Search

The cell wall of Streptomyces rutgersensis var. castelarense contains structurally different chains of 1,3-type glycerol teichoic acid. Part of the molecules consisting of 20-25 monomers, carry on every third glycerol phosphate unit (at C-2) alpha-glucosamine residues, only half of which are N-acetylated. There are chains with O-lysine groups, and free nonsubstituted ones. The chain structure has been ascertained by chemical analysis and 13C- and 1H-NMR spectroscopy.

Thin-layer chromatography coupled with flame ionization detection was used to develop a method to separate and to determine simultaneously three polyether carboxylic ionophore antibiotics (abierixin, nigericin and grisorixin) produced by Streptomyces hygroscopicus NRRL B 1865. Various proportions of chloroform, methanol and formic acid (or acetic acid as a substitute for formic acid) were used in the developing solvent to determine changes in RF values of the antibiotics and to allow conditions for maximum resolution to be obtained. Development on Chromarods SII with chloroform-methanol-formic acid (97:4:0.6, v/v/v) gave satisfactory and reliable separations of the three polyether antibiotics. Under these conditions, the internal standard methyl desoxycholate was found to be suitable for their simultaneous determination in the lipid extracts of Streptomyces hygroscopicus NRRL B 1865.

The structure of WS1279, isolated from Streptomyces sp. as an immunoactive lipopeptide, has been deduced on the basis of chemical and physical evidence as S-[2,3-bis(palmitoyloxy)propyl]-N alpha-palmitoyl-Cys-Asn-Ser-Gly-Gly-Ser- OH. This was confirmed by synthesis.

C-1027-AG, a selective antagonist of antitumor antibiotic C-1027, was isolated by column chromatography on DEAE-cellulose, butyl-Toyopearl and Sephadex G-50 from a culture filtrate of Streptomyces globisporus. The amino acid sequence of purified C-1027-AG was determined with a protein sequencer on the basis of fragment peptides obtained by enzymatic hydrolysis with lysylendopeptidase, V8 protease, endopeptidase AspN and chymotrypsin, after performic acid oxidation. C-1027-AG is shown to consist of a single polypeptide chain cross-linked by two disulfide bonds, and to contain a total of 110 amino acid residues with alanine and glycine as its amino- and carboxyl-termini, respectively; its molecular weight was calculated to be 10,500 daltons. The primary structure of C-1027-AG is indicated to be identical to the protein moiety of C-1027, and is highly homologous to the sequences of antitumor proteins obtained from other Streptomyces species.

The calf thymus DNA (CT-DNA) and poly(dI-dC).poly(dI-dC) binding properties of the natural antitumor antibiotic CC-1065 and selected analogs of CC-1065 were studied by circular dichroism (CD) and absorbance methods. The results indicate that the intense long wavelength DNA-induced CD band of these molecules originates from a chiral electronic transition which is delocalized over the whole molecule. Both the covalently bound species (N-3 adenine adduct) and the reversibly bound species exhibit the characteristic spectral behavior of an inherently dissymmetric chromophore when these agents bind within the minor groove of B-form DNA. This mechanism of optical activity accounts for why CC-1065 shows a weak CD in buffer but a very intense induced CD at long wavelength when bound to DNA, why the intensity of the induced CD of CC-1065 analogs depends upon how many fused ring systems the analog contains, and why covalently bound analogs having the mirror image configuration of the natural configuration also exhibit an intense positive induced CD band at long wavelength.

Tautomycin inhibited the catalytic subunits of protein phosphatase-1 (Kiapp = 0.16 nM) more potently than protein phosphatase 2A (Kiapp = 0.4 nM), and the native forms of these enzymes in mammalian, protozoan and plant extracts were inhibited in a similar manner. Protein phosphatase 2B was inhibited 10,000-fold less potently, while two other phosphatases and six protein kinases were unaffected at 10 microM. Okadaic acid prevented the binding of tautomycin to protein phosphatase 2A, indicating a common binding site for both inhibitors. The different relative potencies of tautomycin and okadaic acid for protein phosphatases 1 and 2A suggest that parallel use of both inhibitors may help to identify physiological substrates for each enzyme.

A procedure is described for the purification of hydrophobic microbial proteins such as streptavidin from Streptomyces avidinii, using Benzyl-DC bead cellulose as the column material. The separation is rapid with a high loading capacity and sufficient resolution for preparative uses. Advantages are discussed especially for industrial purposes.

The three-dimensional structure of an alpha-amylase inhibitor, HAIM, composed of 78 amino acids, was analyzed by two-dimensional NMR techniques. Sequence-specific assignments were made for the amino acid residues from Ile-6 to Cys-72. Distance geometry analysis of the interresidue NOEs revealed that the HAIM molecule consists of two beta-sheets, as is the case in a homologous alpha-amylase inhibitor, Tendamistat, though one of its beta-strands is much shorter than that of Tendamistat. The combination of molecular modeling from Tendamistat and distance geometry analysis was confirmed to be useful for our purpose.

The sequence-specific resonance assignment of apo-neocarzinostatin from Streptomyces carzinostaticus was carried out from two-dimensional proton-NMR spectra. The assignments were obtained for the backbone protons of 111 of the 113 residues of the protein, missing the two C alpha H of one glycine but including 3 of the 4 prolines. The majority of side chain protons were also assigned. The secondary structure derived from the analysis of sequential connections corresponds to ten beta-strands separated by clearly identified loops and turns. Inter-strand connectivities and slowly exchanging amide protons confirm the presence of the two disulfide bridges from Cys37 to Cys47 and from Cys88 to Cys93 and indicate a global folding similar to that of the similar proteins, actinoxanthin and macromomycin, for which crystallographic data are available.

Leinamycin is a recently discovered antitumor antibiotic with an unusual 1,3-dioxo-1,2-dithiolane structure. It preferentially inhibits the incorporation of [3H]thymidine into the acid-insoluble fraction of Bacillus subtilis. In vitro, leinamycin causes single-strand cleavage of supercoiled double-helical pBR322 DNA in the presence of thiol cofactors. Scavengers of oxygen radical did not supress the DNA-cleaving activity. Thiol-activated leinamycin binds calf thymus DNA at 4 degrees C and thermal treatment of the leinamycin-DNA adduct released a chemically modified leinamycin from the complex. The lack of cytotoxicity and DNA-cleaving activity for S-deoxyleinamycin indicates that the 1,3-dioxo-1,2-dithiolane moiety is essential for the activity of leinamycin. Thus, the primary cellular target of leinamycin appears to be DNA. It binds DNA and causes single-strand break at low concentrations, which may account for the potent antitumor activity.

A computer-based comparison of the amino acid sequences of human placental 17 beta-hydroxysteroid dehydrogenase and Streptomyces coelicolor actIII protein, which is important in the synthesis of the antibiotic actinorhodin, gives an alignment score 12.15 standard deviations higher than that of 1000 comparisons of randomized sequences of these proteins. The probability of getting this score by chance is 3 x 10(-34). Comparison of actIII protein with Drosophila melanogaster alcohol dehydrogenase yields a score of 10.3 standard deviations (P = 3.5 x 10(-25)). Based on these similarities, we propose that 17 beta-hydroxysteroid dehydrogenase, actIII protein, and Drosophila alcohol dehydrogenase are derived from a common ancestor.

Probestin, a new inhibitor of aminopeptidase M, has been isolated from the culture broth of Streptomyces azureus MH663-2F6. The 1H and 13C NMR studies and amino acid analysis confirmed the presence of one 3-amino-2-hydroxy-phenylbutanoic acid, leucine and two proline residues in the molecule. Stereochemistries of these amino acids were determined by HPLC analysis. The fragmentation pattern shown in the mass spectrum and the chemical analysis on probestin clarified the amino acid sequence. Thus the structure of probestin was defined as (2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-L-leucyl-L-prolyl-L-pro line.

RK-286C, a new inhibitor of protein kinase C, has been found by the bleb-forming assay using K562 cells. It was produced by Streptomyces sp. RK-286 and purified by solvent extraction, silica gel chromatography and preparative HPLC. Spectrometric analysis revealed that the structure is 4'-demethylamino-4'-hydroxystaurosporine.

Resorthiomycin, a novel antitumor antibiotic, was isolated from the fermentation broth of a strain of Streptomyces collinus by ethyl acetate extraction, silica gel chromatography and HPLC. Resorthiomycin exhibited an in vitro cytotoxic activity against mouse leukemia L5178Y cells (IC50, 15.5 micrograms/ml) and also inhibited the clonogenic activity of a multidrug-resistant mutant of human hepatoma PLC/PRF/5 cells to a greater extent than that of the parental cells. On the other hand, this antibiotic does not possess any antibacterial or antifungal activity.

Probestin has been isolated as part of a program designed to find microorganism-produced inhibitors of aminopeptidase M from Streptomyces azureus MH663-2F6. It was purified by use of column chromatography of Amberlite XAD-4, silica gel, YMC-gel, Toyopearl HW-40, YMC D-ODS-5 (HPLC) and then isolated as colorless powders. Probestin is competitive with the substrate, and the inhibition constant (Ki) of it was 1.9 x 10(-8) M.

Analytical and preparative high-performance liquid chromatography of 3 phenazines and furonaphthoquinone derivative on reversed-phase column are described. The mobile phase was methanol and water. The injected amount of the mixture was about 30 mg for a preparative chromatographic run requiring 80 min. Substances were detected directly in the column effluent by UV detection.

A novel enzyme inhibitor against RNA-directed DNA polymerase of avian myeloblastosis virus was produced by an isolate of a new streptomycete for which the name Streptomyces retrostaticus is proposed. This enzyme inhibitor, which was named retrostatin, did not inhibit DNA-directed DNA polymerase of Escherichia coli and DNA-directed RNA polymerase of Ehrlich ascites tumor cells. Retrostatin was produced by the microorganism together with streptonigrin. These two substances were extracted from the culture broth with ethyl acetate at acidic pH. Retrostatin is an acidic pH indicator and the free acid was recovered as a red powder. Retrostatin had weak antibiotic activities against Gram-positive bacteria and yeasts.

The native or modified alpha-amylase inhibitor Hoe 467A - isolated from the culture medium of Streptomyces tendae 4158 - and overlapping peptides were degraded by the automatic Edman technique. The oxidized or aminoethylated or oxidized and maleoylated inhibitor was digested with trypsin and the native inhibitor with pepsin. Further digestion with Staphylococcus aureus proteinase was also carried out. After peptic digestion two cystin peptides were isolated, which allowed the establishment of the disulfide bonds. The alpha-amylase inhibitor is a polypeptid consisting of 74 amino-acid residues with a molecular mass of 7958 Da. The inhibitor is composed of all naturally occurring amino acids except methionine and phenylalanine and shows no sequence homology to known inhibitors. The clinical and pharmacological importance in respect to the inhibitors ability for inactivation of human salivary and pancreatic alpha-amylase is discussed. Especially the proteinase resistance of the inhibitor enables a clinical application in human (e.g. Diabetes mellitus) per os.

Crude preparations of DNA isolated from Act. rimosus 907, an oxytetracycline-producing organism and Act. fradiae 676, a neomycin-producing organism were analyzed with electrophoresis in agarose gel followed by electron microscopic analysis of DNA isolated from the gel. It was found that Act. rimosus and Act. fradiae contained ring molecules of DNA with the molecular masses of 37 . 10(6) and 59 . 10(6) dalton respectively.

Skeletal structure of copiamycin (1) (C54H95N3O17), a potent antifungal antibiotic, was determined from the physicochemical properties of this compound and of its degradation products. This compound consists of 32-membered polyhydroxy lactone ring, an alpha, beta-unsaturated ester group, as well as a side chain with a disubstituted guanidine moiety as its terminal. One of the hydroxyl groups (presumably at C-19) forms a hemiketal ring with the keto group at C-15, and another (at either C-21 or C-23) forms a hemiester with a malonic acid moiety.

The effects of L-valine and L-isoleucine on the composition of mycelial fatty acids were investigated during growth of differentiating parent strains of Streptomyces hygroscopicus and Streptomyces griseus as well as their non-differentiating derivatives (Amy-strains) on a synthetic medium. Both in the Amy+ and Amy- strains, in the presence of L-valine, the portion of the isopalmitic acid (iC16:0) increased, but the addition of L-isoleucine led to an elevated level of the 12-methyltetradecanoic acid (aC15:0). The results suggest that the genetically determined alterations in the ratio of both fatty acids in the non-differentiating derivatives may be due to specific changes in the biosynthetic pathways of both amino acid precursors rather than due to changes of their catabolism.

A plant pathogenic actinomycete identified as Streptomyces lydicus was isolated from the deep-pitted scab lesions of potato tubers. This strain produces a new polypeptide antibiotic named chandramycin. The antibiotic was isolated from culture broth by extraction with organic solvents and purified by chromatography. The purified antibiotic is a light-yellow crystalline compound soluble in water and in most organic solvents. Amino acid analysis of the acid hydrolysates of chandramycin revealed the presence of glycine, cis-methyl proline, valine, beta, beta-dimethylaminobutyric acid, beta-methyl-phenylalanine, and beta-2-thioazolyl-beta-alanine. The amino acid composition of chandramycin is qualitatively similar to that of a known antibiotic, bottromycin A2. Chandramycin showed activity against several gram-positive and a few gram-negative species of bacteria. It showed a strong activity against anaerobic microorganisms. Oral doses of antibiotic when administered up to 466 mg/kg of body weight failed to produce any observable toxic effect in mice.

A new peptide antibiotic named takaokamycin was isolated from a fermentation broth of Streptomyces sp. AC-1978, a soil isolate. It exhibits antibacterial activity against some Gram-positive bacteria. The molecular weight was found to be 1,130 on the basis of elemental analysis, FD-mass spectrum and 1H and 13C NMR. Acid hydrolysate of takaokamycin contains isoleucine, threonine and unidentified amino acids.

A method for spectrophotometric determination of p-aminoacetophenone (p-AAP) in the mycelium and fermentation broth filtrates of organisms producing polyenic macrolide antibiotics is described. The level of p-AAP accumulation was studied as applicable to the biosynthesis of levorin, a polyenic antibiotic of the heptaen series. The levels of p-AAP under usual conditions of the levorin-producing organism cultivation and under conditions of its stimulation with the products of the vital activity of yeast-like fungi were compared. Higher levels of p-AAP in the cultures grown with stimulation of the antibiotic biosynthesis were noted. Inadequacy of the correlation between production of levorin and p-AAP is discussed.

FR-900482 (4-formyl-6,9-dihydroxy-14-oxa-1,11-diazatetracyclo[7.4.1.02,7, O10,12]tetradeca-2,4,6-triene-8-ylmethyl carbamate), a new antibiotic with antitumor activity was isolated from fermentation broth of Streptomyces sandaensis. Its antitumor activities were studied and compared with that of mitomycin C (MMC) in animals. FR-900482 in doses of 0.32 approximately 10 mg/kg (ip) prolonged the life of mice bearing ascitic P388, L1210, B16, MM46, Ehrlich or EL4 tumors and rats bearing ascitic AH130 or AMC60 tumors. FR-900482 in doses of 5.6 approximately 18 mg/kg (iv) inhibited human LX-1, MX-1, SC-6 and LC-6 tumors xenografted sc in nude mice. FR-900482 was more effective than or equally effective to MMC in all the tumors used. FR-900482 was ineffective against cyclophosphamide-resistant P388, but was effective against MMC- or vincristine-resistant P388. The results suggest that FR-900482 may have clinical potential.

A new antibiotic U-62162 has been isolated from the fermentations of Streptomyces verdensis Dietz, sp. n. (UC-8157). The compound has been characterized and its gross structure has been elucidated. The antibiotic inhibited the growth of Gram-positive bacteria (particularly Staphylococcus aureus) but was inactive in experimentally infected animals.

A new antibiotic complex has been isolated from cultures of Streptomyces strain No. JA 10124. On the basis of taxonomic studies, the producing microorganism is described as Streptomyces griseoflavus (Krainsky, 1914) Waksman et Henrici, 1948, subsp. thuringiensis subsp. nov., type strain JA 10124. The antibiotic complex, designated as streptovirudin, was isolated from extracts of both mycelium and culture filtrate. It is a white amorphous material which consists of ten closely related components including streptovirudins A, B, C, D and E. The streptovirudin complex exhibits antibiotic activity against Gram-positive bacteria, mycobacteria, and various DNA- and RNA-viruses.

Carbazomycins C (III), D (IV), E (V) and F (VI), the minor components of the carbazomycin complex, were isolated from the cultured broth of Streptoverticillium ehimense together with carbazomycins A (II) and B (I). Among them, III and IV were shown to be new substances and their structures were elucidated as 4-hydroxy-3,6-dimethoxy-1,2-dimethylcarbazole and 3,4,6-trimethoxy-12-dimethylcarbazole, respectively, by spectroscopic and chemical means. The other components, V and VI, were found to contain an aldehyde function and were identified as carbazomycinal and 6-methoxycarbazomycinal, respectively. The antimicrobial activity of III and IV are also reported.

The presence of teichoic acids in a number of streptomycetes led to the conclusion that these biopolymers were widely spread in genus Streptomyces. The nature of the teichoic acid present in the mycelium was determined by extracting it with 10% trichloroacetic acid, precipitating it with ethanol and identifying the precipitated polymer by partial acid and alkali hydrolysis to alditol, alditol phosphates and glycosylalditol phosphates. Most strains examined in this survey contained glycerol or ribitol teichoic acids; in some cases neither type was detected. Structurally teichoic acids closely resemble those of other genera of gram-positive bacteria and in many cases represent poly(glycerol phosphate) and poly(ribitol phosphate) chains. The proportion of alditol residues bearing sugar substituents varied widely. Three species of genus Streptoverticillium contained glycerol teichoic acids. It is believed that some of the data presented in this paper might be used with some success in taxonomic studies of streptomycetes.

Aradioimmunoassay for (p)ppGpp was set up and applied to Streptomyces hygroscopicus. The (p)ppGpp specific antibodies obtained from rabbits immunized with ppGpp-human serum albumin could not distinguish between guanosine-5'-triphosphate-3'-diphosphate (pppGpp) and guanosine-5'-diphosphate-3'-diphosphate (ppGpp) whereas the cross reacitivity against structurally related nucleotides was negligible. Therefore, the antibodies were used for determination of (p)ppGpp, i.e. the sum of pppGpp and ppGpp. The pretreatment of biological samples for quantification of (p)ppGpp included formic acid extraction of cell pool followed by step gradient elution on DEAE-Sephadex A25 (Cl-) column for separation of (p)ppGpp from nucleotides with three or less phosphate groups. The very sensitive radioimmunoassay allowed determination of very low amounts: 1--50 pmoles (p)ppGpp per assay tube. The application of the RIA to S. hygroscopicus revealed a basal level in exponentially growing mycelium of about 2 pmoles (p)ppGpp per mg dry weight. The equilibrated pool size after induction of stringent response with serine hydroxamate was found to be about 30 pmoles (p)ppGpp per mg dry weight.

This paper reports the pH titration at 25 degrees C and the assignment of aromatic proton resonances of three tyrosyl residues of each identical subunit of Streptomyces subtilisin inhibitor (Mr 23000) by high-resolution 1H NMR spectroscopy. The complete assignments of the specific tyrosyl resonances were made based on the results of the differential chemical modification of the tyrosyl residues with tetranitromethane followed by peptide analysis, independently of the knowledge of the crystal structure. pKa values of Tyr-7, -75, and -93 were determined in a 2H2O solution to be 10.95, 11.8, and greater than or equal to 12.6, respectively, at 25 degrees C, whereas pKa values of nitrated Tyr-7 and -75 were determined to be 7.3 and 7.9, respectively. Tyr-93 was not nitratable under normal conditions. The strong resistance to nitration, together with the extremely high pKa value and the high-field shifted positions of the ring proton resonances of Tyr-93 at neutral pH, strongly suggests that Tyr-93 takes part in a hydrogen bonding as a proton donor. Tyr-7 is more easily modified with tetranitromethane than Tyr-75, although in the crystal Tyr-75 is more exposed than Tyr-7. The result, together with the pKa value of Tyr-75 significantly higher than that of a normal tyrosine, indicates that the microenvironment of Tyr-75 is more restricted in solution than in solid. These results imply that structural details of a protein may be quite similar in solution to those in the crystal in the rigid hydrophobic region of the protein but that in the surface region of the protein local structures may well differ between the solution and the crystal.

The composition of cell walls was comparatively studied in Streptomyces roseoflavus var. roseofungini 1128 and in its variant 1-68. In the logarithmic phase of growth, the content of teichoic acid in the cell wall of the parent culture was four times as high as in the cell wall of the variant. The cell walls of the parent culture contained 5 to 7 times more O-lysyl residues not only due to a higher content of teichoic acid in the walls but also owing to a lower content of lysyl groups in the teichoic acid of the variant. An additional polysaccharide comprising galactose and glucosamine was found in the cell wall of the variant but not in the parent strain. The peptidoglycan of the both cultures had a structure typical of Streptomyces spp.; its content in the cell walls of the two cultures was identical (ca. 50% of the dry cell wall biomass weight). The results are discussed in connection with the peculiarities of the variant hyphal septation.