Actinomadura Sp Research Articles

Goondolinones A and B: Terpenyl-quinolin-4(1H)-ones from an Australian Volcanic Crater Soil-Derived Actinomadura sp., with Selective Activity against Dirofilaria immitis (Heartworm).

A chemical investigation of the Australian pasture soil-derived Actinomadura sp. S4S-00245B09, supported by chemical, bioactivity and cultivation profiling, yielded a new class of terpenyl-quinolin-4(1H)-ones, goondolinones A and B (1 and 2), featuring an unprecedented fused seven-membered ether. Structures were assigned to 1 and 2 on the basis of detailed spectroscopic analysis, including X-ray analysis of 1, and biosynthetic considerations. Goondolinone A (1) lacks antibacterial or antifungal properties, is noncytotoxic to two human carcinoma cell lines, but exhibits selective inhibition of the motility of heartworm Dirofilaria immitis microfilaria (EC50 5.1 μM) and L4 larvae (EC50 21.4 μM). As a new anthelmintic scaffold, future understanding of the structure activity relationship and mechanism of action of 1 could inform the discovery of new treatments for heartworm and other filarial diseases, capable of safeguarding the health and welfare of companion animals.

Maduraflavacins A-E, Unusual Phenyl Polyene Metabolites Produced by a Rare Marine-Derived Actinomadura sp.

Phenyl polyenes comprise a small family of bacterial natural products with broad and potent bioactivities, primarily found in actinobacteria. Here we report the discovery of five new phenyl polyene metabolites, maduraflavacins A-E (1-5), from a rare, marine-derived actinobacteria strain Actinomadura glauciflava NA03286. The structures of these natural products were determined by NMR spectroscopy, HRESIMS, LC-MS/MS, and chemical derivatization. All of these new maduraflavacins feature methyl substitutions at the polyene side chain, and maduraflavacins A-C (1-3) possessed a 1-N-β-d-glucosamine-(3 → 1)-O-β-d-glucopyranosyl-(3 → 1)-O-β-d-glucopyranosyl-(6 → 1)-O-β-d-glucopyranoside tetrasaccharide moiety via an amido linkage with a phenyl polyene skeleton. Compounds 1 and 2 showed weak antibacterial activities against the Gram-positive bacteria Staphylococcus aureus Sau 16339 and Micrococcus luteus, respectively.

Goondansamycins A-H: Benzenoid Ansamycins from an Australian Volcanic Crater Soil-Derived Actinomadura sp. S4S-00069B08.

A chemical investigation of Australian soil-derived bacteria Actinomadura sp. S4S-00069B08 yielded eight new benzenoid ansamycins, goondansamycins A-H. Goondansamycins feature rare 1,4-benzoxazin-3-one or o-diamino-p-benzoquinone moieties and can exist as both aglycones or 9-O-α-glycosides of either d-rhodinose or d-amicetose. Structures were solved on the basis of detailed spectroscopy, including X-ray analysis.

Madurastatins with Imidazolidinone Rings: Natural Products or Side-Reaction Products from Extraction Solvents?

Madurastatins are a group of pentapeptides containing an oxazoline moiety, and, in a few cases, an imidazolidinone ring as an additional structural feature. In our search for new potential antiparasitic metabolites from natural sources, we studied the acetone extracts from a culture of Actinomadura sp. CA-135719. The LC/HRMS analysis of this extract identified the presence of the known madurastatins C1 (1), D1 (4), and D2 (5) together with additional members of the family that were identified as the new madurastatins H2 (2) and 33-epi-D1 (3) after isolation and spectroscopic analysis. The planar structures of the new compounds were established by HRMS, ESI-qTOF-MS/MS, and 1D and 2D NMR data, and their absolute configuration was proposed using Marfey's and bioinformatic analyses of the biosynthetic gene cluster (BGC). A revision of the absolute configuration of madurastatins D1 and D2 is proposed. Additionally, madurastatins containing imidazolidinone rings are proved to be artifacts originating during acetone extraction of the bacterial cultures.

Enhanced Pentostatin Production in Actinomadura sp. by Combining ARTP Mutagenesis, Ribosome Engineering and Subsequent Fermentation Optimization

The special structure of pentostatin causes it to possess a wide spectrum of biological and pharmacological properties, and it has been extensively employed to treat malignant tumors and is the first-line treatment for hairy cell leukemia. Pentostatin is mainly distributed in several actinomycetes and fungi species. However, its low titer in microbes is not able to meet medical needs. Here, we report a strain improvement strategy based on combined atmospheric and room-temperature plasma (ARTP) mutagenesis and ribosome engineering screening, as well as fermentation optimization, for enhanced pentostatin production. The original strain, Actinomadura sp. ATCC 39365, was treated with ARTP and screened by ribosome engineering to obtain one stable pentostatin high-yield mutant Actinomadura sp. S-15, which produced 86.35 mg/L pentostatin, representing a 33.79% increase compared to Actinomadura sp. ATCC 39365. qRT-PCR analysis revealed that pentostatin biosynthesis-related gene expression was significantly upregulated in Actinomadura sp. S-15. Then, to further enhance pentostatin production, the fermentation medium was optimized in flask culture and the pentostatin production of Actinomadura sp. S-15 reached 152.06 mg/L, which is the highest pentostatin production reported so far. These results demonstrate the effectiveness of combined ARTP mutation, ribosome engineering screening, and medium optimization for the enhancement of pentostatin production, and provide a methodology enabling the sustainable production of pentostatin on an industrial scale.

Isolation, characterization, and bioactivity profiling of oxazoline containing madurastatin siderophores from Actinomadura sp.

Nine new hydroxyphenyloxazolines, madurastatin B4, C2, D3 and D4, E1 and E2, F1 as well as G1 and G2 (8-16), along with two new enantiomers of madurastatin D1 (ent-6) and D2 (ent-7) and two known congeners, madurastatin B1 (2) and C1 (5), were isolated from the liquid culture of Actinomadura sp. ST100801 based on the initial activity against Escherichia coli screened in bicarbonate-supplemented Mueller Hinton II medium and identification via molecular networking. Structure elucidation was achieved by comprehensive 1D and 2D NMR as well as MS/MS fragmentation analyses. Their absolute configuration was determined by Marfey's analysis. Complemented with functionalized hydroxyphenyloxazolines (2, 4,17-18) obtained by total synthesis, the isolated compounds were evaluated for antibacterial activities revealing MICs down to 4 µg ml-1 against Moraxella catarrhalis. Therefore, this study enlarges the family of madurastatin siderophores.

GNPS-Guided Discovery of Madurastatin Siderophores from the Termite-Associated Actinomadura sp. RB99.

In this study, we analyzed if Actinomadura sp. RB99 produces siderophores that that could be responsible for the antimicrobial activity observed in co‐cultivation studies. Dereplication of high‐resolution tandem mass spectrometry (HRMS/MS) and global natural product social molecular networking platform (GNPS) analysis of fungus‐bacterium co‐cultures resulted in the identification of five madurastatin derivatives (A1, A2, E1, F, and G1), of which were four new derivatives. Chemical structures were unambiguously confirmed by HR‐ESI‐MS, 1D and 2D NMR experiments, as well as MS/MS data and their absolute structures were elucidated based on Marfey's analysis, DP4+ probability calculation and total synthesis. Structure analysis revealed that madurastatin E1 (2) contained a rare 4‐imidazolidinone cyclic moiety and madurastatin A1 (5) was characterized as a Ga3+‐complex. The function of madurastatins as siderophores was evaluated using the fungal pathogen Cryptococcus neoformans as model organism. Based on homology models, we identified the putative NRPS‐based gene cluster region of the siderophores in Actinomadura sp. RB99.

Biocontrol of toxinogenic Aspergillus flavus and Fusarium oxysporum f. sp. albedinis by two rare Saharan actinomycetes strains and LC-ESI/MS-MS profiling of their antimicrobial products

Fungi colonizing fruits in the field and post-harvest constitute a major threat to the global food sector. This study focuses on the biocontrol of Aspergillus flavus (aflatoxin-producing mold considered carcinogenic by IARC) and Fusarium oxysporum f. sp. albedinis (FOA) (phytopathogenic agent, causal of El Bayoud in the Algerian and Moroccan Sahara). These molds have a significant economic impact and pose a serious human health problem. The aim of this work is to study the antifungal activity of two rare actinomycetes strains; Saccharothrix sp. COL22 and Actinomadura sp. COL08 strains against toxinogenic A. flavus and F. oxysporum f. sp. albedinis. The strains are isolated from Citrullus colocynthis rhizosphere on different media: ISP2, GLM, TSA, Starch-casein-agar and WYE and with different treatments of the samples (physical, chemical treatment and enrichment). The antifungal tests against the pathogenic microorganisms were performed on ISP2, GLM and TSA medium by means of the agar cylinders method. The kinetics of antibiotic production were performed on ISP medium over 16 days. The characterization of the antimicrobial compounds by LC-ESI/MS-MS showed that the bacterial extracts contain Antibiotic SF 2738C, Tetrodecamycin and Aplysillamide B. The phenotypic and molecular studies showed that Saccharothrix sp. COL22 is closely related to the Saccharothrix longispora strain type and that Actinomadura sp. COL08 is closely related to the Actinomadura hibisca strain type. The two strains are rare and showed an interesting activity against toxinogenic A. flavus and F. oxysporum f. sp. albedinis.

Kumemicinones A–G, Cytotoxic Angucyclinones from a Deep Sea-Derived Actinomycete of the Genus Actinomadura

Chemical investigation of the fermentation products of a deep sea water-derived actinomycete, Actinomadura sp. KD439, identified seven new angucyclinones, designated as kumemicinones A-G (1-7), together with the known SF2315B and miaosporone E. NMR and MS spectroscopic analyses, combined with X-ray crystallography and quantum chemical calculations of NMR chemical shifts and electronic circular dichroism (ECD) spectra, uncovered the structures of new angucyclinones as regioisomers of SF2315B at the allyl alcohol unit (1 and 2), an epoxy ring-opened γ-hydroxy enone isomer (3), a B/C-ring-rearranged product (4), or dimers with a new mode of bridging (5-7), adding new structural variation to this antibiotic group. The absolute configuration of SF2315B was also determined by comparison of ECD spectra with those of 1 and 2. All the angucyclinones exhibited cytotoxicity against P388 murine leukemia cells, with IC50 values ranging from 1.8 to 53 μM.

Revealing the underlying mechanisms mediated by endophytic actinobacteria to enhance the rhizobia - chickpea (Cicer arietinum L.) symbiosis

PurposeThe effects of endophytic actinobacterial strains, Microbispora sp. CP56, Actinomadura sp. CP84B, Streptomyces spp. CP200B and CP21A, on the chickpea-Mesorhizobium symbiosis, were investigated in planta, with the aim of revealing the underlying mechanisms of action.MethodsThe actinobacterial endophytes were co-inoculated with Mesorhizobium ciceri onto chickpea seedlings to study the effect on plant growth parameters, nodulation development and grain yield. The role of actinobacterial exudates on rhizobial growth was investigated, as was the role of root exudates of actinobacteria-colonized plants on the expression of rhizobial nod factors and biofilm formation. Changes in expression of plant flavonoids and bacterial N-fixation genes resulting from actinobacterial co-inoculation were assessed using qPCR.ResultsApplication of actinobacterial endophytes, together with M. ciceri, showed growth promotion of chickpea with an increase in root nodule number and weight. Enhanced nodulation was accompanied by increases in total plant nitrogen, larger total plant weight and a 2–3-fold increase in grain yield. Factors associated with this tripartite symbiosis are promotion of rhizobial growth, earlier nodule formation, increased secondary root formation, up-regulated expression of genes related to flavonoid synthesis and nif genes. In addition, exudates of chickpea roots colonised with actinobacteria increased nodulation-related biological processes - rhizobial chemotaxis, biofilm formation and nod gene expression.ConclusionThese endophytic actinobacteria positively affect many aspects of the chickpea-Mesorhizobium symbiosis resulting in increases in grain yield. Similar improvements recorded in chickpea growing in potted field soils, shows the potential to enhance chickpea production in the field.

Rare actinobacteria isolated from the hypersaline Ojo de Liebre Lagoon as a source of novel bioactive compounds with biotechnological potential.

The Ojo de Liebre Lagoon is a Marine Protected Area that lies within a UNESCO World Heritage Site and is a critical habitat for important migratory species such as the grey whale and bird species. Unique hypersaline environments, such as the Ojo de Liebre Lagoon, are underexplored in terms of their bacterial and chemical diversity, representing a potential source for new bioactive compounds with pharmacological properties. Actinobacteria are one of the most diverse and prolific taxonomic bacterial groups in terms of marine bioactive compounds. This study aimed to identify the culturable actinobacterial community inhabiting the Lagoon, as well as to test their potential as new sources of anticancer compounds with pharmacological potential. A selective isolation approach focused on spore-forming bacteria from 40 sediment samples generated a culture collection of 64 strains. The 16S rRNA gene analyses identified three phyla in this study, the Actinobacteria, Firmicutes and Proteobacteria, where the phylum Actinobacteria dominated (57%) the microbial community profiles. Within the Actinobacteria, nine different genera were isolated including the Actinomadura, Micromonospora, Nocardiopsis, Plantactinospora and Streptomyces sp. We observed seasonal differences on actinobacteria recovery. For instance, Micromonospora strains were recovered during the four sampling seasons, while Arthrobacter and Pseudokineococcus were only isolated in February 2018, and Blastococcus, Rhodococcus and Streptomyces were uniquely isolated in June 2018. Ethyl acetate crude extracts derived from actinobacterial cultures were generated and screened for cytotoxic activity against six cancer cell lines. Strains showed promising low percentages of viability on lung (H1299), cervical (SiHa), colon (Caco-2) and liver (HepG2) cancer lines. Molecular networking results suggest many of the metabolites produced by these strains are unknown and they might harbour novel chemistry. Our results showed the Ojo de Liebre Lagoon is a novel source for isolating diverse marine actinobacteria which produce promising bioactive compounds for potential biotechnological use as anticancer agents.

Nomimicins B-D, new tetronate-class polyketides from a marine-derived actinomycete of the genus Actinomadura.

Three new tetronate-class polyketides, nomimicins B, C, and D, along with nomimicin, hereafter named nomimicin A, were isolated from the culture extract of Actinomadura sp. AKA43 collected from floating particles in the deep-sea water of Sagami Bay, Japan. The structures of nomimicins B, C, and D were elucidated through the interpretation of NMR and MS analytical data, and the absolute configuration was determined by combination of NOESY/ROESY and ECD analyses. Nomimicins B, C, and D showed antimicrobial activity against Gram-positive bacteria, Kocuria rhizophila and Bacillus subtilis, with MIC values in the range of 6.5 to 12.5 μg/mL. Nomimicins B and C also displayed cytotoxicity against P388 murine leukemia cells with IC50 values of 33 and 89 μM, respectively.

Description of the bacterial RNA polymerase inhibitor GE23077-producer Actinomadura sp. NRRL B-65521T as Actinomadura lepetitiana sp. nov.

The filamentous actinomycete that produces the antibiotic GE23077 was isolated by the Lepetit Research Group from a soil sample collected in Thailand, and it was classified as a member of the genus Actinomadura on the basis of its morphology and cell-wall composition. Phylogenetic analysis based on 16S rRNA gene sequences indicated that this strain formed a distinct monophyletic line within the genus Actinomadura, and it was most closely related to Actinomadura bangladeshensis DSM 45347T (99.31 % similarity) and Actinomadura mexicana DSM 44485T (98.94 %). The GE23077-producing strain formed an extensively branched, non-fragmented vegetative mycelium; no pseudosporangia were formed and the arthrospores were organized in slightly twisted chains. The cell wall contained meso-2,6-diaminopimelic acid and the diagnostic sugar was madurose. The predominant menaquinone was MK-9(H6), with minor amounts of MK-9(H8) and MK-9(H4). The diagnostic phospholipids were phosphatidylinositol and diphosphatidylglycerol. The major cellular fatty acids were C16 : 0 and tuberculostearic acid (10-methyloctadecanoic acid), followed by minor amounts of C18:1ω9c, C16:1ω7c and 10-methylheptadecanoic acid. The genomic DNA G+C content was 71.77 mol%. Significant differences in the morphological, chemotaxonomic and biochemical data, and the low DNA-DNA relatedness between the GE23077-producing strain and closely related type strains clearly demonstrate that it represents a novel species of the genus Actinomadura, for which the name Actinomadura lepetitiana sp. nov. is proposed. The type strain is NRRL B-65521T(=LMG 31258T=DSM 109019T).

An Fe2+‐ and α‐Ketoglutarate‐Dependent Halogenase Acts on Nucleotide Substrates

AbstractWhile halogenated nucleosides are used as common anticancer and antiviral drugs, naturally occurring halogenated nucleosides are rare. Adechlorin (ade) is a 2′‐chloro nucleoside natural product first identified from Actinomadura sp. ATCC 39365. However, the installation of chlorine in the ade biosynthetic pathway remains elusive. Reported herein is a Fe2+‐α‐ketoglutarate halogenase AdeV that can install a chlorine atom at the C2′ position of 2′‐deoxyadenosine monophosphate to afford 2′‐chloro‐2′‐deoxyadenosine monophosphate. Furthermore, 2′,3′‐dideoxyadenosine‐5′‐monophosphate and 2′‐deoxyinosine‐5′‐monophosphate can also be converted, albeit 20‐fold and 2‐fold, respectively, less efficiently relative to the conversion of 2′‐deoxyadenosine monophosphate. AdeV represents the first example of a Fe2+‐α‐ketoglutarate‐dependent halogenase that converts nucleotides into chlorinated analogues.

An Fe2+ - and α-Ketoglutarate-Dependent Halogenase Acts on Nucleotide Substrates.

While halogenated nucleosides are used as common anticancer and antiviral drugs, naturally occurring halogenated nucleosides are rare. Adechlorin (ade) is a 2'-chloro nucleoside natural product first identified from Actinomadura sp. ATCC 39365. However, the installation of chlorine in the ade biosynthetic pathway remains elusive. Reported herein is a Fe2+ -α-ketoglutarate halogenase AdeV that can install a chlorine atom at the C2' position of 2'-deoxyadenosine monophosphate to afford 2'-chloro-2'-deoxyadenosine monophosphate. Furthermore, 2',3'-dideoxyadenosine-5'-monophosphate and 2'-deoxyinosine-5'-monophosphate can also be converted, albeit 20-fold and 2-fold, respectively, less efficiently relative to the conversion of 2'-deoxyadenosine monophosphate. AdeV represents the first example of a Fe2+ -α-ketoglutarate-dependent halogenase that converts nucleotides into chlorinated analogues.

Gene Cluster Activation in a Bacterial Symbiont Leads to Halogenated Angucyclic Maduralactomycins and Spirocyclic Actinospirols.

Growth from spores activated a biosynthetic gene cluster in Actinomadura sp. RB29, resulting in the identification of two novel groups of halogenated polyketide natural products, named maduralactomycins and actinospirols. The unique tetracyclic and spirocyclic structures were assigned based on a combination of NMR analysis, chemoinformatic calculations, X-ray crystallography, and 13C labeling studies. On the basis of HRMS2 data, genome mining, and gene expression studies, we propose an underlying noncanonical angucycline biosynthesis and extensive post-polyketide synthase (PKS) oxidative modifications.

MS-Derived Isotopic Fine Structure Reveals Forazoline A as a Thioketone-Containing Marine-Derived Natural Product.

Forazoline A is a structurally complex PKS-NRPS hybrid produced by marine-derived Actinomadura sp. During the course of studies highlighting the application of IFS analysis as a powerful tool for natural products analysis, we were alerted to an earlier misinterpretation with respect to forazoline A structure elucidation. In particular, IFS reveals that forazoline A contains a thioketone moiety rarely seen in secondary metabolites and, thus, constitutes an even more intriguing structure than originally thought.

Genomics-driven discovery of the biosynthetic gene cluster of maduramicin and its overproduction in Actinomadura sp. J1-007.

Maduramicin is the most efficient and possesses the largest market share of all anti-coccidiosis polyether antibiotics (ionophore); however, its biosynthetic gene cluster (BGC) has yet to been identified, and the associated strains have not been genetically engineered. Herein, we performed whole-genome sequencing of a maduramicin-producing industrial strain of Actinomadura sp. J1-007 and identified its BGC. Additionally, we analyzed the identified BGCs in silico to predict the biosynthetic pathway of maduramicin. We then developed a conjugation method for the non-spore-forming Actinomadura sp. J1-007, consisting of a site-specific integration method for gene overexpression. The maduramicin titer increased by 30% to 7.16g/L in shake-flask fermentation following overexpression of type II thioesterase MadTE that is the highest titer at present. Our findings provide insights into the biosynthetic mechanism of polyethers and provide a platform for the metabolic engineering of maduramicin-producing microorganisms for overproduction and development of maduramicin analogs in the future.

Draft genome sequence of Actinomadura sp. K4S16 and elucidation of the nonthmicin biosynthetic pathway.

Actinomadura sp. K4S16 (=NBRC 110471) is a producer of a novel tetronate polyether compound nonthmicin. Here, we report the draft genome sequence of this strain together with features of the organism and assembly, annotation and analysis of the genome sequence. The 9.6 Mb genome of Actinomadura sp. K4S16 encoded 9,004 putative ORFs, of which 7,701 were assigned with COG categories. The genome contained four type-I polyketide synthase (PKS) gene clusters, two type-II PKS gene clusters, and three nonribosomal peptide synthetase (NRPS) gene clusters. Among the type-I PKS gene (t1pks) clusters, a large t1pks cluster was annotated to be responsible for nonthmicin synthesis based on bioinformatic analyses. We also performed feeding experiments using labeled precursors and propose the biosynthetic pathway of nonthmicin.

Complete NMR assignment and absolute configuration of k4610422, a norditerpenoid inhibitor of testosterone-5α-reductase originally from Streptosporangium: rediscovery from a thermophilic Actinomadura.

A norditerpenoid k4610422 (1), an inhibitor of testosterone-5α-reductase originally discovered from a mesophilic rare actinomycete of the genus Streptosporangium, was isolated from the culture extract of a thermophilic actinomycete Actinomadura sp. The complete 1H and 13C NMR assignment and absolute configuration of 1 were addressed by spectroscopic measurements including NOESY and CD spectra coupled with ECD calculation, which allowed to establish the (5 R,9 S,10 R,13 S)-configuration. Compound 1 was moderately cytotoxic against P388 murine leukemia cells with IC50 30 μM.