Micromonospora musae sp. nov., an endophytic actinomycete isolated from roots of Musa species.

Two novel actinobacterial strains, MS1-9T and NGC1-4, were isolated from roots of Musa (ABB) cv. 'Kluai Namwa', collected from Chachoengsao province, and Musa (ABB) cv. 'Kluai Chang', from Suphan Buri province, Thailand, respectively. Comparative analysis of 16S rRNA gene (98.0 to 98.9% similarity), gyrase subunit B (gyrB) gene and whole-genome sequences emphasised that the strains MS1-9T and NGC1-4 showed closely related with Micromonospora peucetia DSM 43363T, M. krabiensis JCM 12869T and M. avicenniae DSM 45758T, respectively. Strains MS1-9T and NGC1-4 contained meso-diaminopimelic acid in cell-wall peptidoglycan. Whole-cell sugars were glucose, xylose, mannose, and ribose. The acyl type of peptidoglycan was glycolyl. MK-10(H6), MK-9(H6), and MK-10(H8) were presented as the major menaquinones. Diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylinositol were detected as predominant phospholipid profiles. The major cellular fatty acids consisted of iso-C15:0, anteiso-C15:0, anteiso-C17:0, iso-C17:0 and C17:0. The DNA G+C content of strains MS1-9T and NGC1-4 were 72.2 and 72.3mol%, respectively. Draft genome sequences indicated by ANI values and digital DNA-DNA hybridisation analysis asserted that the strains MS1-9T and NGC1-4 should be represented as a novel species within the genus Micromonospora for which the name Micromonospora musae sp. nov. is proposed. The type strain is MS1-9T (=JCM 32149T=TISTR 2659T).

Structure elucidation and biological activity of antibacterial compound from Micromonospora auratinigra, a soil Actinomycetes.

AIMS: The aim of this study was to isolate and characterize the bioactive compound of Micromonospora auratinigra, HK-10 and its antibacterial inhibitory mechanism. METHODS AND RESULTS: An oily bioactive compound was extracted from HK-10 (GenBank accession no. JN381554) and found to have promising antibacterial activity. The compound was characterized as 2-methylheptylisonicotinate (1) by (1) H, (13) C NMR and mass spectroscopy. Minimum inhibitory concentration (MIC) of this molecule was tested by micro broth dilution method and was found to be 70, 40, 80, 60, 60 and 50 µg for Staphylococcus aureus, Bacillus subtilis, Proteus vulgaris, Echerichia coli, Pseudomonas aeruginosa and Mycobacterium abscessus respectively. The effects of compound 1 were studied on bacterial membrane structure using scanning electron microscopy. The results indicated a membrane-disrupting mechanism, resulting in the dysfunction of the cytoplasmic membrane structure and cell death of the pathogenic bacterial strains. Kinetics of growth of the test organisms was also analysed and indicated 2-methylheptylisonicotinate 1 as a bactericidal agent. Furthermore, we have studied the binding affinity of 1 towards different membrane proteins of pathogenic bacteria by in silico analysis. CONCLUSIONS: 2-methylheptylisonicotinate was isolated from M. auratinigra, a rare actinobacterial strain possessing antibacterial activity through a membrane-disrupting mechanism, and has MICs similar to standard antibiotic neomycin sulphate. It is the first report about a strain of M. auratinigra, isolated from Indo-Burma biodiversity hotspot of North-east India with new antimicrobial activities. In silico studies have also supported these results performed on various membrane targets of pathogenic bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The antibacterial potential of M. auratinigra is reported for the first time. The results indicate the possible use of 2-methylheptylisonicotinate as a source of antibacterial agent against dreaded human pathogens.

Micromonospora pattaloongensis sp. nov., isolated from a Thai mangrove forest.

An actinomycete, designated strain TJ2-2(T), was isolated from soil collected from a mangrove forest in Pattaloong Province, Thailand, and was subjected to morphological and chemotaxonomic analysis and phylogenetic investigation based on 16S rRNA gene sequences. The data from these analyses indicated that the novel strain should be classified as a member of the genus Micromonospora and that the closest relative was Micromonospora olivasterospora DSM 43868(T) (98.7 % gene sequence similarity). The DNA-DNA hybridization data and some physiological and biochemical properties indicated that the novel strain could be readily distinguished from its closest phylogenetic relatives. On the basis of these phenotypic and genotypic data, strain TJ2-2(T) represents a novel species of the genus Micromonospora, for which the name Micromonospora pattaloongensis sp. nov. is proposed. The type strain is TJ2-2(T) (=JCM 12833(T)=TISTR 1559(T)).

Cryptoendolithic actinomycetes from antarctic sandstone rock samples: Micromonospora endolithica sp. nov. and two isolates related to Micromonospora coerulea Jensen 1932.

Three cryptoendolithic, aerobic actinomycetes (AA-459T, AA-319 and AA-321) from antarctic sandstone were characterised phenotypically and by molecular taxonomic methods. The isolates had single spores on substrate mycelium, meso-diaminopimelic acid (m-DAP) and glycine (cell wall type II), a whole cell sugar pattern D (galactose, xylose, arabinose, glucose or rhamnose) and phospholipids of type PII (diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol). Their predominant fatty acids were iso-16:0 and iso-15:0 or 17:1omega8c, the menaquinone profile was complex with mainly MK10 (H4) and MK10 (H6). A wide variety of sugars and several acids were utilised for growth. The isolates were sensitive to a few antibiotics, but formation and excretion of antibiotics was not observed. Phenotypically, isolates AA-319 and AA-321 were similar. Phylogenetic analysis of 16S rRNA gene sequences revealed close relationship of strains AA-319 and AA-321 with each other (99.5%) and clustering (98.5%) with Micromonospora coerulea DSM 43143T. DNA-DNA hybridisation showed both strains to be genomically highly similar to strain DSM 43143T. Phenotypically they could be viewed as separate taxa, but presently they will be considered as strains of Micromonospora coerulea. Strain AA-459T was phylogenetically close to Micromonospora chersina DSM 44151T (99.1%) and to Micromonospora rosaria DSM 803T, but DNA-DNA similarity with M. chersina DSM 44151T was low with 28.9/33.5 %, indicating the presence of a different and new species. Consequently, isolate AA-459T (DSM 44398T NRRL B-24248T) is described as the type strain of Micromonospora endolithica sp. nov.

IB-96212, a novel cytotoxic macrolide produced by a marine Micromonospora. I. Taxonomy, fermentation, isolation and biological activities.

A novel bioactive macrolide, IB-96212 has been isolated from the fermentation broth of a marine actinomycete, L-25-ES25-008. The strain belongs to the genus Micromonospora. The macrolide showed a very strong cytotoxic activity against P-388, and lower but significant activity against A-549, HT-29, and MEL-28 cell lines. We describe the isolation, taxonomy and fermentation of the producing strain as well as the isolation of IB-96212.

Isolation, antifungal activity, and structure elucidation of the glutarimide antibiotic, streptimidone, produced by Micromonospora coerulea.

The antibiotic Ao58A,which showed strong antifungal activity against some plant pathogenic fungi, was purified from the culture broth and mycelial mats of Micromonospora coerulea strain Ao58 using various chromatographic procedures. The molecular formula of the antibiotic Ao58A was deduced to be C(16)H(23)NO(4) (M + H, m/z 294.1707) by high-resolution FAB mass spectroscopy. Analyses of (1)H NMR, (13)C NMR, and 2D NMR spectral data revealed that the antibiotic Ao58A is the glutarimide antibiotic streptimidone, 4-(2-hydroxy-5, 7-dimethyl-4-oxo-6,8-nonadienyl)-2,6-piperidinedione. The antibiotic Ao58A was very effective in inhibiting growth of Phytophthora capsici,Didymella bryoniae, Magnaporthe grisea, and Botrytis cinerea in the range approximately 3-10 microg mL(-)(1) of MICs. In vivo evaluation of the antibiotic Ao58A under greenhouse condition showed strong control efficacies against the development of P. capsici, B. cinerea, and M. grisea on pepper, cucumber, and rice plants, respectively. The antibiotic Ao58A was equally as effective as metalaxyl, vinclozolin, and tricyclazole in the control of these plant diseases. However, it did not show any phytotoxicity on the plants even when treated with 500 microg mL(-)(1).

LL-E19085 alpha, a novel antibiotic from Micromonospora citrea: taxonomy, fermentation and biological activity.

A new antibacterial antibiotic, designated LL-E19085 alpha, was isolated from the fermentation broth of an actinomycete strain. Based on cultural, physiological, morphological and chemical characteristics, culture LL-E19085 was identified as a new subspecies of Micromonospora citrea. Antibiotic LL-E19085 alpha demonstrated potent activity against a spectrum of Gram-positive aerobic and anaerobic bacteria.

Neihumicin, a new cytotoxic antibiotic from Micromonospora neihuensis. I. The producing organism, fermentation, isolation and biological properties.

A new cytotoxic and antifungal antibiotic, neihumicin, was isolated from the culture broth of a soil isolate identified as Micromonospora neihuensis Wu, sp. nov. Neihumicin shows in vitro cytotoxicity against KB tissue culture cells (ED50 0.94 micrograms/ml) as well as antifungal activity against Saccharomyces cerevisiae ATCC 9763.

Clostomicins, new antibiotics produced by Micromonospora echinospora subsp. armeniaca subsp. nov. II. Taxonomic study of the producing microorganism.

Taxonomic properties of actinomycete strain KMR-593, a soil isolate, which produces new anti-anaerobe antibiotics, clostomicins, were investigated. The strain was identified as a new subspecies of the genus Micromonospora and designated Micromonospora echinospora subsp. armeniaca subsp. nov.

Morphological characteristics of colony development in Micromonospora chalcea.

Colonies of Micromonospora chalcea display two types of mycelia. Vegetative mycelia are nonbranched, have a propensity for parallel disposition, and possess infrequent septa. The reproductive mycelia develop on their apical portion and are characterized by branching, frequent septation, and bearing of spores that are either sessile or at the end of small sporophores.

[Isolation and regeneration of Micromonospora olivoasterospora protoplasts]. / Poluchenie i regeneratsiia protoplastov Micromonospora olivoasterospora.

The conditions for preparation and regeneration of the protoplasts of M. olivoasterospora were developed. It was found that effective formation of the protoplasts required preliminary cultivation of M. olivoasterospora in the medium containing glycine in a concentration inhibiting its growth at least by 60-80 per cent. The strains studied markedly differed in their sensitivity to glycine and were highly sensitive to it. The efficacy of the protoplast formation depended on the culture age and increased with the use of the lytic enzyme 3 of Cytophaga dissolvens. The possibility and advisability of the use of prolonged lysis of the Micromonospora cell walls were shown. A rich organic medium was used for regeneration of the protoplasts.

Studies on a new nucleoside antibiotic, dapiramicin. I. Producing organism, assay method and fermentation.

A new antibiotic, dapiramicin, has been isolated from the fermentation broth of Micromonospora sp. SF-1917. Since the dapiramicin exhibited essentially no in vitro activity by usual bioassays, two assay methods for dapiramicin were developed, HPLC and a new bioassay by growth inhibition of fungal mycelia on a slide glass. Fermentation of dapiramicin is also described.

Mutagenesis of Micromonospora rosaria by using protoplasts and mycelial fragments.

Both mycelial fragments and protoplasts were successfully employed for mutagenesis of Micromonospora rosaria NRRL 3718, and the results were compared. The optimal conditions and effective procedures for mutagenesis of M. rosaria by a chemical mutagen, N-methyl-N'-nitro-N-nitrosoguanidine, have been determined. Mutation was efficiently induced when mycelial fragments were treated with N-methyl-N'-nitro-N-nitrosoguanidine at a concentration of 0.3 to 0.5 mg/ml in the reaction buffer of pH 7.0. Optimal treatment time was 20 to 40 min. Ampicillin treatment was very effective for enrichment of auxotrophs. Protoplasts showed much higher sensitivity to the lethal effect of N-methyl-N'-nitro-N-nitrosoguanidine. Although protoplasts have some advantage of single cell characteristics, the frequency of auxotrophs obtained was somewhat lower. Up to 4% of the colonies were shown to be auxotrophs under the well-defined conditions. This mutagenesis method with protoplasts or fragmented mycelia (or both) should be applicable to other actinomycetes that have limited or no sporulation.

Cell wall composition of Micromonospora olivoasterospora, Micromonospora sagamiensis, and related organisms.

Cell walls of 19 Micromonospora species were analyzed for their components. All the cell walls had xylose and arabinose, but the presence of glucose, galactose, mannose, or rhamnose depended on the strain. Amino acids present in the walls consisted of glycine, glutamic acid, diaminopimelic acid, and alanine, in a molar ratio of approximately 1:1:1:0.6--0.8. 3-Hydroxydiaminopimelic acid, together with meso-diaminopimelic acid, was found in many species and was isolated from Micromonospora olivoasterospora to compare the color constant in an amino acid analyzer with that of meso-diaminopimelic acid. The cell walls of Micromonospora sagamiensis and M. olivoasterospora contained only D-alanine and not L-alanine. All species tested except Micromonospora globosa contained glycolate in an almost equimolar ratio to diaminopimelic acid in their cell walls. Among 45 strains of 12 genera examined, Actinoplanes, Ampullariella, Amorphosporangium, and Dactylosporangium species had a significant amount of glycolate in the whole cells. Based on these results, the primary structure of the peptidoglycan of Micromonospora is discussed.

Fine structure of sporogenesis and septum formation in Micromonospora globosa Kriss and M. fusca Jensen.

Sporogenesis in two species of Micromonospora M. globosa and M. fusca (Actinomycetes) was quite similar. As in fungi, spore formation began as a blowing-out of a hyphal tip with the subsequent centripetal invagination of the plasma membrane. Septal wall material was deposited in a typical three-layered pattern, i.e., two electron-opaque layers separated by an electron-transparent layer. A second electron-opaque wall layer was later formed within the spore and finally a third, less electron-opaque wall was produced. Spore dihiscence was facilitated by the fragmentation of the first-formed wall surrounding the spore. Sporogenesis in Micromonospora is blastic in nature producing terminal, thick-walled spores. In M. fusca, a sporulation process was observed which closely resembled sporangial formation. The process appeared similar to that described for the genus Actinoplanes. Swollen, multiseptate structures were also present. Also in M. fusca, perforate septa with flared pore margins were observed. These septa were similar in appearance to the dolipore septa of Basidiomycetes although they lack a parenthesome and pore plug. Although an extensive membrane system (mesosome) was associated with the finishing septum, its function in the process of septum formation was not determined.