Molecular Details of Actinomycin D-Treated MRSA Revealed via High-Dimensional Data.

Methicillin-resistant Staphylococcus aureus (MRSA) is highly concerning as a principal infection pathogen. The investigation of higher effective natural anti-MRSA agents from marine Streptomyces parvulus has led to the isolation of actinomycin D, that showed potential anti-MRSA activity with MIC and MBC values of 1 and 8 µg/mL, respectively. Proteomics-metabolomics analysis further demonstrated a total of 261 differential proteins and 144 differential metabolites induced by actinomycin D in MRSA, and the co-mapped correlation network of omics, indicated that actinomycin D induced the metabolism pathway of producing the antibiotic sensitivity in MRSA. Furthermore, the mRNA expression levels of the genes acnA, ebpS, clfA, icd, and gpmA related to the key differential proteins were down-regulated measured by qRT-PCR. Molecular docking predicted that actinomycin D was bound to the targets of the two key differential proteins AcnA and Icd by hydrogen bonds and interacted with multiple amino acid residues of the proteins. Thus, these findings will provide a basic understanding to further investigation of actinomycin D as a potential anti-MRSA agent.

Insights into Streptomyces spp. isolated from the rhizospheric soil of Panax notoginseng: isolation, antimicrobial activity and biosynthetic potential for polyketides and non-ribosomal peptides.

BACKGROUND: Streptomycetes from the rhizospheric soils are a rich resource of novel secondary metabolites with various biological activities. However, there is still little information related to the isolation, antimicrobial activity and biosynthetic potential for polyketide and non-ribosomal peptide discovery associated with the rhizospheric streptomycetes of Panax notoginseng. Thus, the aims of the present study are to (i) identify culturable streptomycetes from the rhizospheric soil of P. notoginseng by 16S rRNA gene, (ii) evaluate the antimicrobial activities of isolates and analyze the biosynthetic gene encoding polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) of isolates, (iii) detect the bioactive secondary metabolites from selected streptomycetes, (iv) study the influence of the selected isolate on the growth of P. notoginseng in the continuous cropping field. This study would provide a preliminary basis for the further discovery of the secondary metabolites from streptomycetes isolated from the rhizospheric soil of P. notoginseng and their further utilization for biocontrol of plants. RESULTS: A total of 42 strains representing 42 species of the genus Streptomyces were isolated from 12 rhizospheric soil samples in the cultivation field of P. notoginseng and were analyzed by 16S rRNA gene sequencing. Overall, 40 crude cell extracts out of 42 under two culture conditions showed antibacterial and antifungal activities. Also, the presence of biosynthesis genes encoding type I and II polyketide synthase (PKS I and PKS II) and nonribosomal peptide synthetases (NRPSs) in 42 strains were established. Based on characteristic chemical profiles screening by High Performance Liquid Chromatography-Diode Array Detector (HPLC-DAD), the secondary metabolite profiles of strain SYP-A7257 were evaluated by High Performance Liquid Chromatography-High Resolution Mass Spectrometry (HPLC-HRMS). Finally, four compounds actinomycin X2 (F1), fungichromin (F2), thailandin B (F7) and antifungalmycin (F8) were isolated from strain SYP-A7257 by using chromatography techniques, UV, HR-ESI-MS and NMR, and their antimicrobial activities against the test bacteria and fungus were also evaluated. In the farm experiments, Streptomyces sp. SYP-A7257 showed healthy growth promotion and survival rate improvement of P. notoginseng in the continuous cropping field. CONCLUSIONS: We demonstrated the P. notoginseng rhizospheric soil-derived Streptomyces spp. distribution and diversity with respect to their metabolic potential for polyketides and non-ribosomal peptides, as well as the presence of biosynthesis genes PKS I, PKS II and NRPSs. Our results showed that cultivatable Streptomyces isolates from the rhizospheric soils of P. notoginseng have the ability to produce bioactive secondary metabolites. The farm experiments suggested that the rhizospheric soil Streptomyces sp. SYP-A7257 may be a potential biological control agent for healthy growth promotion and survival rate improvement of P. notoginseng in the continuous cropping field.

Purification and characterization of actinomycins from Streptomyces strain M7 active against methicillin resistant Staphylococcus aureus and vancomycin resistant Enterococcus.

BACKGROUND: The increased rate of resistance among two highly concerned pathogens i.e. methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) necessitates the discovery of novel anti-MRSA and anti-VRE compounds. In microbial drug discovery, Streptomyces are well known source of two-thirds of natural antibiotics used clinically. Hence, screening of new strains of streptomycetes is the key step to get novel bioactive compounds with antimicrobial activity against drug resistant bacteria. RESULTS: In the present study, Streptomyces antibioticus strain M7, possessing potent antibacterial activity against different pathogenic bacteria, was isolated from rhizospheric soil of Stevia rebudiana. 16S rRNA sequence of M7 (1418 bp) showed 96.47-100% similarity with different Streptomyces spp. and the maximum similarity (100%) was observed with Streptomyces antibioticus NBRC 12838T (AB184184). Phylogenetic analysis using neighbor joining method further validated its similarity with Streptomyces antibioticus NBRC 12838 T (AB184184) as it formed clade with the latter and showed high boot strap value (99%). Antibacterial metabolites isolated from the fermentation broth were characterized using NMR, FT-IR and LC-MS as actinomycins V, X2 and D. The purified actinomycins exhibited potent antibacterial activities against test bacteria viz. B. subtilis, K. pneumoniae sub sp. pneumoniae, S. aureus, S. epidermidis, S. typhi, E. coli, MRSA and VRE. Among these actinomycins, actinomycin X2 was more effective as compared to actinomycins D and V. The minimum inhibitory concentration values of purified compounds against a set of test bacterial organisms viz. VRE, MRSA, E. coli (S1-LF), K. pneumoniae sub sp. pneumoniae and B. subtilis ranged between 1.95 and 31.25 µg/ml. CONCLUSIONS: This study demonstrates that actinomycins V, X2 and D produced by S. antibioticus strain M7 hold the potential to be used against multidrug resistant bacteria, particularly VRE and MRSA.

Novel actinomycin group compound from newly isolated Streptomyces sp. RAB12: isolation, characterization, and evaluation of antimicrobial potential.

Streptomyces sp. RAB12 having potential to produce novel actinomycin group compounds was isolated from soil samples collected from CSIR-Indian Institute of Chemical Technology, Hyderabad, India, garden premises using International Streptomycetes Project (ISP) protocols. The 16S rRNA sequence of the strain RAB12 exhibited identity with Streptomyces sp. 13647M and the sequence was deposited in NCBI under the accession number KY 203650 while the strain RAB12 was deposited in The Microbial Type Culture Collection and Gene Bank (MTCC) with accession number MTCC 12747. Cell-free extract of this novel strain revealed two bioactive principles viz., RSP 01 and RSP 02. HR-MS analysis indicated a molecular mass of 1269.61 and 1270.63 m/z g/mol for RSP 01 and RSP 02, respectively. Proton 1H, 13C NMR, 2D NMR and mass spectroscopy analysis revealed a similar fingerprint to that actinomycin D except for a peak at δH3.59 J (1H NMR) and δ 208.88 (13C NMR) for RSP 01 compound suggesting the presence of keto carbonyl at 5-oxo position on the proline moiety which is absent in actinomycin D. Purified RSP 02 depicted a similarity with RSP 01 except a peak in the 1H proton NMR at δH 3.81 J. HR-ESI mass spectra confirmed the molecular formulae for RSP 01 and RSP 02 as C62H84N12O17 and C62H86N12O17, respectively. Antimicrobial activity profile revealed higher antimicrobial activity against bacterial strains (Pseudomonas aeruginosa, Micrococcus luteus, Staphylococcus aureus, Salmonella typhi, and Bacillus subtilis) and Candida albicans compared to standard actinomycin D. MIC and MBC for RSP 01 were observed to be 0.0039 and 0.0078 (µg/ml) against C. albicans, while for actinomycin D, it was found to be 0.031 and 0.62 (µg/ml), respectively indicating a tenfold higher potency. Thus, these RSP 01 and RSP 02 compounds from Streptomyces sp. RAB12 may be promising candidates for industrial and clinical applications.

Antimicrobial investigation of selected soil actinomycetes isolated from unexplored regions of Kashmir Himalayas, India.

The aim of the present study was to isolate and evaluate the antimicrobial potential of soil actinomycetes of Kashmir Himalayas. The secondary metabolites of actinomycetes are the prominent source of antibiotics. A total of 121 morphologically different actinomycete strains were isolated and screened for antimicrobial activity against various human pathogens. The ethyl acetate extract of fermented broth an actinomycete strain, identified as Streptomyces pratensis exhibited significant antimicrobial activity against Staphylococcus aureus ATCC 29213 with MIC 0.25 µg/ml and Mycobacterium tuberculosis Strain H37Rv with MIC 0.062 µg/ml. The strain S. pratensis IIIM06 was grown on large scale and their broth was extracted with ethyl acetate. The extract was subjected to various chromatography techniques which led to the isolation of four compounds whose structures were established as actinomycin C1, actinomycin C2, actinomycin C3 and actiphenol on the basis of spectral data analysis. Actinomycin C1, C2 and C3 exhibited potent antimicrobial activity against S. aureus as well as M. tuberculosis. The isolated indigenous actinomycetes exhibited good antibacterial activity and the study reveals that IIIM06 is a promising strain and could be of great potential for industrial applications.

Antibacterial and Cytotoxic Actinomycins Y6-Y9 and Zp from Streptomyces sp. Strain Gö-GS12.

Four new Y-type actinomycin analogues named Y6-Y9 (1-4) were isolated and characterized from the scale-up fermentation of the Streptomyces sp. strain Gö-GS12, as well as actinomycin Zp (5), which was, for the first time, isolated as a natural product. Structures of the new compounds were elucidated by the cumulative analyses of NMR spectroscopy and HRMS. The 4-hydroxythreonine on the ß-ring of 1 uniquely undergoes both a rearrangement by a 2-fold acyl shift and an additional ring closure with the amino group of the phenoxazinone chromophore, and the α-rings of 4 and 5 contain a rare 5-methyl proline. Compounds 2-5 showed potent antibacterial activities against Gram-positive bacteria that correlated with cytotoxicity against representative human cell lines. The combination of a ß-ring rearrangement and additional ring closure in 1 rendered this actinomycin significantly less potent relative to the nonrearranged comparator actinomycin Y5 and other actinomycins.

Antifungal properties of an actinomycin D-producing strain, Streptomyces sp. IA1, isolated from a Saharan soil.

An actinomycete strain named IA1, which produced an antimicrobial compound, was isolated from a Saharan soil in In Amenas, Algeria. The study of the 16S rDNA sequence of this strain permitted to relate it to Streptomyces mutabilis NBRC 12800(T) (99.93% of similarity). Strain IA1 exhibited strong activity against a wide range of plant pathogenic fungi. One bioactive compound produced in large amounts (46.7 mg L(-1) day(-1) ), named YA, was isolated and purified by TLC and reverse phase HPLC. The structure elucidation of the pure substance, using combined data from UV visible, NMR spectra, and mass spectrometry, permitted to identify it as actinomycin D, and was thus found for the first time in S. mutabilis related species. The biocontrol abilities of the strain IA1 and compound YA were evaluated through two diseases, i.e., chocolate spot of field bean and Fusarium wilt of flax. The occurrence of the two fungal diseases was effectively reduced. The reduction of chocolate spot disease symptoms reached 80 and 91.7% with IA1 and YA seedlings pretreatments, respectively. Soil pretreatment with IA1 or YA also allowed to reduce Fusarium wilt disease impact by almost 60%.

Characterization of Streptomyces padanus JAU4234, a producer of actinomycin X2, fungichromin, and a new polyene macrolide antibiotic.

Strain JAU4234, identified as Streptomyces padanus, was isolated from soil collected in Jiangxi Province, China. It produced actinomycin X2, fungichromin, and a new polyene macrolide compound with antifungal activity, antifungalmycin 702. Antifungalmycin 702 had good general antifungal activity and may have potential future agricultural and/or clinical applications.

Isolation, characterization, anti-MRSA evaluation, and in-silico multi-target anti-microbial validations of actinomycin X2 and actinomycin D produced by novel Streptomyces smyrnaeus UKAQ_23.

Streptomyces smyrnaeus UKAQ_23, isolated from the mangrove-sediment, collected from Jubail,Saudi Arabia, exhibited substantial antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA), including non-MRSA Gram-positive test bacteria. The novel isolate, under laboratory-scale conditions, produced the highest yield (561.3 ± 0.3 mg/kg fermented agar) of antimicrobial compounds in modified ISP-4 agar at pH 6.5, temperature 35 °C, inoculum 5% v/w, agar 1.5% w/v, and an incubation period of 7 days. The two major compounds, K1 and K2, were isolated from fermented medium and identified as Actinomycin X2 and Actinomycin D, respectively, based on their structural analysis. The antimicrobial screening showed that Actinomycin X2 had the highest antimicrobial activity compared to Actinomycin D, and the actinomycins-mixture (X2:D, 1:1, w/w) against MRSA and non-MRSA Gram-positive test bacteria, at 5 µg/disc concentrations. The MIC of Actinomycin X2 ranged from 1.56-12.5 µg/ml for non-MRSA and 3.125-12.5 µg/ml for MRSA test bacteria. An in-silico molecular docking demonstrated isoleucyl tRNA synthetase as the most-favored antimicrobial protein target for both actinomycins, X2 and D, while the penicillin-binding protein-1a, was the least-favorable target-protein. In conclusion, Streptomyces smyrnaeus UKAQ_23 emerged as a promising source of Actinomycin X2 with the potential to be scaled up for industrial production, which could benefit the pharmaceutical industry.

Identification of actinomycin D as a specific inhibitor of the alternative pathway of peptidoglycan biosynthesis.

Peptidoglycan is an indispensable component of bacterial cell walls. We recently discovered an alternative peptidoglycan biosynthetic pathway, which involves two enzymes, MurD2 and MurL, catalyzing the ligation of L-Glu to UDP-MurNAc-L-Ala and epimerization of the terminal L-Glu of the MurD2 product, respectively. Because the pathway operates in Xanthomonas oryze, a pathogen causing bacterial blight of rice, we searched for specific inhibitors from metabolites produced by actinomycetes to obtain a lead compound to function as an agrochemical. Actinomycin D was isolated from Streptomyces parvulus NBRC 13193 as a specific inhibitor of the pathway. In vitro analysis indicated that actinomycin D inhibited the MurD2 reaction.

Actinobacteria from Antarctica as a source for anticancer discovery.

Although many advances have been achieved to treat aggressive tumours, cancer remains a leading cause of death and a public health problem worldwide. Among the main approaches for the discovery of new bioactive agents, the prospect of microbial secondary metabolites represents an effective source for the development of drug leads. In this study, we investigated the actinobacterial diversity associated with an endemic Antarctic species, Deschampsia antarctica, by integrated culture-dependent and culture-independent methods and acknowledged this niche as a reservoir of bioactive strains for the production of antitumour compounds. The 16S rRNA-based analysis showed the predominance of the Actinomycetales order, a well-known group of bioactive metabolite producers belonging to the Actinobacteria phylum. Cultivation techniques were applied, and 72 psychrotolerant Actinobacteria strains belonging to the genera Actinoplanes, Arthrobacter, Kribbella, Mycobacterium, Nocardia, Pilimelia, Pseudarthrobacter, Rhodococcus, Streptacidiphilus, Streptomyces and Tsukamurella were identified. The secondary metabolites were screened, and 17 isolates were identified as promising antitumour compound producers. However, the bio-guided assay showed a pronounced antiproliferative activity for the crude extracts of Streptomyces sp. CMAA 1527 and Streptomyces sp. CMAA 1653. The TGI and LC50 values revealed the potential of these natural products to control the proliferation of breast (MCF-7), glioblastoma (U251), lung/non-small (NCI-H460) and kidney (786-0) human cancer cell lines. Cinerubin B and actinomycin V were the predominant compounds identified in Streptomyces sp. CMAA 1527 and Streptomyces sp. CMAA 1653, respectively. Our results suggest that the rhizosphere of D. antarctica represents a prominent reservoir of bioactive actinobacteria strains and reveals it as an important environment for potential antitumour agents.

First Y-type actinomycins from Streptomyces with divergent structure-activity relationships for antibacterial and cytotoxic properties.

Streptomyces sp. strain Gö-GS12 was found to produce five novel actinomycins Y(1)-Y(5) (). Their amino acid pattern discloses them as members of a new family of this important class of antibiotics. Compounds differ from Z-type actinomycins in their beta-peptidolactone rings which here contain trans-4-hydroxyproline (Hyp) or 4-oxoproline (OPro) amino acids, and from the X-congeners by containing methylalanine (MeAla). Within the new Y-type actinomycins variations are not only in the rare chlorinated or hydroxylated threonine residue. Furthermore, the beta-ring can undergo rearrangement by a two-fold acyl shift (compounds and ) or show a unique additional ring closure with the chromophore (compound ), resulting in metabolites with yet unknown structural motifs, altered conformations and distinct bioactivities. The strongest bioactivity was found for the chlorine containing actinomycin Y(1) (), the most surprising for Y(5) () with cytotoxic and antibacterial effects losing their coherence, which has been observed for the first time here.

Antifungal effects of actinomycin D on Verticillium dahliae via a membrane-splitting mechanism.

Antifungal bioassays led to the isolation of actinomycins D and A1 from Streptomyces luteus TRM45540 collected from Norpo in Xinjiang, and these compounds were identified by nuclear magnetic resonance spectroscopy. The antifungal activity of actinomycin D was higher than that of actinomycin A1. Actinomycin D clearly inhibited the spore germination, hyphal growth and biomass accumulation of Verticillium dahliae in a dose-dependent manner. Flow cytometric analysis with propidium iodide, total ergosterol measurement, cell leakage and scanning electron microscopy experiments demonstrated that the plasma membrane of this fungus was damaged by actinomycin D, resulting in swollen cells and cellular content leakage. Transmission electron microscopy revealed that parts of the plasma membrane infolded after being treated with actinomycin D. The antifungal activity of actinomycin D damaged the fungal plasma membrane of V. dahliae via a membrane-splitting mechanism, which provided new insights into the functional mechanism of actinomycin D.

Cytotoxic antibiotic angucyclines and actinomycins from the Streptomyces sp. XZHG99T.

Three new angucycline-type C-glycosides, grincamycins L-N (1-3), together with the known rabelomycin (4), moromycin B (5), fridamycin D (6), saquayamycin B1 (7), actinomycin X2 (8), and actinomycin D (9), were isolated from the culture of the soil-derived Streptomyces sp. XZHG99T collected from Color desert, Dengpa District, Tibet. The structures of 1-3 were established by detailed analyses of comprehensive spectroscopic data. Compounds 1-9 exhibited significant cytotoxicity against a panel of human cancer cell lines A549, H157, MCF7, MDA-MB-231, and HepG2, while 4, 8, and 9 showed decent antibacterial activity against Mycobacterium smegmatis and Staphylococcus aureus.

Neo-actinomycins A and B, natural actinomycins bearing the 5H-oxazolo[4,5-b]phenoxazine chromophore, from the marine-derived Streptomyces sp. IMB094.

Neo-actinomycins A and B (1 and 2), two new natural actinomycins featuring an unprecedented tetracyclic 5H-oxazolo[4,5-b]phenoxazine chromophore, were isolated from the marine-derived actinomycete Streptomyces sp. IMB094. Their structures were elucidated by spectroscopic analyses. The presence of this ring system was proposed to originate from a condensation between actinomycin D (3) with α-ketoglutarate and pyruvate, respectively. Compound 1 showed potent cytotoxic activities against human cancer HCT116 and A549 cell lines in the nanomolar range (IC50: 38.7 and 65.8 nM, respectively) and moderate antibacterial activities against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) strains.

Actinomycins as proteinase inhibitors.

A novel actinomycin (Act SG3) from a strain of Streptomyces galbus var. C-72, as well as actinomycin D (Act D) were found to act as competitive inhibitors of serine proteinases from microorganisms. The inhibitory properties of Act SG3 and Act D are compared with these of other peptide antibiotics, namely bacitracin A (Bac A) and gramicidin S (Gr S). The last compound has only a weak inhibitory effect. The following order of affinity for the four peptide antibiotics towards subtilisin DY and proteinase K was observed: Bac A > Act D > Act SG3 = Gr S. The affinity towards thermitase changes as follows: Act SG3 = Act D > Bac A > Gr S.

Isolation and characterization of three novel polyether antibiotics and three novel actinomycins as cometabolites of the same Streptomyces sp. X-14873, ATCC 31679.

Streptomyces sp. X-14873 (ATCC 31679) has been found to produce a number of secondary metabolites. Three have been identified as the novel actinomycins, X-14873B, C and D, each of which contains both proline and 3-hydroxy-5-methylproline. Potentially, the most important microbial product from this fermentation is the novel polyether antibiotic X-14873A which differs from lysocellin only in the substituents at carbons C-4 and C-5 in the tetrahydropyranyl ring. The methyl group and proton in lysocellin are replaced by an ethyl and hydroxyl group, respectively in X-14873A. In addition, two other novel polyethers, X-14873H and G, were isolated and shown to differ from 1 in lacking a carboxyl group and in the case of 3, possessing an ether bridge across the terminal tetrahydrofuranyl ring system.

[Individual variability and an error in the method of determining protein thermostability]. / Individual'naia izmenchivost' i oshibka metoda opredeleniia teploustoichivosti belkov.

The individual variability of the heat resistance of two enzymes (aldolase and actomyosine) of the m. gastrochemius of the grass frog Rana temporaria has been quantitatively evaluated by means of analysis of variance. 37--41% of the total variability of the character may be due to errors in the methods of investigation, whereas 59--63% of the variability is due to phenotypic differences between individuals of the same population. The extent of error is primarily associated with the initial stages of the experiment, i. e. the isolation of the enzyme preparations. The individual variability of the heat resistance of proteins is lower than that of the cells or the organisms from which they are derived.

Occurrence and biosynthesis of C-demethylactinomycins in actinomycin-producing Streptomyces chrysomallus and Streptomyces parvulus.

Streptomyces chrysomallus and Streptomyces parvulus produce novel C-demethylactinomycins besides their normal actinomycins when fed with 3-hydroxyanthranilic acid (3-HA). The 3-HA is incorporated into pentapeptide lactone precursors in competition with the regular precursor 4-methyl-3-hydroxyanthranilic acid (4-MHA). The resultant 3-HA pentapeptide lactones can condense with each other, as well as with the continuously formed 4-MHA pentapeptide lactones giving C-demethylactinomycins lacking one or both methyl groups in their phenoxazinone chromophores. In case of C-demethylactinomyins lacking one methyl group, the condensation was shown to be regiospecific directing the 3-HA portion almost exclusively to the α-side of the phenoxazinone chromophore. As 3-HA is a weaker substrate for the 4-MHA-incorporating enzyme actinomycin synthetase I than 4-MHA, C-demethylactinomycins never exceeded 7-8% of total actinomycin formed. Surprisingly, C-demethylactinomycins (up to 0.8%) were also found in the actinomycin mixtures of unsupplemented streptomycete cultures after longer cultivation times, indicating the natural presence of 3-HA. Feeding with 3-hydroxykynurenine (3-HK) induced also formation of C-demethylactinomycins indicating that 3-HK is source of 3-HA. Analysis of tryptophan metabolites in the intracellular pools of the streptomycetes using 5-(3)H-tryptophan as radiotracer revealed formation of 4-MHA, but not of 3-HA. This indicates that intracellular 3-HK is almost exclusively converted to 3-hydroxy-4-methylkynurenine (4-MHK), which has been identified previously as direct precursor of 4-MHA. However, small amount of 3-HK leaking out from the 4-MHA pathway can be prematurely converted to 3-HA all along the cultivation of the streptomycetes resulting in the formation of natural C-demethylactinomycins.