Transformation of Renieramycin M into Renieramycins T and S by Intramolecular Photoredox Reaction of 7-Methoxy-6-methyl-1,2,3,4-tetrahydroisoquinoline-5,8-dione Derivatives.

In connection with our studies of biologically active 1,2,3,4-tetrahydroisoquinoline marine natural products, we describe herein a useful intramolecular photoredox transformation of 7-methoxy-6-methyl-1,2,3,4-tetrahydroisoquinoline-5,8-dione tricyclic models into 5-hydroxy-tetrahydroisoquinol[1,3]dioxoles in excellent yields. We applied this methodology to the transformation of renieramycin M into renieramycins T and S and the transformation of saframycin A. The results of cytotoxicity studies are also presented.

Light-Mediated Transformation of Renieramycins and Semisynthesis of 4'-Pyridinecarbonyl-Substituted Renieramycin-Type Derivatives as Potential Cytotoxic Agents against Non-Small-Cell Lung Cancer Cells.

The semisynthesis of renieramycin-type derivatives was achieved under mild and facile conditions by attaching a 1,3-dioxole-bridged phenolic moiety onto ring A of the renieramycin structure and adding a 4'-pyridinecarbonyl ester substituent at its C-5 or C-22 position. These were accomplished through a light-induced intramolecular photoredox reaction using blue light (4 W) and Steglich esterification, respectively. Renieramycin M (4), a bis-tetrahydroisoquinolinequinone compound isolated from the Thai blue sponge (Xestospongia sp.), served as the starting material. The cytotoxicity of the 10 natural and semisynthesized renieramycins against non-small-cell lung cancer (NSCLC) cell lines was evaluated. The 5-O-(4'-pyridinecarbonyl) renieramycin T (11) compound exhibited high cytotoxicity with half-maximal inhibitory concentration (IC50) values of 35.27 ± 1.09 and 34.77 ± 2.19 nM against H290 and H460 cells, respectively. Notably, the potency of compound 11 was 2-fold more than that of renieramycin T (7) and equal to those of 4 and doxorubicin. Interestingly, the renieramycin-type derivatives with a hydroxyl group at C-5 and C-22 exhibited weak cytotoxicity. In silico molecular docking and dynamics studies confirmed that the mitogen-activated proteins, kinase 1 and 3 (MAPK1 and MAPK3), are suitable targets for 11. Thus, the structure-cytotoxicity study of renieramycins was extended to facilitate the development of potential anticancer agents for NSCLC cells.

5-O-(N-Boc-l-Alanine)-Renieramycin T Induces Cancer Stem Cell Apoptosis via Targeting Akt Signaling.

Cancer stem cells (CSCs) drive aggressiveness and metastasis by utilizing stem cell-related signals. In this study, 5-O-(N-Boc-l-alanine)-renieramycin T (OBA-RT) was demonstrated to suppress CSC signals and induce apoptosis. OBA-RT exerted cytotoxic effects with a half-maximal inhibitory concentration of approximately 7 µM and mediated apoptosis as detected by annexin V/propidium iodide using flow cytometry and nuclear staining assays. Mechanistically, OBA-RT exerted dual roles, activating p53-dependent apoptosis and concomitantly suppressing CSC signals. A p53-dependent pathway was indicated by the induction of p53 and the depletion of anti-apoptotic Myeloid leukemia 1 (Mcl-1) and B-cell lymphoma 2 (Bcl-2) proteins. Cleaved poly (ADP-ribose) polymerase (Cleaved-PARP) was detected in OBA-RT-treated cells. Interestingly, OBA-RT exerted strong CSC-suppressing activity, reducing the ability to form tumor spheroids. In addition, OBA-RT could induce apoptosis in CSC-rich populations and tumor spheroid collapse. CSC markers, including prominin-1 (CD133), Octamer-binding transcription factor 4 (Oct4), and Nanog Homeobox (Nanog), were notably decreased after OBA-RT treatment. Upstream CSCs regulating active Akt and c-Myc were significantly decreased; indicating that Akt may be a potential target of action. Computational molecular modeling revealed a high-affinity interaction between OBA-RT and an Akt molecule. This study has revealed a novel CSC inhibitory effect of OBA-RT via Akt inhibition, which may improve cancer therapy.

Jorunnamycin A Suppresses Stem-Like Phenotypes and Sensitizes Cisplatin-Induced Apoptosis in Cancer Stem-Like Cell-Enriched Spheroids of Human Lung Cancer Cells.

It has been recognized that cancer stem-like cells (CSCs) in tumor tissue crucially contribute to therapeutic failure, resulting in a high mortality rate in lung cancer patients. Due to their stem-like features of self-renewal and tumor formation, CSCs can lead to drug resistance and tumor recurrence. Herein, the suppressive effect of jorunnamycin A, a bistetrahydroisoquinolinequinone isolated from Thai blue sponge Xestospongia sp., on cancer spheroid initiation and self-renewal in the CSCs of human lung cancer cells is revealed. The depletion of stemness transcription factors, including Nanog, Oct-4, and Sox2 in the lung CSC-enriched population treated with jorunnamycin A (0.5 µM), resulted from the activation of GSK-3ß and the consequent downregulation of ß-catenin. Interestingly, pretreatment with jorunnamycin A at 0.5 µM for 24 h considerably sensitized lung CSCs to cisplatin-induced apoptosis, as evidenced by upregulated p53 and decreased Bcl-2 in jorunnamycin A-pretreated CSC-enriched spheroids. Moreover, the combination treatment of jorunnamycin A (0.5 µM) and cisplatin (25 µM) also diminished CD133-overexpresssing cells presented in CSC-enriched spheroids. Thus, evidence on the regulatory functions of jorunnamycin A may facilitate the development of this marine-derived compound as a novel chemotherapy agent that targets CSCs in lung cancer treatment.

Chemistry of Renieramycins. Part 19: Semi-Syntheses of 22-O-Amino Ester and Hydroquinone 5-O-Amino Ester Derivatives of Renieramycin M and Their Cytotoxicity against Non-Small-Cell Lung Cancer Cell Lines.

Two new series of synthetic renieramycins including 22-O-amino ester and hydroquinone 5-O-amino ester derivatives of renieramycin M were semi-synthesized and evaluated for their cytotoxicity against the metastatic non-small-cell lung cancer H292 and H460 cell lines. Interestingly, the series of 22-O-amino ester derivatives displayed a potent cytotoxic activity greater than the hydroquinone derivatives. The most cytotoxic derivative of the series was the 22-O-(N-Boc-l-glycine) ester of renieramycin M (5a: IC50 3.56 nM), which showed 7-fold higher potency than renieramycin M (IC50 24.56 nM) and 61-fold more than jorunnamycin A (IC50 217.43 nM) against H292 cells. In addition, 5a exhibited a significantly higher cytotoxic activity than doxorubicin (ca. 100 times). The new semi-synthetic renieramycin derivatives will be further studied and developed as potential cytotoxic agents for non-small-cell lung cancer treatment.

Jorunnamycin A from Xestospongia sp. Suppresses Epithelial to Mesenchymal Transition and Sensitizes Anoikis in Human Lung Cancer Cells.

Metastasis is a key driving force behind the high mortality rate associated with lung cancer. Herein, we report the first study revealing the antimetastasis activity of jorunnamycin A, a bistetrahydroisoquinolinequinone isolated from a Thai blue sponge Xestospongia sp. evidenced by its inhibition of epithelial to mesenchymal transition (EMT), sensitization of anoikis, and suppression of anchorage-independent survival in human lung cancer cells. Treatment with jorunnamycin A (0.05-0.5 µM) altered the expression of p53 and Bcl-2 family proteins, particularly causing the down-regulation of antiapoptosis Bcl-2 and Mcl-1 proteins. Under detachment conditions for 12 h, jorunnamycin A-treated cells exhibited diminution of pro-survival proteins p-Akt and p-Erk as well as the survival-promoting factor caveolin-1. Corresponding with the inhibition on the Akt and Erk pathway as well as activation of p53, there was an increase in the epithelial marker E-cadherin and a remarkable decrease of EMT markers and associated proteins including vimentin, snail, and claudin-1. As the loss of anchorage dependence is an important barrier to metastasis, the observed inhibitory effects of jorunnamycin A on the coordinating networks of EMT and anchorage-independent growth emphasize the potential development of jorunnamycin A as an effective agent against lung cancer metastasis.

Renieramycin T Induces Lung Cancer Cell Apoptosis by Targeting Mcl-1 Degradation: A New Insight in the Mechanism of Action.

Among malignancies, lung cancer is the major cause of cancer death. Despite the advance in lung cancer therapy, the five-year survival rate is extremely restricted due to therapeutic failure and disease relapse. Targeted therapies selectively inhibiting certain molecules in cancer cells have been accepted as promising ways to control cancer. In lung cancer, evidence has suggested that the myeloid cell leukemia 1 (Mcl-1) protein, an anti-apoptotic member of the Bcl-2 family, is a target for drug action. Herein, we report the Mcl-1 targeting activity of renieramycin T (RT), a marine-derived tetrahydroisoquinoline alkaloid that was isolated from the Thai blue sponge Xestospongia sp. RT was shown to be dominantly toxic to lung cancer cells compared to the normal cells in the lung. The cytotoxicity of this compound toward lung cancer cells was mainly exerted through apoptosis induction. For the mechanism of action, we found that RT mediated activation of p53 protein and caspase-9 and -3 activations. While others Bcl-2 family proteins (Bcl-2, Bak, and Bax) were minimally changed in response to RT, Mcl-1 protein was dramatically diminished. We further performed the cycloheximide experiment and found that the half-life of Mcl-1 was significantly shortened by RT treatment. When MG132, a potent selective proteasome inhibitor, was utilized, it could restore the Mcl-1 level. Furthermore, immunoprecipitation analysis revealed that RT significantly increased the formation of Mcl-1-ubiquitin complex compared to the non-treated control. In conclusion, we report the potential apoptosis induction of RT with a mechanism of action involving the targeting of Mcl-1 for ubiquitin-proteasomal degradation. As Mcl-1 is critical for cancer cell survival and chemotherapeutic failure, this novel information regarding the Mcl-1-targeted compound would be beneficial for the development of efficient anti-cancer strategies or targeted therapies.

5-O-Acetyl-Renieramycin T from Blue Sponge Xestospongia sp. Induces Lung Cancer Stem Cell Apoptosis.

Lung cancer is one of the most significant cancers as it accounts for almost 1 in 5 cancer deaths worldwide, with an increasing incident rate. Management of the cancer has been shown to frequently fail due to the ability of the cancer cells to resist therapy as well as metastasis. Recent evidence has suggested that the poor response to the current treatment drugs and the ability to undergo metastasis are driven by cancer stem cells (CSCs) within the tumor. The discovery of novel compounds able to suppress CSCs and sensitize the chemotherapeutic response could be beneficial to the improvement of clinical outcomes. Herein, we report for the first time that 5-O-acetyl-renieramycin T isolated from the blue sponge Xestospongia sp. mediated lung cancer cell death via the induction of p53-dependent apoptosis. Importantly, 5-O-acetyl-renieramycin T induced the death of CSCs as represented by the CSC markers CD44 and CD133, while the stem cell transcription factor Nanog was also found to be dramatically decreased in 5-O-acetyl-renieramycin T-treated cells. We also found that such a CSC suppression was due to the ability of the compound to deplete the protein kinase B (AKT) signal. Furthermore, 5-O-acetyl-renieramycin T was able to significantly sensitize cisplatin-mediated apoptosis in the lung cancer cells. Together, the present research findings indicate that this promising compound from the marine sponge is a potential candidate for anti-cancer approaches.

Synergistic Cytotoxicity of Renieramycin M and Doxorubicin in MCF-7 Breast Cancer Cells.

Renieramycin M (RM) is a KCN-stabilized tetrahydroisoquinoline purified from the blue sponge Xestospongia sp., with nanomolar IC50s against several cancer cell lines. Our goal is to evaluate its combination effects with doxorubicin (DOX) in estrogen receptor positive MCF-7 breast cancer cells. MCF-7 cells were treated simultaneously or sequentially with various combination ratios of RM and DOX for 72 h. Cell viability was determined using the MTT assay. Synergism or antagonism was determined using curve-shift analysis, combination index method and isobologram analysis. Synergism was observed with pharmacologically achievable concentrations of DOX when administered simultaneously, but not sequentially. The IC95 values of RM and DOX after combination were reduced by up to four-fold and eight-fold, respectively. To gain insights on the mechanism of synergy, real-time profiling, cell cycle analysis, apoptosis assays, and transcriptome analysis were conducted. The combination treatment displayed a similar profile with DNA-damaging agents and induced a greater and faster cell killing. The combination treatment also showed an increase in apoptosis. DOX induced S and G2/M arrest while RM did not induce significant changes in the cell cycle. DNA replication and repair genes were downregulated commonly by RM and DOX. p53 signaling and cell cycle checkpoints were regulated by DOX while ErbB/PI3K-Akt, integrin and focal adhesion signaling were regulated by RM upon combination. Genes involved in cytochrome C release and interferon gamma signaling were regulated specifically in the combination treatment. This study serves as a basis for in vivo studies and provides a rationale for using RM in combination with other anticancer drugs.

Chemistry of Renieramycins. 17. A New Generation of Renieramycins: Hydroquinone 5-O-Monoester Analogues of Renieramycin M as Potential Cytotoxic Agents against Non-Small-Cell Lung Cancer Cells.

A series of hydroquinone 5-O-monoester analogues of renieramycin M were semisynthesized via bishydroquinonerenieramycin M (5) prepared from renieramycin M (1), a major cytotoxic bistetrahydroisoquinolinequinone alkaloid isolated from the Thai blue sponge Xestospongia sp. All 20 hydroquinone 5-O-monoester analogues possessed cytotoxicity with IC50 values in nanomolar concentrations against the H292 and H460 human non-small-cell lung cancer (NSCLC) cell lines. The improved cytotoxicity toward the NSCLC cell lines was observed from the 5-O-monoester analogues such as 5-O-acetyl ester 6a and 5-O-propanoyl ester 7e, which exhibited 8- and 10-fold increased cytotoxicity toward the H292 NSCLC cell line (IC50 3.0 and 2.3 nM, respectively), relative to 1 (IC50 24 nM). Thus, the hydroquinone 5-O-monoester analogues are a new generation of the renieramycins to be further developed as potential marine-derived drug candidates for lung cancer treatment.

Streptomyces actinomycinicus sp. nov., isolated from soil of a peat swamp forest.

A novel actinomycete, strain RCU-197T, was isolated from soil of a peat swamp forest in Rayong Province, Thailand. Using a polyphasic approach, the strain was classified in the genus Streptomyces. It contained ll-diaminopimelic acid in the cell-wall peptidoglycan. No diagnostic sugars were detected in whole-cell hydrolysates and there was a lack of mycolic acids. The major menaquinones were MK-9(H6) and MK-9(H8). The predominant cellular fatty acids were iso-C14 : 0, iso-C15 : 0, anteiso-C15 : 0 and iso-C16 : 0. The polar lipids profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol and phosphatidylinositol mannoside, an unknown aminolipid and two unknown phospholipids. Phylogenetic analysis of 16S rRNA gene sequences showed the strain formed distinct clade within the genus Streptomyces and was closely related to Streptomyces echinatus NBRC 12763T (98.78 % 16S rRNA gene sequence similarity). According to the polyphasic approach as well as DNA-DNA relatedness, the strain could be clearly differentiated from closely related species and represents a novel species of the genus Streptomyces, for which the name Streptomyces actinomycinicus sp. nov. is proposed. The type strain is RCU-197T ( = JCM 30864T = TISTR 2208T = PCU 342T).

Streptomyces andamanensis sp. nov., isolated from soil.

A novel actinomycete, strain KC-112T, was isolated from soil collected from Similan Islands, Phang-Nga Province, Thailand. The strain exhibited morphological and chemotaxonomic characteristics consistent with those of members of the genus Streptomyces. The formation of smooth spiral spore chains was observed on aerial mycelia. ll-Diaminopimelic acid was detected in whole-cell hydrolysates, but no diagnostic sugars were detected and the strain lacked mycolic acids. The N-acyl type of muramic acid was acetyl. The major menaquinones were MK-9(H8), MK-9(H6) and MK-9(H2). The predominant cellular fatty acids were anteiso-C15 : 0, anteiso-C17 : 0, iso-C16 : 0 and C16 : 0. The polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside, an unknown phospholipid, an unknown aminolipid, unknown lipids and an unknown glycolipid. The DNA G+C content was 73 mol%. On the basis of 16S rRNA gene sequence analysis, strain KC-112T was closely related to Streptomyces fumanus NBRC 13042T (98.8 % 16S rRNA gene sequence similarity), Streptomyces anandii NBRC 13438T (98.8 %) and Streptomyces capillispiralis NBRC 14222T (98.8 %). DNA-DNA relatedness values among strain KC-112T and type strains of closely related species were lower than 70 %. On the basis of evidence from this taxonomic study using a polyphasic approach, strain KC-112T represents a novel species of the genus Streptomyces, namely Streptomyces andamanensis sp. nov. The type strain is KC-112T ( = KCTC 29502T = NBRC 110085T = PCU 347T = TISTR 2401T).

Nocardia rayongensis sp. nov., isolated from Thai peat swamp forest soil.

An actinomycete strain, RY45-3T, isolated from a peat swamp forest soil in Rayong Province, Thailand, was characterized using a polyphasic approach. The strain belonged to the genus Nocardia on the basis of morphological, physiological, biochemical and chemotaxonomic properties. Cell-wall peptidoglycan contained meso-diaminopimelic acid. The N-acyl group of muramic acid in the cell wall was glycolyl type. The diagnostic sugars in whole-cell hydrolysates were galactose and arabinose. MK-8 (H4ω-cycl) was the major menaquinone. The major fatty acids were C16 : 0 and C18 : 1ω9c. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannosides. The genomic DNA G+C content was 71 mol%. On the basis of 16S rRNA gene sequence similarity analysis, strain RY45-3T was closely related to Nocardia jiangxiensis JCM 12861T (98.9 %), Nocardia nova JCM 6044T (98.8 %) and Nocardia pseudobrasiliensis JCM 9894T (98.6 %). The strain showed low levels of DNA-DNA relatedness with N. jiangxiensis JCM 12861T, N. nova JCM 6044T and N. pseudobrasiliensis JCM 9894T (range from 3.6 to 55.3 %). On the basis of the phenotypic characteristics and the results mentioned, this strain could be differentiated from closely related type strains and represents a novel species of the genus Nocardia, for which the name Nocardia rayongensis sp. nov. (type strain RY45-3T = JCM 19832T = TISTR 2213T = PCU 334T) is proposed.

Streptomyces verrucosisporus sp. nov., isolated from marine sediments.

Five actinomycete isolates, CPB1-1T, CPB2-10, BM1-4, CPB3-1 and CPB1-18, belonging to the genus Streptomyces were isolated from marine sediments collected from Chumphon Province, Thailand. They produced open loops of warty spore chains on aerial mycelia. ll-Diaminopimelic acid, glucose and ribose were found in their whole-cell hydrolysates. Polar lipids found were diphosphatidylglycerol, phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannoside. Menaquinones were MK-9(H6), MK-9(H8), MK-10(H6) and MK-10(H8). Major cellular fatty acids were anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0. The taxonomic position of the strains was described using a polyphasic approach. blastn analysis of the 16S rRNA gene sequence revealed that these five strains exhibited the highest similarities with 'Streptomyces mangrovicola' GY1 (99.0 %), Streptomyces fenghuangensisGIMN4.003T (98.6 %), Streptomyces barkulensisRC 1831T (98.5 %) and Streptomyces radiopugnans R97T (98.3 %). However, their phenotypic characteristics and 16S rRNA gene sequences as well as DNA-DNA relatedness differentiated these five strains from the other species of the genus Streptomyces. Here, we propose the novel actinomycetes all being representatives of the same novel species, Streptomyces verrucosisporus, with type strain CPB1-1T (=JCM 18519T=PCU 343T=TISTR 2344T).

Micromonospora sediminis sp. nov., isolated from mangrove sediment.

The novel actinomycete, strain CH3-3T, was isolated from mangrove sediment collected from Chonburi Province, Thailand. On the basis of its spore morphology and chemotaxonomic characteristics, the strain belonged to the genus Micromonospora. It contained meso-diaminopimelic acid, glucose, mannose, xylose, ribose and rhamnose in the whole-cell hydrolysate, MK-10(H4), MK-10(H6) and MK-10(H8) as major menaquinones, and iso-C15 : 0, iso-C16 : 0 and iso-C17 : 0 as major cellular fatty acids. blast analysis of 16S rRNA gene sequences revealed that the strain showed highest similarity with Micromonospora palomenae NEAU-CX1T (98.97 %) and Micromonospora coxensis 2-30-b(28)T (98.97 %). Based on phylogenetic tree analysis of the 16S rRNA gene sequence, the strain formed a cluster with M. palomenae NEAU-CX1T, Micromonospora halophytica DSM 43171T, M. coxensis 2-30-b(28)T and Micromonospora purpureochromogenes DSM 43821T. On the basis of phenotypic differences and DNA-DNA relatedness evidence, strain CH3-3T could be clearly distinguished from the closely related species of the genus Micromonospora and represents a novel species of the genus Micromonospora for which the name Micromonospora sediminis sp. nov. is proposed. The type strain is CH3-3T (=JCM 18523T=PCU 350T=TISTR 2396T).

Chemistry of Renieramycins. 15. Synthesis of 22-O-Ester Derivatives of Jorunnamycin A and Their Cytotoxicity against Non-Small-Cell Lung Cancer Cells.

Eighteen 22-O-ester derivatives of jorunnamycin A (2) were prepared via 2, and their cytotoxicity against human non-small-cell lung cancer (NSCLC) cells was evaluated. Preliminary study of the structure-cytotoxicity relationship revealed that the ester part containing a nitrogen-heterocyclic ring elevated the cytotoxicity of the 22-O-ester derivatives. Among them, 22-O-(4-pyridinecarbonyl) ester 6a is the most potent compound (IC50 1.1 and 1.6 nM), exhibiting 21-fold and 5-fold increases in cytotoxicity against the H292 and H460 NSCLC cell lines, respectively, relative to renieramycin M (1), the major cytotoxic bistetrahydroisoquinolinequinone alkaloid of the Thai blue sponge Xestospongia sp.

Chemistry of Ecteinascidins. Part 5: An Additional Proof of Cytotoxicity Evaluation of Ecteinascidin 770 Derivatives.

Eleven 2'-N-acyl derivatives (5a-k) were prepared from ecteinascidin 770 (Et 770: 1b) via known 18,6'-O-bisallyl-protected compound (3) in three steps. Their in vitro cytotoxicities were determined by measuring IC50 values against human cell lines HCT116 and DU145. 5-Isoxazolecarboylamide derivative (5i) and 4-methoxybenzoylamide derivative (5k) were found to be promising leads for further optimization.

Synthesis and Absolute Configuration of Acanthodendrilline, a New Cytotoxic Bromotyrosine Alkaloid from the Thai Marine Sponge Acanthodendrilla sp.

Acanthodendrilline (1), a new bromotyrosine alkaloid, was isolated from the Thai marine sponge Acanthodendrilla sp. The structure of 1 was fully characterized by spectroscopic analysis, in agreement with the synthesized compound used to resolve the single chiral center at C-11. Total synthesis of the enantiomers of 1 allowed for the comparison of specific rotation values and hence the determination of the absolute configuration as 11-S. Cytotoxicity evaluation revealed that (S)-1 exhibited approximately three-fold more potent cytotoxicity against the human non-small cell lung cancer H292 cell line than (R)-1.

Actinomadura rayongensis sp. nov., isolated from peat swamp forest soil.

A novel actinomycete strain RY35-68(T), isolated from a peat swamp forest soil sample in Rayong Province, Thailand, was characterized using a polyphasic approach. The strain belonged to the genus Actinomadura based on morphological and chemotaxonomic characteristics. Cell-wall analysis revealed the presence of meso-diaminopimelic acid and N-acetylmuramic acid in the peptidoglycan layer. The diagnostic sugar in whole-cell hydrolysates was identified as madurose. The predominant menaquinones were MK-9(H6), MK-9(H8) and MK-9(H4). The major cellular fatty acids were C16 : 0 and iso-C16 : 0. The major polar lipids were diphosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannoside. The genomic DNA G+C content was 73.7 mol%. On the basis of 16S rRNA gene sequence similarity analysis, strain RY35-68(T) was closely related to the species Actinomadura atramentaria JCM 6250(T) (97.5 %). The value of DNA-DNA relatedness between strain RY35-68(T) and A. atramentaria JCM 6250(T) was 37.6-42.6 %. On the basis of its phenotypic characteristics and these results mentioned, this strain could be distinguished from the closely related type strain and represents a novel species of the genus Actinomadura, for which the name Actinomadura rayongensis sp. nov. (type strain RY35-68(T) = JCM 19830(T) = TISTR 2211(T) = PCU 332(T)) is proposed.

Micromonospora fluostatini sp. nov., isolated from marine sediment.

The novel actinomycete strain PWB-003T, which produced fluostatins B and C antibiotics, was isolated from nearshore sediment collected from Panwa Cape, Phuket Province, Thailand. Data from the present polyphasic study indicated that strain PWB-003T represented a member of the genus Micromonospora. It produced single spores on substrate mycelia and contained meso-diaminopimelic acid in the cell-wall peptidoglycan. Whole-cell hydrolysate contained ribose, xylose, arabinose, mannose and glucose. The predominant menaquinone was MK-10 (H4). Cellular fatty acids comprised C18 : 1ω9c, iso-C16 : 0, anteiso-C17 : 0, iso-C15 : 0 and iso-C17 : 0. On the basis of 16S rRNA gene sequence similarity analysis, the novel strain was closely related to Micromonospora eburnea LK2-10T (99.38 %), Micromonospora chaiyaphumensis MC5-1T (99.16 %), Micromonospora yangpuensis FXJ6.011T (98.97 %), Micromonospora echinaurantiaca DSM 43904T (98.97 %), Micromonospora pallida DSM 43817T (98.97 %), Micromonospora sagamiensis DSM 43912T and Micromonospora auratinigra JCM 12357T (both 98.97 %). The G+C content of the DNA was 74.5 mol%. DNA-DNA relatedness values among strain PWB-003T and related type strains ranged from 11.3 ± 1.3 to 38.8 ± 1.1 %. On the basis of these observations, strain PWB-003T could be distinguished from its closely related type strains and is considered to represent a novel species of the genus Micromonospora, for which the name Micromonospora fluostatini sp. nov. (type strain PWB-003T = JCM 30529T = PCU 341T = TISTR 2345T) is proposed.