Streptomyces griseolus # 182--a novel organism producing oligomycin antibiotics. Taxonomy, fermentation, and isolation]

Target screening of natural immunosuppressors resulted in isolation of a strain of Streptomyces griseolus (No. 182) producing a complex of antifungal antibiotics. The strain proved to be an aerobe with the growth temperature of 26 to 28 degrees C. Morphological features and physiological properties of the strain were studied. Scanning electron microscopy revealed smooth, oval spores 1.10-1.25 mu in size. The findings showed that the strain belonged to Streptomyces griseolus. Unlike the previously described organisms producing the oligomycin complex the new strain formed straight or twisted sporophores and did not produce melanoid pigment or soluble pigment when grown on the Gauze mineral agar medium No. 1. The procedures for biosynthesis and chemical recovery of the antibiotic complex from the mycelium are described. The complex was shown to include 3 components at a ratio of 80:15:5 identified as oligomycins A, B and C respectively. The oligomycin complex was highly active against Aspergillus niger 137, Tolypocladium inflatum, Fusarium ocsisporum, Curvularia lunata 645 and Trichoderma alba F-32 (MIC 0.1-1.0 mcg/ml). The activity against yeast and bacterial cultures was observed only when the doses were higher than 100 mcg/ml.     

A study of the dicyclohexylcarbodiimide-binding component of the mitochondrial ATPase complex from beef heart

1. The binding of [14C]-dicyclohexylcarbodiimide to membrane proteins of beef heart mitochondria has been investigated using dodecylsulphate/polyacrylamide gel electrophoresis. Upon incubation of submitochondrial particles with low concentrations of dicyclohexylcarbodiimide (5 nmol/mg protein) radioactivity was incorporated into three components with apparent molecular weights of 30000, 18000 and less than 6500. Only the two smaller components were found to be extracted into chloroform/methanol. The same two components were labelled when the isolated ATPase complex or a reconstituted F0F1 system was incubated with low concentrations of dicyclohexylcarbodiimide. High concentrations of dicyclohexylcarbodiimide (20-100 nmol/mg protein) resulted in binding to several mitochondrial proteins. 2. The maximal amount of dicyclohexylcarbodiimide which can bind to submitochondrial particles, the isolated ATPase complex, and the reconstituted F0F1 system was found to exceed the amount required for maximal inhibition of the ATPase activity by several-fold. The distribution of the bound [14C]dicyclohexylcarbodiimide between the different dicyclohexylcarbodiimide-binding components was investigated as a function of dicyclohexylcarbodiimide concentration. The smallest and largest components revealed a high affinity for dicyclohexylcarbodiimide-binding which paralleled the inhibition of ATPase activity. The intermediate component had a markedly lower affinity for dicyclohexylcarbodiimide-binding. 3. The larger dicyclohexylcarbodiimide-binding component of the isolated ATPase complex can be converted into the smaller component by treatment of the ATPase complex with performic acid. Partial conversion can also be achieved by extraction of the band from the dodecylsulphate-polyacrylamide gel after electrophoresis, followed by re-electrophoresis. The observations suggest that the larger component may be an oligomer of the smaller one. 4. Using concentrations of oligomycin and dicyclohexylcarbodiimide which were equal to or greater than those required for maximal inhibition of the ATPase activity, oligomycin was found to diminish the binding of [14C]dicyclohexylcarbodiimide to both dicyclohexylcarbodiimide-binding components of the isolated ATPase complex.     

Amino acid composition of a new mitochondrially translated proteolipid isolated from yeast mitochondria and from the OSATPase complex

A new mitochondrially translated 10000 Mr proteolipid was isolated from yeast mitochondria. This proteolipid was purified by phosphocellulose chromatography, followed by reverse phase HPLC. This proteolipid was also extracted from the oligomycin sensitive ATPase complex and purified by HPLC. Its amino acid composition is different from the Dicyclohexylcarbodiimide binding protein.     

Analysis of the protein subunit structure of the oligomycin sensitive ATPase proteolipid fraction by reverse phase high pressure liquid chromatography.

The proteolipid fraction which is obtained from the bovine mitochondrial oligomycin sensitive ATPase complex by extraction with chloroform:methanol is resolvable into 7 components by preparative reverse phase high pressure liquid chromatography. Each of these 7 components is present together with 4 additionally resolved components in the proteolipid fraction which is obtained by extraction of submitochondrial particles with chloroform: methanol. Of the 7 components derived from the oligomycin sensitive ATPase, 4 have been identified with known protein subunits of the membrane sector of this complex, 2 are newly documented subunits of this complex, and 1 remains uncharacterized.     

Detection and identification of ferricrocin produced by ectendomycorrhizal fungi in the genusWilcoxina

The ectendomycorrhizal fungiWilcoxina mikolae isolates CSY-14 and RMD-947 andW. rehmii isolate CSY-85 were grown in pure culture under iron-limiting conditions. All three isolates tested positive for siderophore formation using both the ferric perchlorate assay and a sensitive HPLC iron-binding assay. A peptide siderophore was isolated from the culture medium by HPLC and shown to contain the amino acids serine, glycine and ornithine in a 1:2:3 ratio. This siderophore was identified as ferricrocin on the basis of electrospray mass spectroscopy and its co-chromatography in two different HPLC systems with ferricrocin isolated fromAspergillus fumigatus. Ferricrocin was the only siderophore isolated from theseWilcoxina cultures. This is the first report of siderophore formation by ectendomycorrhizal fungi.     

Structures of trichovirins II, peptaibol antibiotics from the mold Trichoderma viride NRRL 5243.

From the culture broth of the mold Trichoderma viride NRRL 5243 a mixture of polypeptides, named trichovirins (TV), could be isolated and purified by chromatography on XAD-2 adsorber resin and Sephadex LH-20 gel. Chromatography on silica gel using chloroform/methanol 8:2 as eluent provided a mixture of peptides named TV I. Subsequent elution with chloroform/methanol 1:1 yielded a second group of peptides named TV II. That group could be separated into individual components by repetitive HPLC on an octadecylsilyl and a fluorocarbon stationary phase. The sequences of 12 peptides of TV II could be determined by electrospray ionization tandem mass spectrometry of isolated peptides and gas chromatography-mass spectrometry of methanolysates. The N-termini of the 18-mer peptides are acetylated and the C-termini consist of leucinol. Owing to the presence of alpha-aminoisobutyric acid (Aib) residues and the bactericidal and hemolytic activity, the peptides belong to the family of peptaibol antibiotics.     

Study of the Antifungal Ability of Bacillus subtilis Strain PY-1 in Vitro and Identification of its Antifungal Substance （Iturin A）

A Bacillus strain,denoted as PY-1,was isolated from the vascular bundle of cotton.Biochemical,physiological and 16S rDNA sequence analysis proved that it should belong to Bacillus subtilis.The PY-1strain showed strong ability against many common plant fungal pathogens in vitro.The antibiotics producedby this strain were stable in neutral and basic conditions,and not sensitive to high temperature.From theculture broth of PY-1 strain,five antifungal compounds were isolated by acidic precipitation,methanolextraction,gel filtration and reverse-phase HPLC.Advanced identification was performed by mass spec-trometry and nuclear magnetic resonance spectroscopy.These five antifungal compounds were proved to bethe isomers of iturin A:A2,A3,A4,A6 and A7.In fast atom bombardment mass spectrometry/mass spec-trometry collision-induced dissociation spectra,fragmentation ions from two prior linear acylium ions wereobserved,and the prior ion,Tyr-Asn-Gln-Pro-Asn-Ser-βAA-Asn-CO~ ,was first reported.     

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.     

An acyl-thioesterase from yeast mitochondria

A previously unstudied acyl-coenzyme A thioesterase activity has been demonstrated in submitochondrial particles from Saccharomyces cerevisiae. The preferred substrate for the enzyme activity is oleoyl-coenzyme A. Tests with inhibitors of the thioesterase showed that, in addition to common thiol inhibitors, the oxidative phosphorylation inhibitors oligomycin and venturicidin also blocked thioesterase activity. Purification of the enzyme catalyzing this activity revealed that thioesterase copurified with mitochondrial ATPase. When thioesterase was isolated from oxidative phosphorylation mutants selected for resistance to these two inhibitors, thioesterase activity was also resistant. The results suggest that thioester hydrolysis may be catalyzed by components associated with the isolated ATPase complex. Further attempts to link this activity to in vivo function of ATPase were not successful.     

Interactions of the Complex Secretory Vesicle Binding Protein Chromobindin A with Nucleotides

Chromobindin A is a large, multisubunit protein that binds to chromaffin granule membranes in a Ca2+- and ATP-regulated manner. Ca2+ stimulates binding to the membrane, whereas ATP, in the the absence of Ca2+, is required for release of the protein from the membrane. We now report that spectral and HPLC data indicate that nucleotides are associated with the native chromobindin A complex and that the protein can bind two molecules of [3H]ATP in vitro. Chromobindin A also appears to be a novel nucleotide triphosphatase. ATPase activity was detected in fractions containing chromobindin A isolated by affinity chromatography, gel filtration, or ion exchange chromatography. Kinetic studies indicated that the Vmax is 44 nmol of Pi/mg/min and the Km is 0.115 mM, whereas the nonhydrolyzable ATP analog 5'-adenylylimidodiphosphate acts as a competitive inhibitor of this reaction with a Ki of 0.08 mM. The activity was found to be sensitive to protease treatment or to preincubation at 65 degrees C and was inhibited by Ca2+ or low pH. The ATPase activity was not inhibited by N-ethylmaleimide, N,N'-dicyclohexylcarbodiimide, vanadate, oligomycin, or azide.     

Purification and characterization of a beta-lactamase from Haemophilus ducreyi in Escherichia coli

A pCb plasmid encoding a beta-lactamase from Haemophilus ducreyi was transferred to Escherichia coli, purified, and characterized. The beta-lactamase could be isolated from a culture filtrate and further purified by ammonium sulfate and chelating Sepharose fast flow loaded with Zn(2+). The purified enzyme resulted in a major band at approximately 30-kDa on SDS-PAGE and its pI was determined to be 5.4. The beta-lactamase could hydrolyze both penicillin antibiotics including ampicillin, benzylpenicillin, and carbenicillin as well as cephalosporin antibiotics including nitrocefin, cephalothin, cephaloridine, and cefoperazone. However, benzylpenicillin was the best substrate. The enzyme activity was inhibited by clavulanic acid but not by boric acid, cefotaxime, ethylenediaminetetraacetic acid, or phenylmethylsulfonyl fluoride. The sequence of the beta-lactamase gene was also determined. It confirmed that the enzyme belonged to a class A beta-lactamase which had 99% identity to the ampicillin resistance transposon Tn3 of pBR322. Two nucleotides were different between the E. coli (Tn3) and H. ducreyi (pCb) genes that affected the amino-acid sequence. The valine at position 82 (ABL 84) was changed to isoleucine and the alanine at position 182 (ABL 184) was changed to valine. Genetic homogeneity among beta-lactamases is remarkable. Amino acid sequencing of some beta-lactamases has shown that substitution of only a few amino acids in the bla gene leads to high-level resistance against specific cephalosporins.     

Biosynthesis of edeine: II. Localization of edeine synthetase within Bacillus brevis Vm4.

Edeine-synthesizing polyenzymes, associated with a complex of sytoplasmic membrane and DNA, were obtained from gently lysed cells of Bacillus brevis Vm4. The polyenzymes-membrane-DNA complex, isolated from dells intensively synthesizing edeines (18--20 h culture) contained edeine B. Edeine B was found to be bound covalently t o the edeine synthetase. The amount of edeine bound to polyenzymes was 0.1--0.3 mumol/mg protein, depending on the age of cells. Detachment of deeine synthetase with a covalently bound edeine B from the membrane-DNA complex was accomplished by a treatment with (NH4)2-SO4 at 45--55% saturation or by DEAE-cellulose column fractionation. In contrast to other components of the complex, the edeine-polyenzymes fragment was not adsorbed to the DEAE-cellulose. Sephadex G-200 column chromatography separated the edeine-polyenzymes complex into 3 fractions. Edeine-polyenzymes complex, obtained from lysozyme-Brij-58-DNAase treated cells, contained edeine B bound to two protein fractions of mol. wt 210 000 and 160 000. Edeine-polyenzymes complex detached from the complex with the membrane and DNA contained edeine B, bound only to protein fraction of mol. wt 210 000. Edeine A was not found in the edeine-polyenzymes complex. No accumulation of free antibiotics within 16--22 h old cells of B. brevis Vm4 was detected. The edeine-polyenzymes complex associated with the DNA-membrane complex has shown no antimicrobial activity. By treating of above with alkali, edeine B of specific activity: 80 units/mjmol was released. The complex of DNA-membrane associated with edeine-polyenzymes complex was able to synthesize DNA, under the conditions described for synthesis, directed by a DNA-membrane complex. Edeine when associated with this complex did not effect the DNA-synthesizing activity.     

Dual inhibitory effects of the peptide antibiotics leucinostatins on oxidative phosphorylation in mitochondria

The effects of the hydrophobic peptide antibiotics leucinostatins A and B, originally isolated by Arai, T., Y. Mikami, K. Fukushima, T. Utsumi, and K. Yazawa. (J. Antibiotics (1973) 26: 157-161), on the functions of rat liver mitochondria were examined. At a concentration of 240 nM, these compounds completely inhibited state 3 respiration and ATPase activity that was stimulated by weakly acidic uncouplers. However, at higher concentrations, they induced uncoupling, probably by their protonophoric action. The uncoupling action was potentiated by known phosphoryl transfer inhibitors such as venturicidin, DCCD and oligomycin. The binding site of leucinostatins at lower concentrations was suggested to be located at, or very close to that of venturicidin. The potencies of the two analogues of leucinostatin were almost the same for all their actions. Their effects were very similar to those of the peptide antibiotics A20668's, which have been used as leucinostatins without any chemical and biological confirmation that they are in fact leucinostatins. Thus, the chemical structures of leucinostatins are thought to be analogues to those of the A20668's.     

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.     

The effect of cyclosporin A and oligomycin on the nonspecific permeability of the mitochondria inner membrane

The effect of oligomycin and cyclosporine A on Ca(2+)-induced nonspecific permeability of the inner mitochondrial membrane was under study. Both oligomycin and cyclosporine A were able to prevent the activation of nonspecific permeability; however, but cyclosporine A was the only agent which could restore the initial permeability of the inner mitochondrial membrane. The effect of cyclosporine A was not shown to be mediated through redistribution of Ca2+ ions between different subpopulations of mitochondria.     

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.     

Effect of cyclosporine A and oligomycin on non-specific permeability of the inner mitochondrial membrane

The effect of oligomycin and cyclosporine A on the induction of non-specific permeability of the inner mitochondrial membrane by Ca²⁺ was under study. Both oligomycin and cyclosporine A were able to prevent the activation of non-specific permeability, but cyclosporine A was the only agent which could restore initial permeability of the inner mitochondrial membrane. The effect of cyclosporine A was shown not to be mediated through redistribution of Ca²⁺ between different mitochondrial subpopulations     

Reconstitution of mitochondrial oligomycin and dicyclohexylcarbodiimide-sensitive ATPase

1. Oligomycin and dicyclohexylcarbodiimide-sensitive ATPase was isolated from beef-heart mitochondria and treated with 3.5 M NaBr in order to remove F1. The residue, called F0, was found to consist of seven components. Five of these are stained by Coomassie blue after dodecylsulfate-polyacrylamide-gel electrophoresis. Two of them correspond to the oligomycin-sensitivity-conferring protein and coupling factor F6, with apparent molecular weights of 21,000 and 9,400, respectively. Three additional polypeptides of molecular weights 23,000, 10,500 and 8,600 were not identified with known proteins. Two components not stained with Coomassie blue were detected by autoradiography of the gels of F0 preincubated with [14C]dicyclohexylcarbodiimide. These two components probably represent monomeric and oligomeric forms of the dicyclohexylcarbodiimide-binding protein. 2. F0 induced an oligomycin and dicyclohexylcarbodiimide-sensitive enhancement of K+ + valinomycin-driven proton translocation across the membrane of artificial phospholipid vesicles. 3. The interaction of F0 with purified, soluble beef heart F1 was investigated. F0 was capable of binding F1 and conferring oligomycin and dicyclohexylcarbodiimide sensitivity and cold stability on its ATPase activity. Furthermore F0 was found to diminish the specific activity of F1-ATPase. A comparison of these effects at varying F0/F1 ratios shows that F0 binds F1 in both an oligomycin-sensitive and an oligomycin-insensitive manner, and that both types of binding involve a conferral of cold stability and a decrease in specific activity. High F0/F1 ratios favoured in oligomycin-sensitive type of binding, indicating that F1 binds preferentially to oligomycin-sensitivity-conferring sites. Treatment of ATPase complex with trypsin resulted in an F0 with a decreased proportion of oligomycin-sensitivity-conferring binding sites and a diminished ability to lower the specific activity an cold lability of F1. 4. Reconstitution of F0 treated with trypsin and F1, oligomycin-sensitivity-conferring protein and F6 showed that at a constant amount of F1 bound, both oligomycin-sensitivity-conferring protein and F6 increased the oligomycin sensitivity of ATPase activity. It was therefore concluded that both of these coupling factors are involved in the conferral of oligomycin sensitivity. 5. The effect of the order of addition of F1, oligomycin-sensitivity-conferring protein and F6 to F0 on the reconstitution of oligomycin-sensitive ATPase activity, and of F1 and oligomycin-sensitivity-conferring protein to submitochondrial particles on the reconstitution of respiratory control, was investigated. The highest values of oligomycin sensitivity and respiratory control were obtained when F1 was added as the first component, indicating that F1 plays a directing role in the organisation of the components.     

Studies on the immunological properties of complex V (mitochondrial ATP synthetase complex)

Antibody raised in rabbits against Complex V (miochondrial ATP synthetase complex) purified from beef heart mitochondria cross-reacted with Complex V and submitochondrial particles from beef heart, beef adrenals, and rat liver as shown by double-diffusion and rocket immunoelectrophoresis analysis. Of the various isolated and purified components of Complex V, only the oligomycin sensitivity-conferring protein showed strong reactivity with the anti-Complex V antibody, soluble F₁-ATPase reacted very faintly, while F₆ and ATPase inhibitor protein showed no precipitin lines. Crossed immunoelectrophoresis indicated that antigenic determinants recognized by the antibody were present on OSCP and possibly on the dicyclohexylcarbodiimide-binding protein. The components of Complex V could be precipitated from beef heart submitochondrial particles dissolved in Triton X-100 and pretreated with control IgG. When the composition of the immunoprecipitate was compared to that of purified Complex V, all the constituent polypeptides of the latter were present in the immunoprecipitate, except for one polypeptide in the low-molecular-weight region. Incubation of Complex V or submitochondrial particles with the anti-Complex V antibody in the absence of Triton X-100 caused inhibition of ATP-P_i exchange but not of ATPase activity. In the presence of Triton X-100, oligomycin sensitivity of Complex V was lost and the antibody was able to inhibit also the ATPase activity. The enzymic activity of soluble F₁-ATPase was unaffected by the antibody in the absence or presence of Triton X-100. These results suggest that the anti-Complex V antibody might be a useful tool for identifying and probing the role of Complex V components involved in energy transduction.     

Isolation and physicochemical study of the components of the new antibacterial antibiotic 2562

An antibacterial antibiotic complex consisting of 2 components designated as 2562 A and 2562 B is produced by Streptomyces griseovarabilis. The antibiotic was isolated from the mycelium and purified chromatographically on a column with aqueous silicic acid. The study of the components showed that component 2562 A was chlorbiocin, while component 2562 B differed from the known antibiotics of this group. Physicochemical assays demonstrated that component 2562 B differed from chlorbiocin by the absence of the methyl group in pyrrol, which is probably attached to sugar at beta-position. It was found that component 2562 B is a new representative of the antibiotic cumero-glycoside group.