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PubMed1992

Mutations in the P1 promoter region of Micromonospora echinospora.

We demonstrated previously that the promoters P1a, P1b, and P1c are very closely spaced and are coordinately turned on during stationary phase in Micromonospora echinospora. To determine the nucleotides important for promoter recognition, we characterized mutations that were defective in transcription from the P1b start site, using Streptomyces lividans as the host. Two mutations upstream of the start site resulted in a drastic loss of promoter activity, while two mutations downstream of the start site resulted in a moderate loss of activity. These mutations suggest an unexpected relationship between promoters P1b and P1c. Three of the mutations that caused diminished transcription from promoter P1b simultaneously resulted in an increase in transcription from the P1c promoter initiation site located 15 bp downstream. Despite the proximity and the coordinate regulation of promoters P1b and P1c, they are in competition as transcriptional start sites.

Base Sequence↗
PubMed1992

Cloning, expression, and characterization of the Micromonospora viridifaciens neuraminidase gene in Streptomyces lividans.

We have cloned the Micromonospora viridifaciens neuraminidase (EC 3.2.1.18) gene (nedA) in Streptomyces lividans. This was accomplished by using the vector pIJ702 and BglII-BclI libraries of M. viridifaciens chromosomal inserts created in S. lividans. The libraries were screened for the expression of neuraminidase by monitoring the cleavage of the fluorogenic neuraminidase substrate 2'-(4-methylumbelliferyl)-alpha-D-N-acetyl-neuraminic acid. Positive clones (BG6, BG7, BC4, and BC8) contained the identical 2-kb BclI-BglII fragment and expressed neuraminidase efficiently and constitutively using its own promoter in the heterologous host. From the nucleotide sequence analysis, an open reading frame of 1,941 bp which encodes a polypeptide with an M(r) of 68,840 was detected. The deduced amino acid sequence has five Asp boxes, -Ser-X-Asp-X-Gly-X-Thr-Trp, showing great similarity to other bacterial and viral neuraminidases. We have also identified the catalytic domain by using truncated proteins produced in S. lividans.

Amino Acid Sequence↗
PubMed1996

Translational autoregulation of the sgm gene from Micromonospora zionensis.

The sisomicin-gentamicin resistance methylase gene (sgm) from Micromonospora zionensis (the producer of antibiotic G-52 [6-N-methyl-sisomicin]) encodes an enzyme that modifies 16S rRNA and thereby confers resistance to 4,6-disubstituted deoxystreptamine aminoglycosides. Here, we report that this gene is regulated on the translational level. The Escherichia coli lacZ gene and operon fusion system was used, and it was shown that an extra copy of the sgm gene decreases the activity of the fusion protein. These results suggested that expression of the sgm gene is regulated by the translational autorepression because of binding of the methylase to its own mRNA. It was shown by computer analysis that the same hexanucleotide (CCGCCC) is present 14 bp before the ribosome-binding site and in the C-1400 region of 16S rRNA, i.e., the region in which most of the aminoglycosides act. A deletion that removes the hexanucleotide before the gene fusion is not prone to negative autoregulation. This mode of regulation of the sgm gene ensures that enough methylase molecules protect the cell from the action of its own antibiotic. On the other hand, if all of the ribosomes are modified, Sgm methylase binds to its own mRNA in an autorepressive manner.

Bacterial Proteins↗
PubMed2006

Optimization of critical medium components for the maximal production of gentamicin by Micromonospora echinospora ATCC 15838 using response surface methodology.

Optimization of the fermentation medium components for maximum gentamicin production by Micromonospora echinospora ATCC 15838 was carried out. Response surface methodology was applied to optimize the medium constituents. A 2(4) full-factorial central composite design was chosen to explain the combined effects of the four medium constituents, viz. starch, soyabean meal, K2HPO4, and CoCl2 and to design a minimum number of experiments. A second order model was developed and fitted using least square method. The R2 value of the model was 0.9723, which shows that model is best fit for the present studies. The results of analysis of variance and regression of a second order model showed that the linear effects of starch (p < 0.001697) and CoCl2 (p < 7.99E-13), and cross product effects of starch and soyabean meal (p < 0.029876) and soyabean meal and CoCl2 (p < 0.008909) were more significant, suggesting that these were critical variables having the greatest effect on the production of gentamicin in the production medium. The optimized medium consisting of 9 g/L starch, 3 g/L soyabean meal, 0.9 g/L K2HPO4, and 0.01 g/L CoCl2 predicted 850 mg/L of gentamicin which was almost 110% higher than that of the unoptimized medium. The amounts of starch, soyabean meal, and K2HPO4 required were also reduced with RSM.

Culture Media↗
PubMed1976

Micromonospora-produced sisomicin components.

A sisomicin fermentation carried out in the presence of (methyl-14C)-L-methionine resulted in a crude mixture, composed of methyl-14C-labeled sisomicin as a major component; and two 4''-C-desmethylsisomicin (66-40B and 66-40D) isomer-like components, an unidentified component and a gentamicin A-like antibiotic as minor components. When (methyl-14C)-L-methionine was added in an early stage of the fermentation (24 hours), incorporation of methyl-14C-label into polar components (e.g., gentamicin A-like antibiotic) preceded that into sisomicin. Chromatographic evidence for the bioconversion of (methyl-14C)-gentamicin A to a radioactive sisomicin-like product (possibly (3''-N-methyl-14C)-sisomicin) was seen, when a Micromonospora blocked mutant was incubated in the presence of the former antibiotic.

Anti-Bacterial Agents↗
PubMed1977

Fortimicin A production by Micromonospora olivoasterospora in a chemically defined medium.

A chemically defined medium was devised in order to study the requirements for fortimicin A production by Micromonospora olivoasterospora KY 11515. Soluble starch was the best carbon source; NH4Cl and NH4NO3 were suitable nitrogen sources both for the growth and fortimicin production. Amino acids such as L-asparagine, L-aspartic acid and L-glutamic acid showed some stimulatory effects on both growth and antibiotic production of M. olivoasterospora while L-serine stimulated only antibiotic production and L-citrulline only the growth. K2HPO4, MgSO4.7H2O and CaCO3 were essential especially for the antibiotic production. The most important finding was that vitamin B12, cobalt and nickel showed marked stimulatory effects on fortimicin A production.

Amino Acids↗
PubMed1977

Mutational biosynthesis by idiotrophs of Micromonospora purpurea. I. Conversion of aminocyclitols to new aminoglycoside antibiotics.

By mutation and strain improvement techniques idiotrophs of Micromonospora purpurea, the gentamicin-producing organism, were obtained which require an exogenous source of 2-deoxystreptamine in order to produce gentamicin. Streptamine incorporation afforded a mixture of 2-hydroxygentamicin C as a complex of essentially the C1 and C2 components whereas 2-deoxystreptamine when incorporated by the same idiotroph afforded the same mixture of C1, C2 and C1a gentamicins as the parent (m1) organism. The 2-hydroxygentamicin C complex exhibited broad-spectrum antibiotic activity with an in vitro potency less than that for the gentamicin C complex, but with greater activity against selected gentamicin C resistant organisms. The LD 50 (i.v.) in mice of the 2-hydroxygentamicin C complex indicated that it had approximately half the toxicity of the gentamicin C complex. 2, 5-Dideoxystreptamine affordeda C1, C2, and C1a mixture of 5-deoxygentamicins, which also had broad spectrum activity, and exhibited improved activity against several gentamicin-acetylating strains of resistant bacteria. The LD50 (i.v.) in mice of the 5-deoxygentamicin C complex indicated that it was about 2.5 times more toxic than the gentamicin C complex. Two derivatives of 2,5-dideoxystreptamine afforded the same mixture of 5-deoxygentamicins. 2-Epistreptamine upon supplementation to a broth containing growing cultures of these idiotrophs also produced antibiotic.

Amino Sugars↗
PubMed1977

Metabolites of gentamicin-producing Micromonospora species I. Isolation and identification of metabolites.

From the cultural broth of a Micromonospora species 25 aminocyclitol antibiotics were isolated by repeated ion-exchange chromatographic processes. The main components of the metabolites were identified as gentamicin C1, C2, and C1a. The other previously reported gentamicin type antibiotics and some other degradation products including gentamicin A, B, B1, X2, sisomicin, garamine, gentamines etc., were identified by chemical, PMR, and mass spectroscopic studies. Besides these, seven new gentamicin type antibiotics were isolated and characterized.

Bacteria↗
PubMed1977

Mutational biosynthesis by idiotrophs of Micromonospora purpurea. II. Conversion of non-amino containing cyclitols to aminoglycoside antibiotics.

A mutant of Micromonospora purpurea, which produces the gentamicin complex only when 2-deoxystreptamine is added to the fermentation medium, produces a new antibiotic complex, 2-hydroxygentamicin, when streptamine or 2,4,6/3,5-pentahydroxycyclohexanone is added to the fermentation medium. This mutant also produces the gentamicin complex when 2,4/3,5-tetrahydroxycyclohexanone is added to the fermentation medium. The C1 and C2 components of 2-hydroxygentamicin have broad spectrum in vitro antibacterial activity similar to the gentamicin C1 and C2 components, but with greater activity against some gentamicin-resistant strains.

Bacteria↗
PubMed1978

Macrolide antibiotics M-4365 produced by Micromonospora. III. In vitro antimicrobial activity of antibiotic M-4365G2 (de-epoxy rosamicin).

Antibiotic M-4365G2 (de-epoxy rosamicin) produced by Micromonospora capillata MCRL 0940 is a new basic 16-membered macrolide antibiotic with activity equal to or superior to erythromycin and josamycin against Gram-positive bacteria. Of interest are the high degree of activity against Gram-negative bacilli and mycoplasmas, and striking inhibitory effects against indole-producing Proteus spp. Bactericidal activity of M-4365G2 is also to be noticed.

Aminoglycosides↗
PubMed1980

Izumenolide-a novel beta-lactamase inhibitor produced by Micromonospora. I. Detection, isolation and characterization.

Micromonospora chalcea subsp. izumensis produces a novel beta-lactamase inhibitor izumenolide (EM4615). Isolation of izumenolide was performed by extraction into butanol under acidic conditions and then back extraction into water at neutrality. The compound was precipitated from the aqueous phase by the addition of calcium or barium salts. Further purification was achieved by distribution in BuOH - 1 N NaOH. Izumenolide is a macrolide containing sulfate ester groups.

Chemical Phenomena↗
PubMed1980

Microbial products. IV. X-14847, a new aminoglycoside from Micromonospora echinospora.

Micromonospora echinospora strain X-14847 produces gentamicin A as the major antibiotic together with a new aminoglycoside, termed X-14847, and identified as a 2-amino-2-deoxy-alpha-D-glucopyranosyl myo-inositol. This report describes the taxonomy of the culture, fermentation conditions, the isolation and the identification of X-14847.

Aminoglycosides↗
PubMed1980

New anthracycline glycosides from Micromonospora. II. isolation, characterization and biological properties.

Four glycosides, designated A, B, C and D, are the main components of the anthracycline complex produced by cultures of Micromonospora sp. nov. They were extracted by solvent partition, separated by column chromatography and characterized by chemical and physical methods as 11-deoxy analogues of daunorubicin. Among these new anthracyclines, displaying antibacterial and cytotoxic activity in vitro, 11-deoxydaunorubicin and 11-deoxydoxorubicin are also active against P388 leukemia in mice.

Animals↗
PubMed1981

Isolation of N-(2,6-diamino-6-hydroxymethylpimelyl)-L-alanine from Micromonospora chalcea.

A novel dipeptide, N-(2,6-diamino-6-hydroxymethylpimelyl)-L-alanine, was isolated from the culture broth of a microorganism identified as Micromonospora chalcea. The dipeptide exhibits antimicrobial activity against Escherichia coli on a synthetic medium, and the activity is synergistically enhanced by several cell wall synthesis-inhibitors.

Amino Acids, Diamino↗
PubMed1982

Sagamicin and the related aminoglycosides: fermentation and biosynthesis. I. Biosynthetic studies with the blocked mutants of Micromonospora sagamiensis.

The mutants blocked in the gentamicin C1 production were derived from a sagamicin producing strain of Micromonospora sagamiensis. The intermediates produced by these mutants were isolated and properly identified. Comparing the biotransformation activities in the resting cells of the mutants with those of a DOS idiotroph, KY 11525, the blocked steps in sagamicin and gentamicin biosynthesis were proposed in each mutant. Mutant KY 11564 was found to produce gentamicin C2a (C-6'-epimer of gentamicin C2) together with gentamicin C1a and sagamicin. KY 11525 transformed gentamicin C2a into C2 and C1, whereas KY 11564 lacked the activity. KY 11565 produced gentamicin X2 and antibiotic G-418, and lacked 6'-amino substitution activities. KY 11566 appeared to be partially blocked in 6'-N-methylation activities, and the major products were gentamicin C2a and C1a. From these results, sagamicin biosynthesis in M. sagamiensis is discussed.

Aminoglycosides↗
PubMed1982

Studies on a new antibiotic M-92 produced by Micromonospora. II. Isolation and physicochemical properties of M-92 and its components.

A new antibiotic complex, M-92 was isolated from the whole fermentation broth of Micromonospora verruculosa MCRL 0404. The whole broth was mixed with talc and filtered. The filter cake thus obtained was extracted with acidic methanol to give a crude powder of M-92 complex, which was then separated into A (acidic) and N (neutral or weakly acidic) groups. The A group components are soluble in alkaline water (pH 8.5), while the N group components are not. These components were further separated into six major components designated VA-2, BA-4, BA-5, BN-1, BN-2 and BN-3 by silica gel column chromatography. Components with the letter "V" are reddish violet and those with "B" are blue. The IR and UV spectra of these components suggest that their chromophores may be the same or very closely related to one another. The molecular formula of BN-3 was determined to be C29H23NO9 by mass spectrometry. The results of various spectroscopies on BN-3 suggest that M-92 components consisted of chromophores in which juglone (5-hydroxynaphthoquinone) is conjugated with naphthazarin (5,8-dihydroxynaphthoquinone).

Anti-Bacterial Agents↗