sp. and A. humicola showed low disease incidence of S. sclerotiorum infection (0.3% and 0.2%, respectively), whereas those bacterized with S. atratus showed a moderate disease incidence (7.6%) compared with the positive control (36.8%). In addition, the ability of the studied Actinomycetes to produce extracellular hydrolytic enzymes was verified. The results showed that A. humicola was able to produce chitinase, glucanase, and protease, whereas Streptomyces sp. and S. atratus produced amylase and pectinase at high and moderate levels, respectively. This study highlights the value of the studied isolates in providing bioactive metabolites and extracellular hydrolytic enzymes, indicating their potential application as fungal-biocontrol agents.

Medium optimization and subsequent fermentative regulation enabled the scaled-up production of anti-tuberculosis drug leads ilamycin-E1/E2.

Tuberculosis (TB) and its evolving drug resistance have exerted severe threats on the global health, hence it is still essential to develop novel anti-TB antibiotics. Ilamycin-E1/E2 is a pair of cycloheptapeptide enantiomers obtained from a marine Streptomyces atratus SCSIO ZH16-ΔilaR mutant, and has presented significant anti-TB activities as promising drug lead compounds, but their clinical development has been hampered by low fermentation titers. By applying the statistical Plackett-Burman design (PBD) model, bacterial peptone was first screened out as the only significant but negative factor to affect the ilamycin-E1/E2 production. Subsequent single factor optimization in shaking flasks revealed that the replacement of bacterial peptone with malt extract could not only eliminate the accumulation of porphyrin-type competitive byproducts but also improve the titer of ilamycin-E1/E2 from original 13.6±0.8 to 142.7±5.7mgL-1 , about 10.5-fold increase. Next, a pH coordinated feeding strategy was adopted in 30L fermentor and obtained 169.8±2.5mgL-1 ilamycin-E1/E2, but further scaled-up production in 300L fermentor only gave a titer of 131.5±7.5mgL-1 due to the unsynchronization of feeding response and pH change. Consequently, a continuous pulse feeding strategy was utilized in 300L fermentor to solve the above problem and finally achieved 415.7±29.2mgL-1 ilamycin-E1/E2, representing a 30.5-fold improvement. Our work has provided a solid basis to acquire sufficient ilamycin-E1/E2 lead compounds and then support their potential anti-TB drug development.

The diversity of bacterial endophytes from Iris pseudacorus L. and their plant beneficial traits

This study reports the diversity of cultivable endophytic bacteria associated with yellow iris (Iris pseudacorus L.) by using 16S rRNA gene analysis and their plant beneficial traits. The 16S rRNA sequence similarities of endophytic bacteria isolated from the leaves and roots of yellow iris showed that the isolates belonged to the genera Staphylococcus, Streptomyces, Variovorax, Pantoea, Paenibacillus, Bacillus, Janthinobacterium, Enterobacter, Brevibacterium, Agrobacterium, Rhizobium, Xanthomonas translucens, and Pseudomonas. The endophytic bacteria Pseudomonas gessardii HRT18, Brevibacterium frigoritolerans HRT8, Streptomyces atratus HRT13, and Bacillus toyonensis HST13 exhibited antimicrobial activity against five plant pathogenic fungi Fusarium, Rhizoctonia, Botrytis, Pythium, and Alternaria. They also demonstrated the capability to produce chitinase, protease, glucanase, lipase, HCN, and indole-3-acetic acid (IAA). Thirteen isolates (46%) produced IAA, and the most active IAA producers were Bacillus cereus, Agrobacterium tumefaciens, Agrobacterium vitis, Bacillus megaterium, and Bacillus aryabhattai. The IAA producing bacterial isolates stimulated root and shoot growth of garden cress. Our findings suggest that medicinal plants could be a promising source for isolating plant-beneficial bacteria that can be used to enhance the growth and protect plants against soil-borne pathogens.

Bacterial Isolation Microwell-Plug (μWELLplug) for Rapid Antibiotic Susceptibility Testing Using Morphology Analysis.

The rapid and accurate diagnosis of infectious diseases with high morbidity rates is crucial because it can minimize the misuse and overuse of antibiotics and increase survival rates in dreadful conditions. The conventional antibiotic susceptibility test (AST) systems used to choose appropriate antibiotics require long wait times to obtain results and cannot prevent the misuse or overuse of antibiotics by clinicians who need to quickly treat patients and cannot wait to identify the underlying cause of their symptoms. Therefore, several rapid AST (rAST) methods have been developed to provide quick test results, but they are complicated to operate, require additional equipment or materials, and give less accurate results than the conventional AST methods. In this study, we propose an rAST method that can obtain precise outcomes from a simple process with a short running time using a bacterial isolation microwell-plug (μWELLplug) in a conventional 96-well plate. The specifically designed hydrogel component of the μWELLplug provides a simple process for cell isolation and the observation of bacterial growth and morphological changes induced by a variety of antibiotic concentrations. The μWELLplug is placed over each well of the 96-well plate, and then bacterial or eukaryotic cells are isolated in the microwells and treated with different antibiotic concentrations to observe their effects. Saccharomyces cerevisiae (yeast, eukaryote), Streptomyces atratus (actinomycetes, prokaryote), Escherichia coli, Staphylococcus aureus, and methicillin-resistant S. aureus were cultivated and tested using the μWELLplug. The minimum inhibitory concentration values from this system were obtained in 3-4 h and correlated well with those from the conventional AST methods whose running time is 18-24 h.

Characterization of Regulatory and Transporter Genes in the Biosynthesis of Anti-Tuberculosis Ilamycins and Production in a Heterologous Host.

Ilamycins are cyclopeptides with novel structures that have been isolated from different Actinomycetes. They showed strong anti-tuberculosis activity and could serve as important anti-tuberculosis drug leads. The functions of the pre-tailoring and the post-tailoring genes in the biosynthesis of ilamycins have been elucidated, but the functions of the regulatory and transporter genes remain elusive. We reported herein the functions of four genes in ilamycin biosynthetic gene cluster (ila BGC) including two regulatory genes (ilaA and ilaB) and two transporter genes (ilaJ and ilaK) and the heterologous expression of ila BGC. The IlaA and IlaB were unambiguously shown to be negative and positive regulator of ilamycins biosynthesis, respectively. Consistent with these roles, inactivation of ilaA and ilaB (independent of each other) was shown to enhance and abolish the production of ilamycins, respectively. Total yields of ilamycins were enhanced 3.0-fold and 1.9-fold by inactivation of ilaA and overexpression of ilaB compared to those of in the Streptomyces atratus SCSIO ZH16, respectively. In addition, the ila BGC was successfully expressed in Streptomyces coelicolor M1152, which indicated that all biosynthetic elements for the construction of ilamycins were included in the PAC7A6. These results not only lay a foundation for further exploration of ilamycins, but also provide the genetic elements for synthetic biology.

Antimycobacterial Rufomycin Analogues from Streptomyces atratus Strain MJM3502.

This study represents a systematic chemical and biological study of the rufomycin (RUF) class of cyclic heptapeptides, which our anti-TB drug discovery efforts have identified as potentially promising anti-TB agents that newly target the caseinolytic protein C1, ClpC1. Eight new RUF analogues, rufomycins NBZ1-NBZ8 (1-8), as well as five known peptides (9-13) were isolated and characterized from the Streptomyces atratus strain MJM3502. Advanced Marfey's and X-ray crystallographic analysis led to the assignment of the absolute configuration of the RUFs. Several isolates exhibited potent activity against both pathogens M. tuberculosis H37Rv and M. abscessus, paired with favorable selectivity (selectivity index >60), which collectively underscores the promise of the rufomycins as potential anti-TB drug leads.

Ilamycin E, a natural product of marine actinomycete, inhibits triple-negative breast cancer partially through ER stress-CHOP-Bcl-2.

Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death among women in the worldwide. Triple-negative breast cancer (TNBC) has a poor clinical outcome. The antitumor efficacy of Ilamycins, natural products with anti-tuberculosis activity isolated from deep sea-derived Streptomyces atratus, in TNBC has not been investigated, and the mechanisms remain elusive. Here, we demonstrated that Ilamycin-E, but not -F, decreases cell viability, inhibits G1/S cell cycle progression, and promotes apoptosis in the TNBC cell lines HCC1937 and MDA-MB-468. Ilamycin E promotes apoptosis via activation of endoplasmic reticulum (ER) stress, increasing the expression of CHOP, and down-regulating the expression of anti-apoptotic protein Bcl-2. Depletion of CHOP or overexpression of Bcl2 significantly rescued Ilamycin E-induced apoptosis. These findings indicate that Ilamycin E has anti-cancer activity in TNBC.

Two imide substances from a soil-isolated Streptomyces atratus strain provide effective biocontrol activity against grapevine downy mildew

ABSTRACTGrapevine downy mildew (Plasmopara viticola) is a devastating disease of grapevine. In this study, 151 actinomycete isolates were obtained and tested for antagonistic activity against P. viticola. The assay suggested that 28 isolates displayed antagonistic effects to varying degrees. The greatest reduction in disease severity was observed with isolate PY-1, which reduced disease severity by 92.13% in the detached leaf assay, and by 83% in a field assay. It was identified as Streptomyces atratus using the 16S rDNA sequence analysis. To elucidate the antagonistic mechanism of PY-1 against P. viticola, scanning electron microscopy showed that major damage to the pathogens sporangia and sporangiophores was observed after treatment for PY-1. Furthermore, PY-1 showed antagonistic activity against other pathogens, including: Botrytis cinerea, Colletotrichum orbiculare, Fusarium oxysporum, Phytophthora capsici and Phytophthora infestans. Two imide compounds were purified from the fermentation liquid using silica gel chromatography and high-performance liquid chromatography and identified as 5-acetoxycycloheximide and cycloheximide using nuclear magnetic resonance. Both compounds showed significant antagonistic activity against P. viticola, determining a reduction in disease severity by 65% and 84%, respectively. In conclusion, 5-acetoxycycloheximide and cycloheximide were identified for the first time in a new S. atratus strain able to effectively control grapevine downy mildew.

소나무재선충에 대한 방선균 균주의 살선충 및 증식억제 효과

Thirty-two actinomycetes isolates from Korean forest soil were screened for their nematicidal and reproduction suppression activity against pine wood nematode (PWN) which is widely spread in Korea. Culture filterates of 21 isolates showed more than 90% mortality at 2-fold concentration. Among them, AM210, SG16, YD116 and YD315 were more effective than others on reproduction of PWN. The YD116 isolate was identified as Streptomyces atratus by morphological and 16S rDNA analyses. Hydrazine hydrate, similar to hydrazidomycin which has cytotoxicity among substances from S. atratus against PWN, was tested for its nematicidal activity. Ten ppm of the hydrate showed 60.8% mortality. Additional studies are needed for practical use of the S. atratus YD116 isolate.

Impact of Matric Potential and Pore Size Distribution on Growth Dynamics of Filamentous and Non-Filamentous Soil Bacteria

The filamentous growth form is an important strategy for soil microbes to bridge air-filled pores in unsaturated soils. In particular, fungi perform better than bacteria in soils during drought, a property that has been ascribed to the hyphal growth form of fungi. However, it is unknown if, and to what extent, filamentous bacteria may also display similar advantages over non-filamentous bacteria in soils with low hydraulic connectivity. In addition to allowing for microbial interactions and competition across connected micro-sites, water films also facilitate the motility of non-filamentous bacteria. To examine these issues, we constructed and characterized a series of quartz sand microcosms differing in matric potential and pore size distribution and, consequently, in connection of micro-habitats via water films. Our sand microcosms were used to examine the individual and competitive responses of a filamentous bacterium (Streptomyces atratus) and a motile rod-shaped bacterium (Bacillus weihenstephanensis) to differences in pore sizes and matric potential. The Bacillus strain had an initial advantage in all sand microcosms, which could be attributed to its faster growth rate. At later stages of the incubation, Streptomyces became dominant in microcosms with low connectivity (coarse pores and dry conditions). These data, combined with information on bacterial motility (expansion potential) across a range of pore-size and moisture conditions, suggest that, like their much larger fungal counterparts, filamentous bacteria also use this growth form to facilitate growth and expansion under conditions of low hydraulic conductivity. The sand microcosm system developed and used in this study allowed for precise manipulation of hydraulic properties and pore size distribution, thereby providing a useful approach for future examinations of how these properties influence the composition, diversity and function of soil-borne microbial communities.

ChemInform Abstract: Hydrazidomycins, Cytotoxic Alkylhydrazides from Streptomyces atratus.

AbstractThree unusual alkylhydrazide natural products, named hydrazidomycin A (Ia), B (Ib), and C (Ic), are isolated from the chloroform extract of a Streptomyces atratus culture.

Screening of New Antibiotics Produced by Actinomycetes and Their Production.

I have studied actinomycetes and their antibiotics over thirty-eight years except eight years for which I had been engaged in the research on the quality control for sterilization of microorganisms. My colleagues and I were successful to discover twelve new species of actinomycetes and fourteen new group antibiotics. In this review, I would like to describe rufomycins, enduracidins, validamycins, maridomycins, T-2636 antibiotics and ansamitocins in terms of their discovery, producers and/or industrial development.Rufomycins (cyclic peptides) with a specific activity to mycobacteria was discovered from Streptomyces atratus, a new species, in our screening program targeting anti-tuberculosis antibiotics. Enduracidins (Enramycins; cyclic peptides with a fatty acid side chain) were screened as antibiotics with low toxicity and strong bactericidal spectrum including multiple drug -resistant streptococci. As to aminocyclitol antibiotics, validamycins, new assay methods (reversed layer method and dendroid-test method) were established in order to develop an industrial fermentation for their agricultural use. As to T-2636 antibiotics with macrolacton ring, the esterase catalyzing the transformation of T-2636 C to T-2636 A was identified in the producing organism, S. rochei subsp. volubillis and then the industrial enzymatic transformation was established. Maridomycins, new macrolide antibiotics, were discovered as a complex of about twenty components from S. hygroscopicus No. B-5050. Selective and improved production of maridomycin III was established by strain improvement by mutation on the basis of the regulation of amino acid metabolism involved in maridomycin biosynthesis. Ansamitocins, new antitumor antibiotics, were discovered form a new species of Actinosynnema and turned out to be similar in structure to maytansine with a strong mitosis-arrest activity from tropical plants.

Purification and N-terminal amino-acid sequences of bacterial malate dehydrogenases from six actinomycetales strains and from Phenylobacterium immobile, strain E.

Malate dehydrogenases from Streptosporangium roseum (DSM 43021), Planomonospora venezuelensis (DSM 43178), Microtetraspora glauca (ATCC 23057), Actinoplanes missouriensis (DSM 43046), Streptomyces atratus (ATCC 14046), Kibdelosporangium aridum (ATCC 39323), and from Phenylobacterium immobile, strain E (DSM 1986) were purified to homogeneity. The N-terminal amino-acid sequences were determined and compared with known prokaryotic and eukaryotic sequence data. The partial sequences from Actinomycetales enzymes include a string of amino acids which is also present in the N-terminal region of malate dehydrogenases from Thermus flavus and from mammalian cytoplasm.

Studies on Streptomycetes

Investigation was made on the mycological and antibacterial properties of a strain No. 46408 isolated from a soil sample collected in Shimoueda, Wakayama Prefecture. The strain No. 46408 was compared with similar strains, St. hygroscopicus and St. halstedii, and it was judged to belong to a new species and therefore named Streptomyces atratus nov. sp. Also a strain A-165-Z1, which produces ilamycin, was referred to on its mycological properties and assumed to resemble St. atratus. St. atratus was found to produce new antibiotics, rufomycin A and B, specially active against acid-fast bacteria. Investigation was made on the isolation procedure, physico-chemical properties and biological activities of antibiotic 46408 produced by Streptomyces atratus nov. sp. The solvent extract from the whole culture of this strain gave two antibiotic substances. Both are yellow, neutral and sparingly soluble in water and active against acid-fast bacteria, especially Mycobacterium tuberculosis and Mycobacterium smegmatis but inactive against most other bacteria, fungi and yeast examined. From the physico-chemical and microbiological properties they were found to be new substances and named rufomycin A and B, respectively. Recently Umezawa et al. reported on new antibiotics, ilamycins, active against acid- fast bacteria. The properties of rufomycins and ilamycins suggest that they really are very much like. Rufomycin A was nearly as effective as streptomycin in experimental tuberculosis in mice.

Studies on Streptomycetes

Investigation was made on the isolation procedure, physico-chemical properties and biological activities of antibiotic 46408 produced by Streptomyces atratus nov. sp. The solvent extract from the whole culture of this strain gave two antibiotic substances. Both are yellow, neutral and sparingly soluble in water and active against acid-fast bacteria, especially Mycobacterium tuberculosis and Mycobacterium smegmatis but inactive against most other bacteria, fungi and yeast examined.From the physico-chemical and microbiological properties they were found to be new substances and named rufomycin A and B, respectively.Recently Umezawa et al. reported on new antibiotics, ilamycins, active against acidfast bacteria. The properties of rufomycins and ilamycins suggest that they really are very much like. Rufomycin A was nearly as effective as streptomycin in experimental tuberculosis in mice.

Studies on Streptomycetes

Investigation was made on the mycological and antibacterial properties of a strain No.46408 isolated from a soil sample collected in Shimoueda, Wakayama Prefecture. The strain No.46408 was compared with similar strains, St. hygroscopicus and St. halstedii, and it was judged to belong to a new species and therefore named Streptomyces atratus nov. sp. Also a strain A-165-Z1, which produces ilamycin, was referred to on its mycological properties and assumed to resemble St. atratus. St. atratus was found to produce new antibiotics, rufomycin A and B, specially active against acid-fast bacteria.