Frequent loss and restoration of antibiotic production by Streptomyces lasaliensis.

Antibiotic nonproducing variants of Streptomyces lasaliensis NRRL 3382R, which makes the polyether antibiotic lasalocid A (Las) and the quinoxaline antibiotic echinomycin (Ech), arose at a frequency of 3-11% after treatment with three different mutagens or regeneration of protoplasts compared with a spontaneous frequency of less than 0.1%. Cosynthesis of lasalocid A was not observed upon testing a large number of Las- mutants in different pair-wise combinations, nor did these mutants accumulate probable intermediates of lasalocid A biosynthesis. These results suggest that loss of the las genes or their expression is induced at a high frequency by mutagenic treatments. In fusions of protoplasts of a strain with the las+ ech+ spo+ nic-1 rif-3 markers with strains bearing the Las- LasS Ech- Bld- (or spo+) str-1 markers, Las+ Ech+ Spo+ StrR progeny were produced at a 61-89% frequency compared with a 1-9% frequency of StrR antibiotic producing progeny with the nic-1 or rif-3 genotypes. The more frequent restoration of antibiotic production than prototrophy or rifampicin sensitivity indicates that these antibiotic characters did not behave as normal chromosomal markers. Therefore the genetic instability might be due to the involvement of a plasmid in antibiotic production. The apparent lack of infectious transfer of the Las+ character to Las- parents in conjugal matings between the few strains tested and no correlation between the presence of a large plasmid, pKSL, and lasalocid A production in several strains of S. lasaliensis do not favor the latter hypothesis, but they do not conclusively disprove it. Consequently, we suggest that a plasmid or another mobile genetic element is controlling antibiotic production in S. lasaliensis.

Production of quinomycin A in Streptomyces lasaliensis.

In addition to lasalocid, an oligoether coccidiostatic compound, other compounds are synthesized by Streptomyces lasaliensis. Mutants producing either of two antibiotics, lasalocid A or quinomycin A (an antibiotic of quinoxaline character), were obtained by natural selection and by mutagenesis. Methods of isolation, purification and estimation of both compounds were established.

Studies on antibiotic biosynthesis by protoplasts and resting cells of Streptomyces echinatus. Part II. Effect of chromophore precursors.

Washed cell and protoplast suspensions from Streptomyces echinatus A8331, which produces the quinoxaline antibiotic echinomycin, have been used to study the effects of analogues of the natural chromophore upon antibiotic biosynthesis. Addition of quinoline-2-carboxylic acid caused a decrease in the labelling of echinomycin from L-[methyl-14C]methionine and an increase in labelled chloroform-extractable material. Quinoxaline-2-carboxylic acid increased the incorporation of radioactivity into both fractions. Thieno[3,2-b]pyridine-5-carboxylic acid, 6-methylquinoline-2-carboxylic acid, and quinoline-2-carboxylic acid (also to a lesser extent 7-chloroquinoxaline-2-carboxylic acid) increased markedly the incorporation of radioactivity into chloroform-extractable material and virtually abolished echinomycin synthesis. Autoradiographs of extracts from suspensions supplemented with the latter four analogues revealed bis-substituted metabolites not found in unsupplemented cultures. When protoplast suspensions were incubated with L-[U-14C]serine, L-[U-14C]valine, or DL-[benzene ring-U-14C]tryptophan, quinoline-2-carboxylic acid, thieno[3,2-b]pyridine-5-carboxylic acid, and 6-methylquinoline-2-carboxylic acid directed the synthesis of antibiotically active bis derivatives at the expense of echinomycin. When analogues of quinoxaline-2-carboxylic acid previously found unsuitable for incorporation by growing cultures were tested in protoplast suspensions, only isoquinoline-3-carboxylic acid caused a large increase in the incorporation of radioactivity from L-[methyl-14C]methionine into chloroform-extractable material. With DL-[benzene ring-U-14C]tryptophan as the radiolabel, benzotriazoline-2-acetic acid and 6-bromoquinoxaline-2-carboxylic acid as well as isoquinoline-3-carboxylic acid sharply reduced the labelling of echinomycin.

Incorporation of fluorotryptophan into triostin antibiotics by Streptomyces triostinicus.

The quinoxaline chromophores of the antibiotics produced by Streptomyces triostinicus are derived from tryptophan. Protoplasts of this organism made novel products when they were incubated with DL-5-fluorotryptophan or DL-6-fluorotryptophan. When added to batch cultures of the organism, DL-5-fluorotryptophan, at concentrations as low as 10 microM, inhibited both mycelial growth and triostin production, but gave rise to novel products. These have been characterized, using fast atom bombardment mass spectrometry, as novel triostins in which one or both of the quinoxaline rings contain an atom of fluorine. The chromatographic properties of the triostins arising from the incorporation of DL-5-fluorotryptophan are very similar to those of triostins containing chlorine or bromine at position 6 of the quinoxaline ring; they are clearly different from those having a chlorine atom at position 7. Accordingly, it is suggested that the carbon atom at position 5 of the indole ring of tryptophan ends up at position 6 of the quinoxaline ring system in triostins A and C.

Studies on antibiotic biosynthesis by protoplasts and resting cells of Streptomyces echinatus. Part I. The synthesis of echinomycin.

Washed suspensions of Streptomyces echinatus, and protoplasts derived from them, have been shown to synthesise echinomycin in the absence of growth. Protoplast suspensions free from significant contamination with unlysed mycelia are obtained by incubation with lysozyme followed by filtration through layers of tightly packed glass wool. Although physiologically young cells produce a better yield of protoplasts, optimal antibiotic biosynthesis is achieved with protoplasts prepared from mycelia about to enter the stationary phase of growth i.e., approximately 24 h after inoculation into a nutrient broth--salts seed medium. As judged by the incorporation of label from L-[methyl-14C]methionine, echinomycin synthesis proceeds for about 1 h after preparation of washed suspensions, but the kinetics of incorporation by intact cells and protoplasts are different. Uptake of labelled methionine by protoplasts is critically dependent upon the presence of sucrose as osmotic stabiliser and is drastically reduced if galactose, calcium, or magnesium is omitted from the suspending buffer. Uptake by intact, washed cells is essentially independent of nutrients in the medium. Small quantities of 11 materials other than echinomycin are detectable in chloroform extracts after labelling with L-[methyl-14C]methionine; some of these may represent precursors in the biosynthesis of the antibiotic. All amino acid constituents of echinomycin as well as tryptophan, a putative precursor of the quinoxaline chromophores, are actively incorporated into echinomycin by protoplasts and resting cells, but not with equal efficiency.

Directed biosynthesis of novel derivatives of echinomycin. II. Purification and structure elucidation.

New antibiotics produced by Streptomyces echinatus A8331 cultured in the presence of heterocyclic aromatic acids can be separated and purified by high-performance liquid chromatography using reversed phase columns. Natural quinoxaline antibiotics and certain quinoline derivatives can also be efficiently separated in normal phase systems. Details of purification procedures are described together with experiments to characterise the new antibiotics by field desorption mass spectrometry and proton magnetic resonance. Mono- and bis-substituted derivatives of echinomycin containing the following replacement chromophores have been isolated: 7-chloroquinoxaline-2-carbonyl, thieno[3,2-b]pyridine-5-carbonyl, and 6-methylquinoline-2-carbonyl. With a 6-methylquinoline-2-carboxylic acid precursor the analogues containing one or two replacement chromophores are each separable into two distinct components. One of the bis-substituted 6-methylquinoline products appears inactive in an antibacterial assay and behaves as a triostin analogue, presumably an immediate precursor of the corresponding echinomycin derivative.

Directed biosynthesis of novel derivatives of echinomycin by Streptomyces echinatus. I. Effect of exogenous analogues of quinoxaline-2-carboxylic acid on the fermentation.

Streptomyces echinatus A8331 cultured on a maltose minimal salts medium normally produces a single antibiotic, echinomycin (quinomycin A), containing two quinoxaline-2-carbonyl chromophores. Echinomycin is powerfully active against experimental tumours and can be assayed by its activity against Gram-positive bacteria. Grown in the presence of aromatic carboxylic acids related to quinoxaline, S. echinatus responds in favourable circumstances by incorporating the added material into analogues of the natural antibiotic having replacement chromophores. Both mono- and bis-substituted derivatives are formed. With quinoline-2-carboxylic acid as precursor, large quantities of analogues are produced, and the time course of synthesis, extraction, purification, assay, and characterization of the derivatives are described. Twenty-two other aromatic acids have been tested as potential substrates for antibiotic analogue biosynthesis. Half of them did not significantly affect growth and echinomycin production. Five appeared to stimulate antibiotic synthesis, while the remainder proved inhibitory. New biologically active antibiotics were detected in cultures supplemented with 7-chloroquinoxaline-2-carboxylic acid; 1,2,4-benzo-as-triazine-3-carboxylic acid; thieno[3,2-b]pyridine-5-carboxylic acid; and 6-methylquinoline-2-carboxylic acid.

A 15N nuclear magnetic resonance study of the biosynthesis of quinoxaline antibiotics.

Uniformly 15N-labelled triostin A and echinomycin have been prepared by growing the producing organisms on enriched media and their 15N nuclear magnetic resonance spectra partially assigned by a combination of nuclear Overhauser effect and scalar coupling constant measurements. Selective feeding experiments using unlabelled L-tryptophan-supplemented media have shown that N-1 and N-4 of the quinoxaline rings have their origins in the indole and amino groups of tryptophan, respectively.

Conversion of triostins to quinomycins by protoplasts of Streptomyces echinatus.

Protoplasts of Streptomyces echinatus have been used to investigate the biosynthesis of echinomycin (quinomycin A). It has been shown that this organism has the capacity to convert a series of triostins to the corresponding quinomycins by a mechanism involving methylation. Evidence is presented which suggests that triostin A is the natural precursor of echinomycin. Conversion of tetra-N-demethyl analogues of triostin A to corresponding analogues of echinomycin was not detected.

Effect of phosphate and amino acids on echinomycin biosynthesis by Streptomyces echinatus.

Streptomyces echinatus produces only echinomycin (quinomycin A), in contrast to other streptomycetes, which produce families of quinoxaline antibiotics differing in the amino acid composition of the oligopeptide (quinomycins A, B, B0, C, D, and E) or the structure of the sulfur-containing cross bridge (triostins A, B, and C). Attempts were made to establish conditions for directed biosynthesis with S. echinatus. The lability of the peptide lactone to alkaline pH was obviated by using high levels of phosphate or HEPES [4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid] buffer in the production medium. Maintaining the pH below 7.5 resulted in an apparent stimulation of production. Amino acids which serve as structural components or as precursors of echinomycin were employed singly or in combination with nitrate in a chemically defined medium. Based on specific yield (micrograms of echinomycin per milligram of mycelia [dry weight]), D- and L-serine, D-alanine, L-valine, and L-phenylalanine produced equivalent yields of antibiotic which were approximately twofold greater than yields obtained with nitrate alone. In contrast, L-alanine, beta-alanine, and L-threonine produced a three- to fourfold stimulation of production. Although the other amino acids diminished antibiotic production, L-isoleucine, which ostensibly was inhibitory to production, supported the accumulation of a quinoxaline antibiotic in which the cross-bridge sulfur lacked a methyl group.

Preparation and DNA-binding properties of substituted triostin antibiotics.

Novel derivatives of the triostin group of antibiotics were prepared by supplementing cultures of the producing organism Streptomyces triostinicus with a variety of aromatic carboxylic acids. Five new antibiotics, each having both the natural quinoxaline chromophores replaced by a substituted ring system, were purified to homogeneity and characterized by high-pressure liquid chromatography and nuclear magnetic resonance. Their antibacterial activities and DNA-binding properties were investigated. Addition of a halogen atom at position 6 of the quinoxaline ring or an amino group at position 3 had little effect on either the biological activity or the DNA-binding characteristics. The bis-3-amino derivative is fluorescent, and its fluorescence is strongly quenched by calf thymus DNA and polydeoxyguanylate-polydeoxycytidylate but not by polydeoxyadenylate-polydeoxythymidylate, suggesting that it binds preferentially to guanosine-cytosine-rich sequences in natural DNA. Binding constants for the bis-6-chloro and bis-3-amino derivatives do not differ greatly from those of unsubstituted triostin A. The analogs having two quinoline chromophores or a chlorine atom in position 7 of the quinoxaline ring display little or no detectable antibacterial activity, in marked contrast to the other congeners. Bis-7-chloro-triostin A binds conspicuously more tightly to polydeoxyadenylate-polydeoxythymidylate than to any other polynucleotide tested.