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[Isolation of antibiotic-producing Actinomycetes from soil samples exposed to UV light].

The use of nonroutine means in isolation of microorganisms from natural substrates extended the possibilities of detecting new cultures which often appear to be producers of previously unknown antibiotics. A new procedure for isolating actinomyces of definite groups was developed. It implies preliminary exposure of soil suspensions to UV light. With the use of the procedure, 2539 strains of actinomycetes belonging to different genera were isolated. There was a marked decrease after the irradiation in isolation of cultures belonging to Streptomyces, a genus most widely distributed in nature and studied in detail while isolation of cultures belonging to other genera, promising as sources of novel antibiotics, increased. Micromonospora, Amycolatopsis and Nocardia proved to be the most stable to the effect of UV light. With the use of the procedure it is possible to increase 2-3-fold isolation of cultures belonging to Micromonospora, a genus known as a producer of many antibiotics including those used clinically.

Actinomycetales↗

[Use of selective media with lincomycin for the directed screening of antibiotic producers].

Relation between lincomycin resistance of Micromonospora cultures freshly isolated from soil samples and their capacity for production of antibiotics related to lincomycin by the structure or mode of action was shown. 32 cultures of Micromonospora were isolated from soil platings containing 50--100 microgram/ml of lincomycin. Crude antibiotic substances were recovered with the method of organic solvent extraction from 10 cultures possessing pronounced antibiotic activity. Selective inactivity (MIC more 1000 microgram/ml) of the crude substances with respect to the lincomycin resistant variant of Staph. aureus 209 p was observed, 2 of them having no inhibitory effect on the erythromycin resistant variant of the staphylococcus. The crude antibiotics inhibited the growth of the initial strain of the staphylococcus and its other antibiotic resistant variants in concentrations of 0.5--10 microgram/ml. It was demonstrated with the use of gas chromatography and mass spectrometry that one substance was lincomycin and 4 substances were known macrolides. Efficiency of the simple method of directed screening of antibiotics belonging to definite groups is indicated. Resistance of actinomycetes freshly isolated from natural substrates to various antibiotics is used as the criterion for antibiotic screening. The method provides detection of various antibiotics which are analogs in the structure or mode of action of the selecting antibiotic used for the screening.

Anti-Bacterial Agents↗

CHEMICAL COMPOSITION OF CELL-WALL PREPARATIONS FROM STRAINS OF VARIOUS FORM-GENERA OF AEROBIC ACTINOMYCETES.

Cell-wall preparations were made from more than 140 strains of aerobic actinomycetes representing most of the form-genera that have been proposed. All cell-wall preparations contained as major constituents glucosamine, muramic acid, alanine, and glutamic acid. In addition, cell-wall preparations from various types of streptomycetes and strains of Microëllobosporia contained glycine and ll-alpha,epsilon-diaminopimelic acid; those from strains of most Actinoplanaceae and micromonosporae contained glycine and meso-alpha-epsilon-diaminopimelic acid; those from strains of Thermoactinomyces, Microbispora, Dermatophilus, and nocardiae of the madurae-pelletieri group contained meso-alpha,epsilon-diaminopimelic acid; and those from strains of Thermomonospora, Micropolyspora, and most nocardiae contained meso-alpha,epsilon-diaminopimelic acid, arabinose, and galactose. All the strains used were also studied morphologically.

Actinomycetales↗

COMPARISON OF THE CELL-WALL COMPOSITION OF MORPHOLOGICALLY DISTINCT ACTINOMYCETES.

Yamaguchi, Tatsuro (The University of Tokyo, Tokyo, Japan). Comparison of the cell-wall composition of morphologically distinct actinomycetes. J. Bacteriol. 89:444-453. 1965.-Cell-wall composition of various morphologically distinct actinomycetes was studied to determine the relationship, if any, between cell-wall composition and morphological criteria in actinomycete taxonomy. The methods used were similar to those of Cummins and Harris. At least five types of cell-wall composition were obtained; however, these were not always correlated with groupings by the conventional classification system. For instance, the sporangium-forming actinomycetes, Actinoplanaceae, had three types of cell-wall composition; the composition of cell walls of Promicromonospora, Micromonospora, and Microbispora was the same as, or similar to, that of Actinomyces, Actinoplanes, and Streptosporangium, respectively; Chainia, Actinopycnidium, Actinosporangium, and Microellobosporia had the same cell-wall composition as Streptomyces, whereas that of Streptoverticillium was slightly different. Possible implications of cell-wall composition and morphological differentiation of hyphae for the taxonomy and phylogeny of actinomycetes are also discussed.

Actinobacteria↗

Total solid-phase synthesis of the azathiocoraline class of symmetric bicyclic peptides.

Thiocoraline is a potent antitumor agent isolated from the marine organism Micromonospora sp. This symmetric bicyclic depsipeptide binds the minor groove of DNA. Here we report two solid-phase strategies for the syntheses of azathiocoraline and its analogues. The thioester linkage was replaced by an amide bond to improve the compound's pharmacokinetic properties. The first strategy is based on a convergent (4+4) approach, whilst the second is a stepwise synthesis, cyclizations in both approaches occurring on the solid support. These two strategies were designed to overcome problems caused by the presence of consecutive noncommercial N-methyl amino acids, to avoid epimerization during cyclization and/or fragment condensation, and to form the disulfide bridge under solid-phase conditions. The heterocyclic moiety was added in the last step of the synthesis to assist the preparation of libraries of new compounds with potential therapeutic applications.

Amino Acids↗

[Enzyme inhibitors from actinomycetes. II. Acid protease inhibitors].

In the course of a screening program directed toward the isolation and evaluation of inhibitors of carboxyl (acid) proteinases, we have found 0.39% of the investigated strains of microorganisms as producers of pepsin inhibitors. In more than 1543 strains which were isolated from various geographical areas and deposited with the IMET Culture Collection, Jena-GDR, and which belong to different genera of actinomycetes such as Streptomyces, Actinomadura, Actinoplanes, Micromonospora and non-identified strains, we have found 6 Streptomyces strains only. Surprisingly, the screening efficiency of the used casein agar spot test and the fibrin-blue-technique was very low. As compared with the inhibitor activity of pepstatin the yield of the fermentation broth of 3 wild type strains ranged from 6 to 27 micrograms/ml. A significant influence of the composition of culture fluids on the inhibitor yield was observed. Supplements by crude glutamine or mixtures of amino acids in culture fluids increased the yield from 3 to 15 times as compared with that of fermentation broth without supplements. After NMG-mutagenic treatment and use of familiar selection procedures 3.1% inhibitor-blocked mutants were detected among 480 strains selected.

Actinomycetales↗

Selection and characterization of a neuraminidase-minus mutant of influenza virus and its rescue by cloned neuraminidase genes.

A neuraminidase (NA)-deficient mutant, designated NWS-Mvi, of the reassortant influenza virus A/NWS/33HA-A/tern/Australia/G70c/75NA (H1N9), was selected by passaging virus in MDCK cells in a medium containing neuraminidase from the bacterium Micromonospora viridifaciens and polyclonal antiserum against the influenza NA. Growth of the resulting mutant virus is dependent on the addition of neuraminidase to the medium. Western blot analysis showed that the neuraminidase protein was absent from the mutant virus particles, and Northern hybridization showed that RNA segment 6, which contains the coding information for the NA, had undergone massive deletion. Viral protein synthesis in cells infected with the mutant virus was not dependent on the addition of neuraminidase. In the absence of a functional NA, the NWS-Mvi mutant virus can infect MDCK cells with normal cytopathic effects. This neuraminidase-minus influenza virus serves as an excellent source of parent virus for reverse genetics experiments involving genes that encode a functional neuraminidase.

Animals↗

Growth and product formation of actinomycetes cultivated at increased total pressure and oxygen partial pressure.

Influence of pressure (P) and oxygen partial pressure (PO2) on cultivation of various Streptomyces spp. and Micromonospora purpurea was examined in pressurized air-lift and stirred tank fermenters. The maximum PO2 was 2100 mbar. Growth and product formation of all cultures tested were markedly influenced by PO2 higher than 1000 mbar. There is evidence that wild strains are more oxygen tolerant than production strains. At a certain PO2 the metabolic activities of all cultures were inhibited. However, results obtained with S. aureofaciens and S. rimosus indicated an increase in specific product formation rate at elevated pressure. With increase in oxygen tension incorporation of oxygen into tetracycline molecules was enhanced. Since elevated oxygen tension can either show inhibiting effects or may be used for regulation of product formation and selectivity, the influence of PO2 should be determined in an appropriate experimental set-up for each process.

Aerobiosis↗

Protoplasts of Trichoderma viride: formation and regeneration.

High yields of protoplasts from the 18-hr old mycelium of Trichoderma viride were obtained by using the lytic system, produced by Streptomyces venezuelae RA and Micromonospora chalcea grown on a synthetic medium containing laminarin and chitin, when 0.7 M MgSO4 or (NH4)2SO4 were used as osmotic stabilizers. Regeneration of these protoplasts occurred through the production of an abortive tube and direct germination of the protoplasts. Regeneration could also take place in the medium used to produce protoplasts, but the process was different in many details.

Bacteriolysis↗

Chemical and structural differences in mycelial and regeneration walls of trichoderma viride.

When incubated in Winge medium, protoplasts from Trichoderma viride obtained by treatment with Micromonospora chalcea or Streptomyces venezuelae RA lytic systems first synthesized an aberrant wall, different from the normal one; it was aseptate, larger and irregular in size and length. They then regenerated a new wall, similar to the original one from which they were liberated. Analysis showed that the wall polymers were mainly beta-(1-3) glucan, beta-(1-6) glucan and chitin in the normal walls, whereas chitin was absent in aberrant tubes. These results are discussed below together with electron micrographs of aberrant and normal walls.

Carbohydrates↗

Cell wall enzymatic lysis of the yeast form of Pullularia pullulans and wall regeneration by protoplasts.

During the process of degradation of the cell wall of the yeast form of Pullularia pullulans by the lytic system of micromonospora chalcea samples were withdrawn at different times and observed under phase contrast and electron microscope. The progressive lysis of the walls reveals a fibrillar component inside the apparently amorphous wall. Freeze etched preparations of cells during the formation and regeneration of protoplasts show that the cellular membrane is split and this method allows the smooth external face of the membrane and other internal face covered by particles to be seen. The fact that the smooth face of the membrane is only visible during the preparation or the regeneration of protoplasts and very rarely when intact cells are fractured, suggests a strong adherence between cell wall and this external layer of the membrane. During the regeneration which takes place as in most of the yeasts and moulds, a special study of the extension of the cell wall is made and a possible mechanism for this extension of the regenerated cell wall is proposed.

Cell Wall↗

Improved processes for the production and isolation of dynemicin A and large-scale fermentation in a 10,000-liter fermentor.

Supplementing the culture of Micromonospora chersina sp. nov. No. M956-1 with NaI (0.5 mg/l) enhanced the production of dynemicin A by 35-fold in shake flask culture. Homogeneous dynemicin A was obtained from the whole broth extract by Dicalite chromatography, Sephadex LH-20 chromatography and vacuum liquid chromatography. Gram quantities of dynemicin A were obtained from the fermentation of M. chersina sp. nov. No. M956-1 in a 10,000-liter fermentor.

Anthraquinones↗

Taxonomy and biotransformation activities of some deep-sea actinomycetes.

Deep-sea soft sediments from trench systems and depths in the northwestern Pacific Ocean ranging from less than 300 to 10,897 m in depth have been analyzed for three target genera of actinomycetes: Micromonospora, Rhodococcus, and Streptomyces. Only culturable strains, recovered at atmospheric pressure on selective isolation media, have been examined to date. Maximum recoveries of culturable bacteria were greater that 10(7)/ml wet g sediment, but actinomycetes comprised a small proportion of this population (usually less than 1%). The target actinomycetes were isolated at all depths except from the Mariana Trench sediments. Actinomycete colonies were defined initially on the basis of colony morphologies, and preliminary identification then was made by chemotaxonomic tests. Pyrolysis mass spectrometry (PyMS) of deep-sea mycolic acid-containing actinomycetes gave excellent correspondence with numerical (phenetic) taxonomic analyses and subsequently was adopted as a rapid procedure for assessing taxonomic diversity. PyMS analysis enabled several clusters of deep-sea rhodococci to be distinguished that are quite distinct from all type strains. 16S rRNA gene sequence analysis has revealed that several of these marine rhodococci have sequences that are very similar to certain terrestrial species of Rhodococcus and to Dietzia. There is evidence for the intrusion of terrestrial runoff into these deep trench systems, and the inconsistency of the phenotypic and molecular taxonomies may reflect recent speciatiion events in actinomycetes under the high-pressure conditions of the deep sea. The results of DNA-DNA pairing experiments point to the novelty of Rhodococcus strains recovered from hadal depths in the Izu Bonin Trench. Biotransformation studies of deep-sea bacteria have focused on nitrile compounds. Nitrile-metabolizing bacteria, closely related to rhodococci, have been isolated that grow well at low temperature, high salt concentrations, and high pressures, suggesting that they are of marine origin or have adapted to the deep-sea environment.

Actinomycetales↗

Bioconversion of 19-deformyl-5-O-desosaminyl tylonolide to 19-deformyl-4'-deoxydesmycosin (TMC-016).

Bioconversion of 19-deformyl-5-O-desosaminyl tylonolide (1) to 19-deformyl-4'-deoxydesmycosin (3) was performed by using a mycinamicin-producing Micromonospora species. Compound 1 at low concentrations was completely converted to compound 3, however, the conversion to compound 3 was suppressed at high concentrations and 19-deformyl-4'-deoxy-2", 3'-di-O-demethyl-desmycosin (2), an intermediate leading to compound 3 accumulated.

Biotransformation↗

Gentamicins.

Gentamicin is a new broad-spectrum antibiotic, basic and water-soluble, produced and developed by Schering Corporation-Bloomfield, New Jersey (1967 and 1968). It is produced by Micromonospora purpurea, a member of a genus of microorganisms from which no other antibiotics have been derived. Paper chromatographic techniques showed the components of gentamicin complex designated as C', C'a, and C2. Gentamicins are bactericidal antibiotics, active in vivo in low concentrations against a wide spectrum of both Gram-positive and Gram-negative organisms. Among the responsive Gram-positive groups of microorganisms are Staphylococcus aureus including many resistant penicillinase producing strains and group A betahemolytic Streptococci. Among the clinically more important species of Gram-positive organisms responsive to gentamicin are both indole-positive and indole-negative Proteus, Pseudomonas, Escherichia coli, Aerobacter, Klebsiella, Salmonella, and Shigella. The production of gentamicins was improved by adding cobalt to the growth medium.

Bacteria↗

The three domains of a bacterial sialidase: a beta-propeller, an immunoglobulin module and a galactose-binding jelly-roll.

BACKGROUND: Sialidases, or neuraminidases, have been implicated in the pathogenesis of many diseases, but are also produced by many non-pathogenic bacteria. Bacterial sialidases are very variable in size, often possessing domains in addition to the catalytic domain. The sialidase from the non-pathogenic soil bacterium Micromonospora viridifaciens is secreted in two forms with molecular weights of 41 kDa or 68 kDa, depending on the nature of the carbohydrate used to induce expression. RESULTS: We report here the X-ray crystal structures of the 41 kDa and 68 kDa forms of the sialidase from M. viridifaciens at 1.8 A and 2.5 A resolution respectively. In addition, we report a complex of the 41 kDa form with an inhibitor at 2.0 A resolution, and a complex of the 68 kDa form with galactose at 2.5 A. The 41 kDa form shows the canonical sialidase beta-propeller fold. The 68 kDa form possesses two additional domains, one with an immunoglobulin-like fold that serves as a linker to the second, which is homologous to the galactose-binding domain of a fungal galactose oxidase. CONCLUSIONS: The presence of the additional carbohydrate-binding domain in the 68 kDa form of the bacterial sialidase reported here is a further example of a combination of carbohydrate binding and cleaving domains which we observed in the sialidase from Vibrio cholerae. This dual function may be common, but only to other bacterial and parasitic sialidases, but also to other secreted glycosidases involved in pathogenesis. The bacterium may have acquired both the immunoglobulin module and the galactose-binding module from eukaryotes, as the enzyme shows a remarkable similarity to a fungal galactose oxidase which possesses similar domains performing different functions and assembled in a different order.

Amino Acid Sequence↗

Mutagenesis of the conserved active-site tyrosine changes a retaining sialidase into an inverting sialidase.

Mutagenesis of the conserved tyrosine (Y370) of the Micromonospora viridifaciens sialidase changes the mechanism of catalysis from retention of anomeric configuration to an unprecedented inverting mechanism in which water efficiently functions as the nucleophile. Three mutants, Y370A, Y370D, and Y370G, were produced recombinantly in Escherichia coli, and all are catalytically active against the activated substrate 4-methylumbelliferyl alpha-D-N-acetylneuraminide. The Y370D mutant was also shown to catalyze the hydrolysis of natural substrate analogues such as 3'-sialyllactose. A comparison of the pH-rate profiles for the wild-type and the Y370D mutant sialidase reveals no major differences, although with respect to the kinetic term k(cat)/K(m), an ionized form of the aspartate-370 enzyme is catalytically compromised. For the wild-type enzyme, the value of the Brønsted parameter beta(lg) on k(cat) is 0.02 +/- 0.03, while for the Y370D mutant sialidase beta(lg) = -0.55 +/- 0.03 for the substrates with bad leaving groups. Thus, for the wild-type enzyme, a nonchemical step(s) is rate-limiting, but for the tyrosine mutant cleavage of the glycosidic C-O bond is rate-determining. The Brønsted slopes derived for the kinetic parameter k(cat)/K(m) display a similar trend (beta(lg) -0.30 +/- 0.04 and -0.74 +/- 0.04 for the wild-type and Y370D, respectively). These results reveal that the tyrosine residue lowers the activation free energy for cleavage of 6'-sialyllactose, a natural substrate analogue, by more than 24.9 kJ mol(-1). Evidence is presented that the mutant sialidases operate by a dissociative mechanism, and the wild-type enzyme operates by a concerted mechanism.

Amino Acid Sequence↗

Structure and mechanism of action of an inverting mutant sialidase.

Mutagenesis of the conserved tyrosine (Y370) of the Micromonospora viridifaciens sialidase to small amino acids changes the mechanism of catalysis from retention of anomeric configuration to inversion [Watson, J. N., et al. (2003) Biochemistry 42, 12682-12690]. For the Y370G mutant enzyme-catalyzed hydrolysis of a series of aryl sialosides and 3'-sialyllactose, the derived Brønsted parameters (beta(lg)) on k(cat) and k(cat)/K(m) are -0.63 +/- 0.05 and -0.80 +/- 0.08, respectively. Thus, for the Y370G enzyme, glycosidic C-O bond cleavage is rate-determining. Analysis of the activity of the Y370G mutant and wild-type enzymes against a substrate [3,4-dihydro-2H-pyrano[3,2-c]pyridinium alpha-d-N-acetylneuraminide (DHP-alphaNeu5Ac)] whose hydrolysis cannot be accelerated by acid catalysis is consistent with these reactions proceeding via S(N)1 and S(N)2 mechanisms, respectively. The overall structure of the Y370G mutant sialidase active site is very similar to the previously reported wild-type structure [Gaskell, A., et al. (1995) Structure 3, 1197-1205], although removal of the tyrosine residue creates two significant changes to the active site. First, the anomeric oxygen atom of the hydrolysis product (beta-N-acetylneuraminic acid) and four water molecules bind in the large cavity created by the Y370G mutation. Second, the side chain of Asn310 moves to make a strong hydrogen bond to one of the bound water molecules.

Aspartic Acid↗