Streptomyces pyxinae sp. nov. and Streptomyces pyxinicus sp. nov. isolated from lichen Pyxine cocoes (Sw.) Nyl.

Two novel actinobacteria, designated as LP05-1T and LP11T, were isolated from the lichen Pyxine cocoes (Sw.) Nyl. collected in Bangkok, Thailand. Genotypic and phenotypic analyses revealed that both strains represented members of the genus Streptomyces. The 16S rRNA gene of LP05-1T showed the highest similarity to the genome of Streptomyces gelaticus (98.41 %), while the 16S rRNA gene of LP11T was most similar to that of Streptomyces cinerochromogenes (98.93 %). The major menaquinones in LP05-1T were MK-9(H8), MK-9(H6), MK-9(H4) and MK-9(H2), and in LP11T, they were MK-9(H8) and MK-9(H6). Both strains exhibited the major fatty acids iso-C15 : 0, anteiso-C15 : 0, iso-C16 : 0 and anteiso-C17 : 0, with LP05-1T also possessing iso-C17 : 0. The polar lipids of LP05-1T included phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside and an unidentified lipid, while those of LP11T consisted of phosphatidylethanolamine, lyso-phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside, an unidentified aminolipid and an unidentified glycolipid. The digital DNA-DNA hybridisation (dDDH) and average nucleotide identity (ANI) values indicated that both strains are distinct from each other with values below 70 and 95 %, respectively. dDDH, ANI by blast (ANIb) and ANI by MUMmer (ANIm) values between LP05-1T and its closely related type strains were 26.07-26.80 %, 81.24-82.01 % and 86.82-86.96 %, respectively, while those for LP11T and its closely related type strains were 30.70-31.70 %, 84.09-85.31 % and 88.02-88.39 %, respectively. The results of the taxonomic investigation, including dDDH and ANI values, indicate that LP05-1T and LP11T are novel type strains of two novel species within the genus Streptomyces. The names proposed are Streptomyces pyxinae sp. nov. for strain LP05-1T (=TBRC 15494T, =NBRC 115434T) and Streptomyces pyxinicus sp. nov. for strain LP11T (=TBRC 15493T, =NBRC 115421T).

Phytohabitans aurantiacus sp. nov., an actinomycete isolated from soil.

A novel actinomycete, designated RD004123T, was isolated from a soil sample collected in Hokkaido, Japan, and its taxonomic position was investigated by a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strain RD004123T fell within the cluster of the family Micromonosporaceae but did not form a reliable cluster with any member of the family. The similarity values between strain RD004123T and the type species of 29 genera in the family Micromonosporaceae were 91.7-97.7 %. Meanwhile, phylogenomic analyses indicated that strain RD004123T was closely related to members of the genus Phytohabitans. Strain RD004123T contained both meso-diaminopimelic acid and l-lysine as the diagnostic diamino acids of the peptidoglycan. The predominant isoprenoid quinones were MK-10(H8) and MK-10(H6), and the major fatty acids were anteiso-C17 :  0, iso-C16 :  0, iso-C15 :  0 and C17 :  0. The detected polar lipids were phosphatidylinositol mannosides, phosphatidylinositol, phosphatidylethanolamine and diphosphatidylglycerol. These chemotaxonomic features corresponded to those of the genus Phytohabitans. Meanwhile, the results of genome comparison analyses and phenotypic characterizations distinguished strain RD004123T from the other members of the genus Phytohabitans. Therefore, strain RD004123T should be assigned as representing a novel species of the genus Phytohabitans, for which the name Phytohabitans aurantiacus sp. nov. is proposed. The type strain is RD004123T (=NBRC 114997T=DSM 114330T).

Iseoic acids and bisiseoate: three new naphthohydroquinone/naphthoquinone-class metabolites from a coral-derived Streptomyces.

Two new naphthohydroquinone derivatives designated iseoic acids A (1) and B (2) and a new symmetrical glycerol bisester of naphthoquinonepropanoic acid designated bisiseoate (3) were isolated from the culture extract of a marine-derived actinomycete Streptomyces sp. DC4-5. The structures of 1-3 were determined by analyzing one- and two-dimensional NMR data and MS analytical data. The absolute configurations were determined by NOESY analysis and the phenylglycine methyl ester (PGME) method for 1 and by considering the structural similarity and biosynthesis for 2 and 3. Compound 3 exhibited modest cytotoxicity against P388 murine leukemia cells with an IC50 value of 19 µM.

Isolation and structure determination of allopteridic acids A-C and allokutzmicin from an unexplored actinomycete of the genus Allokutzneria.

Two classes of new polyketides, allopteridic acids A-C (1-3) and allokutzmicin (4), were isolated from the culture extract of an actinomycete of the genus Allokutzneria. The structures of 1-4 were elucidated through the interpretation of NMR and MS analytical data. Compounds 1-3 possess the same carbon skeleton with pteridic acids but their monocyclic core structures are distinct from the spiro-bicyclic acetal structures of pteridic acids. Compound 4 is a linear polyketide of an unprecedented class, featured by a guanidino-terminus and an epoxide modification. Compounds 1-3 promoted the root elongation of germinated lettuce seeds by ca. 10-40% at 1~10 µM whereas 4 retarded the seed growth. Compound 4 exhibited weak antimicrobial activity against Candida albicans with MIC 25 µg mL-1.

Microbispofurans A-C, plant growth-promoting furancarboxylic acids from plant root-derived Microbispora sp.

Microbispofurans A-C (1-3), new alkyl/alkenyl furancarboxylic acids, were isolated from the culture extract of the plant root-derived Microbispora sp. RD004716. The planar structures of 1-3 were determined by extensive analysis of 1D and 2D NMR spectroscopic data. Although 1-3 showed no appreciable antimicrobial activity or cytotoxicity, strong plant growth-promotion activity of the germinated red leaf lettuce seeds was observed at 10 µM. Furancarboxylic acids and their methyl esters were found in actinomycetes and fungi; however, the isolation of furandicarboxylic acid was unprecedented.

Bacterial microbiome in tropical lichens and the effect of the isolation method on culturable lichen-derived actinobacteria.

Ten samples of tropical lichens collected from Doi Inthanon, Thailand, were explored for the diversity of their bacterial microbiomes through 16S rRNA-based metagenomics analysis. The five predominant lichen-associated bacteria belonged to the phyla Proteobacteria (31.84%), Planctomycetota (17.08%), Actinobacteriota (15.37%), Verrucomicrobiota (12.17%), and Acidobacteriota (7.87%). The diversity analysis metric showed that Heterodermia contained the highest bacterial species richness. Within the lichens, Ramalina conduplicans and Cladonia rappii showed a distinct bacterial community from the other lichen species. The community of lichen-associated actinobacteria was investigated as a potential source of synthesized biologically active compounds. From the total Operational Taxonomic Units (OTUs) found across the ten different lichen samples, 13.21% were identified as actinobacteria, including the rare actinobacterial genera that are not commonly found, such as Pseudonocardia, Kineosporia, Dactylosporangium, Amycolatopsis, Actinoplanes, and Streptosporangium. Evaluation of the pretreatment method (heat, air-drying, phenol, and flooding) and isolation media used for the culture-dependent actinobacterial isolation revealed that the different pretreatments combined with different isolation media were effective in obtaining several species of actinobacteria. However, metagenomics analyses revealed that there were still several strains, including rare actinobacterial species, that were not isolated. This research strongly suggests that lichens appear to be a promising source for obtaining actinobacteria.

Species-specific secondary metabolism by actinomycetes of the genus Phytohabitans and discovery of new pyranonaphthoquinones and isatin derivatives.

To further exploit secondary metabolic potential of a minor actinomycete genus Phytohabitans within the family Micromonosporaceae, metabolite profiling by HPLC-UV analysis, combined with 16S rDNA sequence-based phylotyping were attempted on seven Phytohabitans strains available at the public culture collection. The strains were grouped into three clades and each exhibited unique and distinct metabolite profiles, which were highly conserved among strains within the same clade. These results were consistent with previous observations on two other actinomycetes genera, reconfirming species-specificity of secondary metabolite production, which were conventionally thought to be strain-specific. A strain RD003215, belonging to the P. suffuscus clade, produced multiple metabolites, some of which were presumed to be naphthoquinones. Liquid fermentation followed by chromatographic separation of the broth extract led to the discovery of three new pyranonaphthoquinones, designated habipyranoquinones A-C (1-3), and one new isatin derivative, (R)-N-methyl-3-hydroxy-5,6-dimethoxyoxindole (4), along with three known synthetic compounds, 6,8-dihydroxydehydro-α-lapachone (5), N-methyl-5,6-dimethoxyisatin (6), and 5,6-dimethoxyisatin (7). Structures of 1-4 were unequivocally elucidated by NMR, MS, and CD spectral analysis, with assistance of density functional theory-based NMR chemical shift prediction and ECD spectral calculation. Compound 2 displayed antibacterial activity against Kocuria rhizophila and Staphylococcus aureus with MIC 50 µg/mL and cytotoxicity against P388 murine leukemia cells with an IC50 value of 34 µM. Compounds 1 and 4 also showed cytotoxicity against P388 cells with IC50 values of 29 and 14 µM, respectively.

Bulbiferamide, an Antitrypanosomal Hexapeptide Cyclized via an N-Acylindole Linkage from a Marine Obligate Microbulbifer.

UV absorption spectroscopy-guided fractionation of the culture extract of a marine obligate bacterium of the genus Microbulbifer yielded a novel cyclic hexapeptide, bulbiferamide (1). NMR spectroscopic and mass spectrometric analyses revealed the structure of 1 to be a cyclic tetrapeptide appending a ureido-bridged two amino acid unit. Notably, Trp is a junction residue, forming on one hand a very rare N-aminoacylated indole linkage for cyclization and on the other hand connecting the ureido-containing tail structure, which is an unprecedented way of configuring peptides. The component amino acids were determined to be l by the advanced Marfey's method. Compound 1 displayed growth inhibitory activity against Trypanosoma cruzi epimastigotes with an IC50 value of 4.1 µM, comparable to the currently approved drug benznidazole, while it was not cytotoxic to P388 murine leukemia cells at 100 µM.

1-(6-Methylsalicyloyl)glycerol from stony coral-derived Micromonospora sp.

A new natural product, 1-(6-methylsalicyloyl)glycerol (1) was isolated from the culture extract of the stony coral-derived Micromonospora sp. C029. The structure of 1 was determined by extensive analysis of 1D and 2D NMR spectroscopic data. The absolute configuration was determined to be S by comparison of specific rotation with synthetic (R)- and (S)-1. Compound 1 showed weak antimicrobial activity against Kocuria rizhophila. Structurally related benzoyl glycerol is not reported from actinomycetes, suggesting that isolation of actinomycetes from little studied environments should be important for the discovery of novel natural products.

Krasilnikolides A and B and Detalosylkrasilnikolide A, Cytotoxic 20-Membered Macrolides from the Genus Krasilnikovia: Assignment of Anomeric Configuration by J-Based Configuration Analysis.

A chemical investigation of strain RD003821, belonging to the underexplored actinomycetes genus Krasilnikovia, led to the discovery of three novel polyketides: two 20-membered glycomacrolides, krasilnikolides A (1) and B (2), and an aglycone of 1, detalosylkrasilnikolide A (3). A major challenge in the structure elucidation of 1 was to determine the anomeric configuration of the α-l-6-deoxytalose (6dTal) unit, which was achieved by J-based configuration analysis (JBCA) that incorporated anomeric carbon- and proton-specific two-bond 13C-1H spin-spin coupling constants as diagnostic parameters. The updated criteria for the conformation/configuration assignment facilitated discrimination of three out of four stereochemical variants at the anomeric and the adjacent C2 positions, which expanded the scope of the JBCA method to determination of the anomeric configuration of aldohexopyranoses. Compounds 1 and 2 are the first macrolides decorated by 6dTal. Compounds 1-3 exhibited cytotoxicity against P388 murine leukemia cells with IC50 values of 14, 8.4, and 3.9 µM, respectively. In addition, 1-3 were antibacterial against the Gram-positive bacterium Kocuria rhizophila with MIC values of 25, 50, and 100 µg/mL. 1 was inhibitory against Staphylococcus aureus with an MIC of 50 µg/mL.

Bisprenyl naphthoquinone and chlorinated calcimycin congener bearing thiazole ring from an actinomycete of the genus Phytohabitans.

A bisprenyl naphthoquinone, phytohabinone (1), and a calcimycin congener with unusual modifications, phytohabimicin (2), were isolated from the culture extract of Phytohabitans sp. RD003013. The structures of 1 and 2 were determined by NMR and MS analyses, and the absolute configuration of 2 was established by using electronic circular dichroism (ECD) calculation. The prenylation pattern of 1 was unprecedented among the known prenylated naphthoquinones. Compound 2 represents a spiroacetal core of polyketide origin substituted with a thiazole carboxylic acid and a dichrolopyrrole moiety, which is an unprecedented modification pattern in the known calcimycin family natural products. Remarkably, 2 showed moderate antimicrobial activity against a Gram-negative bacterium Ralstonia solanacearum while calcimycin was inactive. Additionally, 2 inhibits the migration of EC17 cancer cells at noncytotoxic concentrations.

Cyclic enaminones and a 4-quinazolinone from an unidentified actinomycete of the family Micromonosporaceae.

Four novel cyclic enaminones, designated RD4123A-D (1-4), and a new 4-quinazolinone metabolite, RD4123E (5), were isolated from the culture extract of an unidentified actinomycete strain RD004123, which belongs to the family Micromonosporaceae. Structures of 1-5 were determined by spectroscopic analyses using NMR, MS, and electronic circular dichroism (ECD), combined with quantum chemical calculations of ECD and NMR chemical shifts and biosynthetic consideration. Compounds 1-5 showed weak to modest cytotoxicity against murine leukemia P388 cells, while being inactive against bacteria and fungi.

Induced production of a new antioxidant phenylpropanoid from Streptomyces sp. by protoplast formation/regeneration.

Streptomyces sp. RD007556 regenerated from protoplast was found to produce p-coumaric acid 3,4-dihydroxybenzoate, propla acid (1) which is not observed in the wild-type strain. The structure of 1 was determined by NMR and MS analyses. Compound 1 showed antioxidant activities in DPPH and superoxide dismutase-like assays.

Catellatolactams A-C, Plant Growth-Promoting Ansamacrolactams from a Rare Actinomycete of the Genus Catellatospora.

Catellatolactams A-C (1-3), three novel ansamacrolactams, were isolated from the culture extract of an underexplored rare actinomycete of the genus Catellatospora. Spectroscopic and spectrometric analyses by NMR and MS elucidated the structure of 1 to be a lactamized pentaketide presumably extended on a 3-amino-5-hydroxybenzoic acid starter unit. Compounds 2 and 3 further received epoxidation and intramolecular cross-linking to incorporate a 2-indolinone unit, with a 3-amino-5-hydroxybenzoic acid pendant on 3. The absolute configurations of 2 and 3 were unequivocally established to both be 2S,6R,7R by comparison of the experimental NMR chemical shifts and ECD spectra with those predicted by DFT-based quantum chemical calculation. While 1-3 showed no appreciable antimicrobial activity or cytotoxicity, root elongation of germinated lettuce seeds was promoted by 2 and 3 at 1-10 µM.

Marinoquinolones and Marinobactoic Acid: Antimicrobial and Cytotoxic ortho-Dialkylbenzene-Class Metabolites Produced by a Marine Obligate Gammaproteobacterium of the Genus Marinobacterium.

Chemical investigation of the culture extract of a marine obligate proteobacterium, Marinobacterium sp. C17-8, isolated from scleractinian coral Euphyllia sp., led to the discovery of three new o-dialkylbenzene-class metabolites, designated marinoquinolones A (1) and B (2) and marinobactoic acid (3). Spectroscopic analysis using MS and NMR revealed the structures of 1 and 2 to be 4-quinolones with an o-dialkylbenzene-containing side chain at C3 and 3 to be a fatty acid bearing an o-dialkylbenzene substructure. The 4-quinolone form of 1 and 2 was unequivocally determined by comparison of the 1H, 13C, and 15N chemical shifts of 1 with those predicted for 2-methyl-4-quinolone A and its tautomer 2-methyl-4-quinolinol B by quantum chemical calculation. Compound 1 was proven to be racemic by X-ray crystallographic analysis and chiral-phase HPLC analysis of its chemical degradation product. Compounds 1-3 exhibited antimicrobial activity against bacteria and filamentous fungi at MIC of 6.3-50 µg/mL. In addition, all compounds showed cytotoxicity against P388 murine leukemia cells at micromolar ranges.

Phytohabitols A-C, δ-Lactone-Terminated Polyketides from an Actinomycete of the Genus Phytohabitans.

Phytohabitols A-C (1-3), new terminally δ-lactonized linear polyketides, were isolated from the culture extract of a rare actinomycete of the genus Phytohabitans. The structures of 1-3, substituted with multiple methyl and hydroxy groups on a conjugated and a skipped diene-containing backbone, were elucidated by NMR and MS spectroscopic analyses. The absolute configuration of 1 was determined by chemical derivatization and chiral anisotropic analysis, coupled with ROESY and J-based configuration analysis. In addition, closely similar 1H and 13C NMR data and optical rotations among 1-3 supported the same stereochemistry of these polyketides. The related streptomycetes metabolites lagunapyrones B, C, and D have α-pyrone rings on the linear part in place of the δ-lactone, but their chirality at the C19-C21 stereocenters were opposite from those described here, posing a question on the previous assignment made solely by comparison of the optical rotations of four possible diastereomers. Compounds 1-3 inhibited migration of cancer cells with IC50 values of 15, 11, and 8.3 µM, respectively, at noncytotoxic concentrations. In addition, 1-3 displayed potent antitrypanosomal activity against Trypanosoma cruzi with IC50 values of 12, 6.4, and 18 µM, comparable to a commonly used therapeutic drug, benznidazole (IC50 16 µM).

Trehangelin E, a bisacyl trehalose with plant growth promoting activity from a rare actinomycete Polymorphospora sp. RD064483.

Trehangelin E (1), a new bisacyl trehalose, was isolated from the culture extract of an actinomycete Polymorphospora sp. RD064483, along with three known congeners, trehangelins A, B, and D. Compound 1 is a new trehalose derivative acylated with (Z)-2-methyl-2-butenoic acid (angelic acid) at 3- and 6'-positions, as determined by NMR and MS analyses. Compound 1 promoted root elongation of germinated lettuce seeds by 30% at 1 µM and 90% at 10 µM compared to the nontreated seeds. Similar promoting activity of root elongation was also observed with trehangelins A and B at the same level.

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.

Streptomyces corallincola and Kineosporia corallincola sp. nov., two new coral-derived marine actinobacteria.

Two new marine actinobacteria, designated as J2-1T and J2-2T, were isolated from a coral, Favites pentagona, collected from Rayong Province, Thailand. The taxonomic positions of the two strains were identified based on polyphasic taxonomy. Based on morphological characteristics and chemotaxonomy, strains J2-1T and J2-2T were identified as members of the genus Streptomyces and Kineosporia, respectively. Strains J2-1T and J2-2T showed the highest 16S rRNA gene sequence similarity to Streptomyces broussonetiae T44T (98.62 %) and Kineosporia babensis VN05A0415T (98.08 %), respectively. Strain J2-1T had chemotaxonomic properties resembling members of the genus Streptomyces. ll-Diaminopimelic acid, glucose and ribose were detected in the whole-cell hydrolysate. Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositolmannoside, unidentified aminolipid and five unidentified phospholipids were detected as the polar lipids. The major cellular fatty acids were C16 : 0 iso, C15 : 0 anteiso, C15 : 0 iso, C16 : 0, C17 : 0 anteiso, C14 : 0 iso and C17 : 0 iso. Strain J2-2T a showed similar cell composition to members of the genus Kineosporia. Both isomers of ll- and meso-diaminopimelic acid were detected in the peptidoglycan. Arabinose, galactose, madurose and xylose were observed in the whole-cell hydrolysate. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside, phosphatidylcholine, an unidentified phospholipid and an unidentified glycolipid. The major cellular fatty acids were C16 : 0, C18 : 1 ω9c, C18 : 0 10-methyl, tuberculostearic acid, C18 : 0 and C17 : 0. Both strains could be distinguished from their closely related type strains according to their phenotypic characteristics. Comparative genome analysis indicated the delineation of two novel species based on digital DNA-DNA hybridization and average nucleotide identity values, which were below 70 and 95 %, respectively. The names proposed are Streptomyces corallincola sp. nov. (J2-1T=TBRC 13503T=NBRC 115066T) and Kineosporia corallincola sp. nov. (J2-2T=TBRC 13504T=NBRC 114885T).

Tenacibactins K-M, cytotoxic siderophores from a coral-associated gliding bacterium of the genus Tenacibaculum.

HPLC/DAD-based chemical investigation of a coral-associated gliding bacterium of the genus Tenacibaculum yielded three desferrioxamine-class siderophores, designated tenacibactins K (1), L (2), and M (3). Their chemical structures, comprising repeated cadaverine-succinic acid motifs terminated by a hydroxamic acid functionality, were elucidated by NMR and negative MS/MS experiments. Compounds 1-3 were inactive against bacteria and a yeast but displayed cytotoxicity against 3Y1 rat embryonic fibroblasts and P388 murine leukemia cells at GI50 in submicromolar to micromolar ranges. Their iron-chelating activity was comparable to deferoxamine mesylate.