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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.

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.

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.

Evidence of the presence of polysaccharide polymers is shown in Streptomyces sp. for the first time. Cytochemical methods revealed the occurrence of polysaccharide granules in sporulating hyphae of Streptomyces viridochromogenes. Onset of the sporogenesis coincided with the appearance of the granules, which reached a maximum number during the early stages of maturation. The later stages of maturation showed a decrease of these granules, and in mature spores no granules were observed.

Culture fluids of Streptomyces tendae 4158 (ATCC 31210) contain a new kind of polypeptide alpha-amylase inhibitor, tendamistat (HOE 467). Several methods of isolating this inhibitor are described, including two rapid crystallisation methods, which produce homogeneous material. A characteristic of tendamistat is its tight-binding, pH-independent inhibition kinetics and the specific inhibition of the mammalian alpha-amylase form a stoichiometric 1:1 complex, which cannot be separated into its individual components by sodium dodecyl sulphate or molecular sieve chromatography. Studies of the mode of action reveal that the alpha-amylase-inhibiting activity is linked to the intact disulphide bridges of the inhibitor. It is assumed that the multipoint protein-protein bond exists between the enzyme and tendamistat. It is shown that extracellular tendamistat inhibits amylase formed by streptomyces. We therefore assume a regulatory function in the microorganism. By-products of tendamistat, which possess similar enzyme-inhibiting properties, are also described.

High-performance liquid chromatography analysis of the p-bromophenacyl esters of mycolic acids from whole organisms gave chromatographic patterns that were useful in differentiation of Rhodococcus and Nocardia species. Rhodococcus equi, R. erythropolis, and R. rhodochrous contained more-polar mycolic acids and were easily separated from the less-polar mycolic acid-containing species of R. sputi, R. bronchialis, R. corallinus, R. rubropertinctus, and R. terrae. The less-polar mycolic acid-containing Rhodococcus species showed chromatographic patterns that partially overlapped (in elution times) the patterns of Nocardia asteroides, N. otitidiscaviarum, and N. brasiliensis, but the larger number of peaks in the last species made separation between the genera possible. Distinct chromatographic patterns were found for most species, except for R. equi strains that showed two different patterns. Strains of R. rubropertinctus and R. terrae appeared identical. N. asteroides and N. otitidiscaviarum showed similar mycolic acid patterns.

Penicillin-binding proteins of three Streptomyces strains, S. cacaoi, S. olivaceus, and S. clavuligerus, were examined by gel electrophoresis and fluorography. In a beta-lactamase producer, S. cacaoi, at least five membrane-bound penicillin-binding proteins were detected, but in two beta-lactam producers, S. olivaceus and S. clavuligerus, fewer penicillin-binding proteins were detected. Mecillinam and methicillin bound selectively to some penicillin-binding proteins in S. cacaoi, whereas they did not bind at all to those in S. olivaceus and S. clavuligerus. Clavulanic acid bound to penicillin-binding proteins only at a very high concentration in S. cacoi. This compound did not bind to those of S. olivaceus and S. clavuligerus. Penicillin-binding proteins in culture supernatant and cytoplasm and minimum inhibitory concentrations of these strains against benzylpenicillin were also examined.

Coresistance to macrolide, lincosamide, and streptogramin B-type (MLS) antibiotics by a common biochemical mechanism characterizes clinically resistant pathogens. Of 10 streptomycetes tested for resistance to macrolide, lincosamide, and streptogramin B-type antibiotics, only 1, Streptomyces erythreus, the organism used for production of erythromycin, was found resistant to all three classes; moreover, it was the only streptomycete in the series tested found to contain N6-dimethyladenine (m62A) in 23S ribosomal ribonucleic acid, the structural alteration of ribosomal ribonucleic acid associated with clinical resistance. Of the seven streptomycetes tested for the presence of m62A and N6-methyladenine (m6A), two, S. fradiae and S. cirratus, which produce the macrolide antibiotics tylosin and cirramycin, respectively, were found to contain m6A, but not m62A. The remaining strains tested, including strains which produce lincomycin and streptogramins, contained neither m6A nor m62A.

The historical background of studies in Japan on chemical carcinogenesis from environmental sources is described from personal experience.

A method is described for the retrieval of streptavidin from the culture broth of Streptomyces avidinii. The key step in this procedure is the adsorption of streptavidin from culture concentrates to an affinity column in which iminobiotin is attached to AH-Sepharose 4B. This column binds streptavbidin at pH 11 and releases the protein at pH 4. The recovery of streptavidin is practically quantitative. The pH dependence of the iminobiotin-avidin affinity, discovered by Green [Green, N. M. (1966) Biochem. J. 101, 774-779], has thus found practical application. The streptavidin bound 4.07 +/- 0.02 mol of [14C]biotin per mol and was essentially homogeneous as judged by disc and slab gel electrophoresis. Streptavidin was extensively succinoylated without loss of biotin-binding capacity. The observations that 125I-labeled streptavidin and 125I-labeled succinoylstreptavidin are retained by iminobiotin-AH-Sepharose 4B columns at pH 7.5 and are eluted at pH 4.0 provides a convenient purification method for these iodinated proteins. The technique employed for the retrieval of streptavidin is generally applicable to the isolation of iminobiotinylated molecules.

The production, isolation, and some structural studies of an antifungal, antibacterial, and antiviral substance from cultures of Streptomyces hygroscopicus are described. This material, designated pantomycin, appears to be a polypeptide antibiotic with inclusion of fatty acids and carbohydrate residues. Amino acid analysis of pantomycin acid hydrolysates indicates that it contains threonine, serine, proline, glycine, alanine, valine, alloisoleucine, and an as-yet-unidentified amino acid which appears to be different from types encountered in proteinaceous materials. In addition to the aforementioned compounds, the antibiotic was shown to contain alpha-aminobutyric acid after hydrogenation. Analysis of ether extracts of the hydrolysate mixture indicated the presence of several fatty aicds; myristic, isotridecanoic, lauric, and undecylic acids. The amino and fatty acid composition of pantomycin is similar to the known antibiotic stendomycin. Pantomycin appears to also have at least one carbohydrate-like residue incorporated into its structure. The presence of carbohydrate was indicated by periodic acid-Schiff base staining of electrophoretic patterns as well as positive color formation in the phenol-sulfuric and Molisch tests, but the carbohydrate did not appear to be either a hexose or a pentose. The antibiotic, which appears to be dissociated in alcoholic solution, forms stable aggregates under aqueous conditons.

Cairomycin B in the fermentation broths of Streptomyces sp. strain AS-C-19 accompanied cairomycin A and cairomycin C. The cairomycins are peptides with potent activity against gram-positive bacteria. On acid hydrolysis, cairomycin A yielded valine and aspartic acid, whereas cairomycin C yielded lysine, glycine, valine, leucine, and aspartic acid, as identified by paper and gas chromatography. These amino acids were found to exist in their alpha-L form. Cairomycin A was tentatively assigned a 6-isopropyl-2,5-diketopiperazine-3-acetic acid structure. The three cairomycins were distinct from each other in their ultraviolet, infrared, and mass spectra; elemental analyses; and their chromatographic behavior in different developing solvents.

Covalently closed circular deoxyribonucleic acid (DNA) with a molecular weight of 20 X 10(6) was identified in strains of Streptomyces coelicolor A3(2) of various fertility types. Hybridization studies and digestion by various restriction endonucleases indicated that the circular DNAs (pSH1) were identical regardless of the fertility type (UF, IF, or NF) of the strain from which it was isolated. The pSH1 DNA was cleaved to many fragments by the endonucleases HincII, SmaI, and SalI and to three or four fragments by BamHI and PstI. Plasmid pSH1 carries single sites for each of the two restriction enzymes, EcoRI and HindIII. These sites are 7.6 X 10(6) daltons apart. Attempts to isolate the fertility factor SCP1 as covalently closed circular DNA were unsuccessful. These data suggest that the biochemically isolated plasmid pSH1 is not identical to the genetically characterized fertility factor SCP1, which has been identified in an autonomous state in IF-type strains and in an integrated state in NF-type strains.

The cell walls of Streptomyces antibioticus 39 contain a glycosylated poly(glycerol phosphate), in which the repeating monomeric unit is O-alpha-D-galactopyranosyl-(1--3)-O-2-acetamido-2-deoxy-beta-D-galactopyranosyl-(1--1)-glycerol monophosphate. The localization of the phosphodiester linkages between hydroxyl groups at positions 2 and 3 of the adjacent glycerol residues and the structure of the glycoside were established by 13C nuclear-magnetic-resonance spectroscopy. The spectral data are also in accordance with the results of the methylation analysis and enzyme degradation of the glycoside.

A liquid chromatographic method is described for analysis of aerobic actinomycetes for isomers of diaminopimelic acid. One or two colonies of organism were hydrolyzed with 6.0 mol of HCl per liter at 121 degrees C for 15 min. The hydrolysate was neutralized and buffered with an NaOH solution (3 mol/liter) containing 0.15 mol of sodium borate per liter. Precolumn derivatization with dansyl chloride was used to form a fluorescent product for detection. Analysis was performed by reversed-phase, ion-pair chromatography. The L-diaminopimelic acid isomer was detected in all 10 strains of Streptomyces tested, and the meso-diaminopimelic acid isomer was detected in all 10 strains of Nocardia tested. Liquid chromatography was compared simultaneously with thin-layer chromatography in the analysis of three strains of aerobic actinomycetes. Liquid chromatography required less growth of the organisms, and analysis was completed within 1 h, compared with the 3 to 5 days required by thin-layer chromatography.

The genome sizes of Streptomyces coelicolor and Streptomyces rimosus as calculated by deoxyribonucleic acid reassociation kinetics are approximately 10.5 X 10(6) nucelotide pairs.

Gamma-aminobutyric acid was isolated from ruptured cells of Streptomyces lincolnensis. Its isolation and purification by preparative liquid-solid chromatography and subsequent identification by gas chromatography-mass spectroscopy are reported.

Covalently closed circular deoxyribonucleic acid (DNA) was isolated from two strains of Streptomyces coelicolor A3(2), representing two of the known fertility types. In each of the two strains circular DNA of about 20 times 10-6 daltons could be detected, amounting to about 1.5% of the total cellular DNA. The possible function of this DNA is discussed.

A new type-II restriction endonuclease SphI, has been partially purified from Streptomyces phaeochromogenes. SphI recognizes the hexanucleotide sequence 5'-GCATGC and cleaves it at the position marked by the arrow. This nucleotide sequence is present twice in SV40 DNA, four times lambda DNA and only once in the cloning vehicles pBR322, pBR325, pBR327 and pBR328.

Crystallization conditions are reported for Streptomyces avidinii streptavidin with and without bound biotin. X-ray examination of the free and bound crystal forms shows the streptavidin-biotin complex crystals to be most suitable for high resolution structure analysis. A complete x-ray data set to 2.6 A resolution was collected for the streptavidin-biotin crystals using a two-dimensional area detector. Reduction and analysis of the x-ray diffraction pattern show that the complex crystallizes in the tetragonal space group I4(1)22 (a = b = 98.4 A, c = 125.8 A), with half of the streptavidin tetramer in the crystallographic asymmetric unit.

The ribosomal proteins from 11 Streptomyces strains representing various numerical taxonomic clusters were compared by two-dimensional PAGE. The protein patterns were specific for each species and were unaffected by acridine dye treatment, suggesting genetic stability of ribosomal proteins. An attempt was made to identify one strain of Streptomyces by both traditional taxonomic methods and analysis of the ribosomal protein patterns. Both methods identified the strain as Streptomyces lavendulae, and protein pattern analysis also showed that Streptomyces avidinii was closely related to this species. The practical application of ribosomal protein patterns in Streptomyces taxonomy was therefore demonstrated.

A streptomycete strain (FERM-P1185), isolated from soil, secreted a slime on glucose-asparagine agar, and produced viscous growth in liquid media containing peptone as nitrogen source. A purified polysaccharide isolated from the culture broth was composed of glucose and mannose units (molar ratio 1-87:1). Periodate oxidation, Smith degradation, methylation analysis, infrared and 13c nuclear magnetic resonance spectra indicated that this mannoglucan had a linear structure consisting of alpha-1,3- and alpha-1,4-linked glucopyranose and mannopyranose units.

Streptomyces venezuelae is a filamentous bacterium with branching vegetative hyphae embedded in the substrate and aerial hyphae bearing spores. The exterior of the spore is inlaid with myriads of tiny rods which can be removed with xylene. The spore wall is approximately 30 nanometers thick. Occasionally, it can be seen that the plasma membrane and the membranous bodies within a spore are connected. The spore's germ plasm is not separated from the cytoplasm by a nuclear envelope. The cell walls of the vegetative hyphae, which are about 15 nanometers thick, are structurally and chemically similar to those of gram-positive bacteria. The numerous internal membranous bodies, some of which arise from the plasma membrane of the vegetative hypha, may be vesicular, whirled, or convoluted. Membranous bodies are usually prominent at the hyphal apices and are associated with septum formation. The germ plasm is an elongate, contorted, centrally placed area of lower electron density than the hyphal cytoplasm. The spores differ from the vegetative hyphae, not only in fine structure, but also in the arginine and leucine contents of their total cellular proteins.

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.

Simple thin-layer chromatographic procedures are outlined for the separation, isolation, and characterization of a complex of lipophilic naphthoquinones. Procedures are also described for the quantitative recovery of naphthoquinones from thin-layer plates. The general usefulness of the described methods is demonstrated by their application in the analysis of menaquinones from several microorganisms. The methods allow distinction between menaquinones varying in side-chain length, degree of saturation, and geometry, as well as in the presence or absence of ring methyl groups.

To achieve a rapid identification of the cell wall components of actinomycetes, several modifications of the procedure of Cummins and Harris were developed. Purified cell walls were prepared by extraction of whole cells with 0.1 N NaOH. A 2-hr 2 N HCl hydrolysate was prepared for identification of sugars. A 2-hr 6 N HCl hydrolysate was prepared for amino acid analysis. Two-dimensional paper chromatography was run on 19-cm papers. Total time required for a single analysis was approximately 24 hr. This procedure gave qualitative results which were completely satisfactory for differentiation of certain species in the genus Actinomyces and in related genera.

The relationships among selected streptomycetes, nocardiae, and mycobacteria have been determined, based upon the base composition of their deoxyribonucleic acid (DNA) and upon the ability of their denatured DNA to anneal with single-stranded reference DNA. The streptomycetes constituted a homogeneous group whose DNA contained between 69 and 73 mole% guanine + cytosine (% GC). Moreover, the streptomycetes examined showed 37 to 88% homology with the Streptomyces venezuelae and S. rimosus reference DNA. The nocardial and mycobacterial DNA both contained 62 to 69% GC. The nocardial strains studied fell into either a 62 to 64% GC group or a 68 to 69% GC group, indicating that they should not be assigned to a single species. The nocardiae having 68 to 69% GC showed 24 to 44% homology with S. venezuelae reference DNA. In competition experiments, wherein unlabeled heterologous DNA interfered with binding of labeled homologous DNA, the nocardial DNA with 68 to 69% GC showed a greater degree of homology with the streptomycetes than did the nocardial DNA with 62 to 64% GC. In addition, the DNA from spores of S. venezuelae was cursorily examined, and interactions between S. venezuelae denatured DNA and polyribonucleotides were sought. The buoyant density of the DNA from S. venezuelae spores was distinctly less than that from mycelia. Moreover, denatured S. venezuelae DNA formed a dense complex with polyriboguanylate.