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PubMed1994

A gene encoding mycinamicin III O-methyltransferase from Micromonospora griseorubida.

A DNA fragment of 42 kb encompassing one of the mycinamicin II (Mm)-resistance-encoding genes, myrB, from a Mm-producing strain, Micromonospora griseorubida, was cloned in Escherichia coli using the cosmid vector pJB8. Nucleotide sequencing of the neighboring region of myrB and a computer-aided analysis of the sequence predicted the presence of an open reading frame (ORF) with 254 amino acids which showed great similarity to the macrocin O-methyltransferase (tylF gene product) in tylosin (Ty)-producing Streptomyces fradiae. When a 1.0-kb AluI fragment containing the complete ORF was fused to the lacZ promoter in the correct orientation and expressed in E. coli, a mycinamicin III (MIII) O-methyltransferase (MOMT) activity was detected only upon induction with isopropyl-beta-D-thiogalactopyranoside (IPTG). All these data indicate that this ORF codes for the structural gene of MOMT, and it is designated mycF.

Amino Acid Sequence↗

PubMed1992

Transcriptional mapping of the promoter of the aminoglycoside acetyltransferase gene (aacC9) of neomycin-producing Micromonospora chalcea.

We have studied the promoter of the gene encoding aminoglycoside acetyltransferase (aacC9) in neomycin-producing Micromonospora chalcea. S1 nuclease mapping showed that the transcription initiation point of this gene is at the translation start point, with no evidence of a conventional ribosome-binding site. The aac of paromycin-producing Streptomyces rimosus forma paromomycinus shows the same characteristic; there is no homology in the promoter regions of the two genes, whereas the coding sequences are very similar.

Acetyltransferases↗

PubMed2004

A new compound from Micromonospora sp. SA246, 9-hydroxycrisamicin-A, activates hepatitis B virus replication.

A new compound from Micromonospora sp. SA246, 9-hydroxycrisamicin-A (9-HCA-A), showed potential for activating hepatitis B virus (HBV) replication. To define the mechanism of 9-HCA-A, we used HepG2 2.2.15 cells which support HBV replication. 9-HCA-A activated HBV replication, increased episomal and integrated HBV DNA content, and increased secretions of HBV antigens (HBsAg and HBeAg) into culture medium. 9-HCA-A also activated HBV transcription in Hep2 2.2.15 cell line. To examine transcriptional control mechanisms, we analyzed the effect of 9-HCA-A on four different HBV promoters (Core, PreS1, PreS2, and X) in hepatoma cell line. 9-HCA-A responsive element was located at HBx promoter. By EMSA, we showed that 9-HCA-A activated the HBx promoter by detaching the 9-HCA-A responsive element binding protein (9H-REBP). Protein phosphatase (PP2A1) treatment detaches the 9H-REBP from the HBx promoter, similar to 9-HCA-A, while protein kinase A treatment does not detach the 9H-REBP from the HBx promoter. Our results showed that 9H- REBP functions as a repressor of HBV replication while 9-HCA-A activated protein phosphatase released the BP on the HBx promoter, thus activating HBV replication.

Anti-Bacterial Agents↗

PubMed2004

Contribution of the active site aspartic acid to catalysis in the bacterial neuraminidase from Micromonospora viridifaciens.

A recombinant D92G mutant sialidase from Micromonospora viridifaciens has been cloned, expressed and purified. Kinetic studies reveal that the replacement of the conserved aspartic acid with glycine results in a catalytically competent retaining sialidase that possesses significant activity against activated substrates. The contribution of this aspartate residue to the free energy of hydrolysis for natural substrates is greater than 19 kJ/mol. The three dimensional structure of the D92G mutant shows that the removal of aspartic acid 92 causes no significant re-arrangement of the active site, and that an ordered water molecule substitutes for the carboxylate group of D92.

Aspartic Acid↗

PubMed2004

The targeted inactivation of polyketide synthase mycAV in the mycinamicin producer, Micromonospora griseorubida, and a complementation study.

Mycinamicin is a 16-membered macrolide antibiotic produced by Micromonospora griseorubida A11725, which shows strong antimicrobial activity against gram-positive bacteria. Recently, the nucleotide sequences of the mycinamicn biosynthetic gene cluster in M. griseorubida have been completely determined. Mycinamicin non-producer M7A21 was isolated by mycAV inactivation, which encodes the module 7 of mycinamicin polyketide synthase (PKS) required for the biosynthesis of the mycinamicin biosynthetic intermediate protomycinolide-IV (PML-IV). When the bioconversion to mycinamicin II (M-II) from PML-IV was performed using M7A21 and the feeding culture method, the productivity of M-II was the same as that of M-II in wild-type strain A11725. p446M7 containing mycAV was constructed using the Escherichia coli-Streptomyces shuttle vector pGM446. The mycinamicin productivity of M7A21 was restored by the introduction of p446M7 into the M7A21 cell, but almost all p446M7 was integrated into the chromosome of M7A21 because the plasmid was unstable in M7A21. The feeding culture and the introduction of the complement gene for M7A21 would be powerful tools to perform combinatorial biosynthesis for the production of new macrolide antibiotics.

Gene Silencing↗

PubMed1978

Mode of action of gentamicin antibiotics produced by Micromonospora purpurea.

Gentamicin antibiotics were produced fermentatively by Micromonospora purpurea. They were separated into gentamicin C1, C1a and C2 by paper chromatographic technique, using chloroform, methanol, and 17.0% of NH4OH (2: 1: 1 v/v) as developing solvent. The different antibiotics showed variable antimicrobial activities. The gentamicin antibiotics inhibited the biosynthesis of DNA and RNA of the proteins, present in the cells of Staphylococcus aureus.

Bacteria↗

PubMed1999

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).

Anti-Bacterial Agents↗

PubMed2002

Isolation of streptonigrin and its novel derivative from Micromonospora as inducing agents of p53-dependent cell apoptosis.

Streptonigrin (1) and its novel natural derivative 7-(1-methyl-2-oxopropyl)streptonigrin (2) were isolated from an actinomycete strain, Micromonospora sp. IM 2670. The inductions for 1 and 2 are more potent in the human neuroblastoma SH-SY5Y cells that contain wild-type p53 than in SH-SY5Y-5.6 cells that overexpress a dominant negative mutant of p53, thus suggesting that they induce apoptosis through a p53-dependent pathway.

Apoptosis↗

PubMed2004

Diazepinomicin, a new antimicrobial alkaloid from a marine Micromonospora sp.

The structure of a new dibenzodiazepine alkaloid, diazepinomicin (1), isolated from the culture of a marine actinomycete of the genus Micromonospora was characterized using spectroscopic methods. Diazepinomicin represents a unique molecular class composed of a dibenzodiazepine core linked to a farnesyl side chain.

Alkaloids↗

PubMed2004

Modulation of glycogen and trehalose levels in Micromonospora echinospora (ATCC 15837).

The growth of Micromonospora echinospora was studied in high and low C/N ratio medium using both batch and continuous culture. Asparagine was consumed rapidly in batch cultures where it served as both a nitrogen and carbon source. Glucose consumption was low suggesting that asparagine functions as the major carbon source under these conditions. The effect of nutrient limitation on the accumulation of storage carbohydrate in batch culture revealed an intimate association between nitrogen limitation and the accumulation of carbonaceous reserves. This study revealed that glycogen constituted the major carbohydrate reserve associated with the onset of sporulation. Intracellular trehalose levels were found to be relatively low and may have been affected by the availability of carbon. Continuous culture studies revealed a correlation between glycogen accumulation and increasing growth rate. It was also found that elevated cellular ATP levels correlated with the increase in glycogen, and reduced glycolytic activity. At the higher growth rates cellular ATP levels were elevated and coincided with reduced activity of the key glycolytic enzyme, phosphofructokinase, suggesting that glycogen can act as a convenient energy reservoir when excess carbon flux dictates.

Adenosine Triphosphate↗

PubMed2005

Retymicin, galtamycin B, saquayamycin Z and ribofuranosyllumichrome, novel secondary metabolites from Micromonospora sp. Tü 6368. I. Taxonomy, fermentation, isolation and biological activities.

A new xanthone compound named retymicin (1) was isolated together with galtamycin B (2) and saquayamycin Z (3), new members of the galtamycin and saquayamycin families, respectively, and the new lumichrome derivative 1-(alpha-ribofuranosyl)-lumichrome (4) from Micromonospora strain Tü 6368, isolated from a soil sample collected in Romania. Retymicin, galtamycin B and saquayamycin Z show cytostatic effects to various human tumor cell lines whereas saquayamycin Z is also active against Gram-positive bacteria.

Anthracyclines↗

PubMed2005

Retymicin, galtamycin B, saquayamycin Z and ribofuranosyllumichrome, novel secondary metabolites from Micromonospora sp. Tü 6368. II. Structure elucidation.

A detailed screening of the secondary metabolite pattern from Micromonospora sp. strain Tü 6368 resulted in the isolation of ten compounds belonging to five different structural families. The structures of the novel compounds 1-(alpha-ribofuranosyl)-lumichrome (3), retymicin (7), galtamycin B (11) and saquayamycin Z (14) were assigned by spectroscopic methods and chemical transformations. This strain fits our hypothesis that the metabolite analysis of biosynthetically talented strains leads readily to novel compounds.

Anthracyclines↗

PubMed2001

Characterization of the biosynthetic gene cluster for the oligosaccharide antibiotic, Evernimicin, in Micromonospora carbonacea var. africana ATCC39149.

Evernimicin (EV) belongs to the orthosomycin class of antibiotics and consists of several modified L- and D-deoxysugars containing unusual orthoester and glycosyl linkages and two orsellinic acid groups, one that is halogenated. The EV biosynthetic gene cluster from Micromonospora carbonacea var. africana ATCC39149 was localized by hybridization to a dTDP-D-glucose 4,6-dehydratase probe and a 120-kb region containing the EV biosynthetic cluster and surrounding regions has been sequenced. BLAST analysis has identified a type I polyketide synthase for orsellinic acid biosynthesis as well as enzymes required for L- and D-deoxyglucose and D-deoxymannose synthesis. In addition, genes involved in glycosyltransfer and resistance were identified. Insertional mutations in several biosynthetic genes blocked EV production, indicating a role for these genes in EV biosynthesis.

Anti-Bacterial Agents↗

PubMed2000

Characterization of an extracellular enzyme system produced by Micromonospora chalcea with lytic activity on yeast cells.

Growth of Micromonospora chalcea on a defined medium containing laminarin as the sole carbon source induced the production of an extracellular enzyme system capable of lysing cells of various yeast species. Production of the lytic enzyme system was repressed by glucose. Incubation of sensitive cells with the active component enzymes of the lytic system produced protoplasts in high yield. Analysis of the enzyme composition indicated that beta(1-->3) glucanase and protease were the most prominent hydrolytic activities present in the culture fluids. The system also displayed weak chitinase and beta(1-->6) glucanase activities whilst devoid of mannanase activity. Our observations suggest that the glucan supporting the cell wall framework of susceptible yeast cells is not directly accessible to the purified endo-beta(1-->3) glucanase and that external proteinaceous components prevent breakdown of this polymer in whole cells. We propose that protease acts in synergy with beta(1-->3) glucanase and that the primary action of the former on surface components allows subsequent solubilization of inner glucan leading to lysis.

Candida↗

PubMed2000

Response of Micromonospora echinospora (NCIMB 12744) spores to heat treatment with evidence of a heat activation phenomenon.

The effects of heat treatment on spores of the actinomycete Micromonospora echinospora were investigated. The percentage of culturable spores in untreated spore stocks was found to be approximately 20%. A 60 degrees C treatment of spores in phosphate buffer for 10 min led to an approximately five-fold increase in the number of culturable units. This indicated that a large proportion of the spores were constitutively dormant. Within 10 min and in the absence of an external energy-yielding substrate, the heat treatment was found to stimulate spore respiration suggesting that endogenous storage compounds were being utilized. Heating spores at 70 degrees C shortened the time period required for activation; holding times greater than 10 min, however, resulted in a reduction of culturable cells. Classic thermal death characteristics were seen at temperatures of 80 degrees C and above with D-values of 21.43, 2.67, 0.45 and 0.09 min being recorded at 70, 80, 90 and 100 degrees C, respectively. Spores of this organism, while being weakly heat resistant in comparison with bacterial endospores, are significantly more resistant than vegetative cells.

Colony Count, Microbial↗

PubMed2003

The importance of amino acids as carbon sources for Micromonospora echinospora (ATCC 15837).

AIMS: This study set out to investigate the effect of amino acids on the uptake of glucose by Micromonospora eichinospora (ATCC 15837). METHODS AND RESULTS: The specific rate of glucose uptake was found to be reduced when organic nitrogen components were present in the medium. Radioactive uptake studies revealed that the Km for glucose in this organism was 53 mm, indicating a low affinity for uptake compared with other actinomycete sugar transport systems. Individual amino acids negatively influenced the rate of glucose transport, suggesting a relationship between amino acid metabolism and glucose uptake in this organism. The sugar transport system was found to be an active process being inhibited by ionophores and KCN. CONCLUSIONS: The data suggest a direct link between amino acid metabolism and glucose uptake at the level of sugar transport. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that the uptake of glucose, a major carbon source for many antibiotic fermentations, is significantly reduced in the presence of amino acids. This fact should inform the medium design and feeding regimes of fermentations involving similar actinomycetes.

Amino Acids↗

PubMed2005

Isolation of three new naturally occurring compounds from the culture of Micromonospora sp. P1068.

Bioassay guided isolation of an antibacterial extract prepared from the fermentation broth of a Micromonospora sp. P1068 led to the isolation of eight compounds identified as (3R) 3,4',7-trihydroxy-isoflavanone (1), 3-hydroxydehydrodaidzein, daidzein (2), 3-methyl-1H-indole-2-carboxylic acid (3), 1H-indole-3-carboxaldehyde (4), 3-(p-hydroxyphenyl)-N-methylpropionamide, N-methylphloretamide (5), phenyl acetic acid (6), 2-hydroxy phenyl acetic acid (7) and 4-hydroxy-5-methoxy-benzoic acid (8). Compounds 1 and 5 were found to be novel chemical entities while 3 was isolated from a natural source for the first time. All compounds were evaluated for their antimicrobial activities against a panel of clinically significant microorganisms. Compound 4 was active against Staphylococcus aureus (MIC, 32 microg/ml), Enterococcus faecium (MIC, 32 microg/ml) and Escherichia coli (MIC, 64 microg/ml).

Biological Assay↗

PubMed1996

16S ribosomal DNA analysis of the genera Micromonospora, Actinoplanes, Catellatospora, Catenuloplanes, Couchioplanes, Dactylosporangium, and Pilimelia and emendation of the family Micromonosporaceae.

In order to determine the phylogenetic structure of the actinomycete family Micromonosporaceae, the 16S ribosomal DNA sequences of 17 type species of the genera Actinoplanes, Dactylosporangium, and Pilimelia were compared with the 16S ribosomal DNA sequences of species of the genera Catellatospora, Catenuloplanes, and Couchioplanes and with those of species of the genus Micromonospora and other actinomycete genera for which the sequences have been previously determined. All genera of the family, together with the genera Catellatospora, Catenuloplanes, and Couchioplanes, form a phylogenetically coherent cluster that is well separated from other families of the order Actinomycetales. Except for one species of the genus Catellatospora, all species cluster according to their genus affiliation. The inclusion of the genera Catellatospora, Catenuloplanes, and Couchioplanes into the family broadens the phenotypic and chemotaxonomic heterogeneity of this taxon. An emendation of the family Micromonosporaceae is given.

Base Sequence↗