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PubMed2004

Gene cluster in Micromonospora echinospora ATCC15835 for the biosynthesis of the gentamicin C complex.

Gentamicin is a 4,6-disubstituted aminocyclitol antibiotic complex synthesised by some members of the actinomycete genus Micromonospora. In a search for the gentamicin biosynthetic gene cluster we identified, using a cosmid library approach, a region of the M. echinospora ATCC15835 chromosome that encodes homologues of aminoglycoside biosynthesis genes including gntB-a close homologue of the 2-deoxy-scyllo-inosose synthase gene (btrC) from butirosin-producing Bacillus circulans. Insertional inactivation was achieved by homologous recombination with an internal gntB fragment-containing suicide plasmid, delivered by conjugal transfer from Escherichia coli. gntB disruptants were gentamicin nonproducing mutants as assayed by an ELISA antibiotic detection system, proving the association of gntB (or a downstream region) with gentamicin biosynthesis. The function of some open reading frames within the cluster, predicted by nucleotide database homology searching, is discussed with regards to their potential roles in gentamicin biosynthesis. The discovery of this genetic region represents the first report of a gene cluster involved in the biosynthesis of a 4,6-disubstituted aminocyclitol antibiotic.

Gentamicins↗
PubMedDate not supplied

[Degradation of naphthalene-2-sulfonate by strains of Micromonospora].

It was possible to isolate several strains of Micromonospora sp. from waters of Rio Reconquista. They all were filamentous bacteria with lateral spores, highly resistant to Cr(III) and another heavy metal cations. All these strains were able to grow on naphthalene-2-sulphonate as sole carbon source in a mineral medium. The biodegradation of the xenobiotic proceeds via the formation of salicylate and gentisate. These compounds have been isolated and mass spectrometry identified.

Argentina↗
PubMed2001

Antibiotic production, accumulation of intracellular carbon reserves, and sporulation in Micromonospora echinospora (ATCC 15837).

The physiology of the actinomycete Micromonospora echinospora was examined during growth. Biphasic accumulation of glycogen occurred, initially during the early exponential growth phase, and again following the onset of sporulation at 120 h. Lipid levels increased during growth eventually representing 25% of the cell mass. A significant proportion of the lipid was found to be in the form of triacylglycerols, which were found to accumulate markedly during the sporulation phase. The disaccharide trehalose was also found to accumulate during growth with levels rising to 5% of the dry weight during the mycelial production phase, then remaining constant during sporulation. Antibiotic was produced transiently by the cultures over the period preceding sporulation.

Anti-Bacterial Agents↗
PubMed2004

Molecular cloning and characterization of a 2-deoxystreptamine biosynthetic gene cluster in gentamicin-producing Micromonospora echinospora ATCC15835.

The organization of the 2-deoxystreptamine (DOS) biosynthetic gene cluster of Micromonospora echinospora has been determined. Sequencing of a 14.04 kb-region revealed twelve open reading frames (ORFs): four putative DOS biosynthetic genes (gtmA, B, C, and D), five amino sugars biosynthetic genes (gtmE, G, H, I, and gacB), two aminoglycoside resistance genes (gtmF and J) as well as a hypothetical ORF (gacA). One of the putative DOS biosynthetic genes, gtmA, was expressed in Escherichia coli, and the purified protein was shown to convert glucose-6-phosphate (G-6-P) to 2-deoxy-scyllo-inosose (DOI), a key step in DOS biosynthesis. In addition gtmJ was expressed in Streptomyces lividans and shown to confer gentamicin resistance. Thus gtmA and gtmJ are implicated in the biosynthesis of gentamicin and in resistance to it, respectively.

Anti-Bacterial Agents↗
PubMed2006

Optimization of nutritional requirements for gentamicin production by Micromonospora echinospora.

Effect of various fermentation media, carbon sources, nitrogen sources, phosphate concentration and culture requirements includes inoculum levels and age were determined on gentamicin production and biomass dry weight production for Micromonospora echinospora, a gentamicin producing strain. Of the substrates tested, starch as a sole carbon source promoted maximal gentamicin production, while maltose promoted maximal growth. Yeast extract as a sole nitrogen source promoted maximal growth, while soyabean meal for gentamicin production. Increasing phosphate concentration enhanced gentamicin production and observed optimum production at 1.2 g/1 (6% v/v) of phosphate having 72 h old inoculum in the medium. Highest gentamicin production was obtained after cultivation with shaking for 120 h in a medium containing starch 0.75% (w/v), soyabean meal 0.5%, K2HPO4 0.12%, CaCO3 0.4%, FeSO4 0.003% and CoCl2 0.0001%. The gentamicin production was 1.2-fold in this medium as compared to basal medium.

Anti-Bacterial Agents↗
PubMed1990

Structure of the DNA of five bacteriophages infecting Micromonospora.

The physical maps of the DNA of five bacteriophages (Mm1, OM2, OM3, Mm4 and Mm5) which infect Micromonospora are presented. The restriction analyses showed that all of them had linear, double-stranded DNA, but only four (Mm1, OM2, Mm4 and Mm5) presented cohesive ends. The phages showed no relationship in terms of their restriction maps or of DNA-DNA hybridization, with the exception of Mm4 and Mm5, which resulted to be very similar. Phage Mm5 presented a high level of resistance to chelating agents, although deletion mutants, all of them showing a single detection of 1.4 kb, were obtained by using extremely selective conditions.

Bacteriophages↗
PubMed1989

[Production of lincomycin by Micromonospora halophytica culture].

A Micromonospara culture designated as 991/78 with activity against gram-positive cocci and bacteria was isolated from samples of silt-covered substrates from the Amu-Darya. Directed screening on a selective medium supplemented with lincomycin in an amount of 50-100 micrograms/ml was used. Identification of the antibiotic produced by the culture showed it to be lincomycin. By its taxonomic features the culture was classified as belonging to Micromonospora (subgroup II, Cinnamomea) and in particular to M. halophytica (Weinstein, Luedemann, Oden, Wagman, 1968). Up to now, it was known that lincomycin was produced only by Streptomyces cultures.

Agar↗
PubMed1989

[Direct screening for producers of antibiotics among the Micromonospora].

A test system was developed for screening organisms producing antibiotics of definite chemical groups or mechanisms of action. The system includes efficient selection of cultures belonging to a definite microbiological taxon (genus Micromonospora), investigation of their biological and taxonomic features, the use of specific selective media with high concentrations of definite antibiotics for isolating antibiotic-producing cultures from natural substrates, the use of specific methods for antibiotic chemical isolation at the initial stages of the screening and chromatographic study of the screened compounds. The system provided efficient screening of valuable antibiotics in a short period.

Aminoglycosides↗
PubMed1987

[Formation of A-factor-type regulators by representatives of the genus Micromonospora].

It is known that substances similar in their chemical structure and biological action to A-factor, a regulator of Streptomyces griseus differentiation are widely observed in streptomycetes. The present paper is concerned with studies on capacity for producing A-factor-like regulators in 13 strains belonging to 11 species of genus Micromonospora. It was shown that 7 strains had an A-factor-deficient test mutant effect analogous to the effect of A-factor. Relation of A-factor-like substance production to biosynthesis of sisomicin and sporogenesis was investigated in M. fusca var. sisomicini, strain RIA 1572. A-Factor-like substances were isolated from submersion culture of strain 1572 on the 6th-8th day of the culture growth. By the time it corresponded to the highest number of the spores in the medium. The highest levels of sisomicin were detected on the 3rd day of the fermentation process. After that period its concentration markedly lowered. Exogenic addition of A-factor had no effect on sisomicin biosynthesis but resulted in increased numbers of the spores.

4-Butyrolactone↗
PubMed1984

[Isolation and regeneration of Micromonospora olivoasterospora protoplasts].

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.

Cell Fractionation↗
PubMed1977

[Effect of cobalt on the growth of a Micromonospora purpurea var. violacea 1935 culture and on the biosynthesis of gentamincin].

The effect of cobalt on growth of Micromonospora purpurea var. violacea 1935 and biosynthess of gentamicin in complex soy-bean meal or "synthetic" gelatin medium was studied. In optimal concentrations cobalt increased the activity level of the culture fluid almost 6 times in both media having practically no effect on the amount of the mycelium formed. Addition of cobalt to the medium resulted in retardation of the organism growth and consumption of the nutrients. Stimulation of gentamicin biosynthesis by cobalt was inversely proportional to the time of the trace element addition. The cobalt effect depended on the aeration conditions, inorganic phosphorus level in the medium and physiological condition of the mycelium.

Aerobiosis↗
PubMed1976

Properties of proteolytic enzymes isolated from a thermophilic strain of Micromonospora vulgaris 42.

Two proteolytic enzymes, protease A and protease B, were isolated in homogeneous state from the cultural broth of the thermophilic actinomycete Micromonospora vulgaris 42. Their physicochemical properties were studied, i.e., molecular weight (50 000 for protease A and 30 000 for protease B), amino acid composition, N-terminal amino acids (phenylalanine for protease A and alanine for protease B). The specificity of the action of these enzymes was assayed by splitting the B chain of oxidized insulin. Both enzymes are neutral proteases of the thermolysine type.

Alanine↗
PubMed1986

Crisamicin A, a new antibiotic from Micromonospora. I. Taxonomy of the producing strain, fermentation, isolation, physico-chemical characterization and antimicrobial properties.

A microorganism, designated as RV-79-9-101 and now identified as Micromonospora purpureochromogenes subsp. halotolerans, isolated from a mud sample in the Philippines, has been shown to produce a complex of antibiotics called crisamicins. Thin-layer chromatography and bioautography, employing solvent extracts of whole fermentation broths, revealed a minimum of five antimicrobial components. The major biologically-active component of the antibiotic complex, crisamicin A, was obtained in pure form after preparative silica gel column chromatography followed by crystallization. Based on physico-chemical data crisamicin A has been identified as a novel member of the isochromanequinone group of antibiotics. It exhibits excellent in vitro activity against Gram-positive bacteria but little or no activity towards Gram-negative bacteria or fungi.

Anti-Bacterial Agents↗
PubMed1986

Isolation and structure determination of crisamicin A, a new antibiotic from Micromonospora purpureochromogenes subsp. halotolerans.

A new antibiotic, crisamicin A, with in vitro activity against Gram-positive bacteria, B16 murine melanoma cells, and herpes simplex, vaccinia, and vesicular stomatitis viruses, has been isolated from Micromonospora purpureochromogenes subsp. halotolerans. On the basis of 1H and 13C NMR spectroscopic, high resolution field desorption mass spectrometric, and circular dichroism studies of the antibiotic and several of its derivatives, the structure of crisamicin A has been assigned.

Anti-Bacterial Agents↗
PubMed2002

Kosinostatin, a quinocycline antibiotic with antitumor activity from Micromonospora sp. TP-A0468.

Kosinostatin, a quinocycline antibiotic was isolated from the culture broth of an actinomycete strain TP-A0468 along with isoquinocycline B. The producing strain was isolated from the seawater sample collected in Toyama Bay and identified as Micromonospora sp. based on the taxonomic study. Kosinostatin was obtained from the culture fluid by solvent extraction and ODS column chromatography. Kosinostatin inhibited the growth of Gram-positive bacteria strongly (MIC=0.039 microg/ml) and Gram-negative bacteria and yeasts moderately (MIC= 1.56 approximately 12.5 microg/ml). It showed cytotoxicity against various cancer cell lines with the IC50 of 0.02 approximately 0.6 microm and inhibited human DNA topoisomerase Ila with the IC50 of 3 approximately 10 microM.

Aminoglycosides↗
PubMed2002

NMR analysis of quinocycline antibiotics: structure determination of kosinostatin, an antitumor substance from Micromonospora sp. TP-A0468.

A quinocycline antibiotic, kosinostatin, was isolated from the culture broth of Micromonospora sp. TP-A0468 along with isoquinocycline B. Structure of kosinostatin was determined to be the stereoisomer of isoquinocycline B regarding to the stereochemistry at the C-2' spiro carbon by NMR analysis. Kosinostatin isomerizes to isoquinocycline B through the inversion of the stereocenter at C-2'. Comparison of physico-chemical properties indicated that kosinostatin is presumably identical with quinocycline B isolated by CELMER et al. from Streptomyces aureofaciens.

Aminoglycosides↗