Comparative characteristics of some actnomycetes, producers of antifungal antibiotics related to chondamycin]

Three actinomycetous strains designated as LIA-0773, LIA-0783 and LIA-0780 were isolated from various soil samples. The cultures actively inhibited the growth of Trichophyton gipseum and produced a non-polyenic antibiotic of the chondamycin type. The strains were identified with Act. griseochromogenes Fukunaga et. al., 1955. The cultures differed within the species by some morphological, cultural, physiological and antibiotic properties, as well as by the component composition of the antibiotic produced. Thus, strain LIA-0773 had larger spiral sporophores, satisfactorily hydrolized starch and inverted sucrose. The strain inhibited the growth of not only the fungi but also grampositive bacteria and mycobacteria and produced an antibiotic composed of 6 components. Strain LIA-0780 had small sporophores with close spirals and low amilolytic activity. It inhibited only the growth of the fungi and produced a monocomponent antibiotic. Strain LIA-0783 was intermediate. By its biological properties it was closer to strain LIA-0780. The antibiotic produced by it consisted of 6 components, while by its physico-chemical properties the antibiotic was close to that produced by strain LIA-0780. All the 3 actinomycetous cultures were considered as different variants of Act. griseochromogenes Fukunaga et al., 1955.     

Investigation of the antibiotic complex produced by <Emphasis Type="Italic">Lactococcus lactis</Emphasis> subsp. <Emphasis Type="Italic">lactis</Emphasis> 194, Variant K

Phase variation in the culture of the environmental strain Lactococcus lactis subsp. lactis 194 resulted in the formation of two types of colonies differing by 15% in antibiotic activity. The active variant 194-K produced an antibiotic complex with a broad spectrum of antibacterial and antifungal activity. Five components (194-A, B, C, D, and E) were isolated from the complex by solid-phase extraction and thin-layer chromatography. Components 194-A and 194-B were hydrophobic neutral compounds soluble in organic solvents. Component 194-A possessed fungicidal activity, whereas component 194-B exhibited only bactericidal activity. Physicochemical studies of the isolated components 194-A and 194-B revealed that they had no analogs in the Berdy database of biologically active substances (BNPD) and appeared to be novel antibiotics. Component 194-C was a hydrophilic polar compound inhibiting growth of gram-positive and gramnegative bacteria. Component 194-D belonged to peptide antibiotics; it inhibited growth of only gram-positive bacteria and was similar to nisin A in biological properties but differed in electrophoretic mobility and molecular mass.     

New heptaene antibiotic, flavomycin, formed by Act. flavus var. geptinicus var. nov]

An actinomycetous culture LIA-0734 was isolated from a soil sample. By its morphologo-cultural properties it was close to Act. flavus and differed from the latter in the sporophores, colour of the substrate mycelium on synthetic media amd markedly pronounced antagonism with respect to yeasts and yeast-like fungi. The culture was classified as Act. flavus var. geptinicus var. nov. The actinomycete produced new aromatic heptaens: flavomycins A and B. Their physico-chemical and biological characteristics and singularity are presented.     

Streptomyces coerulatus, a producer of the nonaromatic heptaene, LIA-0735

An actinomycete designated as Streptomyces coerulatus LIA-0735 was isolated from a soil sample collected in the Alma-Ata region. The strain produces a nonaromatic heptaenic antibiotic with a very low inhibitory effect on the growth of gram positive bacteria, yeasts and actinomycetes. By a number of features antibiotic LIA-0735 differs from the known heptaens of the nonaromatic group.     

Streptoverticillium griseoviridum n. sp., a producer of the candidin-amphotericin B group, antifungal heptaene nonaromatic antibiotic 0185]

An actinomyceteous strain LIA-0185 producing a heptaenic non-aromatic antibiotic of the candidin type was isolated from a soil sample taken in the Georgian SSR under the programme of screening antifungal antibiotics. The taxonomic study of the strain showed that it belonged to the series of viridoflavum and had the following main taxonomic features: the sporophores in the whorls, straight, remote: the aerial mycelium from yellow to dark-olive-grey; the substrate mycelium olive; the soluble pigment absent; the melanine pigment was produced on the peptone medium; the culture formed H2S; assimilated glucose, mannose, inozide and to a lesser extent fructose; did not assimilate arabinose, xylose, sucrose, lactose, ramnose and raffinose. The strain inhibited the growth of yeast and fungi, grampositive bacteria and actinomycetes and produced a complex of non-aromatic heptaenic antibiotics. The actinomycete differed from the other whorl cultures. It was classified as a new species Sv. griseoviridum sp. nov. The antibiotic complex was a mixture of 2 components, i. e. I and II present approximately in equal amounts. Component II was analogous to candidin. Component I was a new original substance.     

Actinomyces fulvoviolaceus ver. achromogenes var. nov., a producer of the new heptaene complex, fulvomycin]

An actinomycetous culture designated as LIA-0721 was isolated from a soil sample. It was close to Act. fulvoviolaceus by its morphologo-cultural features and differed from it in production of melanoid pigments and the spectrum of carbohydrate assimilation. This justified its classification as Act. fulvoviolaceus var. achromogenes var. nov. The culture produced new aromatic heptaens, i. e. fulvomycins A, B and C. Their physico- chemical and biological characteristics are presented.     

Antitumor proteins of Streptomyces macromomyceticus: purification and characterization of auromomycin, macromomycin A, and macromomycin D

Macromomycin A and the two related proteins auromomycin and macromomycin D were isolated from the culture filtrates of Streptomyces macromomyceticus by chromatography on columns of DEAE-cellulose, Amberlite XAD-7, and decylagarose. Antibodies prepared against macromomycin A showed antigenic identity by Ouchterlony double diffusion between the three purified proteins. This similarity was further demonstrated by their behavior on disc gel electrophoresis, the amino acid compositions, and comparative peptide mapping of the aminoethylated derivatives. They differed, however, in other chemical and biological properties. Auromomycin and macromomycin A, pI 5.4, have antibiotic activity, which is absent in macromomycin D, pI 5.2. This antibiotic activity was associated with chromophore groups that were extractable by methanol. High-pressure liquid chromatography of the methanol extracts gave difference profiles for each of the purified proteins. The differences in the three proteins extended to their ultraviolet-visible spectra, fluorescence and circular dichroism, and the changes of these properties with heating. The heat denaturation, with auromomycin and macromomycin melting at 70.5 degrees C and macromomycin D at 57.0 degrees C, was reversible. Changes were noted in the spectra both during and following heating at 80 degrees C; the antibacterial activity was lost in auromomycin and only partially reduced in macromomycin A. The properties of the three proteins support the general similarities in their polypeptide structures, modifications in the properties of which are endowed by the differences in the associated nonprotein chromophores.     

Results of the long laboratory storage of Streptoverticillium hachijoense (Jamaguchi) Baldacci, the producer of trichomycin]

Viability, morphologo-cultural features and antibiotic properties of Sv. hachijoense, strain LIA-0052 stored for 10 years in a dry state and in the state of a resting culture were studied. Spores and mycelium of 2-week strains most stable to some chemical and physical factors were used for drying. It was found that viability of strain LIA-0052 was maintained for a longer period of time after lyophilization, in garden soil and agar culture under a layer of mineral oil. By the end of the observation period the viability of the soil culture decreased and the morphologo-cultural properties were stabilized. When the strain was cultivated on media with sucrose, the level of its antibiotic activity increased.     

Purification of Antibiotics from Physarum Gyrosum by High Pressure Liquid Chromatography

A family of five antibiotic substances was isolated from the slime mold Physarum gyrosurn by high pressure liquid chromatography (HPLC). For this purpose, mold was cultured for two weeks in a liquid medium. Soluble products were harvested by rotary evaporation of medium and extraction with 1-butanol. Paper chromatography in ethyl acetate :pyridine:water (2:2:1 v/v) was used for preliminary fractionation. Active components were separated by HPLC with a reverse-phase column packed with Bondapack C₁₈/Porasil B (Waters Associates) and were eluted with a linear gradient of methanol:water increasing from 70 to 100% methanol over 90 minutes. Puri-fication was completed by rechromatographing individual fractions. Purity of the active components was verified by HPLC and thin layer chromatography. Activity assays against Bacillus cereus showed these materials to be bacteriostatic rather than bacteriocidal.     

云南红豆杉内生放线菌TAR11活性代谢产物的初步研究

目的：初步研究从云南红豆杉植株根部筛选到的一株抗植物病害的内生放线菌TAR11的活性代谢产物性质。方法：以枯草芽孢杆菌为指示菌、以抑菌活性为指标,测定TAR11发酵液的最小抑菌浓度;用不同温度、pH值处理,了解活性物质的稳定性;用有机溶剂对活性物质萃取和溶解,并用纸层析对活性物质进行初步分类。结果：TAR11发酵液抗菌物质的最小抑菌浓度为0．78％,对温度敏感,在酸性和中性条件下稳定,可被三氯甲烷萃取,能溶于水、甲醇、乙醇、丙酮、乙醚。结论：低浓度的放线菌TAR11代谢产物能强烈抑制枯草芽孢杆菌活性,经纸层析实验初步鉴定为一类碱性抗生素。放线菌TAR11有望开发成为新一代生物药物。     

Detection and characterization of the Gloeosporium gloeosporioides growth inhibitory compound iturin A from Bacillus subtilis strain KS03

The Bacillus subtilis strain KS03 was isolated, and identified as a biological control agent that inhibits the anthracnose disease fungus Gloeosporium gloeosporioides. The antifungal compound was purified from its culture broth through butanol extraction, diethylaminoethyl (DEAE) Sepharose CL-6B chromatography, and preparative thin layer chromatography. Tandem mass spectrometric analyses (MS/MS), with matrix-assisted laser desorption ionization (MALDI) time-of-fight/time-of-flight (TOF/TOF) mass spectrometry, showed that the antifungal compound was iturin A, a cyclic lipopeptide antibiotic. The major compound, with a molecular mass of 1042 Da, was identified as iturin A(2).     

New species of the genus Nocardia and its antibiotic properties]

A culture of a new species of Nocardia, i.e. N. indigoensis producing an antibiotic close to celicomycins was isolated from a soil sample of Kazakhstan plated on the selective medium with kanamycin. By a number of chemical properties and biological activity the antibiotic differed from celicomycins. Probably it is a new natural substance.     

Streptomyces multispiralis nov. sp. and a New Antibiotic,Neohumidin

A streptomycete isolated in our laboratories was found to produce a new antibiotic effective for the control of sheath blight of the rice plant. As a result of taxonomical study it was proved to belong to a new species and therefore it was named Streptomyces multispiralis nov. sp. From its fermented broth, the antibiotic was isolated and crystallized from benzene. After the comparison of its physicochemical and biological properties with those of known antibiotics, it was considered to be a new antibiotic and named neohumidin. It inhibited the growth of certain phytopathogenic fungi, saprophytic fungi, yeasts and gram-positive bacteria.     

Purification and Properties of Component B of 2,4,5-Trichlorophenoxyacetate Oxygenase from Pseudomonas cepacia AC1100

Pseudomonas cepacia AC1100 degrades 2,4,5-trichlorophenoxyacetate (2,4,5-T), an herbicide and chlorinated aromatic compound. Although some progress has been made in understanding 2,4,5-T degradation by AC1100 by molecular analysis, little is known about the biochemistry involved. Enzymatic activity converting 2,4,5-T to 2,4,5-trichlorophenol in the presence of NADH and O(inf2) was detected in cell extracts of AC1100. Phenyl agarose chromatography of the ammonium sulfate-fractionated cell extracts yielded no active single fractions, but the mixing of two fractions, named component A and component B, resulted in the recovery of enzyme activity. Component B was further purified to homogeneity by hydroxyapatite and DEAE chromatographies. Component B had a native molecular weight of 140,000, and it was composed of two 49-kDa (alpha)-subunits and two 24-kDa (beta)-subunits. Component B was red, and its spectrum in the visible region had maxima at 430 and 560 nm (shoulder), whereas upon reduction it had maxima at 420 (shoulder) and 530 nm. Each mole of (alpha)(beta) heterodimer contained 2.9 mol of iron and 2.1 mol of labile sulfide. These properties suggest strong similarities between component B and the terminal oxygenase components of the aromatic ring-hydroxylating dioxygenases. Component A was highly purified but not to homogeneity. The reconstituted 2,4,5-T oxygenase, consisting of components A and B, converted 2,4,5-T quantitatively into 2,4,5-trichlorophenol and glyoxylate with the coconsumption of NADH and O(inf2).     

Bacereutin, an antifungal antibiotic isolated from metabolites of Bacillus cereus CHU 130

An antifungal metabolite, bacereutin, was isolated from culture filtrate of Bacillus cereus CHU 130. The bacterium was isolated from soils collected in Changhwa County, Taiwan, and was grown in soybean meal-mannitol broth for production of the antibiotic metabolite. The antibiotic metabolite was isolated by adsorption column chromatography of Amberite XAD-2 and was purified by passing through the chromatographic columns packed with Dowex 50W-X8, Sephadex LH 20 and Biogel P-2. The antibiotic metabolite was soluble in water and 87% acetone, and was slightly soluble in methanol, but was not dissolved in n-propanol, n-butanol, acetone, benzene and ethyl acetate. The antibiotic metabolite was a heat-stable and ninhydrin-positive substance. The antibiotic activities of bacereutin were tested by means of the agar-diffusion plate method. The antibiotic metabolite inhibited the growth of Saccharomyces cerevisiae CHU 1, Paecilomyces variotii CHU 6, Rhizomucor miehei CHU 40 and Fusarium oxysportum CHU 98. Bacereutin was a ninhydrin-positive antifungal antibiotic.     

Preparation and Ultraviolet Light-Induced Transformation of an Antifungal Mixture of Heptaene Antibiotics of Streptomyces surinam

Conditions for the production and isolation of an antifungal antibiotic mixture (DJ400) were investigated. Different preparations of DJ400 may contain at least 12 different heptaenes, which were characterized by partition chromatography (peak number) and ultraviolet (UV) spectra (types A, B, C). Irradiation with UV light transformed the predominant UV spectrum of type B into A. Comparison of untreated and UV-irradiated products indicated that the main components may exist in two forms with identical structures except for an all trans-heptaene system in A compounds and one internal cis double bond in B compounds. The ratio of the major components 6B and 8B depended on the media composition. Component 4B is probably a precursor of 6B; 10B and 12 may be precursors of 8B.     

Conversion of biosynthetic human proinsulin to partially cleaved intermediates by collagenase proteinases adsorbed to isolated rat adipocytes.

Studies of the biological activity of proinsulin have resulted in widely varying conclusions. Relative to insulin, the biological activity of proinsulin has been reported from less than 1% to almost 20%. Many of the assays in vitro for the biological potency of proinsulin have utilized isolated rat adipocytes. To examine further the interaction of proinsulin with rat adipocytes, we prepared specifically-labelled proinsulin isomers that were iodinated on tyrosine residues corresponding to the A14, A19, B16 or B26 residue of insulin. These were incubated with rat adipocytes and their metabolism was examined by trichloroacetic acid precipitation, by Sephadex G-50 chromatography, and by h.p.l.c. chromatography. By trichloroacetic acid-precipitation assay, there was little or no proinsulin degradation. By G-50 chromatography and subsequent h.p.l.c. analysis, however, we found that the labelled proinsulin isomers were converted rapidly and almost completely to materials which eluted differently on h.p.l.c. from intact proinsulin. This conversion was due primarily to proteolytic activity which adsorbed to the fat cells from the crude collagenase used to isolate the cells. Two primary conversion intermediates were found: one with a cleavage at residues 23-24 of proinsulin (the B-chain region of insulin), and one at residues 55-56 in the connecting peptide region. These intermediates had receptor binding properties equivalent to or less than intact proinsulin. These findings show that isolated fat cells can degrade proinsulin to intermediates due to their contamination with proteolytic activity from the collagenase used in their preparation. Thus the previously reported range in biological activities of proinsulin in fat cells may have arisen from such protease contamination. Finally, the present findings demonstrate that a sensitive assay for degradation of hormones is required to examine biological activities in isolated cells.     

The antineoplastic antibiotic polypeptide 308 produced by Actinomadura recticatena. Its isolation and physicochemical properties

A antitumor antibiotic belonging to the group of polypeptide antibiotics containing chromophore was isolated from the culture of Actinomadura recticatena Terekhova, Preobrazhenskaya et Galatenko, 1984, strain 308. Biosynthesis, isolation, physicochemical and biological properties of the antibiotic are described. The results of elemental analysis, the melting point, optical properties, UV, IR and NMR spectra and the data on acid hydrolysis showed that antibiotic 308 was most closely related to antibiotic BBM-928 A.     

黄曲霉拮抗菌Bacillus amyloliquefaciens B10-6-1产生的脂肽类抗生素的分离和鉴别

本文旨在对黄曲霉拮抗菌Bacillus amyloliquefaciens B10-6-1的脂肽类抗生素相关基因进行克隆,对其所产生的脂肽类抗生素进行组分分离,对有抑菌活性的脂肽类抗生素进行结构鉴别。以解淀粉芽孢杆菌B10-6-1基因组DNA为模板,采用PCR方法对ItuA、ItuB、ItuC、ItuD、FenA、FenB、FenD、Sfp、bmyA、bmyB、bmyC合成基因进行DNA扩增。将该菌发酵液经过离心、酸沉淀、甲醇抽提等步骤得到脂肽类抗生素的粗提物,采用高效液相色谱法分离,对收集的洗脱峰组分进行体外抑菌试验,对有抑菌活性的组分进行液质（HPLC-ESI-MS）分析。PCR扩增检测结果显示黄曲霉菌拮抗菌B10-6-1能扩增出ItuA、ItuC、ItuD、FenA、FenD、Sfp、bmyA、bmyB、bmyC合成基因,未能扩增出ItuB、FenB合成基因。高效液相色谱法收集到7种组分,体外抑菌试验证明组分5和7有抑菌活性。经液质测定,组分5和组分7分别为脂肽类抗生素C₁₄Bacillocnycin D和C₁₅Bacillocnycin D。本研究探明了黄曲霉菌拮抗菌B10-6-1所产生的天然抑菌活性成分,为该菌用于黄曲霉菌的生物防治奠定了理论基础。     

Trapping of an intermediate in the reaction catalyzed by 5-oxoprolinase

Bacterial 5-oxoprolinase is composed of two protein components: Component A, which catalyzes 5-oxoproline-dependent ATP-hydrolysis and Component B, which couples the hydrolysis of ATP with the decyclization of 5-oxoproline to form glutamate (Seddon, A. P., Li, L., and Meister, A. (1984) J. Biol. Chem. 259, 8091-8094). Studies on this unusual enzyme system have led to evidence that an intermediate is formed by Component A. Application of the isotope-trapping method demonstrated an activated 5-oxoproline intermediate, whose formation requires ATP, Mg2+, and Component A. The amount of ATP-dependent trapping was close to the number of enzyme active sites. The intermediate formed by Component A was shown to be reducible by potassium borohydride to proline in low yield; when Component B was added, the formation of proline was abolished. Treatment of reaction mixtures containing Component A, 5-oxoproline, and [gamma-32P] ATP with diazomethane led to appearance of a 32P-labeled compound (found on thin layer chromatography), whose formation was significantly reduced when Component B was present. The new compound, which is labile, breaks down to form dimethyl[32P]phosphate. The total amount of dimethyl[32P]phosphate formed after breakdown is close to the number of active sites of Component A. The data are consistent with the conclusion that a phosphorylated form of 5-oxoproline is formed by Component A and suggest that Component B is required for conversion of this intermediate to glutamate.