Purification, characterization and antimicrobial properties of an antibiotic (wassumycin) from Streptomyces species 215.

A yellow amorphous compound, wassumycin, was isolated from an unidentified strain of Streptomyces. Wassumycin inhibited the growth of several species of human and animal pathogenic bacteria especially Clostridia. Wassumycin also inhibited the growth of HeLa cells, and showed good antitumour activity against experimental mouse tumours such as L1210, P388 leukaemias, and B16 melanoma. On the basis of the chemical and antimicrobial properties, the antibiotic appeared similar to the chromomycins and olivomycins, but its ultraviolet, infrared, and proton magnetic resonance spectra differed from theirs significantly.

Antifungal macrodiolide from Streptomyces sp.

Aerobic fermentation cultures of Streptomyces sp. produced an antifungal macrodiolide. This new antibiotic consists of two units of homononactic acid linked to form a cyclic diester. An unknown polypeptide was also isolated in trace quantities. The antibiotic with polypeptide complex showed high levels of antifungal activity compared with that of the macrodiolide alone. The macrodiolide also showed a stimulatory effect on some species of fungi. The production, purification, and characterization of these compounds are reported.

Quantification of azo-coupled lysine in azo proteins by amino acid analysis.

Hydrochloric acid hydrolysis of azo proteins in which lysine residues are azo coupled, results in conversion of modified lysines to alpha-amino-epsilon-hydroxy caproic acid plus alpha-amino-epsilon-chloro caproic acid. The latter can interfere with the determination of tyrosine by amino acid analysis. This potential problem can be avoided either by making basic and then neutralizing HCl hydrolysates or by hydrolyzing the protein in methane sulfonic acid.

Characterization of an antibiotic produced by a strain of Pseudomonas fluorescens inhibitory to Gaeumannomyces graminis var. tritici and Pythium spp.

The production, isolation, and characterization of an antibiotic substance from cultures of Pseudomonas fluorescens 2-79 (NRRL B-15132) is described. P. fluorescens 2-79 originally was isolated from the roots of wheat and is suppressive to the wheat root disease take-all caused by Gaeumannomyces graminis var. tritici. The antibiotic was isolated from potato glucose broth cultures of strain 2-79 by solvent extraction. It was purified by silica gel column chromatography and was a greenish yellow, needle-shaped crystal with a melting point of 242 degrees C (decomposition). It was soluble in methylene chloride, chloroform, acetone, 2 N sodium hydroxide, and 2 N hydrochloric acid and was insoluble in water, methanol, ethyl acetate, tetrahydrofuran, diethyl ether, carbon tetrachloride, hexane, and petroleum ether. On the basis of UV, infrared, 1H-nuclear magnetic resonance, 13C-nuclear magnetic resonance, mass spectral analysis, and elemental analysis, the structure of the antibiotic is proposed to be a dimer of phenazine carboxylic acid. Lithium aluminum hydride reduction of the antibiotic yielded hydroxymethyl phenazine as a major product which retained most of the biological characteristics of the parent molecule. There were no toxic symptoms when mice received this antibiotic by oral doses up to 464 mg/kg. The antibiotic showed excellent activity against several species of fungi, including the wheat pathogens Gaeumannomyces graminis var. tritici, Rhizoctonia solani, and Pythium aristosporum; and it may have a role in suppression of take-all in vivo by strain 2-79.

Treponemycin, a nitrile antibiotic active against Treponema hyodysenteriae.

Two strains of Streptomyces sp. (isolates 275 and 124) were isolated from soil samples collected from the fields around Lewiston, Idaho. Based on their cellular morphology and physiology, the two isolates were identified as Streptomyces albovinaceous. Both isolates produced an antibiotic when grown in liquid culture medium containing homogenized oats. The antibiotic (treponemycin) was isolated from the culture broth by solvent extraction and purified by silica gel column and preparative thin-layer chromatographies. Treponemycin is a crystalline light-yellow compound with an mp of 93 to 95 degrees C, levorotatory, and soluble in most organic solvents. It is sparingly soluble in water but insoluble in petroleum ether. On the basis of elemental analysis and mass spectral data, the molecular formula of the antibiotic was deduced to be C28H43O6N. The infrared spectrum of the antibiotic indicated the presence of unsaturation, nitrile, lactone, or ester or all three functions, and carbonyl functions in the molecule. A sharp infrared absorption band for nitrile at 2,220 cm-1 and the presence of an unsaturated group indicated that the nitrile function may be attached to an unsaturated carbon atom. The presence of dienenitrile functions was further supported by the UV absorption spectrum of the antibiotic, which gave a UVmax at 257 nm. The proton magnetic resonance spectrum of the antibiotic did not give any peak which could be exchanged with deuterated water, which is an indication of the absence of carboxylic and hydroxyl functions in the molecule. All of the functional groupings indicated by the infrared and UV spectra of the molecule were further confirmed by the 13C-magnetic resonance spectrum of the compound. A brief hydrogenation of the antibiotic yielded a biologically active tetrahydro derivative, whereas extended hydrogenation produced an inactive primary amine. Mild alkaline hydrolysis and subsequent esterification of the antibiotic with diazomethane produced an inactive dimethyl ester. Apparently both the nitrile and the lactone functions are essential for the treponomycin molecule to show antimicrobial activity. The antibiotic showed inhibitory activity against several species of bacteria, especially Treponema hyodysenteriae, the causative agent of swine dysentery. In view of the oral 50% lethal dose of 400 mg/g and its low MIC against four stains of T.hyodysenteriae, the antibiotic may have value as a swine dysentery therapeutic.

Production, purification, and characterization of chandramycin, a polypeptide antibiotic from Streptomyces lydicus.

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.

Grahamimycins: antibiotics from Cytospora sp. Ehrenb. W.F.P.L. 13A.

Aerobic fermentation of cultures of Cytospora sp. Ehrenb. W.F.P.L. 13A produces a group of novel, broad-spectrum, dilactone, macrocyclic antibiotics. These compounds, named grahamimycins, were isolated from the culture medium by extraction with organic solvents and separated by chromatography. Three active components have been obtained in crystalline form and have been designated grahamimycins A, A1, and B. The most active of these, grahamimycin A, exhibited activity against 36 species of bacteria, 8 species of blue-green algae (cyanobacteria), and 2 species of green algae, and antifungal activity against 5 fungi. Studies on the growth of the Cytospora with respect to formation of antibiotic activity, and the isolation, characterization, and structure elucidation of the grahamimycins are described. These studies indicate that the grahamimycins are distinct from other known antibiotics and represent a new class of antibiotic structures.

Some chemical and physical characteristics of pantomycin, and antiobiotic isolated from Streptomyces hygroscopicus.

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.

Immunological Comparisons of Chymotrypsin Inhibitor I among Several Genera of the Solanaceae.

Microcomplement fixation was employed to compare the immunological differences that occur between purified inhibitor I from potato tubers, the four purified protomers that comprise it, and inhibitor I from tuber and leaf extracts. Total inhibitors of chymotrypsin and trypsin in leaves of seven genera of the Solanaceae were identified by enzymatic assay. In leaves of three genera, Solanum, Lycopersicum, and Datura, chymotrypsin inhibitor I was identified immunologically. In petals of all seven genera inhibitor I was also identified immunologically. With the microcomplement fixation technique inhibitor I from leaf or petal extracts of eight Solanaceae genera were compared. An immunological relationship of inhibitor I among seven of these genera was established.